PRODUCTION AND STUDY OF HUMAN PARATHYROID HORMONE

ANTIBODIES

A thesis presented for the degree of doctor of philosophy

by

IBRAHIM ALI MOHAMED BSc, MSc, AIMLS

23rd December 1988 University of Surrey

Division of Clinical Biochemistry Department of Biochemistry

Guildford, Surrey England

ProQuest Number: 10804275

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ABSTRACT

In this study, the immunogenidty of hPTH(l-34) was assessed in mice and rats, using different routes of immunization and two forms of hPTH(l-34), free and conjugated. The responder rats and mice were used for the production of monoclonal antibodies to hPTH(l-34). Several mouse-mouse and rat-mouse hybridoma cell lines were pro­duced. The antibodies secreted were examined for various characteristics and used for the detection of hPTH(l-34) in biological specimens.

Polyclonal antibody was also produced in goat against the free hPTH(l-34). Lymphocytes isolated from goat blood were thereafter used in the production of monoclonal antibodies. Fusion of goat B- lymphocytes with both mouse NS1 myeloma cell line and hetero­myeloma (ovine-murine hybridoma) resulted in five cell lines secreting goat monoclonal antibodies to hPTH(l-34).

To m y parents

ACKNOWLEDGMENT

I am deeply indebted to my supervisors Dr. R. Hubbard and Dr. J. Chakraborty for their guidance and advice throughout the period of this project.

I also would like to thank the following: Dr. S. Hampton for help with antisera production and helping with some of the materials used in this work, Guildhay antisera Ltd. for the supply of some materials used in this project, Dr. J. M. Zanelli for help with iodination, Dr. G. Carter for providing the labelled bovine parathyroid hormone, and Mr N. K. Cheikh for assistance with computing and typing.

I wish to thank all my colleagues, friends and all members of the Biochemistry Departement who made the right environment for this work to be carried out, and with whom I have the pleasure of working with.

Finally my thanks are due to the Higher Institute of Technology for providing the financial support for this project.

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Table of Contents

ABSTRACT............................................................................................. i

DEDICATION......................................................................................... ii

ACKNOWLEDGMENT ......... iii

CONTENT .............................................................................................. iv

CHAPTER 1: INTRODUCTION .............................................. 1

1.1 INTRODUCTION................................................................ 2

1.2 IMMUNE SYSTEM............................................................. 3

1.2.1 Functional cells of the immune system ........................... 3

1.2.2 Immune response ..................................................... 4

1.2.3 Antibody structure and function ..................................... 6

1.3 MONOCLONAL ANTIBODY ................ 9

1.3.1 Immunization ............................................................... 11

1.3.1.1 In vivo immunization ....................................................... 11

1.3.1.2 In vitro immunization ....................................................... 13

1.3.2 Fusion partners ................................................................... 14

1.3.2.1 Murine myeloma cell lin e s ............................................... 14

1.3.2.2 Human myeloma cell lin es ............................................... 14

1.3.3 Fusogen.......................................................................... 15

1.3.3.1 Virus fusion ........................................................................ 17

1.3.3.2 Polyethylene glycol ........................................................... 18

1.3.3.3 Electro-fusion..................................................................... 18

1.3.4 Hybridization ...................................................................... 19

1.3.4.1 Selection.............................................................................. 19

1.3.4.2 Fusion protocols ................................................................. 20

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1.3.4.3 Feeder layer ............... 20

1.3.4.4 Production of monoclonal antibody in v iv o .................. 21

1.3.4.5 Production of monoclonal antibody in v i tro ................. 21

1.4 PARATHYROID HORMONE.............................................. 22

1.4.1 Biosynthesis and secretion ................................................. 22

1.4.2 Heterogeneity and metabolism .......................................... 27

1.4.3 Physiology of PTH .............................................................. 29

1.4.4 Structure function relationship ......................................... 34

1.4.5 Measurement of parathyroid hormone ............................. 36

1.5 GENERAL AIMS OF THE PRESENT WORK .................. 43

CHAPTER 2: DEVELOPMENT OF SCREENING ASSAYS FOR MONOCLONAL ANTIBODIES ............................................................. 45

2.1 INTRODUCTION................................................................. 46

2.2 RADIOIMMUNOASSAY (RIA )......................................... 50

2.2.1 Chemicals and reagents ...................................................... 50

2.2.2 Iodination of hPTH(l-34) ................ 50

2.2.3 M ethod................................................... 54

2.3 ENZYME LINKED IMMUNOSORBENT ASSAY(ELISA)....................................................................... 54

2.3.1 Chemicals and reagents ............................ 55

2.3.2 M ethod.......................... 55

2.3.3 Testing the solid phase....................................................... 58

2.3.4 Glutaraldehyde sensitization ............................................. 58

2.3.5 Testing the effect of different coating buffers ................. 58

2.3.6 Testing different blocking agents ...................................... 59

2.3.7 Testing the effect of detergent on antigen antibodybinding ..................................................................................................... 59

2.4 RESULTS..................... 59

2.4.1 R IA .................................................................................... 59

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2.4.1.1 Iodination ..................................................................... 59

2.4.1.2 Optimization.................. 62

2.4.2 ELISA.................................................................................... 67

2.4.2.1 Solid phase support........................................................... 67

2.4.2.2 Blocking agent ................................................................... 67

2.4.2.3 Effect of Tween 20 on antigen antibody binding 73

2.5 DISCUSSION........................................................................ 73

2.5.1 R IA ................... 73

2.5.2 ELISA.................................................................................... 73

CHAPTER 3: MOUSE AND RAT MONOCLONAL ANTIBODY TO hPTH(l-34) ......................... 76

3.1 INTRODUCTION................................................................. 77

3.2 MATERIALS AND METHODS ......................................... 78

3.2.1 Reagents and chemicals ............. 78

3.2.2 Cell culture media ........................................................ 78

3.2.3 Preparation of thymocyte conditioned media ................. 79

3.2.4 Conjugation of hPTH( 1-34) ........... 79

3.3 IMMUNIZATION....................................................... 79

3.3.1 Mice immunization ............................................................. 79

3.3.2 Rat immunization ......................................................... 80

3.3.3 Secondary in vitro immunization.................................... 80

3.3.4 Monitoring of serum polyclonal response ....................... 83

3.4 MAINTENANCE OF MYELOMA CELL LINES............. 83

3.4.1 NS1 mouse myeloma cell lin e ............................................ 83

3.4.2 Y3 rat myeloma cell line ................................................... 84

3.5 FUSION .................... 85

3.5.1 Preparation of feeder layer ................................................ 85

3.5.2 Fusogen................................................................................ 85

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3.5.3 Preparation of immune spleen cells ................................. 85

3.5.4 Mouse-mouse fusion .......................................................... 86

3.5.5 Rat-rat fusion .................................................................... 86

3.5.6 Rat-mouse fusion ......................... 87

3.5.7 Screening ........................................................................ 87

3.5.7.1 Mouse monoclonal screening.......................................... 87

3.5.7.2 Rat monoclonal screening.................................................. 88

3.5.7.3 Freezing and thawing of cells .................................... 88

3.5.7.4 Cloning................................................................................ 88

3.6 CHARACTERIZATION OF MONOCLONAL ANTI­BODY 89

3.6.1 Immunoglobulin class ....................................................... 89

3.6.2 Purification of monoclonal antibody ............................... 90

3.6.2.1 Sephacel anion exchange column ...................................... 90

3.6.2.2 Protein A colum n ................................................. 90

3.6.2.3 Mono Q anion exchange column ....................................... 91

3.6.2.4 Ascites production ............................................................. 91

3.7 RESULTS.................................................................... 92

3.7.1 Immunization................................................................... 92

3.7.2 Myeloma cell line growth characteristics................... 92

3.7.2.1 Mouse NS1 myeloma cell l in e .................................. 101

3.7.2.2 Rat Y3 myeloma cell l in e .............. 101

3.7.3 Fusion ............................ 101

3.7.3.1 Mouse-mouse fusion .......................................................... 101

3.7.3.2 Rat-rat fusion .......... 106

3.7.3.3 Rat-mouse fusion ............................................................ 106

3.7.3.4 Screening m ethods.............................................................. 108

3.7.3.5 Characterization of monoclonal antibody....................... I l l

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3.7.3.6 Purification of monoclonal antibody............................... 117

3.8 DISCUSSION AND CONCLUSION .................................. 117

3.8.1 Mouse monoclonal antibodies............................................. 117

3.8.2 Rat monoclonal antibodies.................................................. 125

CHAPTER 4: GOAT POLYCLONAL AND MONOCLONAL ANTIBODY TO hPTH(l-34) ............ 126

4.1 INTRODUCTION................................................................. 127

4.2 MATERIALS AND METHODS ............ 129

4.2.1 Conjugation of hPTH( 1-34) ............................................... 129

4.2.2 Immunization....................................................................... 129

4.2.3 Monitoring of serum antibody .......................................... 129

4.3 CHARACTERIZATION OF ANTIBODY .......................... 131

4.3.1 Specificity ............................................................................. 131

4.3.2 Affinity ................................................................................. 132

4.3.3 Displacement and standard curves ........................... 132

4.4 GOAT MONOCLONAL ANTIBODY ................... 133

4.4.1 Myeloma cell line ................................................................ 133

4.4.2 Blood collection.................... 133

4.4.3 Lymphocyte separation.................................................. 134

4.4.4 Fusion and plating out ....................................................... 134

4.4.5 Goat-hetero-myeloma fusion ........................ 134

4.4.6 Goat-mouse NS1 myeloma fusion ..................................... 135

4.4.7 Screening............................................................................... 135

4.4.8 Cloning ................................................................................. 136

4.5 RESULTS............................................................... 136

4.5.1 Serum polyclonal antibody................................................ 136

4.5.2 Characterization of an tibody ............................ 137

4.5.3 Monoclonal antibody........................ 148

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4.6 DISCUSSION AND CONCLUSION.................................... 151

4.6.1 Polyclonal antibody ................................. 151

4.6.2 Monoclonal antibody ........................................................ 152

CHAPTER 5: APPLICATION OF MONOCLONAL ANTIBO­DIES ........................................................................................................ 153

5.1 INTRODUCTION................................................................ 154

5.2 IMMUNOCYTOCHEMISTRY................................. 154

5.2.1 M aterials.................................... 154

5.2.2 Buffers.................................................................................. 156

5.2.3 Tissue samples .................................................................... 156

5.2.4 Procedure for alkaline phosphatase staining .................... 157

5.2.5 Procedure for horseradish peroxidase staining................. 157

5.3 SANDWICH ELISA............................................................. 158

5.3.1 M aterials.............................................................................. 159

5.3.2 Purification of goat anti-PTH antibody........................... 159

5.3.2.1 Caprylic acid and ammonium sulphate precipita­tion method ........... 159

5.3.2.2 Anion exchange method ..................................................... 159

5.3.3 Purification of mouse monoclonal antibody 160

5.3.4 Conjugation of monoclonal antibody with HRPOaseenzym e.................................................................................................... 160

5.3.5 M ethod...................................... 160

5.3.5.1 Sandwich ELISA procedure............................................... 160

5.4 RESULTS.............................................................................. 161

5.4.1 Immunocytochemistry........................................................ 161

5.4.2 Sandwich ELISA.................................................................. 162

5.4.3 Assay .................................................................................... 172

5.5 DISCUSSION AND CONCLUSION............... 185

CHAPTER 6: DISCUSSION AND CONCLUSIONS............................ 187

FUTURE WORK

REFERENCES .....................

- 1 - CHAPTER 1

CHAPTER 1

INTRODUCTION

INTRODUCTION

- 2 - CHAPTER 1

1.1. INTRODUCTION

It was only about 60 years ago that the antibody was accepted as serum globulin although long before that the presence of antibody activity in the serum had been known. Soon after their recognition as part of the serum immunoglobulins, antibodies were characterized and studied in terms of their interaction with the antigen and structure.

Antibodies are regarded by immunologists as sensitive and specific tools on which many immunological methods depend. Antigen- anti­body reactions constitute the basis of numorous procedures devised for identification, quantitative measurement and isolation of particular molecules from a mixture. Approximately 108 different B-lymphocyte are present in the spleen of mice and man. Although they were derived from a common stem cell, each developed its own capacity to make an antibody that recognizes different antigenic determinants. As the separation of of various antibodies can be almost impossible at times, conventional antisera contain a mixture of antibodies which differ from animal to animal, and even between animals which are genetically identical. The characteristics of the antibody may also differ in the same animal, between different bleeds. Serum immuno­globulin levels vary with genetic background, antigen load and age.

The idea of one cell-one antibody was proposed in 1959 (Burnet 1959). This theory predicted that antibodies made by the same cell are homogeneous. The mode of production of monoclonal antibodies sup­ports this theory. Monoclonal antibody is the result of collective characteristics of two different cells, one secretes the antibody and the other has the capability of growing in culture indefinitely. Thus the resulting cell inherits both characteristics, and so produces and secretes antibody continuously and reproducibly. . The ability to raise, and select specific antibody to individual determinant

demon stratesthe utility of monoclonal antibody technique. Antigen purityis no longer a necessity. The resulting antibodies have the fine specificity to the determinant recognized on the antigen, and so the

INTRODUCTION

- 3 - CHAPTER 1

cross-reactivity should be minimal.

Monoclonal antibody has been gradually replacing the polyclonal variety in many fields of application. It is chosen, in the main for specificity, homogeneity and continuity. However, polyclonal antibody will still continue to be used and in certain situations may even be pre­ferred. For example, polyclonal antibody would be chosen when high affinity antibodies are required, as the monoclonal antibody usually displays a low affinity (Goding 1986).

1.2. THE IMMUNE SYSTEM

The immune system acts as a third line of defence. Compared with lower animals, vertebrates have sophisticated immune systems, which respond in a specific manner, distinguishing self from non-self. The flexibility and effectiveness of the specific immune response to an antigen are provided by the specificity, memory and non-self recogni­tion characteristics of immune system.

1.2.1. Functional cells of the immune system

The immune system is composed of a number of cells types, and between them they neutralize, destroy, and produce antibody against the foreign material. The different cells were derived from a common stem cell.

There are different immune cells which cooperate in combating the foreign antigen. They originally inhabit different primary lym­phoid tissue from which they get their names. Those abundant in the bursar of fabricius in birds or equivalent probably the foetal liver, and bone marrow in mamma]s(Miller 1966, Wiessman et al., 1974) are called B-cells, and those abundant in the thymus are called the T-cells.

The development and maturation of these cells in the primary lymphoid organs is antigen-independent. They migrate from the pri­mary lymphoid organs, through the blood and lymph network system, the B- cells mostly dwell in the primary follicle and T-cells in the diffuse cortex (Howard 1972, Sprent 1973). The secondary lym-

INTRODUCTION

- 4 - CHAPTER 1

phoid organs provide the environment necessary for the presentation and processing of antigen, leading to an antigen dependent mature immune response.

Small lymphocytes are the principal cells involved in immune response (Hunt et al., 1972, Howard and Gowans 1972), and the depletion of lymphocytes abolishes such response. The cooperation of the T-cells and B-cells is essential for immune response. Clinical diseases which manifest these situations have for many years been kown to occur in humans. The Di-George’s syndrome is characterized by the absence of T-cells and B-cells are normal or near normal. Burton’s syndrome, on the other hand, presents with normal T- cells and reduced B-cells.

Although B and T cells are functionally different, they are indistinguishable morphologically under light microscopy (Miller 1974). However, they can be identified by reaction specificity as in sheep red blood cell rosette formation (Jondal et al., 1972) and by a variety of surface markers (Raff 1971, Loor and Roelants 1975, Kieseilow et al., 1975).

It is the functional properties of B and T cells which has been utilized in the hybridoma technology. Since the introduction of monoclonal antibody technique many B and T cell lines have been used in the production of hybridoma and thymoma respectively. The cooperation between B and T cells that finally leads to antibody pro­duction is the target in hybridoma technology.

1.2.2. Immune response

The exposure of the immune cells to a foreign antigen result in the stimulation of these cells to combat the antigen. The stimulation of the different cells to secret antibody requires the recognition, stimula­tion, proliferation and differentiation of these cells.

the total antibodiesAlthough it was estimated that about l-5x 107 represent ~ repertoire

in an inbred mouse, only 1000-8000 recognize any particular antigenic determinant (Kohler 1970,Kreth and Williamson 1973) and

INTRODUCTION

- 5 - CHAPTER 1

of these only 5-10 appear in the antisera in response to each antigenic determinant (Schreier et al., 1980). This indicates the degree of the heterogeneity of conventional antisera and the difficulty in the reproduction of animal response.

The clonal selection theory was accepted by immunologists to explain the heterogeniety of antibody (Burnet 1959). The heterogene­ous nature of the immune response was explained by the existence

.whichof cell population in each cell is capable of producing only one anti­body. This theory thus emphasizes that the whole cell is the unit of selection. Only a small number of cells can be stimulated because they possess a receptor for the antigen, while the majority of them cannot be stimulated. Later it was confirmed that the receptors are immunoglobulins on the curface of the B-lymphocyte (Ada 1970, Raff1971), and they could be exact copies of the secreted antibody for B-cell (Mitchison 1967).

B-cell activation, proliferation and differentiation has been recently reviewed (Weigle 1987). It is concluded that two pathways are involved in the antigen stimulation of B-cells, MHC-restricted and MHC-unrestricted. The activation of MHC-restricted pathway seems to be the major route responsible for the significant part of the total antibody response, and different sub-population of B-cells dictate the response of each pathway.

The acceptance that myeloma proteins are indistinguishable from normally secreted immunoglobulin proteins (Weissman 1978, Potter1972) and the induction of mouse myeloma tumors producing homogeneous immunoglobulins (Potter 1972) confirm the essentiality of one cell one antibody specificity prediction.

The stimulation of the body immune response involves the stimulation of B and T lymphocytes. Events leading to the destruction of the antigen involve two immune response pathways depending on what type of cell is involved. Humoral immune response involves B- cell activation and production of antibodies to neutralize antigens.

INTRODUCTION

- 6 - CHAPTER 1

When the antigen accumulates in the secondary lymphoid tissue, stimulation and expansion of antigen specific clones take place accompanied with B-T cell cooperation and the formation of memory cells which are primed and ready for fast action in the second exposure to the antigen. Then antibody is produced and released to bind the antigen. When T cells are involved the response is known as cell-mediated immune response. They differentiate into different specialized types of T cells which destroy the antigen.

1.2.3. Antibody structure and function

The antibody is a large molecule of glycoprotein, consisting of two identical heavy chains and two identical light chains linked by disul­phide bonds (Porter 1967, Edelman 1969, 1970); the two H-L chains are also linked by a disulphide bridge. The molecule has different regions of constant and variable amino acid sequences. The struc­ture of immunoglobulin G (IgG) is shown in figure 1.1.

The elucidation of the structure of antibody and the amino acid sequence revealed that the light chains are composed of 220 amino acids and have a molecular weight of 23000 daltons. The heavy chain consists of 440 amino acids and has a molecular weight of 55000 dal­tons.

The biological properties of the antibody molecule are determined by the heavy chain constant region, and the antibody specificity is provided by the variable regions of the chain. Table 1.1 summarizes the different properties of immunoglobulins. There are only two types

INTRODUCTION

- 7 - CHAPTER 1

3F Jz z

CO CO

COCO

S-S

O

CO

oOX

Figure 1.1: The structure of prototypical Ig. VL vari­able Light, VH variable Heavy and CL Constant Light domain. The first domain of each chain is the variable region and the remaining domains comprise the constant region of the chain. The disulphide bonds (S-S) connecting the L and H chains are always between the CL and CHI domains with the precise location differ between classes and species . The hinge region is the boundary between the CHI and CH2 domains, this also differs according to class and species. (Adapted from Clark 1986).

INTRODUCTION

- 8 - CHAPTER 1

Table 1.1

Physical properties of human immunoglobulins

Ig G IgA IgM IgD IgE

No. of subclasses 4 2 - - -

No. of basic units 1 1/2 5 1 1

Heavy chain class 1.2,3,4 1.2 u

Light chain k / \ k / \ k / \ k / \ k / \

Molecular weight 150kd 160kd 900kd 185kd 200kd

Concentration in nor­

mal serum in mg/ml

8-16 1.4-4 0.5-2 0-0.4 17-450 ng/ml

% of total Ig in serum 80 13 6 0-1 0-0.002

%Carbohydrate content 3 8 12 13 12

Half life in serum 23 5.8 5.1 2.8 2.5

Notes:

1-IgG3 half life in serum is only 10 days

2-IgE binds basophils and mast cells and

have a longer half life in serum.

INTRODUCTION

- 9 - CHAPTER 1

of light chain constant region, kappa and lambda, and only one of them present in any one antibody molecule.

1.3. MONOCLONAL ANTIBODY

An immu ne response to an antigen involves the activation of many cells which secrete antibodies to various epitopes on the antigen. These antibodies are called polyclonal antibodies. The antibo­dies in the serum are a mixture of different antibodies to the same antigen and to unrelated antigens to which the animal has had an immune response. '

The antibodies secreted by clone of cells derived from a single cellare called monoclonal antibodies. Under normal circumstances theisolation and propagation of an antibody-producing cell is impossiblebecause these cells cannot grow in tissue culture. Hence the fusion

anof antibody-producing cell with an immortal myeloma cell was used to circumvent the growth and isolation problems. The resultant cells are capable of both growing indefinitely in tissue culture and producing and secretingantibody. Thus, the isolation and propagation of antibody- producing cellsbecame possible.

Although myeloma cells have been known for some time, only ; few antigens were found to be bound with the antibody secreted by these cells. Their use in the production of monoclonal antibody to pre- determined specificity was only introduced thirteen years ago (Kohler and Milstein 1975). Since then the technique has been used in many laboratories and monoclonal antibodies have been produced against a wide range of compounds. A general scheme of monoclonal antibody is shown in figure 1.2

In the last decade thousands of articles have appeared dealing with different aspects of monoclonal antibody. Improvements in the technique, optimization of different steps, manipulation and use of monoclonal antibodies have been reviewed extensively (Goding 1986, Reading 1982, Foster 1982, James and Bell 1987, Samilovich et al., 1987, Hubbard 1983).

INTRODUCTION

- 1 0 - CHAPTER 1

CELLPREPARATION

CLONING

FUSION

SELECTION OF HYBRIDOMAS

SELECTION OF ANTIBODY

PRODUCERS

4 days

8 mm.

24 h

10 to 15 days

1 to 2 weeks

PRODUCTION OF MONOCLONAL Ab.

Antigen

M yelom a H G P R T ©

Balb/c

cells)S p leen ( io 8 cells)

P olyethylene g lyco l j

M icroculture (■<- serum )

4 0 0 to 6 0 0 m icro cu ltu resT

A ddition of se le c to r m ed iu m : H.A.T.

T

________________ J ____________S p ec ific sc r e en in g

S p ec ific sc r e en in g : s e le c tio n of c lo n e s

Culture

I.P. Injection of c e lls

P reservation of c e l ls by freezin g

Figure l .2: The different stages of lymphocyte hybridiza­tion. The timing of each stage is shown at the left side of the diagram.

INTRODUCTION

-11 - CHAPTER 1

In this review the different steps leading to the production of monoclonal antibody will be described in brief, giving the relevant literature.

1.3.1. Immunization

Immunization usually implies the administration of an antigen in-vivo to specifically activate and differentiate B-lymphocytes. Efficient immunization is seen as a key factor for a successful pro­duction of monoclonal antibody. It depends on the interaction between and contribution of different factors. The factors controlling the immunogenicity are still poorly understood. The physiological state of the host is important in obtaining a good response (Crump­ton 1974). The recognition of antigen by macrophages is the initial event in the immune response (Opitz et al., 1976, Pierce and Klin- man 1981, Unanue 1979). Antibody characteristics are deter­mined at an early stage of B-cell differentiation, where the antibody diversity is generated (Leder 1982, Tonegawa 1983),and the function effectors are determined late in a process called "class switching" (Hon jo 1983).

Theoretically, the intensity of response is not very important in the production of monoclonal antibody. On the other hand many workers report time and again, that the fusion efficiency depend on the response of an animal, and the immunization schedule.

Two methods of immunization are generally used in the generation of monoclonal antibody, those which depend on the stimulation of B- cells in- vivo and those which depend on the stimulation of B-cells in- vitro.

1.3.1.1. In-vivo immunization

Many of the monoclonal antibodies produced were derived from in vivo immunization. Despite many years of research immuni­zation is still regarded as an art than a science, and factors control­ling it are still poorly understood.

INTRODUCTION

- 1 2 - CHAPTER 1

Many immunization protocols and schedules have been used successfully in the production of monoclonal antibodies. However, it is well known that the successful production of monoclonal antibody require the stimulation and activation of the antigen specific B- cells. Hence, lengthy protocols and complicated schedules may not be necessary. For example, intrasplenic immunization for nine days have been shown to be successful in producing monoclonal antibodies (Spitz 1986).

The dependence of the fusion specific efficiency on the number of specific B-cells in the spleen prompted the use of different ways to increase the number of the desired B-cells. The antigen can be adsorbed to a matrix, which is then injected in the host (Sternick and Sturmer 1984, Knudsen 1985). Splenocytes from immunized animal can be transferred into another X-irradiated animal, and the spleen of the second animal used for fusion (Siraganian et al., 1983). Selective reduction of response toward the determinants which are not of interest, is possible by using cytotoxic agents (Matthew and Petterson 1983), by passive immunization of the recipient with antibody directed toward the other determinants (Thalhamer and Freund 1985), by immunization with antigen-antibody complex (Eager and Kennett 1986) and by the blocking of unwanted deter­minants by binding them with antibody, and the antigen antibody complex used for immunization (Foster 1982).

The selection of animals high titre of antibody in the serum for fusion does not always result in high specific fusion (Stahli et al., 1983), and more important is the time between the boosting and the fusion. It was shown that the specificity of antibody produced as well as the fusion specific effeciency depend on the time between boosting and fusion, and has been found to be between three and four days. The repeated stimulation with a potential immunogen seems to reduce the initial response (Lee and Kohler 1974). However, high concentration of circulating antigen up to the day of fusion has been shown to be a prerequisite for successful fusion (Stahli et al., 1980).

INTRODUCTION

- 1 3 - CHAPTER 1

The properties of antibodies are known to be affected by the form and amount of antigen,the choice of adjuvant and the regimen of immunization. IgE antibodies were shown to be produced when alumi­num hydroxj de gel was used as an adjuvant (Tung 1983, Levine and Vaz 1970), while the use of Freunds adjuvant supressesIgE antibody production (Tung et al., 1978). Oral administration of bac­terial cell wall antigen (Morisaki et al., 1983) and long term adminis­tration of antigen intravenously (Colwell et al., 1986) resulted in IgA antibody production. IgM antibody was usually obtainable by taking the spleen 3-4 days after the first immunization (Trucco et al., 1978).

1.3.1.2. In-vitro immunization

The exposure of dissociated spleen cells to an antigen in tissue cul­ture is called in vitro immunization. Before the introduction of mono­clonal antibody technique, in vitro immunization was used, and responses were demonstrated (Marbrook 1967, Mishell and Dutton 1967). The cell culture conditions, from the viewpoint of antibody synthesis have been studied (Click et al., 1972).

Immediately after the introduction of monoclonal antibody techniques(Kohler and Milstein 1975), in vitro immunization has been applied to the production of monoclonal antibody. Since then this tech­nique has been applied successfully for the production of monoclonal antibody to highly conserved proteins (Pardue et al., 1983) as well as many other antigens (Luben et al., 1982, Jonak and Kennett 1984). The application of in vitro immunization has been reviewed by Reading (Reading 1982, 1986).

Crucial importance of in vitro immunization lies in the fact that producing human-human hybridomas in the usual way is not ethically feasible. This technique can be also of particular value in the pro­duction of monoclonal antibody to precious and rare antigens. Other advantages include, short immunization time, escaping the MHC con­trol, circumventing the route of immunization problems and better chance of spleen cell size monitoring and control if needed. Finally,

INTRODUCTION

- 1 4 - CHAPTER 1

with the increased demand of human monoclonal antibody, it is at present the only method well-suited for this purpose.

1.3.2. Fusion partners

1.3.2.1. Murine myeloma cell lines

From the work of Kohler and Milstein (1975)it was indicated that, the successful marriage between normal spleen cell and immortal myeloma partner depend on the differentiation state of both cells, and the use of the same species myeloma cells enhances the chance of hybrid rescue.

A number of mouse myeloma cell lines have been used as fusion partners. The choice between the different myeloma partners depend on the fusion properties and the secretion and synthesis of immunoglobulin by the cells. Those cell lines which support the fast growth of their hybrids and do not produce and/or secrete antibody themselves are usually the best.

The majority of the commonly used myeloma cell lines are either of mouse (Balb/c) or rat (Lou/c) origin . The mouse cell lines are fre­quently used both in mouse-mouse and cross-species fusions. Rat myeloma cell lines on the other hand are very seldom used. This could be due to the problems encountered in maintaining the cell line as well as the rat hybridoma (Samoilovich et al., 1987).

1.3.2.2. Human myeloma cell lines

The potential of human-human monoclonal antibodies is obvi­ous, especially in therapy where the murine monoclonal antibodies usually stimulate the production of antibody when given in-vivo. In addition, human antibodies and/or autoantibodies can be used to pro­duce anti- idiotypic antibodies, which will be useful in the study of immune system regulation (Kozbor and Roder 1983).

The production of human monoclonal antibody has been recently reviewed (James and Bell 1987). There have been several cell lines established as fusion partners, only few of these are myeloma

INTRODUCTION

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and the rest are Epstein-Barr virus transformed cells. The murine myeloma cell lines are very successful fusion partners, the human myeloma cell lines in comparison grow very slowly and are rarely used.

The mouse-human fusion, though useful did not eliminate the sensitization problems encountered in in vivo use of antibody. Furthermore, the resulting hybridomas are not stable due to chromosomal loss shortly after fusion. One way to circumvent this problem is using the mouse-human hybrid as fusion partner (James and Bell 1987), and the same principle can be used to improve the growth characteristics of human myeloma lines (Kozbor et al., 1984).

Despite the existence of a number of human myeloma cell lines, none of them offer any advantages over others in terms of fusion fre­quency and cloning efficiency. Nonetheless, the unabated research no doubt will result in better myeloma cell lines. The murine and human myeloma cell lines available for fusion are shown in table 1.2.

1.2.3. Fusogen

There are many ways to fuse cells and membranes; some are effective, others not. Spontaneous cell fusion is known to occur at a very low level, therefore, chemical, viral and electrical methods were developed to accelerate the fusion.

Fusion of many biological materials is easy to accomplish with any of the above mentioned methods, but the viability and the survival of the cells or the fused material is important as much as the fusion. In the field of hybridoma production, different methods were applied with different degrees of success, they include virus, PEG and electrofusion. Laser cell fusions have been shown to be effective, but they need expensive equipment which is beyond the capability of most laboratories.

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Table 1.2

Myeloma cell lines used for hybridoma production

Species Cell line Ig secreted Selectable marker References

Mouse

P3-x63-Ag8.653 - HPRT- Kearney et al., 1979

SP2/0-Agl4 - HPRT- Shulman et al., 1978

FO - HPRT-.

Fanekas de st.Groth and Scheidegger 1980

S 194/5XX0.BU.5 - TK- Trowbridge 1978

FOX-NY - APRT-,HPRT- Taggart and Samloff 1983

Rat IR983F - HPRT- Bazin 1982

Y3-Ag 1.2.3 K HPRT- Galfre et al., 1979

Human

SKO-007 E/C HPRT- Olsson and Kaplan 1980

GM-1500 6TG-2 v2/C HPRT- Croce et al., 1980

KR-4 v/C HPRT-fOua Kozbor et al., 1982

LICR-LON-HMy2 vl/C HPRT- Edwards et al., 1982

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1.3.3.1. Virus fusion

Viruses of the RNA-containing lipid-envelope type are most com­monly employed in cell fusion. The presence of specific receptor on the cells is thought to be essential for the binding of fusogenic viruses (Okada 1969). However, Sendai virus has been found to bind artificial membranes without receptors (Haywood 1974), and the Sam- liki Forst Virus (SFV) infects cells lacking histocompatibility antigen (Helenius et al., 1980).

The chemical structure of the viral spike glycoproteins and the lipid composition of the cell membranes are of critical importance (Gallaher et al., 1973, Gething et al., 1978, Fries and Helenius 1979), while temperature and pH are not very important in fusion (Okada 1969, Foster 1982).

Different types of viruses were used in cell fusion, to induce con­tinuous synthesis of specific antibodies. Simian virus was used to pro-

/duce antibody to type III pneumococcal polysaccharides (Strosberg et al., 1974), Abelson virus was used to transform mouse lymphocytes to Ig-producing lines (Abelson and Rabstein 1970), Epstein Barr virus was used to transform human lymphocytes (Fahey et al., 1966). Since then several lines have been established secreting antibodies to acetylcholine receptor (Kamo et al., 1982), phosphorylcholine (Yoshie and Ono 1980), bacterial and viral antigens (Rosen et al., 1983, Crawford et al., 1983, Seigneurin et al., 1983), haptens (Steinitz et al., 1977, Kozbor et al., 1979) and rheumatoid factor (Steinitz et al., 1980, Steinitz et al., 1982).

Despite the success of viruses in transforming cells, and pro­duction of specific antibodies, it appears that the amount of anti­body secreted is relatively small and the lines established lose their ability to produce specific antibody in vitro after long term culture (Kozbor and Roder 1981, Crawford et al., 1983). Viral cell fusion was also found to be low (Yelton et al., 1981) and standard preparation is needed for long term success (Pentecorvo 1975).

INTRODUCTION

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1.2.3.2. Polyethylene glycol (PEG)

PEG was first applied to somatic cell hybridization by Pon- tecorvo (Pontecorvo 1975) after it had been shown to fuse plant cells (Kao and Michayluk 1974). The precise mechanism by which PEG promote cell fusion is not known, although during the initial stages of hybridization structural changes in membrane were observed (Knutton and Pasternak 1979), and membranes were found to make a contact.

PEG as well as its commercial contaminants are necessary for fusion. The removal of these impurities render PEG non- fusogenic over 1 minute cellular exposure (Westerwoudet 1985), and purified PEG was found to fuse hen erythrocytes over 15 minute exposure. The fusion is enhanced at least to a lesser extent by the inclusion of DMSO in PEG solution (Norwood et al., 1976, Fazekas de st. Groth and Scheidegger 1980).

Although PEG is toxic to cells at high concentrations, 30 to 55% w /v were usually used to fuse cells, with the fusion optimum being at 50%. High and low molecular weight grades of PEG can be used in fusion, the most common two were 1500 and 4000 molecular weight. The fusion efficiency has been shown to be affected by the molecu­lar weight, the manufacturer and even the lot number (Davidson et al., 1976, Fazekas de st. Groth and Scheidegger 1980, Goding 1983).

1.2.3.3. Electro-fusion

This technique has been in use for sometime for the fusion of plant cells. More recently it was developed and applied to the production of murine and human monoclonal antibodies (Zimmermann et al., 1982, Zimmermann et al., 1985, Vienken and Zimmermann 1982, Bischoff et al., 1982).

The electro-fusion depends on the establishment of close contact between cell membranes by the action of electrical force, and an intense electrical field pulse of short duration to trigger the fusion between adjacent cells.

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This technique offers many advantages over the other two common methods. Fusion of pairs of cells is possible, it permits immediate cloning of fused cells and it gives high fusion efficiency. However, despite these advantages this method requires expensive equipment, it is cumbersome as only a few cells are handled each time. Hence the technique cannot be used to the full unless the steps are automated and the equipment is available at a lower price.

1.3.4. Hybridization

1.3.4.1. Selection

Considering the low percentage of cells fused, the newly formed hybrids have to be protected and well cared for. The fact that fusion mixtures contain three different types of cells and only one type is desired, necessitate selection if the hybrids are to survive. The spleen cells usually die within a week,so they do not pose a threat to the hybrid cells. Myeloma cells on the other hand will not die in normal media and have to be selected.

The selection of the desired cells is achieved by the continuous selection of myeloma cells in media containing metabolic poisons such as 8-azaquanine and 6-thioquanine. The cells grown in these selec­tion media develop a deficiency in enzymes important for the synthesis of DNA and RNA by the salvage pathway. Therefore, when grown in media containing a blocker for the de novo pathway they die.

The commonly used system was the hypoxanthine- aminopterin- thymidine (HAT) selection system devised by Littlefield (1964). The myeloma cells are made deficient in the enzyme hypox- anthine phosphoribosyltransferase (HPRT) or thymidine kinase (TK) by growing them in 8-azaquanine. Those surviving in 8-azaquanine lack HPRT or TK enzymes and depend on the de novo synthesis of nucleotide precurors for nucleic acid synthesis. This de novo pathway is blocked by the addition of aminopterin to the media.

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1.3.4.2. Fusion protocols

Many fusion protocols have been used in hybridoma production (Goding 1986). Only minor modifications were introduced to the origi­nal technique (Galfre et al., 1977), but the essentials were the same.

Improvements in the fusion efficiency seem to be dependent on the type of myeloma cell line, and the type and concentration of PEG. The duration of exposure seems to be around one minute, although in a recent study it was found to be less. The pH of the PEG solution has been shown to be important (Sharon et al., 1980), and the optimal was found to be between pH 8-8.2. Temperature, cell number and the ratio of spleen cells to myeloma cells seem to be of less importance (Goding 1986).

1.3.4.3. Feeder layer

Unknown factors support the growth of hybrids and single cell during cloning; these factors have been provided by the addition of peritoneal macrophages (Fazakas de st.Groth and Scheideggen 1980), splenocytes (Goding 1986), thymocytes (Oi and Herenberg 1980) and human endothelial culture supernatant (Astaldi 1983) to the fused or cloned seeded cells. The addition of cells which provide the growth factors although supporting the growth of cells, may act as a source of contamination. Feeder layer cells may kill or select the grow­ing hydrid cells either by exhustion of media nutrients on which the hybrid live or secretion of toxic materials which kill the hybrids.

The use of alternatives, like macrophage-conditioned media have been shown to support the growth of plasmacytoma cells (Nordan and Potter 1986) and hybridoma cloning (Rathein and Greczy1986). In mouse-rat fusions, T-cell-derived lymphokines were shown to be essential for the growth of the resulting hybrids (Van Snick et al., 1986).

The development of defined additives could lead to serum free media used in all stages of cultivation (Samoilovich et al., 1987).

INTRODUCTION

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1.3.4.4. Production of monoclonal antibody in-vivo

The injection of hybridoma cells into the peritoneal cavity for the in vivo production of monoclonal antibody is a common practice. It is fast, needs only 2 weeks for the ascitic fluid to accumulate in the peri­toneal cavity, and yield a concentrated antibody sometimes up to 50 mg/ml of ascites (Samoilovich et al., 1987). The mice normally prepared by injection of 0.5 ml prestain (2,6,10,14- tetramethylpentadecane ) intraperitoneally 7 days before the cells injected into the mice, and two to three weeks afterward ascitic fluid is collected. However, the production of antibody in-vivo, without prior preparation of mice is also possible (Samoilovich et al., 1987), and the use of adjuvant instead of prestain was shown to decrease the prepara­tion time with no loss in antibody activity or decrease in ascites volume.

The only obstacle is that the cells should be compatible with that of the host, although suppression of the immune system by immune suppressive drugs or X-irradiation is used for the non­compatible cells. The passage (Truitt et al., 1984, Kozbor et al., 1985) and growth of cells in liver tissue (Hirsch et al., 1985) enhance their stabilization and secretion.

The antibody produced in vivo may not be suitable for use in therapy, and its purification and treatment could change its characteristics. Moreover, purification was shown to reduce the yield with each step (Dalchau and Fabre 1982, Bazin et al., 1984).

1.3.4.5. Production of monoclonal antibodies in-vitro

Hybridoma cells are originally grown in-vitro. The concentration of antibody is low compared to in vivo production. However, in many applications highly purified antibody is needed. In both in vivo and in-vitro systems the antibody is contaminated with complex mix­tures of proteins and contain many unknown components. Hence, cells were grown in synthetic serum free media to ease the purification (Clevdand and Erlanger 1983, Kovar and Franek 1984). The growth

INTRODUCTION

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of cells as well as the increase in antibody production were of prime importance in the in-vitro production of antibody. From the commer­cial point of view in vivo production of antibody is not a feasible pro­position. Many additives to the media were used to support the growth and increase the antibody production with claimed successes. These include different proteins, hormones, growth factors, and mix­tures of all (Chang et al., 1980, Darfler and Insel 1982).

The large scale needed for the commercial viability of antibody production necessitates the development of many devices where large numbers of cells can be cultivated, and large amounts of antibody obtained daily. The cells have been grown in many devices depending on the resources and needs, ranging from an ordinary T-flask, to fer- minters , hollow fiber devices and microencapsulation (Galfre and Milstein 1981, Fazekas de st.Groth and Scheidegger 1980, Hopkinson 1985, Grinda and Jarvis 1984, Scheirer et al., 1984).

Some of these techniques are being exploited currently for in vitro production of monoclonal antibodies by many commercial institu­tions, and adapted as routine practice for the cultivation of many chosen cell lines. There are even claims that the antibody concentration in vitro may have reached the corresponding in vivo levels.

1.4. PARATHYROID HORMONE

1.4.1. Biosynthesis and secretion

The 84 aminoacid PTH polypeptide arises from a larger precursor of 115 amino acids PrePro-Parathyroid hormone (PreProPTH) by two successive cleavages of small amino-terminal sequences (Habener and Kronenberg 1978, Habener and Potts 1978 and Habener et al.,1978). According to the ’signal’ hypothesis (Blobel and Dobberstein 1975 ) the leader sequence of PreProPTH passes through the mem­brane of the endoplasmic reticulum whilst translation of the growing peptide chain continues behind it. Within the rough endoplasmic reticulum the first 25 amino acids are then cleaved to form Pro­

INTRODUCTION

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parathyroid hormone (ProPTH). In the golgi complex the other six amino acids cleaved resulting in the formation of parathyroid hor­mone (PTH) ( Habener et al., 1979) Secretion of the hormone seems coupled in someway to synthesis as only small amounts of PTH were stored in the gland. The PTH structure is shown in figure 1.3, and the synthesis and secretion is shown in figure 1.4.

Calcium surprisingly appears to have small or no effect on the conversion of ProPTH to PTH (Habener et al., 1975), although paradoxically it is the major regulator of parathyroid activity and expected to have some effect on the rate of PTH synthesis. High extra­cellular calcium concentration stimulates and low concentration inhibits the degradation of PTH within the parathyroid gland (Habener et al., 1975). The stimulation and inactivation of chief cells of the parathyroid gland to synthesize PTH is affected by hypo and hypercalcaemia respectively. Recent studies have shown that some PTH is stored in granules and released by exocytosis (Habener et al., 1979) though contrary to other secretory cells, where exocytosis is stimulated by calcium,PTH secretion is inhibited (Brown et al., 1980 and Habener et al., 1976). The preferential and rapid release of PTH in response to stimuli suggested the direct synthesis and tran­sport of PTH to the periphery of the cell without packaging (Mac Gre­gor et al., 1975 and Morrissey and Cohn 1979).

Cyclic AMP (cAMP) is evidently involved in the secretion of PTH. Adenylate cyclase activity is inhibited by calcium and it is present in parathyroid gland . cAMP concentration has been found to be increased in parallel with PTH secretion caused by adrenaline,isoprenaline, dopamine, secretin, prostaglandin E2 and hypocalcaemia (Blum et al., 1978, Brown et al., 1980, Brown et al., 1979, Brown et al., 1977, and Gardner et al., 1978). Decreased con­centration of cAMP was also noted with agents suppressed PTH secre­tion such as a -adrenergic agonists (Brown et al., 1978) and prosta­glandin F2 a ( Grander et al., 1978). Studies of dibutyryl cyclic AMP and other inhibitors of phosphodiesterase in vitro caused increased

INTRODUCTION

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HUMAN- Q B O V IN E - ^ ) PORCINE-

Figure 1.3: The structure of human parathyroid hormone. The structure of bovine and porcine PTH is shown with the positions indicated above and below the human structure. (Adapted from Keutmann et al., 1978).

INTRODUCTION

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N U C L E U S

DNA RNP3" Y Y - i $ ^ V r

s ,5‘

- 3 mRNA

RIBOSOMES

A AAA 3 '0M ETH(ONYLAMNOPEPTJOASE

vrz? ;;;;;;/ /77T77Pre-ProPTH

PARATHYROIDCELL

ZZZ22222ZZZ>pt hm b ProPTH b m r iV,

CISTERNA ___ . ., ( 3 ) t r y p t k :

«> CPASE B

ENDOPLASMIC RETICULUM

< I m n l5-20m m

PERIPHERALCIRCULATION

CYTOPLASMIC MATRIX

IIHISECRETORYGRANULEGOLGI

30rr*n

Figure 1.4: The proposed intracellular pathway of the biosynthesis of parathyroid hormone. Pre-Pro-Parathyroid hormone (Pre-Pro-PTH), Pro-Parathyroid hormone (Pro­PTH). The time needed for these events to occur is given below the scheme. (Adapted from Amaud 1983).

INTRODUCTION

- 2 6 - CHAPTER 1

PTH secretion

The calcium concentration in the blood profusing the gland has been found to be inversely related to PTH concentration in vivo and in vitro ( Habener and Potts 1976) using bioassay and radioimmunoas­say. However, the relation between PTH secretion and plasma calcium is not a simple one; mild hypocalcaemia initially produces a steep increase in the rate of secretion , which becomes maximal and remains so with increased hypocalcaemia. The inverse relationship also exists between magnesium concentration and the rate of PTH secretion both in vivo and in vitro ,but it is two to three times less potent on a molar basis (Habener and Potts 1976). Therefore,compared to calcium, magnesium contribution to PTH release is small under phy­siological conditions. However, very low concentrations are associated with reversible failure of PTH secretion (Anast et al., 1976 )

The relationship between Vit D and PTH secretion is not very easy to establish, a number of studies intended to clarify the relationship gave conflicting results. 1,25-DHCC has been shown to have no effect (Hurst et al., 1979), to stimulate (Canterbury et al., 1978) or to suppress (Chertow et al., 1975, and Dietel et al., 1979) the secretion of PTH in vivo (Canterbury et al., 1978, and Hurst et al., 1979) and in vitro (Dietel et al., 1979). 24,25-DHCC was reported to have no effect (Dietel et al., 1979) and suppress PTH secretion (Canter­bury et al., 1978, and Care et al., 1976), and 25,26-DHCC reduced PTH secretion (Care et al., 1978).

Catecholamines, according to some evidence, are involved in PTH secretion. PTH release was found to be stimulated by beta adren­ergic agonists (Kukreja et al., 1976, and Williams et al., 1977). The specific receptors on parathyroid cell are of the B-2 subtype, and isoprenaline stimulates PTH release and cAMP production greater than noradrenaline (Brown et al., 1977). The PTH secretory response to hypocalcaemia has not been modified when the effect of adrenaline was inhibited by propranolol (Blum et al., 1978, and Brown et al.,

INTRODUCTION

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1978) indicating the dissociation of the two receptor response sys­tems. Adrenergic agonists augment secretion when the secretory route is already high (Blum et al., 1978, and Mayer et al., 1979) but have little or no effect during normocalcaemia.

1.4.2. Heterogeneity and metabolism

The interpretation of PTH immunoassay results has been complicated by the heterogeneity of circulating PTH (Berson and Yalow 1968,and Habener and Segre 1979). Intact hormone (molecular weight 9500 daltons) is the main form secreted by the glands. Other fragments may also be secreted especially during hypercalcaemia ( Mayer et al., 1979). Biologically inert carboxy terminal PTH fragment (molecular weight 7500 daltons) has been demonstrated in large quantities in the serum and significant quantities of biologically active amino-terminal (molecular weight 4000 daltons) moiety has also been detected ( Segre et al., 1977). Most of the immunoassays used will measure carboxy terminal fragments (D’Mour etal 1979), intact hor­mone and small quantities of amino terminal peptides 1-34 or 1-36 (Hunziker etal., 1977).

The cleavage of the hormone to carboxy terminal and amino terminal fragments may be a form of degradation or activation of the hormone prior to receptor binding (Segre et al., 1981). However, the cleavage of intact hormone has been suggested to be not necessary for the expression of full biological activity in bone and kidney (Goltzman 1978).

The half life of the fragments may determine the hetero­geneity of PTH in serum. The amino terminal fragment has a half life of 2-5 minutes the shortest, while that of carboxy terminal pep­tide may be in hours (Segre et al., 1976, Flueck, et al., 1977, Habener et al., 1976, and Hunziker et al., 1977). The metabolism in the peri­phery mainly liver, kidney and bone is the main source of hetero­geneity under normal homeostasis. Liver selectively takes up intact hormone but not amino or caboxy terminal fragments (Martin et al.,

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1979); this uptake was demonstrated in dog, rat, chicken and humans in vivo and in vitro (Martin et al., 1979), with the hypocalcaemia accelerating the process.

The kidney may act as the source of heterogeneity, especially in hypocalcaemia (Hruska et al., 1977, and Hanono et al., 1978) as well as removing the intact hormone and fragments from the circulation. The carboxy terminal fragments are removed by glomerular filtration and tubular reabsorption (Martin et al., 1979), and the intact hormone and amino terminal fragments are removed by peri­tubular uptake and also by glomerular filtration with subsequent peroximal tubular reabsorption. The high value of serum carboxy ter­minal PTH assays in chronic renal failure patients (Freitag et al., 1978) indicate impaired glomerular filtration. The degradation of PTH by the liver and kidney is responsive to serum calcium level (Kleeman and Kleeman 1979) and there is a weak positive correlation between serum calcium and metabolism of PTH in the kidney but not in the liver or the limb.

Bone metabolizes the intact hormone with the release of carboxy terminal fragments and complete degradation of amino terminal fragments, indicating the contribution of skeletal metabolism of PTH to the heterogeneity of circulating fragments. Intact PTH stimulate renal cortical membrane, adenylate cyclase in fetal rabbit bone (Goltzman 1978), and fetal rat and isolated bone cells (Freitag et al.,1979). The 24-48 peptide which is a competitive inhibitor to PTH degradation has been found not to inhibit renal cortical adenylate cyclase activation by the intact hormone (Resenblatt et al., 1977). Moreover, the amino terminal fragment (1-34) but not the intact hormone were found to be extracted by dog tibia (Martin et al.,1978), and cAMP production was stimulated with the amino terminal fragment 1-34 but very little with intact hormone. Perifusion of cat limb bone and hen femur with PTH shows no calcium mobilization from bone..

INTRODUCTION

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1.4.3. Physiology of PTH

The principal target tissues where PTH has a direct effect are kid­ney and bone.

In the kidney, early studies showed PTH to produce a phospha- turic effect which was later found to be the result of impurities affecting the renal dynamics. Improved laboratory procedures and the availability of highly purified parathyroid hormone demonstrated that PTH decreased phosphate reabsorption in proximal and distal tubules causing phosphaturia ( Pastoriza et al., 1978). Phosphate tran­sport paralleled closely that of sodium in proximal tubule under the influence of PTH. Some suggest that PTH, through regulation of sodium reabsorption, indirectly modifies phosphate reabsorption in the proximal tubule; according to others they are dissociated. PTH adminstration increases bicarbonate excretion which is largely due to decreased proximal tubular reabsorption.

After PTH administration the urinary excretion of calcium was decreased via complex actions on renal tubules. Calcium reabsorption is enhanced in the thick limb of Henle’s loop (Bourdean and Burg1979), in the distal convoluted tubule and the cortical collecting ducts (Agus et al., 1975 and Shareghi and Stoner 1978) but inhibited in the proximal tubule. The full expression of the hypocalcaemic effect of thiazide diuretics requires PTH ( Parsons 1976), and PTH adminstration reduces magnesium excretion. Primary hyperparathy­roidism patients have high rates of magnesium excretion even though they may be hypomagnesaemic which can be explained by the overriding of the retentive effect of PTH and inhibition of mag­nesium reabsorption.

One of the important actions of PTH on the kidney is to stimulate the conversion of 25-hydroxycholecalciferol to the active hormone 1,25-dihydroxycholecalciferol through its effect on renal 1 a -

hydroxylase enzyme. It is also shown that primary hyperparathy­roidism patients have higher levels of 1,25-DHCC than normal

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subjects (Haussler etal., 1976).

The mechanism of action of PTH on the kidney has been studied with a combination of methods. It activates adenylate cyclase through its binding to cell surface receptors. Early studies were ham­pered by PTH iodination problems, as chloramine T is thought to pro­duce some damage which renders the hormone biologically inactive. Hence, later studies used PTH labelled with 125 I using lactoperoxidase method ( Health and Aurbach 1975 ) or 15Semethionine or ?H -tritium (Zull et al., 1977). PTH receptor was suggested to be a single high affinity site present in relatively high concentration (Nissenson and Arnaud 1979). The binding of PTH to its receptor was not affected by many peptides, but was displaced by insulin in some studies and inhibited by high concentrations of ACTH in others (Zull et al.,1977).

PTH generated cyclic AMP is thought to activate cytosolic and membrane bound kinases (Kinne et al., 1975). Both cyclic AMP and PTH increase the permeability of proximal tubules ( Lorentz 1976 and Puschett et al., 1976). Cyclic AMP or dibutyryl cyclic AMP were shown to cause phosphaturia in the dog, rat and man . Cyclic AMP were shown to mimic PTH in decreasing proximal tubular reabsorption of phosphate and sodium in the dog and r a t .

While phosphate reabsorption was shown to be inhibited by cyclic AMP, dibutyryl cyclic AMP and PTH (Goldberg et al., 1976), cyclic AMP and its analogue dibutyryl cyclic AMP were mimicking some or all the effects of PTH on the renal tubules. They cause decrease in fractional excretion of calcium (Burnatowsk et al., 1977), reduce calcium reabsorption in proximal tubule, increase calcium reabsorption in the cortical thick ascending loop of Henle (Bourdean and Burg1979), and the distal convoluted tubule (Costanzo and Windhager1978). Thus the action of PTH on the kidney is mediated by cyclic AMP.

In bone, PTH inhibits bone formation ( Parsons 1976, Raisz

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1977 and Raisz et al., 1978), and its osteolytic action was found to be inhibited by a diffedency of many steroid hormones, caldtonin and vitamin D (Atkins and Peacock 1975, and Raisz 1977). PTH is also shown to have an anabolic action on bone (Parsons 1976, and Herrman-Erlee 1976). Bone formation is also accelerated by low doses of synthetic human PTH (1-34) in animals (Hafti et al., 1982), and to increase bone mass in man (Reeve et al., 1976). Bone cyclic AMP (Heerche et al., 1978, and Smith and Johnson 1975),as well as tissue cyclic AMP, all rise after exposure of bone to PTH having bone cyclic AMP peaking within thirty minutes. The rise reaches thirty to fourty fold with large doses of PTH in rat (Nogata et al., 1975) but not in man (Tomlison et al., 1975). Some or all actions of PTH on bone are mediated by cyclic AMP. In rats cyclic AMP and dibu­tyry l cyclic AMP were shown to increase urinary excretion of pep- tidyl hydroxyproline. In bone cell culture in vitro cyclic AMP and dibutyryl cydic AMP was shown to mimic PTH action by inducing bone resorption and enhancing lactate, citrate and lysosomal enzyme formation. However, the effects of dibutyryl cyclic AMP differ from that of PTH as the effects are produced only at high concentration and only after a long delay, and in addition it antagonizes PTH over a wide range of concentrations.

PTH regulates caltium flow from the bone to the extracellular fluid by direct interaction with the bone (Talmage and Mayer 1976). Mineralization and bone matrix formation were found to be inhibited and bone resorption and cacium release were stimulated with high doses of PTH (Raisz 1976). PTH is also shown to increase hyaluronate synthesis (Severson 1978), and to inhibit citrate decarboxylation and collagen synthesis (Dietrich et al., 1976). The effectors of PTH- induced bone resorption is believed to be the multinucleated osteoclasts. Mononucleated cells may also contribute to bone resorption (Mundy et al., 1977, Kahn et al., 1978 and Galasko 1976). The effects of PTH on osteoclasts starts with an increase in RNA synthesis, raising the number of nuclei per osteoclast (Addison 1980) and the increase in

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osteoclast number (Fallon et al., 1981).

The stimulatory effect of PTH on the lysosomal enzyme system has been studied using histochemical methods and enzyme assays of bone medium (Eilon and Raisz 1978). It shows a correlation between the stimulation of enzyme activity and relase of radiolabelled calcium from bone. PTH also inhibited osteoblast activity; it decreases the synthesis of collagen by bone (Dietrich et al., 1976 and Kream et al.,1980) and by osteoblastic osteosarcoma cells (Kream and Majeska1981). PTH also decreases citrate decarboxylation. Organ cultures have contributed much to the current understanding of the final physiological effects of PTH. Among the currently used models are; neonatal tissue subjected to enzymatic digestion ( Peck et al., 1977, Smith et al., 1975, Rao et al., 1977, Chen and Feldman 1978, Wong and Cohn 1975, Nijweide and Van der Plas 1979 and Pliam et al., 1981) and normal and abnormal cells derived from osteosarcomas (Majeska et al., 1978 and Martin et al., 1976). The target cells for PTH in bone is illustrated by the presence of PTH specific receptors. Binding of biologically active .125/ -PTH to osteoblasts and pre­osteoblasts in chicken calvaria (Slive et al., 1981) and in chicken bone cells (Pliam et al., 1981) are in agreement with the bioassay results from mouse bone cells (Smith et al., 1975, Rao et al., 1977, Wong and Cohn 1975 and Peck and Kohler 1981) and osteosarcoma cells (Majeska et al., 1978 and Martin et al., 1976) in which PTH eli­cited large increases in cellular cAMP content and rapid morpholo- giocal changes (Miller et al., 1976 and Jones and Boyde 1976). The large increase in cAMP seen in PTH treated bone cell studies suggest that it largely comes from the osteoblasts, as it correlates with activa­tion of cAMP dependent kinases (Portridge et al., 1981).

Calcium is required for the action of PTH in the kidney (Rasmussen et al., 1976) as well as in bone (Wong et al., 1978). Cal­cium may affect PTH action through changing the conformation of the receptor on the cell surface (Chen et al., 1980 and Zull et al., 1976) or regulating adenyl-cyclase-associated calmodulin activity (Peck

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and Kohler 1981). PTH-treated bone is shown to increase calcium uptake (Dziak and Stern 1975). However, calcium entry to the cells (OB cells) may be a direct result of PTH binding, as PTH induction of cAMP and calcium uptake may be unconnected (Chen et al., 1980). Moreover, neither cAMP nor l,25(OH)2 D3 is shown to increase bone cell calcium uptake (Dziak 1978). Thus there may be two mediators of PTH action on bone , normally cAMP and calcium.

The calcium inophore A23187 has been shown to stimulate cal­cium release from bone at the appropriate concentration (Dziak and Stern 1975) and the calcium channel blocker verapamil inhibits PTH effect on bone (Herrman-Erlee et al., 1977). However, the ability of PTH to increase cyclic AMP and calcium entry to the target cells is shown to be dissociated (Dziak and Stern 1975).

PTH has been shown to have anabolic effects on bone. Prolonged treatment with low concentration of PTH has been found to enhance bone formation in vivo , and the same was observed with the 1-34 bio­logically active fragment in vitro (Herrman-Erlee et al., 1976) mimick­ing the in vivo action of the 1-84. There is evidence that 1-34 fragment is generated in vivo but has not been found when bPTH(l-84) was incubated with bone cells or bone (Freitage et al., 1979). Thus it seems that the effect of PTH on bone is dose dependent (Majeska and Rodan 1981).

Several studies have demonstrated the binding in vitro of labelled PTH to normal plasma membrane of chicken (Chansel et al., 1977, and Nisseneon and Amaud 1979), rat (Rosenblatt et al., 1976), cow (Zull et al., 1977), dog (Segre et al., 1979, and Bellorin et al., 1981) and man (Abbott et al., 1981). The biological significance of this has been attributed to PTH binding sites if they appear to be coupled to activation of adenylate cyclase, since physiological effects are not demonstrable.

cAMP play an intermediary role in PTH induced inhibition of renal phosphate reabsorption ( Burnatowska et al., 1977),PTH stimulation

INTRODUCTION

- 3 4 - CHAPTER 1

of distal tubular calcium reabsorption ( Burnatowska et al., 1977) and l,25(OH)2D vitamin D3 production (Horiuchi et al., 1977). Some evidence shows that renal adenylate cyclase system is equally sensi­tive to PTH in vitro versus in vivo (Nissenson et al., 1980). PTH bind­ing sites have been shown in solubilized bovine cortical plasma mem­branes (Malbon and Zull 1977) and found to be > 100,000 daltons by gel filtration.

bPTH(l-84) and (1-34) were reported to have similar potencies in stimulating renal adenylate cyclase (Nissenson and Arnaud 1979 and Nissenson et al., 1981) while only bPTH(l-34) increased cyclic AMP production in isolated perfused canine bone, but not bPTH(l-84) (Martin et al., 1978).

1.4.4. Structure function relationship

Dark-field electron microscopic study (Fiskin et al., 1977) and a theoretical study of PTH secondary structure (Zull and Lev 1980) suggest a structure consisting of two stabilized spherical clusters linked by a straight chain region.

In many assay systems synthetic bPTH(l-34) behave identically to PTH(l-84) isolated from natural sources (Parsons et al., 1975, Segre et al., 1979, Herrmann-Erlee et al., 1976, Herrmann-Erlee et al., 1978, Neuman and Schneider 1980 and Bringhurst and Potts 1981). However, in many others the synthetic bPTH(l-34) was shown to be less active than the native hormone (Herrmann-Erlee et al., 1976, Bringhurst and Potts 1981, Martin et al., 1980, Herrmann-Erlee et al., 1980 and McGowan et al., 1981). In many assays these differences could be attributed to greater stability of the 1-84 possibly due to resistance to proteolytic influences (Herrmann-Erlee et al., 1978 and Bringhurst and Potts 1981).

The removal of the amino acids from the carboxy terminal of the PTH(l-34) leads to a decrease in binding affinity and hence in activity (Segre et al., 1979). However, in sensitive bioassays fragments as small as 1-26 sequence are found to bind and activate PTH receptors

INTRODUCTION

CHAPTER 1

at high concentrations (Martin et al., 1980 and Goltzman et al., 1978) whereas the peptides with 1-25 sequence or smaller show no binding or activation.

The removal of the amino terminal residues from bPTH(l-34) has been shown to drastically reduce the bioactivity ( Herrmann-Erlee et al., 1976 and Goltzman et al., 1978). bPTH(3-34) is a competitive inhibitor of PTH action( Herrmann-Erlee et al., 1976 and Goltzman et al., 1978), and thus the structure 3-34 has all the binding require­ments, while position 1 and 2 are essential for the activation of the receptor. The replacement of the carboxy terminal 34 of PTH(l-34) leads to an increase in biological activity (Parsons et al., 1975, and Martin et al., 1981). The substitution of phenylalanine in position 34 in bPTH(l-34) was found to be very useful in iodinating the frag­ment with no loss of biological activity (Rosenblatt and Potts 1977, Goltzman et al., 1975 and Rosenblatt et al., 1976). The oxidation of methionine-containing species and analogues of PTH results in loss of binding affinity and inactivation of the hormone (Martin et al., 1981, Rosenblatt et al., 1976, Coltrera et al., 1980, and Goltzman et al., 1975). The substitution of methionine with norleucine in position 8 and 18 resulted in a small decrease in biological activity in vitro (Rosenblatt and Potts 1977, Goltzman et al., 1975 and Chen et al.,1980),concomitant tyrosinamide substitution in position 34 resulted in increase in activity in vitro, and the 3-34 analogue was a power­ful antagonist at equimolar concentration (Rosenblatt et al., 1976 and Rosenblatt et al., 1977). The norleucine substitution leads to a decrease in biological activity in vivo (Rosenblatt et al., 1981). The substitution of valine in position 2 with D-valine resulted in total loss of biological activity (Coltrera et al., 1980 and Rosenblatt et al.,1981).

Human hormone and its fragments were shown in the early in vitro studies to be less active than the corresponding bovine sequence peptides (Goltzman et al., 1975). In the PTH molecule posi­tion 1 and 2 are associated with activation of the receptors and

INTRODUCTION

- 3 6 - CHAPTER 1

biological activity. The bovine hormone contains alanine in position 1 whereas the human hormone has serine there, and exchange of posi­tion 1 between the two hormones showed that the difference between the two hormones is totally due to the amino acid in position 1 (Zull and Lev 1980, Parsons et al., 1975, Goltzman et al., 1978, Goltzman 1975 and Tregear and Potts 1975). In comparing hPTH(l- 34) and bPTH(l-34) in vitro using the renal plasma membrane model system from various species, hPTH(l-34) was found to be less active in most cases (Goltzman et al., 1975 and Martin et al., 1975).

The hormone-degradative activity seems to be different among animal species, dependent on the assay tissue. hPTH(l-34) was inactive in the rat system where the degradative activity is high (Neuman and Schneider 1980 and Goltzman et al., 1975) and equipotent with bPTH(l-34) in the chick where PTH-active peptidases are low (Martin et al., 1975). It was also shown on the basis of chick hypercalceamia assay that PTH fragments which where less active in vitro were equipotent with the 1-84 sequence in vivo (Parsons et al., 1975, and Neuman and Schneider 1980). The 1-D-alanine substitution which leads to loss of activity in vitro resulted in increased activity in vivo. In various systems bPTH(3-34) analogue was found to be a weak antagonist of PTH in human giant cell tumors of bone, (Goldring et al., 1979, Ausiello et al., 1980 and Goldring et al., 1981) in embryonic calvarial systems (Herrmann-Erlee et al., 1980) and in skin fibroblast system (Goldring et al., 1979).

1.4.5. Measurement of parathyroid hormone

Parathyroid activity was measured by rat bioassays and various in vitro bioassays . However, these assays lack the sensitivity to detect the small quantities of hormonal activity in serum. Comple­ment fixation assay was also used to measure PTH activity. Although it is not sensitive enough to measure PTH activity in serum, it was found useful in studying immuno-chemical changes in the PTH molecule, and it is the first assay to detect PTH-like agents in non­

INTRODU CTION

- 3 7 - CHAPTER 1

parathyroid neoplasms associated with hypercalcaemic syndrome.

The purification of the hormone, and the confirmation of amino- acid sequence led to the development of PTH assays. Until the introduction of radioimmunoassay, the measurement of PTH in serum was not possible. The first radioimmunoassay for PTH was described by Berson in 1963 (Berson et al., 1963). With the improvement in the bioassays, later generations of such systems were also capable of measuring PTH in serum.

The introduction of multi-channel analyzers in clinical biochemistry laboratories led to increased detection of hyper- calcaemia, and consequently to a greater demand for PTH assay in serum. Thus the need for more sensitive assays, which measure particular fragments in serum. Some of the PTH fragments are present in very low concentration, others are devoid of biological activity. Hence, the usefulness of results depends on the type of assay used for the measurement of PTH.

In the last decade several assays have been used in the measurement of PTH in serum. At present three assay types are capa­ble of measuring PTH in serum; these are ( l) Immunoassays (2) Bioassays (3) High Performance Liquid Chromatography (HPLC) .

Immunoassays

Since the introduction of RIA, PTH assays have undergone dramatic changes. The first RIA for PTH (Berson et al., 1963) used antisera raised against bovine PTH (bPTH) and ,131/ -bPTH. However, the assay fails to distinguish between normals and

hyperparathyroidism patients. In subsequent years, many assays have been developed, yet this same problem of overlap between nor­mals and groups of patients persists. The failure of these assays and many others is due to the heterogeneity of PTH in circulation ( Berson and Yalow 1968). It is now known that there are at least four different forms of PTH present in circulation: intact molecule, C- ter­minal, mid-molecule and N-terminal fragments. The demonstration

INTRODUCTION

- 3 8 - CHAPTER 1

that different forms of PTH are present in the circulation, changed the approach and the design of assays. Together with the availability of synthetic fragments, more specific assays were developed.

There are four immunoassays utilized for the measurement of PTH in serum. The availability of a good antibody reagent usu­ally determines which assay to use. However, in reality each assay is useful in certain clinical situations. The different assays currently used are:

Intact molecule assay:

Antisera raised against crude PTH can be obtained with specificity for different regions of the amino-acid chain of PTH. Two types of antisera to PTH have been produced: antisera with specificity largely to the C-terminal fragment and antisera with specificity largely for intact parathyroid hormone (D’Amour et al., 1984), the later antisera are seemingly of two sub-populations; thosespecific to the N-terminal and to the whole molecule with nocross- reactivity to the N or C-terminals.

The new assays for the whole molecule use: (a) antisera against PTH (24-48) region since this is the cleavage region, (b) sequen­tial immunoextraction of N-terminal fragments followed by mid region assay ( Mallette 1983 and Lindall et al., 1983) and (c)immunometric assays, where two different antibodies raisedagainst two different fragments (i.e C-terminal and N-terminal) are utilized (Brown et al., 1987, Nussbaum et al., 1987). These assays were shown to be very sensitive and fast (Nussbam et al., 1987). The use of non-isotopic labels was shown

to achieve high sensitivity assays for PTH (Brown et al.,1987). These assays are new and await evaluation and clinical assessment.

C-terminal assays

Assays for the C-terminal uses antisera raised against the C- terminal fragment. Generally, the antisera have a high affinity for

INTRODUCTION

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the C-terminal. However, it may react with the whole molecule to a lesser extent, but has no cross-reactivity with the N- terminal fragment. Although the C-terminal assays did not usually show an inverse relationship with serum ionized calcium, they have a good sensitivity and discriminates between the different groups of abnormal and normals patients better than other assays. This is presumably due to their long half life and high concentration in circulation. In chronic renal failure patients, C-terminal assays give a high value because the normal renal clearance is severely reduced.

The extensive use of these assays is mainly due to the scarcity of other good assays which measure the biologically active forms of the hormone.

Mid-molecule assays

The availability of synthetic fragments led to the development of this type of assay. Antisera are usually raised against the (43- 68) amino-acid sequence of the hormone. Many assays able to detect the 44-68 fragment have been described. These assays meas­ure the intact molecule as well as mid region fragments which did not contain the C-terminal sequence of PTH ( Roos et al., 1981, Marx et al., 1981, and Mallette et al., 1982). They have no cross­reactivity with the N-terminal fragment, thus apparently there is no biological activity attached to the fragments measured by these assays.

N-terminal assays

Many assays are described with antisera specific to the N-terminal fragment (Silverman and Yelow 1973, Martin et al., 1978, Segre et al., 1977, Canterbury et al., 1975, Neuman et al., 1975, Segre et al., 1981a, Segre et al., 1981b, Morrissey et al., 1980, MacGregor et al., 1979 Martin et al., 1979, Martin et al., 1977, Hruska et al., 1977, Vieira et al., 1984, Vieira et al., 1986,). These assays have a poor discriminating ability. The antisera are usually raised against

INTRODUCTION

- 4 0 - CHAPTER 1

crude preparation of bovine or human PTH, and they seem to recognize the whole molecule as well as the N-terminal fragment. Because the N-terminal fragment contains the amino-acid sequence required for biological activity, the results of the assays were expected to correlate with the clinical status of the patients. However, to date these assays have not shown a good correlation with primary hyperparathyroidism. The newer assays perform much better (Potts et al., 1983, Hawker et al., 1984,Fischer et al., 1982), which suggests that the poor perfor­mance of past assays might have been due to the non-specificity of the antisera.

On the other hand, N-terminal assays have been very helpful in many other situations. They have been used in chronic renal failure patients, where all other assays give high values because of renal impairment(Segre et al., 1981b, Morrissey et al., 1980, MacGregor et al., 1979). They have also been used in the measure­ment of administered hPTH(l-34) (Zanelli et al., 1980). Another use of the N-terminal assay was in the catheterization studies for the localization of hyperplastic parathyroid glands ( Hruska et al., 1977, Martin et al., 1978, and Marx et al., 1981). The main reason that prevents extensive use of N-terminal assays is their poor sensitivity. However, with the availability of monoclonal antibodies together with other analytical developments moire sensi­tive assays may be forthcoming in the near future.

High performance liquid chromatography (HPLC)

This technique has not been used routinely in the laboratory, however, it has the sensitivity needed to detect hPTH in serum . HPLC with reversed phase has been used to purify and analyse PTH (Bennet et al., 1981, Zanelli et al., 1981, Zanelli et al., 1983). Generally,the assay involves extraction of material on octadecylsilyl-silica, and the extracts were chromatographed using reversed phase HPLC. In most HPLC methods PTH in the collected fractions is assayed using radioim-

INTRODU CTION

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munoassay (Zanelli et al., 1983). Three peaks were evident in the immuno-reactivity profile of parathyroid extract, C-terminal, N- terminal and intact molecule.

HPLC assay is dependent on micro-scale chemical analysis and biological assessment, it has not been used routinely in the assessment of patients samples.

Bioassays

The old generation of the different in vitro and in vivo bioas­says are not sensitive enough to measure PTH in normal subjects serum. However, at present there are two bioassays which have sufficient sensitivity to detect PTH in serum.

a Receptor bioassay

This assay depends on the activation of adenylate cyclase in can­ine renal cortical membranes (Abbott et al., 1980, Nissenson et al., 1981). This in vitro bioassay measures both intact and bio­logically active hPTH(l-34) (Abbott et al., 1980) and the sensi­tivity can be enhanced by the addition of the analog of guanosine-triphosphate, 5’-guanylimidodiphosphate. The detection limit of the original assay was too high to detect PTH in normal serum, it has subsequently been reported that the assay could be applied to measure PTH in normal serum (Avioli et al., 1981). The use of human instead of canine membranes giving the same sensitivity has also been reported (Niepel et al., 1981)

b Cytochemical Bioassay

This assay depends on the stimulation of glucose-6-phosphate dehydrogenase activity in the distal convoluted tubules of guinea-pig kidney maintained in vitro ( Chambers et al., 1978). It has been developed using techniques used in the ACTH cyto­chemical bioassay (Chayen et al., 1972). The enzyme activity is measured by microdensitometry ( Chambers et al., 1978). This technique has been used by different groups for the measurement of PTH (Chambers et al., 1978, Fenton et al., 1978, Goltzman et

INTRODUCTION

- 4 2 - CHAPTER 1

al., 1980) and a modification of the original technique has also been reported (Posillico et al., 1981). Human and bovine synthetic biologically active fragments gave a parallel response (Goltzman et al., 1980) and equimolar to that for intact bovine hormone. It was estimated that biologically active hormone present in the circula­tion of normal individuals ranged between 1-30 ng/L (1-30 pg/ml) (Goltzman et al., 1980, Fenton et al., 1978, Chambers et al.,1978) as biologically active PTH in serum was reported to con­stitute only 10% of that measured by RIA (Allgrove et al., 1983).

The assay is capable of distinguishing between hypo-, hyper­parathyroid patients and normal patients (Goltzman et al., 1980, Fenton et al., 1978, Allgrove et al., 1983). Despite the high sensitivity of this assay, it is not expected to be used rou­tinely in the laboratory, because it is not practical and is cumbersome, It could, however, serve as a reference method for comparing the different assays, and provide another means of confirming the diagnosis in the difficult cases.

The methods mentioned above are theoretically divided into two groups; those which measure the biologically active forms of PTH, which are expected to correlate with the clinical situation of the patients, and those which measure the fragments which display no biological activity but are present in high concentration in the plasma, and have 10 to 15 times longer half-life than the biologically active fragments. It has been easy to measure the inactive fragments, because of their high concentration and longer half-life. On the other hand, measurement of biologically active fragments presents a problem, because they demand a very highly specific and sensitive assay. Despite the common use of assays which measure biologically inactive fragments, the assay of biologically active fragments is needed for the understanding of the physiology of PTH. Biologically inactive fragments give no information about the physiology and the minute to minute change in the secretion of the hormone. Their assay correlates with glomerular filtration rate, on which their clearance

INTRODUCTION

- 4 3 - CHAPTER 1

depends.

For the measurement of PTH in body fluids, immunoassays are preferable because they are fast, easy and can handle a large number of samples. The main disadvantages of many of these procedures avail­able currently in that fragments devoid of biological activity are measured. Hence, there is a need for improved immunoassays that will apply to biologically active fragments of PTH.

The antibody plays the major role in the characteristics of immunoassays. The sensitivity and specificity of the assay is usually determined by the antibody, hence, assays which use polyclonal antibody tend to suffer from a high cross-reactivity and modest sensitivity.The introduction of monoclonal antibodies is expected to yield more specific and sensitive assays for the different fragments of PTH.

1.3. AIMS OF THE PRESENT WORK

The measurement of parathyroid hormone plays a key role in the differential diagnosis of hypercalcaemia. The procedure for the assess­ment of parathyroid status has improved over the years, thus also con­tributing to the understanding of the etiology of hypercalcaemia. Dur­ing the past decade many assays have been developed which measure one form or another of PTH in serum. However, the methodologies, tend to be diverse and unlike other peptide hormones, the antibodies are rather limited from the viewpoint of quality and assay availability. A better assay could be developed, standardized, and put into a wider use. In particular, application of such assay to the biologically active frag­ment of PTH might also assist in the laboratory investigation of PTH- related diseases.

We therefor undertook the raising of polyclonal and monoclonal antibodies to the hPTH(l-34). In the early stages, polyclonal antibodies were raised in the goat and with that material various detection sys­tems for the antibody were devised and the assay procedure developed. A maior part of the project was dedicated to the development of

INTRODUCTION

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monoclonal antibody to hPTH(l-34). Attempts were made to identify the most effective immunization schedule and animal species combina­tion for cell fusion. The latter included mouse-mouse, rat-rat and rat- mouse systems, and was further expanded to goat-mouse and goat- heteromyeloma (ovine-murine hybridoma) fusions.

The antibodies produced as a result of this work were applied tothe detection of PTH-like molecules in tissues and to the construction

wjthof an immunoassay for use'body fluids.

INTRODUCTION

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CHAPTER 2

DEVELOPMENT OF SCREENING ASSAYS FOR MONOCLONAL ANTIBODIES

SCREENING ASSAYS

-4 6 - CHAPTER 2

2.1. INTRODUCTION

The success or failure of the production of a monoclonal anti­body is largely governed by the screening procedures employed. The concentration of monoclonal antibody that can be produced is theoretically very small, especially in the early stages,where only a few cells of those growing produce the desired antibody.

A good screening assay should not just be able to detect an antibody, it must be rapid, sensitive, reliable, and recognize all anti­bodies of all types of immunoglobulins. Such a system is an essential pre-requisite during the screening stages for the detection of hybrid cell lines secreting the required antibody. The detection and selection of each antibody is limited by the assay, and it is important that, the assay chosen for this purpose has the following features.

SensitivityThe assay must be very sensitive to allow the detection of a small concentration of antibody secreted by the hybrids in the very early stages. It is one of the steps which severely limit the possible selection of antibody.

SpecificityIt is desirable to select only those cells which produce an anti­body specific to the antigen. The assay chosen has to be accordingly capable of meeting this requirement. It may not however,be possi­ble to select for an absolute specificity from the beginning.

SimplicityThe simplicity of the assay as well as the efficiency to handle a large number of samples are the other requirements for screening systems. It is necessary to detect and select the desired clone very early. Time is very important, especially when large number of clones per well are expected. The hybrids exert a very diverse growth characteristics, some very important hybrids may be lost in the detection and selection step, and careful selection of a rapid, simple assay will minimize the loss.

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- 4 7 - CHAPTER 2

ReliabilityThe whole feature of the screening assay depends on the relia­bility and accuracy of the assay. The detection rate of the assay must be consistent, and must be accurate to avoid the waste of valuable time pursuing false positive cultures, or most important the loss of very valuable positive cultures.

It is appropriate to say that, the assay must be chosen with regard to the nature of antigen and the uses for which the antibodies are intended(Edwards 1981, Galfre and Milstein 1981, Catty et al., 1981, Foster 1982).

In the last decade many assays have been used to detect monoclonal antibodies, some of which are described in the study of antibody formation at the single cell level (Cunningham 1973). Others were those used with the polyclonal antibody adapted for the detec­tion of monoclonal antibodies. Generally it is possible that many immunological assays can be adapted for the detection of monoclonal antibody within the limits of screening assays.

A brief account of the most commonly used assays for screening hybridoma supernatants is given below, with more attention paid to those used in the screening of antibodies to soluble antigen.

Binding assays are the most popular, and commonly used assays in hybridoma screening, because of their simplicity and ability to detect all types of immunoglobulins. These are particularly useful for the detection of monoclonal antibodies against soluble antigens.

Radioimmunoassay (RIA)

Since the introduction of RIA (YaJow and Berson 1960) various modifications have been used for screening monoclonal antibodies (Soos and Siddle 1982,and Baxter et al., 1982).

These have also been adapted for the same purpose, to microtitre plate in conjunction with solid phase immunoassay (Goding 1986, Catty et al., 1981, Galfre and Milstein 1981, and Hudson and Hay1980). In this type of assay, the antigen is adsorbed on to a solid

DEVELOPMENTOF SCREENING ASSAYS

-4 8 - CHAPTER 2

phase support. The antibody solution is added to the solid phase antigen followed by the labelled antiglobulin. This type of assay eliminates the separation step of the classical RIA, and this is accomplished by washing the plate. The labelled antiglobulin serves as a general reagent, detecting all types of immunoglobulins.

Enzyme linked immunosorbent assay (ELISA)

ELISA was first described in the early seventies (Van Weeman and Schunrs 1971, Engval et al., 1971),since then, the ELISA assay has expanded and the application have been covered in several publications ( Bidwell et al., 1976, Voller et al., 1976 ,1979 and 1981, Voller and Bidwell 1980).

The use of ELISA in the detection of monoclonal antibody was demonstrated (Kennett 1981, Sundar Raj et al., 1982, and Douillard et al., 1980). Enzyme labelled and radiolabelled protein A was used as a general reagent (Suter 1980, and Nowinski et al., 1981). The ELISA makes use of the microtiter plate involving the same steps as those described above in the microtiter plate solid phase RIA, except that the radiolabel is substituted by enzyme labelled antibody. After separation of bound from free antibody by washing, a colourless substrate is added, and a soluble, coloured product is formed. The optical density of the solution is then measured in a spec­trophotometer.

Immunofluorescence assay (IFA)

Those are a group of assays which use a fluorescence-labelled antibody instead of enzyme or radio- labels. Fluorescein-labelled anti­body was first described by Coons et al (1941), since then a whole range of assays based on fluorescein- labelled antibody has been reported (Johnson et al., 1979 and London and Kamel 1981). Fluorescein-labelled antibody has been employed in the simultaneous screening and cloning of hybridomas using fluorescein-activated cell sorter (FACS) (Park et al., 1979, Dangl and Herzenberg 1982). These labels are commonly used in the identification of monoclonal

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- 4 9 - CHAPTER 2

antibodies directed against cellular antigens (Epstein 1981).

Biological assays

These assays are based on the effect of monoclonal antibody on the biological activity of the antigen. They are not very popular in hybridoma screening as they are cumbersome and cannot fulfill the requirements for a screening assay. The main features of the assay are that (i) the antigen does not have to be purified for the assay ,and (ii) the inhibitory effect gives some information about the antibody binding site on the biologically active molecule.

Haemagglutination assays

This type of assay, first described by Coombs (1981), was adapted for hybridoma screening (Galfre and Milstein 1981, Catty et al., 1981). They are qualitative assays, where the antigen is chemi­cally attached to the surface of red blood cells. In the presence of anti­body haemagglutination occurs. They are usually less sensitive than the binding assays, fail to detect some clones, and require a large amount of antigen. Direct and indirect haemagglutination assays can be used (Fraster et al., 1982)

Immunocytochemical assay: This assay is not considered suitable for screening assays of hybridomas, because it is very tedious and time consuming. However,if the antibody is finally going to be used in immunocytochemistry, this assay could be used in conjunction with other primary assays, where those found positive could be subjected to this assay when the time is not critical in selection.

The assay uses tissue which has been fixed and treated with different solutions. This sometimes dramatically changes the antigen, which may leads to different results ( Milstein et al., 1983)

Having considered the various posibilites of suitable screening system, available reagents and the cost of assays. In this chapter, the development of indirect ELISA for the detection of monoclonal anti­body to hPTH(l-34), and the optimization of liquid phase RIA ( Zanelli et al., 1980) are presented. The outline of direct and indirect

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- 5 0 - CHAPTER 2

ELISA systems are shown in figure 2.1.

2.2. RADIOIMMUNOASSAY (RIA)

2.2.1. Chemicals and reagents

hPTH(l-34) and bovine serum albumin(BSA) RIA, grade, were supplied by Sigma chemicals Ltd. Sodium iodide was sup­plied by Amersham chemicals Ltd. All other reagents were of analyt­ical grade from BDH chemicals Ltd.

2.2.2. Iodination of hPTH(l-34)

The incorporation of iodine 125 into proteins has been used for labelling antigens since the early days of radioimmunoassay. The iodine is substituted onto the aromatic side- chain of tyrosine resi­dues yielding a stable compound. It is also possible to substitute onto phenylalanine and histidine residues , although the substitution is 30 - 80 % times less than that for tyrosine.

The incorporation of iodine into hPTH(l-34) was found to be difficult and accompanied by other problems, not least the loss of activity.

Chloramine T method is the most common procedure used for the iodination of proteins. Other methods offer some advantages but the chloramine T method, for its simplicity and short time needed to accomplish the labelling is favourable. hPTH(l-34) has been iodinated by various other methods, but these showed no advantage over the chloramine T method. The only method which has not been used for the iodination of PTH, so far, is the Iodogen method.

In this study chloramine T method as well as the iodogen method were used to iodinate hPTH(l-34).

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- 51 - CHAPTER 2

Antigen adsorbed on the plate wells by addition of the antigen in coating buffer to the wells and incubatation.

AJLAddition of sample containing the anti­body to the antigen coated wells, the antibody should bind the solid phase antigen.

1

Addition of enzyme labelled anti-species antibody to bind the first antibody.

■ • ♦ 2 d♦ *oo

1

Colourless substrate for the enzyme is added and a colour product is formed which can be measured in spectropho­tometer.

Figure 2.1 a: Representation of the steps involved in indirect Volfef etel°C 197?) aSSay °f antibody (adaPted from

DEVELOPMENTOF SCREENING ASSAYS

- 5 2 - CHAPTER 2

Antibody to the desired antigen is adsorbed on the wells of ELISA plate by adding the antibody diluted in coating buffer to the wells and the wells incu­bated.

The free sites blocked with gelatin or other protein, and the sample containing the antigen is added and incubation is continued. The antigen should bind the solid phase antibody.

After washing the wells, antibody binds different determinant of the antigen and labelled by an enzyme is added to the wells and incubated.

o 0 •o a o ■ ♦ £ • °

A # -Substrate is added to the wells, and a colour product is formed, which can be measured spectrophotometrically.

Figure 2.1 b: Representation of the steps involved in double antibody sandwich ELISA for measuring antigen (adapted from Voller et al., 1979).

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- 5 3 - CHAPTER 2

Chloramine T method : The method used was that of Zanelli ( Zanelli et al., 1980). Briefly, hPTH(l-34) was dissolved in 0.1 M acetic acid to the concentration of 100 ug/ml. Aliquots (20ul-2ug) were snap frozen and stored at -70°C until needed for use. The stored material was used over two years.

Antigen (2ug) brought to room temperature and 100 ul of phos­phate buffer 0.2 M was added to it. The contents of the tube were mixed and 1.5 or 0.5 mCi of sodium iodine was added. Chloramine T (20ul,2.66mg) was added to the tube, mixed for 20 seconds and the reaction stopped by the addition of 50 ul of 3.6 mg/ml sodium meta­bisulphate. The tube mixed and 1 ml of phosphate buffer 0.02 M containing 0.5% BSA was added followed by 20 ul of 2 mg/ml potas­sium iodide. The mixture was then subjected to purification. The purification was done either with sephadex G15 or Quiso . Early purification was done using QUISO, later work was done on sephadex G15. The iodinated hormone was eluted with phosphate buffer 0.02 M pH 7.4 containing 0.5% BSA. The fractions containing the iodi­nated hormone eluted first and those which give high counts were further assayed for immunoreactivity. The best fraction was ali- quoted in 50 - 100 ul aliquots and stored at —40°C .

Iodogen method : The method used was similar to that of Hampten ( Hampten 1982). Iodogen was dried on the inner walls of polyethylene tubes as described by Salcinski (Salcinski et al., 1979).1,3,4,6 Tetrachloro 3 6 diphenylglyco uril was dissolved in analar dichloromethane to the concentration of 1 mg/ ml and 20 ug were adsorbed to the walls of polypropylene tubes.

To a tube containing 20 ug of iodogen, 0.5 mci sodium iodide was added followed by 2 ug of hPTH(l-34). The tube mixed occasionally for 10 minutes. Then 20 ul of 1 mg/ ml sodium metabisulphate was added followed by 1 ml of phosphate buffer pH 7.4 containing 0.5 % BSA. Potassium iodide (20ul, 2mg/ml)was then added, and then the mixture was transferred onto a G15 sephadex column. The labelled-

DEVELOPMENTOF SCREENING ASSAYS

- 5 4 - CHAPTER 2

peptide was eluted using phosphate buffer pH 7.4 containing 0.5 % BSA. Fractions of approximately 1 ml were collected. The fractions were counted, and those with high counts were tested for immunoreac- tivity.

2.2.3. Method

Buffers

Barbitone buffer pH 8 .6 was made as follows: 10.39 g of sodium barbital,3.722 g of ethylenediamine tetra-acetic acid sodium salt (EDTA), 0.1 mg thiomersal were made up to one liter with distilled water. The pH adjusted to 8 .6 using 0.1 N HC1. The buffer kept at 4°C

for not more than four weeks.

Working buffer

0.5 % BSA and 0.1 % Tween 20 were added to the barbital buffer before use. This buffer was kept at 4°C for not more than two weeks. Labelled antigen and antibodies were diluted in the same buffer.

Procedure

To duplicate LP3 tubes 200 ul of working buffer was added, fol­lowed by 100 ul of sample or control. The tubes were shaken, and 100 ul of labelled hPTH( 1-34) was added to each. The contents were mixed and incubated at 4°C overnight. Next day, 100 ul of pre­cipitating antibody was added to all tubes except totals, the tubes mixed and incubated at 4°C for 2.5 hours. Polyethylene glycol 6000 (200ul, 12% w /v) was added to all tubes except totals, the tubes were mixed and incubated at 4°C for 1/2 hour. The tubes were centrifuged at 4°C in Beckman J 6 refrigerated centrifuge for 45 minutes at 3000 rprrr. The supernatant was aspirated and the pellet counted for 100

seconds in LKB multigamma counter.

2.3. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

DEVELOPMENTOF SCREENING ASSAYS

- 5 5 - CHAPTER 2

2.3.1. Chemicals and reagents

Donkey anti-mouse serum, donkey anti-mouse horseradish peroxi­dase labelled (D x M HRPOase), affinity purified donkey anti-mouse IgG, donkey anti-goat horseradish peroxidase labelled (D x G HRPOase),were all supplied by Guildhay Antisera Ltd. Mouse IgG was supplied by Sigma Diagnostics Ltd. Polyvinyl chloride (PVC) and polystyrene plates were supplied by Dynatech. Sheep anti-rat horseradish peroxidase (S x R HRPOase) was supplied by Serotec Ltd.

2.3.2. Method

Buffer

Coating buffer

Carbonate/bicarbonate buffer,0.1M, pH 9.6,was prepared by dis­solving 1.59 g of Na2C 03 and 2.93 g of NaHC03 in one litre distilled water and the pH was adjusted to 9.6. The buffer was stored at 4°C

for up to four weeks.

Washing diluent buffer

0.15 M phosphate buffered saline pH 7.4; 8 g of NaCl, 0.2 KC1. 1.15 g of N a2H P04 and 0.2 g of KH2P 04 were dissolved in one litre of distilled water. The pH was adjusted to 7.4. Usually, this buffer is prepared 10 X concentrated, and the working buffer diluted when needed. Gelatin (0.1%) and Tween 20 (0.05%) were added before use, and the buffer used within two days.

Substrate buffer

Phosphate citrate buffer pH 5.3; 4.666g of citric acid,7.298g of Na 2HPO 4 were dissolved in a litre of distilled water. The pH was adjusted to 5.3. Generally, 500 ml was prepared each time and kept at 4°C for not more than four weeks.

Substrate solution

To 100 ml of substrate buffer 40 mg of ortho phenylenediamine (OPD) was added, mixed and 40 ul of H 202 (30 % v/v) was added to

it. The solution was prepared just before use, and usually not more

DEVELOPMENTOF SCREENING ASSAYS

- 5 6 - CHAPTER 2

than 1 hour. The solution was always made up in a dark bottle as the substrate was light sensitive.

Procedure A

1: A PVC plate was coated with antigen diluted in coating buffer, tothe concentration of 2 ug / ml. The plate incubated at 4°C over­night or 2 hours at 37° C .

2: The plate washed once with PBS, and blocked with the addition of200 ul of PBS-1 % BSA or 0.1 % gelatin for 1 hour at 37° C .

3: The plate washed again with PBS, and the samples or controls wereadded to it and incubated at 4°C overnight or for 2 hours at 37° C .

4: The plate washed 6-9 times with washing buffer, shaken a fewtimes to remove any fluid, and 200 ul of HRPOase labelled anti­body diluted in washing buffer was added to all wells. The plate incubated at 37° C for 2 hours.

5: The plates were washed 6-9 times with washing buffer, shakendry and 150 ul of substrate solution was added to all wells. Theplates were then incubated for 30 minutes at 37° C .

6: The reaction was stopped by the addition of 50 ul of 2.5 M H 2S 04,

and the plates read at 490 nm in an Automatic ELISA Plate Reader.

The outer wells were not used as they might have given greater variability in absorbance (Kirka et al., 1980). The format was shown in figure 2.2 a.

Procedure B

The procedure was basically the same as in procedure A with the format being somewhat different. Each sample and control was added to two different wells, one containing antigen, the other without antigen ( used as blank), the positive result then is the difference in

DEVELOPMENTOF SCREENING ASSAYS

- 5 7 - CHAPTER 2

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DEVELOPMENTOF SCREENING ASSAYS

- 5 8 - CHAPTER 2

absorbance between the the antigen well and the blank ( non antigen well). This measure has been taken to avoid false positive results, as some immunoglobulins stick to plastic very strongly even in the presence of high concentration of other proteins, or the blocking with high concentration of other irrelevant proteins. The format of the assay is shown in figure 2.2 b.

2.3.3. Testing of solid phase

Using ELISA procedure A, different solid phase supports were assessed for their suitability for use in the indirect ELISA system.

Mainly two different types of solid phases were employed , polys­tyrene and polyvinyl chloride plates. Polystyrene and polyvinyl chloride plates were supplied by Dynatech and other polystyrene plates by Alpha .

Using ELISA procedure A and positive polyclonal serum, the plates were tested for their efficiency in adsorbing the antigen. The antigen was diluted to the concentration of 2 ug /ml. The plate were coated with 200 ul per well of antigen overnight at 4°C . The positive antiserum was serially diluted, and applied to the plate. The plates were incubated for 2 hours at 37° C and developed as mentioned in procedure A.

2.3.4. Glutaraldehyde sensitization

The glutaraldehyde sensitization method (Stafford and Kilgallan 1980) was used to enhance the efficiency of adsorption. Glu­

taraldehyde solution, 200ul(l%) was added to polystyrene plate wells and incubated for 3 hours at 37° C . The plate was washed extensively with PBS and used in indirect ELISA method as mentioned above.

2.3.5. Testing the effect of different coating buffers

Although originally the coating buffer used was carbonate/bicarbonate buffer, a various ELISA methods use different buffers mainly because they enhance the adsorption more effectively than the carbonate/bicarbonate buffer. For hPTH(l-34) using

DEVELOPMENTOF SCREENING ASSAYS

- 5 9 - CHAPTER 2

radiolabelled hormone the optimum pH was found to be 7.4 (Nussbam et al., 1981). The buffers tested were carbonate/bicarbonate buffer pH9.6 and phosphate buffered saline pH 7.4. PVC plate was coated with antigen diluted in each buffer, and a positive polyclonal serum was used to assess the amount of antigen adsorbed to each plate, exactly as men­tioned above for the testing of solid phase support.

2.3.6. Testing different blocking agents

The two standard blocking agents BSA and gelatin were tested for their efficiency in reducing the non specific binding. BSA and gelatin were supplied by Sigma chemicals and the concentration ranged between 1-2 %. They were dissolved in PBS and applied to the plate. The plates were incubated for 1 hour at 37°c .

2.3.7. Testing the effect of detergent on antigen antibody binding

The hPTH(l-34) polyclonal antiserum was diluted in washing buffer containing detergent Tween 20, and diluted in PBS without the detergent Tween 20. It was then applied to PVC plates coated with antigen. The plates were developed as mentioned above in procedure A.

2.4. RESULTS

2.4.1. RIA

2.4.1.1. Iodination

The iodination of hPTH(l-34) with Chloramine T method was very simple. It takes only one hour and a half to accomplish. Purification of the labelled hormone using Quiso or sephadex G15 was very easy, and reproducible, even on small column of 5 cm long as shown in figure 2.3a and 3.2b.

The reproducibility of the method, however, was very poor, despite the use of high concentration of sodium iodide, up to 1.5 mCi. Because of the constraints of the University safety regulation, and a lower levels of radioiodinating had to be used in the routine procedure.

DEVELOPMENTOF SCREENING ASSAYS

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- 6 0 - CHAPTER 2

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DEVELOPMENTOF SCREENING ASSAYS

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-61 - CHAPTER 2

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DEVELOPMENTOF SCREENING ASSAYS

- 6 2 - CHAPTER 2

The effect of using high concentration of sodium iodide on the iodina­tion was shown in figure 2.4. It appears that 0.5 mCi can produce agood labelled, and sufficient for iodination. From those results the use of 0.5 mCi was adapted, and used throughout the period of this project.

From six iodinations using Chloramine T method and 0.5 mCi of radioactivity each time, only one produced labelled product of high specific activity which gave a maximum binding higher than 50%. However, because of the lack of its reproducibility the method was abandoned in favour of other mild methods.

It appears that, Iodogen method has not been used in the iodina­tion of PTH. It is a more gentle method, the oxidation is very mild and causes little damage to the hormone. It was first described by Sci- lacinsk (Scilacinsk et al.,1979). When used here for hPTH(l-34) for example, the radiolabelled hPTH(l-34) produced in four separate runs, each gave 85-95% binding under comparable assay conditions. This method was found to be very consistent. The elution profile of the labelled hormone from sephadex G15 is shown in figure 2.5. The immunoreactivity of the different fractions assayed using antiserum to hPTH(l-34) was shown in figure 2.6.

2.4.1.2. Optimization

The assay have been adapted for polyclonal antibody measure­ment. The dilution of second antibody and the normal serum were titrated to give the best combination for antigen-antibody complex precipitation. The optimized assay, was then compared with the precipitation of the complex using dextran coated charcoal. Donkey anti-goat serum was diluted 10,20,30 and 40 times, each dilution was tested for precipitating the labelled hPTH(l-34) and goat ati-PTH(l- 34)anti-serum complex. The results obtained are shown in figure 2.7. Normal goat serum was added to give initially 1 in 10 dilution of don­key anti-goat serum and subsequently final dilution of 1:100, 1:200, 1:500, 1:1000. The precipitation of the complex with 0, 1:500 and 1:1000 normal goat serum dilutions was almost twice as that with

DEVELOPMENTOF SCREENING ASSAYS

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DEVELOPMENTOF SCREENING ASSAYS

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- 6 5 - CHAPTER 2

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1:100 and 1:200 as can be seen in figure 2.8. The use of second anti­body was compared with 5% dextan coated charcoal, where 100 ul of charcoal was added to each tube and incubated for 10 minutes. As shown in figure 2.9, although the percentage binding of dextran coated charcoal was higher , the NSB was very high 22%. The second antibody and PEG method give NSB of less than 2% .

The same procedure was used to titrate the second antibody and the normal serum for the rat antibody.

2.4.2. ELISA

The ELISA method A was used in the early work. The blank value was found to be more useful as a negative control, and has been used in all later work.

2.4.2.1. Solid phase support

The two different solid phases available were tested for their adsorption of hPTH(l-34). It is evident from figure 2.10 that polyvinyl chloride (PVC) plates adsorb hPTH(l-34) better than polystyrene plates. Glutaraldehyde sensitization of polystyrene plates increased the adsorption of antigen to the sensitized plates over the non-sensitized but less than that adsorbed by the PVC plates. From the results in figure 2.10, it was decided to adapt PVC plates for use in antibody screening. The results of chequerboard ELISA for the goat serum using PVC plates were shown in Figure 2.11.

2.4.2«2. Blocking agent

The two most commonly used blocking agents were tested. The results in figure 2.12 show that there is no difference between BSA and gelatin in reducing the non-specific binding. Casein was found to block all the activity if used as blocking agent or used in the washing and dilution buffer.

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2.4.2.3. Effect of Tween 20 on Antigen Antibody binding

When the goat polyclonal anti-serum was diluted in PBS with and without Tween 20, no difference could be seen between these two ways of diluting serum in the standard ELISA B method. When the same was applied to rat positive serum, the results were clearly different. Figure 2.13 shows the results obtained with the rat serum.

2.5. DISCUSSION

2.5.1. RIA

The liquid phase RIA adopted in this study is very sensitive for the measurement of antibodies to hPTH(l-34), it detected antibody in serum diluted many times . It is expected that the method will be sensitive enough to detect monoclonal antibody in tissue culture super­natant.

The liquid phase RIA has different characteristics from solid phase RIA. The liquid phase assay has been shown to have advantages over the solid phase. Moreover, the solid phase ELISA developed util­ize the same principle with the many advantages of using an enzyme label instead of radiolabel. As mentioned before, the use of both sys­tems in the screening for hybridoma secreting antibody is a precaution step in case of the failure of either system, as well as the expectation that monoclonal antibody specificity will allow each system to detect antibodies different from each other.

2.5.2. ELISA

The many advantages ELISA has over the RIA explains the exten­sive use of it in the detection of antibody and especially in screening for monoclonal antibody over the past decade.

The ELISA developed in this study has been shown to exhibit the characteristics required for the screening of hybridoma products.

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The assay was first set up with polyclonal antiserum, but is expected also to be applicable to monoclonal antibodies, may be with some minor modifications. The solid phase has been optimized, and the PVC plates were found very suitable. The sensitized polystyrene could also be used, although the NSB was found to be higher than that for PVC plates. The optimum concentration of hPTH(l-34) was in the range of lug/m l for the polyclonal antibody. However, because of the affinity of monoclonal antibody and the concentration in the super­natants 2ug/ml is adapted for screening hybridoma products.

The assay as it stands at this stage, is cheap, fast, capable of han­dling a large number of samples, and is easy to perform. The uses of it as well as some of the modifications and problems encountered will be discussed in the relevant sections of this work.

DEVELOPMENTOF SCREENING ASSAYS

- 7 6 - CHAPTER 3

CHAPTER 3

MOUSE AND RAT MONOCLONAL ANTIBODYTO hPTH(l-34)

MOUSE AND RAT MONOCLONALS

- 7 7 - CHAPTER 3

3.1. INTRODUCTION

The univalent antibody is the product of a committed spleen cell fused with an immortal myeloma cell. The fusion of B-lymphocyte from any species with myeloma cell line from another species is theoretically possible. However, the stability of the resultant hybridoma cells, and their secretion characteristics would be governed by many factors which are not clearly understood at present.

To date only mouse and rat myeloma cell lines are available. The mouse myeloma cell lines originate from Balb/c mice and the rat myeloma cell lines from Lou/c rats. The in vivo production of monoclonal antibody requires the use of compatible species, and that restricts the choice of animals which can be used for the produc­tion of monoclonal antibody. The cross-species fusion is possible, but the success has so far been limited to certain species. Of these the rat- mouse combination is probably the most important.

Historically the mouse system was the first to be used in the production of monoclonal antibody to pre-determined specificity( Kohler and Milstein 1975). Since then the technology expanded and the methodology has been refined. The mouse system has been used extensively as it has some advantages over the rat system. The mice are easier to handle, they are generally good responders,and there are several cell lines available; these cell lines have been well- studied and the fusion methodology optimized.

The success of the mouse system can be demonstrated by the wealth of literature and the commercial products available on the market.

Rat cell lines on the other hand, should be theoretically better than the mice cell lines. Because of their large size, the rats give twice the spleen cell number of mice, they are easier to bleed and more blood is obtainable. They also produce more ascites than mice (about 5-10 times more than mice). The cell lines, in particular, grow slower than the mice cell lines, hence, do not necessitate early cloning. The size

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 78 - CHAPTER 3

of the cell lines is smaller than that of mice; this allows the use of ratios near 1:1 spleen to myeloma,and this is expected to increase the number of positive clones.

Despite the apparent advantages the rat system have over the mouse system, the use of rat in monoclonal antibody work appears to have been surprisingly limited. A literature survey shows there are not many reports of rat-rat fusions, although it was claimed that rat-rat fusion has a greater stability (Clark et al., 1983) and have the same efficiency as the mouse system.

3.2. MATERIALS AND METHODS

3.2.1. Reagents and chemicals

hPTH(l-34),BSA-RIA grade,Carbodimide,were all supplied by Sigma. Rat typing sera. Rat monoclonal typing Kit, and mouse typing sera were from Serotec Ltd, agarose grade A, and saphacel anion exchange resin from Pharmacia chemicals.

3.2.2. Cell culture media

Media used were RPMI and DMEM both supplied from Gibco. Fetal calf serum (FCS), penicillin/streptomycin, L-glutamate, sodium pyruvate, Hypoxanthine Aminopterin Thymidine (HAT) all purchesed from Gibco.

Tissue culture plates ( 96, 24, 6 well/plate) and Flasks ( 25 and 75 ml flasks) were purchased from Falcon, Costar or Gibco.

Preparation of media

Standard culture media and its concentrated form (X10) were sup­plied without L-glutamate; L- glutamine was added to it prior to use. The media was diluted with sterile distilled water as required. The pH of the media was adjusted after the addition of sodium carbonate with sodium hydroxide to bring the pH near neutrality. The media used throughout was either serum-free containing sodium bicarbonate and neutral in pH or another media with 10% FCS ( fo r example RPMI 10%

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 7 9 - CHAPTER 3

FCS). The later media also contained sodium pyruvate, L-glutamine and penicillin/streptomycin. The selective media was that with 10% FCS and HAT added to it.

3.2.3. Preparation of thym ocyte conditioned media

Thymus glands from 4-5 week old mice were used. The mouse was rinsed in ethanol, the thymus glands removed and were washed in serum free media(RPMI). The thymus glands were then pressed through 50 mm mesh stainless steel screen into a 10 cm petri dish containing 5ml serum free RPMI. The capsule was discarded and the clumps of cells dispersed using a pasteur pipette. Then the cells were washed twice in serum-free RPMI. The cells were counted and resuspended in RPMI containing 20% fetal calf serum, at a density of 5x 106 cells/ml. The cells were cultured for four days at 37°C in humidified atmosphere of 5%C02 • The cell debris were removed by centrifugation and the supernatant aliquoted in 10 ml tubes and kept frozen at — 70° C until used. Four to five mice were processed at a time.

3.2.4. Conjugation of hPTH(l-34)

hPTH(l-34) were conjugated to BSA using the carbodimide method. hPTH(l-34),173ug was dissolved in 3ml of dimethyl sul- phoxide. Solutions of BSA(881ug) and carbodimide(585ug) were made in 3ml and 4 ml of distilled water respectively. The BSA solution was mixed with the hPTH solution for a few minutes, then the carbodi­mide solution was added to them. The components were mixed and then left stirring on a magnetic stirrer at a low speed overnight. Then the solution kept at 4°c for another 24hours before it was freeze dried.

3.3. IMMUNIZATION

3.3.1. Mice immunization:

Mice, 8-10 weeks old (supplied by the Animal Unit University of Surrey) were divided into four groups for immunization. hPTH(l-34)

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 0 - CHAPTER 3

in the free form or conjugated to BSA as mentioned above was used as immunogen.

The mice were immunized with the immunogen through one of three routes, intra-peritoneally (i.p), intra-muscularly (i.m) in three sites in the back legs, or intra-dermally (i.d) in different sites along the back. The immunogen was mixed 1:1 in complete Freunds adju­vant in the first injection, then with the immunogen dissolved in sterile distilled water in the other two boosts. The mice were left to rest for three to four weeks between immunization and for the same period before they were boosted for fusion. Full details of the immunization schedule and materials used are shown in table 3.1.

3.3.2. Rat immunization:

Lou/c rats, 6-8 weeks old ( supplied by the Animal Unit University of Surrey) were divided into three groups for immunization. The rats were immunized with hPTH(l-34) in the free form mixed 1:1 with complete Freunds adjuvant in the first immunization, then twice with the hPTH(l-34) in the free form dissolved in sterile dis­tilled water . The rats were left for four weeks between immunization and for the same length of time before they were boosted for fusion.

The rats were given 0.5 ml of the immunization mixture through one of three routes, intra-peritoneally(i.p), intra-dermally (i.d) in different spots or intra-muscularly (i.m) in both back legs. The detailed schedule and materials used for immunization are given in table 3.2.

3.3.3. Secondary in-vitro immunization

After primary immunization of the mouse, spleen was removed aseptically into 5ml of serum-free RPMI. The spleen was pressed through a 50mm mesh and the clumps of cells were dispersed by using a pasteur pipette. The cells were left standing for 5 minutes to allow the clumps

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 81 - CHAPTER 3

Table 3.1

Mouse immunization schedule

Group One

Immunization No.of animals Immunogen ug/mice Adjuvant ratio BCG ul Route of immunization

1 6 10 ug free 1:1 50 i.p

2 6 5 ug free - - i.p

3 6 5 ug free - - i.p

Group Two

1 6 10 ug free 1:1 50 i.d

2 6 5 ug free - - i.d

3 6 5 ug free - - i.d

Group Three

1 6 10 ug free 1:1 50 i.m

2 6 5 ug free - - i.m

2 6 5 ug free - - i.m

Group Four

1 13 13.5 ug conjugated to BSA 1:1 50 i.p

2 13 5.3 ug free - - i.p

3 12 5 ug free - - i.p

i.p: Intra-peritoneal

i.m: Intra-muscular

i.d: Intra-dermal

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 82 - CHAPTER 3

Table 3.2

Rat immunization schedule

Group One

Immunization No. of animals Immunogen ug/rat Adjuvant ratio BCG ul Route of immunization

1 2 10 ug free 1:1 50 i.p

2 2 5 ug free - - i.p

3 2 5 ug free - - i.p

Group Two

1 2 10 ug free 1:1 50 i.d

2 2 5 ug free - - i.d

3 2 5 ug free - - i.d

Group Three

1 2 10 ug free 1:1 50 i.m

2 2 5 ug free - - i.m

3 2 5 ug free - - i.m

i.p: Intra-peritoneal

i.d: Intra-dermal

i.m: Intra-muscular

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 83 - CHAPTER 3

to settle down. The cell suspension was decanted into another tube, centrifuged for 5 minutes at 1000 rpm and the pellet was resuspended in 10ml RPMI medium containing 20% fetal calf serum. The antigen was dissolved in 10ml of RPMI medium containing 20% fetal calf serum, and then filtered through 0.22u sterile filter .Thymocyte-condi­tioned media (10 ml) was thawed out and made ready. The antigen solution was added to the spleen cells and incubated for 1 hour at 37° C in humidified atmosphere of 5%C02 . The thymocyte-conditioned media was added to the mixture, the mixture seeded in six well tis­sue culture plate, and incubated in humidified atmosphere of 5%C<92

for five days.

3.3.4. M onitoring of serum polyclonal response

Mice were bled retro-orbitally or by cardiac puncture after each immunization. For groups 1,2 and 3, all mice were bled retro-orbitally after each immunization, whereas from group 4, one mouse was bled by cardiac puncture after the second and third immunization.

Blood was collected in small Eppendrof polyethylene tubes, kept at 4°C overnight, then spun in a bench top centrifuge for 10 minutes. The serum was decanted, aliquoted and kept frozen at — 20° C until required.

All rats were tail bled after each immunization, and the blood treated in the same way as for mice.

Mice and rats sera were screened for antibody to hPTH(l-34) using ELISA and liquid phase RIA.

3.4. MAINTENANCE OF MYELOMA CELL LINES

3.4.1. NS1 mouse myeloma cell line

The NS1 myeloma cell line, of Balb/c origin, was grown in RPMI medium containing 10% fetal calf serum. The cells initially were grown in 10ml tissue culture flask, and the concentration of cells kept at 5x 105 cell/ml, then the cells expanded to a 75ml tissue culture flask maintaining the same concentration. The cells were allowed to

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 4 - CHAPTER 3

grow up to lx 106 cell/ml whenever needed.

The logarithmic growth of the cells were determined by incubating lx io5 cells/ml in a 10ml tissue culture flask and counting the number of cells for five days.

3.4.2. Y3 Rat myeloma cell line

The rat Y3 cell line was kindly donated by Prof. Milstein. The cells were thawed at 37°C and washed in Dulbecco serum free media twice, then resuspended in Dulbecco suplmented with 10% FCS, glutamine,sodium pyruvate,penicillin and streptomycin.The cells were plated in 75cm tissue culture flask and allowed to grow at 37° C in 5% CO 2 in humidified atmosphere. Y3 were anchor cells, and so they stick to plastic. When the cells fill the confluent of the flask, they start growing in suspension,‘those grown in suspension were harvested by centrifugation and allowed to continue growth in suspension flasks .The other cells were also dissociated from the surface using trypsin EDTA at 37° C , and grown in suspension flask. In the early phase the cells were incubated in suspension flasks incubated at 37° C without C 02 in dry atmosphere. The media was checked every day and the pH neutral­ized with 10% HC1 whenever it needed adjustment. The media was changed every other day or every three days depending on how fast the cells were growing. In the early phase they were growing slowly and the media was changed every three days. At a later stage the media was changed every other day, because the cells were growing faster. The cells were monitored by counting them using trypan blue exclusion method. These cells were used in the first two rat-rat fusions.

The cells were grown inahot room rather than 37°C incubator pri­marily because of contamination, and also because the magnetic stirrers needed a lot of space in the incubator. At a later stage the cells were transferred to C 0 2 incubator, and the suspension flasks were kept at 37° C in humidified atmosphere of 5%C02 . The media was changed when the nutrients depleted, the cells were pelleted and resuspended in new media to a concentration of lx 105 cells/ ml. They

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 5 - CHAPTER 3

were always kept at a log phase growth.

3.5. FUSION

3.5.1. Preparation of feeder layer

A mouse which did not recive any immunogen was used. The mouse was drenched in ethanol and the spleen taken aseptically and immersed in 10 ml RPMI free serum. All other steps were carried out in sterile laminar flow cabinet. The spleen was pressed through a stainless steel mesh and the clumps of cells were dispersed. The cells were transferred to 10 ml centrifuge tube and left standing for 5 minutes. When the large clumps settled at the bottom of the tube, the suspended cells were transferred using sterile plastic Pasteur pipette into another 10 ml tube and kept at 37° c 5%C02 until used. Usually 4ml out of 10 ml suspension was used per 100 ml of media as feeder for fusion or cloning.

Mouse feeder layer was used in both mouse and rat fusions and cloning, and also sometimes in growing some slow growing cells which needed to be supported by feeder layer.

3.5.2. Fusogen

Polyethelyene glycol,2g (PEG) of M.W 1500 (BDH) was auto- claved. Serum free RPMI (2 ml) was added to it while it was still warm. The solution was either used immediately or stored frozen at —20° C . Before fusion the PEG solution was usually kept at 37° C in a water bath for at least half hour.

3.5.3. Preparation of immune spleen cells:

The spleen from mouse/or rat after primary immunization was processed in the same way as for the feeder layer. Finally, the total number of cells were counted in haemocytometer. When spleen from animals after secondary in vitro immunization was used, the cells were collected from the tissue culture wells into a 50 ml sterile plastic tube, and counted as mentioned above.

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 6 - CHAPTER 3

3.5.4. Mouse-mouse fusion:

Preparation of myeloma cell line: The NS1 myeloma cells in a log­arithmic phase growth were counted and the volume needed was transferred into 50 ml sterile centrifuge tube.

The spleen cells were harvested by centrifugation as were the myeloma cells. The two cell populations were mixed together in the ratio of 5:1 spleen to myeloma cells in 50 ml sterile plastic tube. A negative control for each fusion was set up as follows; 50 ul of the cell mixture (immune-spleen cells mixed with myeloma cells before they were subjected to PEG fusion) were plated in four wells of 24 well plate. The cells were spun down at 1500 rpm in bench top MSE centrifuge. The pellet was drained from fluid using sterile plas­tic pasteur pipette, and the cells loosened by knocking the edge of the tube on the bench. Warmed PEG (0.8 ml) was added slowly with continuous shaking over 1 minute. The cells were left standing undisturbed for another minute, then 10 ml RPMI free serum was added over 5 minutes, and left standing undisturbed for another 10 minutes. The cells were harvested by centrifugation, and resuspended in 100 ml RPMI containing 10% fetal calf serum and 2 times the nor­mal concentration of HAT.

Plating out: In fusions where 24 well tissue culture plates wereused, 1ml of fusion cell mixture was added to each well, then another ml of feeder layer was added. In fusions where 96 well tissue culture plates was used, the fusion cells were resuspended in 8-10 ml; and 0.1 ml was taken in each well, to which 0.1 ml of feeder layer was added. The plates were incubated at 37° C , 5%C02 in humidified atmosphere for a week before they were inspected for growth, and the media was changed.

3.5.5. Rat-Rat fusion:

Preparation of myeloma cell line: The Y3 myeloma cells in a loga­rithmic phase growth were counted and the volume needed was transferred into 50 ml sterile centrifuge tube.

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 7 - CHAPTER 3

Immune spleen cells were prepared as above mixed with Y3 rat myeloma cells in the ratio of 2:1 spleen to myeloma. The mixture of cells were pelleted by centrifugation. The pellet was drained of any fluid, and the bottom of the tube was knocked on the bench to release the cells. A small volume (0.8 ml)of pre-warmed PEG 1500 was added slowly to the cells over 1 minute with slight mixing. The cells were left to stand for another 1 minute on the bench, and 10 ml Dul­becco serum free was added to the cells over 6 minutes with mixing. The cells were left standing on the bench for another 10 minutes, then they were centrifuged at 1000 rpm for 5 minutes in top bench centrifuge,and the pellet was resuspended in 100 ml of Dul­becco containing 10 % FCS and two times the usual concentration of HAT. The cells were seeded in eight 24 well plates. The plates were incubated at 37° C in humidified atmosphere o f CO 2 .

3.5.6. Rat-Mouse fusion:

The fusion was carried out as for the rat-rat fusion experiment except that the NS1 mouse myeloma cell line was used instead of Y3 rat myeloma cell line. The rat spleen cells were mixed with NS1 mouse myeloma cells in the ratio of 5:1 spleen to myeloma cells, and fused using 50% PEG. The fused cells were seeded in 24-well plates and incubated at 37°C humidified atmosphere 5% C 02 .

3.5.7. Screening

3.5.7.1. Mouse monoclonal screening

Primary microscopical screening was done after 10 days post fusion. The wells which show a positive growth under the micro­scope were marked and after 2-3 days of media change they were assayed using indirect ELISA and liquid phase RIA to examine for the presence of antibody specific to hPTH(l-34). The wells which prove to be positive in any of the screening assays were expanded in 6 well tissue culture plates for cloning and freezing. However, a second screening was usually done a day before cloning to make sure that the

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 8 - CHAPTER 3

cells were still secreting antibody.

3.5.7.2. Rat monoclonal screening

The plates were inspected microscopically after 10 days for growth. Those showing growth were further assayed for specific antibody to hPTH(l-34) using indirct ELISA and RIA. The screening assays were those described in chapter 2.

3.5.7.3. Freezing and thawing of cells

The cells were allowed to grow upto a density of to6 cells/ml in 10 ml tissue culture fiask, then the cells were pelleted by centrifu­gation. The pellet was resuspended in RPMI containing 10% fetal calf serum and 10% dimethylsulphoxide(DMSO), to the density of 1—2xl06 cells/ml. The cells were aliquoted in 1 ml portions in freezing vials. The vials were frozen at -20°c for 1 hour, then transferred to —70°C for another 2 hours, before they were put into liquid nitrogen at —196° C . When the cells were needed, a vial was taken out of liquid nitrogen and thawed quickly at 37° C . The cells were diluted with RPMI 10% FCS and pelleted by centrifugation. The pellet was washed once with the same media and the cells cultured in a 10 ml tissue culture flask at 37°C , in humidified atmosphere of 5%C<92

3.5.7.4. Cloning

Using the dilution method, the positive clones were counted and diluted to the concentration of 50,10 and 5 cells/ml and seeded in 96 well tissue culture plates. A small volume(100 ul) of the cell suspension was added to each well followed by 100 ul of feeder layer. The plates were incubated for a week at 37° C 5%C02 in humidified atmosphere before they were inspected for positive growth. Those wells which showed positive growth of one clone were assayed for antibody secretion. Positive antibody secretors were recycled again through the same process once more. Aliquotes of the positive clones were stored frozen in liquid nitrogen.

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 8 9 - CHAPTER 3

3.6. CHARACTERIZATION OF MONOCLONAL ANTIBODY

3.6.1. Immunoglobulin class

Two methods were used in the determination of immunoglobu­lin class and subclass of mouse and rat monoclonal antibodies.

Immundifusion: Agarose was supplied by Pharmacia, sheep and rabbit anti mouse immunoglobulins and subclasses were from Nordic or Sero- tec. 3% PEG 6000 in 1% agarose was prepared as follows: Ig of agarose and 3g of PEG 6000 were added to 100 mis of phosphate buffered saline pH 7.4 and heated on a hot plate until boiling, then the solution cooled down to 56° C . The solution was either kept at 56° C

until used or aliquoted in 10 ml volumes and stored at 4°C until needed. Glass slides,8x8 cm, were cleaned using methanol, and wiped with clean tissue. They were put on a levelling table, and 10 ml of 1% agarose 3% PEG was poured on the glass slide giving a thickness of 1.5-1.8 mm. Using the appropriate gel cutters suitable holes were cut in the gel. The samples and the typing sera were added to the holes and the slides were incubated at room temperature for 24-48 hours until a precipitin line could be seen. The slides were washed with saline for 2 hours, then pressed by covering them with W ath- man no.l filter paper and some weight was placed on top of them. Finally they were dried and stained using coomassie blue R250 for 5 minutes and de-stained using acetic acid and methanol until the back­ground was clear. The slides were then viewed against a bright back­ground.

Rat monoclonal antibody typing kit was also used in typing rat monoclonal antibody. Radial immune diffusion plates as well as typing sera were provided in the kit and the procedure recommended by the manufacturer was adhered to.

ELISA: The screening ELISA method was used until the stage of HRPOase labelled antibody addition. The typing sera, at a dilution of 1:1000,was added and the plate was incubated at 37°C Tor 2 hours. The plate was washed with washing buffer, HRPOase-labelled

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 9 0 - CHAPTER 3

antibody against sheep, goat or rabbit at 1:5000 dilution was added and the plate re-incubated at 37° C for another 2 hours. After washing, 150ul of substrate was added and the plate was incubated at 37° C for 30 minutes. The reaction was stopped by the addition of 50ul of 2.5M H 2SOa , and the plate read at 490nm in an automatic ELISA reader.

3.6.2. Purification of monoclonal antibody

3.6.2.1. Saphacel anion exchange column

Saphacel ready to use pre-swallowed ion exchange resins was sup­plied by Pharmacia. A 10 cm long column was packed using Pharma­cia peristaltic pump. The column was then washed extensively with phosphate buffer pH 8.6. Samples precipitated by ammonium sul­phate were used, the concentration of antibody was measured in a spectrophotometer at 280nm, and calculated according to Hudson and Hay 1980 (Hudson and Hay 1980). The samples were diluted in phos­phate buffered saline to the concentration of lOmg/ml, further diluted in phosphate buffered saline 1:10 and applied to the column at a flow rate of 0.5ml/minute. The eluate was monitored at 280nm, until no protein was seen emerging from the column. A step gradient was prepared from the washing buffer added to it 500mM sodium chloride in the other chamber. The gradient ran from 0 to 500 mM N ad at 0.5 ml/minute, with eluate collected in 5 ml fractions. The fractions were assayed for immunoreactivity by ELISA. The fractions with good immunoreactivity were pooled together, and dialysed against phosphate buffered saline, pH 7.4, overnight with buffer changed three times. The dialysate was freeze dried and kept at 4° C .

3.6.2.2« Protein A column

Protein A column was packed and washed with phosphate buffer 0.03M pH 8.0. The elution buffer was citrate/phosphate buffer pH 3.5. The samples were either concentrated supernatant or ascitic fluid. The column was washed with washing buffer for 1/2 hour and then

MOUSE AND RAT MONOCLONAL ANTIBODIES

-9 1 - CHAPTER 3

the sample applied containing not more than 2mg total protein. The sample was washed with washing buffer until no protein could be detected emerging from the column judged by absorbance at 280 nm. The elution buffer was then applied and fractions collected manually where all the protein emerging out of the column in one peak was collected in one tube. The fractions were quickly diluted with 0.3M phosphate buffer to neutralize the pH, and dialysed against phosphate buffer pH 7.4 overnight with three changes of buffer. The dialysate was assayed for immunoreactivity, then freeze dried and kept at 4°C until reconstituted and used.

3.6.2.3. MONO Q anion exchange column

The MONO Q column was supplied by Pharmacia packed and ready to use. The ascites was centrifuged at 3000 rpm for 15 minutes,and an aliquot of ascitic fluid (200 ul) was filtered through 0.22 u filter and injected to the column. The column was washed with Tris-Hcl buffer pH 7.2. The immunoglobulins were separated using a sodium chloride gradient from 0-500 mM. The column was run at 1 ml /minute, and fractions of 1 ml were collected. The different fractions were assayed for immuno-reactivity using indirect ELISA. The fractions which showed good immunoreactivity were pooled together and dialysed against PBS pH 7.4 and freeze dried.

3.6.2.4. Ascites production

Using prestaine: Balb/c mice were used as the antibody is of Balb/c origin. The mice were primed with the injection of 0.5ml of prestaine intraperitoneally and then left for 10 days. The cells were grown in 75ml tissue culture flask and counted. The required volume was centrifuged to harvest the cells and then resuspended in RPMI serum free or PBS to the density of of 1-2* 106 /0.2ml. The mice were injected with 0.2 ml of this cell suspension intra-peritoneally. When the mice showed maximum enlargement, they were sacrificed by cervical dislocation and the ascites sucked using 19G syringe. The ascites were centrifuged at 3000 rpm in J6 Beckman refrigerated

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 9 2 - CHAPTER 3

centrifuge for 15 minutes, the pellet was discarded and the superna­tant aliquoted in 1 ml aliquotes and stored at -2 0 ° C .

3.7. RESULTS

3.7.1. Im m unization

In this study the two main factors affecting the response of an animal upon challenge with antigen or immunogen were investigated. First, the form of antigen (free or conjugated to a carrier protein) and second, the route of immunization. The choice of species was also of interest because of lack of information about the two species concerned (Balb/c mice and Lou/c rats) from the viewpoint of raising monoclonal antibody to PTH-like hormones.

Mice im m unization: The results of Balb/c mice immunization and the polyclonal response monitored using ELISA are shown in figures 3.1- 3.4. The results show that only Balb/c mice immunized with the conjugated hPTH(l-34)-BSA responded positively. No difference could be seen between the different routes of administration when the free hPTH(l-34) was used as immunogen, which suggest that the route of immunization may not affect the response of Balb/c mice to hPTH(l- 34).

Rat im m unization: Rats showed a different result from mice. They were tested for the route of immunization using the free fragment hPTH(l-34). The sera from rats were assayed using ELISA and RIA. The results in figures 3.5-3.7 show that rats responded to the immunization with the free hPTH(l-34) only when immunized intra- muscularly (i.m). The other routes did not result in any detectable antibody in the serum. When the two non-responder groups were left for 8-10 weeks and re-immunized with hPTH(l-34) intra­muscularly only one rat responded.

3.7.2. Myeloma cell lines grow th characteristics

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 93 - CHAPTER 3

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MOUSE AND RAT MONOCLONAL ANTIBODIES

- 101 - CHAPTER 3

3.7.2.1 Mouse NS1 myeloma cell line

The growth characteristics of NS1 myeloma cell line was demonstrated in figure 3.8. The cultures were maintained under non­limiting nutrient conditions (i.e the cells were spun and resuspended in fresh medium every day). The viability of the cells declined rapidly when the cell density approached lx io6 cells/ml regardless of the nutrient.

3.7.2.2. Rat Y3 myeloma cell line

The Y3 cell line growth characteristics in stationary phase plastic flasks are shown in figure 3.9. The cells were usually thawed and grown in plastic flask and then transferred to a spinner. However, when the cells were thawed and grown directly in the spinner they grew much slower, even when they were seeded in the container without spinning for 24 hours as shown in figure 3.10.

The growth of cells at 37° C inahumidified atmosphere of 5% C 02

in the incubator was better than in the hot room at 37° c . The viabil­ity of cells declined slightly in the first two days of suspension as shown in Figure 3.10, unlike that observed when plastic flask was used( Figure 3.9). When the Y3 cells were grown in a hot room, their growth characteristics were the same as those grown in C 02 incubatorsas can be seen in figure 3.11.

3.7.3. Fusion

3.7.3.1. Mouse-mouse fusion

The fusion protocol followed was the same in all fusions. The SP2 myeloma cell line was used only because the NS1 was suspected of contamination in that particular fusion. No attempt has been made to investigate the fusion efficiency of other myeloma cell lines.

In all fusions, except NOs 1,4,6 and 7, spleen cells from in vivo immunization were fused with the NS1 myeloma cell line. In fusions NOs 1,4,6 and 7 spleen cells immunized in vitro were used for fusion. The results of all fusions are given in Table 3.3.

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MOUSE AND RAT MONOCLONAL ANTIBODIES

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The results were calculated in reference to the total number of wells seeded per fusion, and the percentage that secreted specific anti­body to hPTH(l-34) were determined from the first screening assay. The clones which continue to produce antibody were much less than quoted in the table. The established antibody secreting clones all ori­ginated from in vitro immunized spleen cells. The clones with low O.D at 490 nm in ELISA lost the secretion of antibody shortly after the first screening, and in general the clones resulting from primary in vivo immunization usually had a low O.D at 490 nm in ELISA than these from the in vitro immunization.

3.7.3.2. Rat-Rat fusion

The Y3 myeloma cell line grows well on plastic, but as they stick to it, it becomes necessary to use trypsin EDTA to detach them. When Y3 cells detached from plastic flask using trypsin-EDTA were used for fusion, no growth on microscopical examination or change of media colour was observed to indicate positive growth.

When the cells grown in a spinner flask in hot room at 37° C for three weeks were used for fusion no positive growth was seen for more than six weeks using the criteria stated above. Then it become necessary to grow the cells in C 02 incubators in humidified atmosphere. However, the fusions carried out with these cells did produce some positive growth, though the efficiency was found to be low. Unfor- tunatly due to the shortage of time and the constraints of expenses, optimization of rat-rat fusion conditions was not persued.

3.7.3.3* Rat-mouse fusion

After the rat-rat fusions were found to be disappointing interms of fusion efficiency, the rat spleen cells were fused with the mouse NS1 myeloma cell line. The results of all rat-rat and rat-mouse fusions are presented in table3.4.

MOUSE AND RAT MONOCLONAL ANTIBODIES

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4:

- 107 - CHAPTER 3

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 108 - CHAPTER 3

3.7.3.4. Screening methods

In mouse-mouse fusions, ELISA and RIA for anti-hPTH(l- 34) antibody were used whenever the amount of supernatant available allowed both assays to be run in parallel. When 96 well plates were used for plating out the fusion, the amount of superna­tant was not enough for both assays, and only ELISA was used first followed by RIA 3 to 5 days later.

In rat fusions, ELISA and RIA for anti-hPTH(l-34) antibody and for bPTH(l-84) antibody were used for screening. As all fusions were plated in 24 well plates the amount of supernatant was sufficient for both assays to be carried out in parallel.

whjchELISA: The positive result was defined as tha tAgave O.D at 490 nm higher than twice the negative control. All the mouse-mouse fusions were assayed and the positive clones selected by ELISA. Both methods of ELISA were used, method A was used in all fusions, while method B only in the last two fusions and in further work with the anti­body. Because of the background, method B usually relied on for the selection of positive clones, eventhough sometimes the blanks gave a high result because of the immunoglobulin type of the antibody. Table 3.5 gives an example of the results of both methods.

Rat fusions were assayed using ELISA method B. In all assays the positive result is taken as that gave O.D at 490 nm higher than twice the negative control and higher than the blank. When the immunoglo­bulin class of the antibody is known to have higher affinity to the solid phase the blank becomes higher and the negative control criteria is used, because the cut off point between negative and positive becomes difficult to establish. Table 3.6 gives an example of rat fusion ELISA results.

whichRIA: The positive was taken as thatAgave percentage binding of at least 2% higher than the NSB. The screening of antibody to hPTH(l-34) and bPTH(l-84) was the same with respect to procedure, solutions and buffers used.

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 109 - CHAPTER 3

Table 3.5Mouse monoclonal antibody screening using ELISA.

Clone ELISA method A O.D at 490 nm ELISA method B O.D at 490 nmTest Blank Test

4/PTH/NS1 1.025 0.765 1.4654/PTH/NS2 1.265 0.629 1.0724/PTH/NS3 1.023 0.712 1.0005/PTH/SP1 1.325 0.427 1.2055/PTH/SP2 1.252 0.814 1.100RPMI 10% 0.095 0.098 0.110N.M.S IK 0.325 0.275 0.2622° Ab 0.055 0.062 0.070Substrate 0.032 0.042 0.058

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 1 1 0 - CHAPTER 3

Table 3.6ELISA results of some rat monoclonal antibody

Clone ELISA method A O.D at 490 nm ELISA method B O.D at 490 nmTest Blank Test

R/PTH/6 1.225 0.965 1.265R/PTH/9 0.965 0.729 0.972R/PTH/14 0.823 0.712 0.835R/PTH/22 1.125 0.627 1.325R/PTH/22,2 1.052 0.714 1.003RPMI 10% 0.125 0.098 0.105N.R.S IK 0.225 0.175 0.2522° Ab 0.035 0.062 0.060Substrate 0.022 0.042 0.038

MOUSE AND RAT MONOCLONAL ANTIBODIES

- I l l - CHAPTER 3

In mouse-mouse fusion the phase separation was accomplished using donkey anti-mouse serum neat or diluted 1:10 as well as dextran coated charcoal. The incubation time of the phase separation was also varied from 3 hours at 4°C to overnight at 4°C for the anti-mouse serum. No binding was seen in any of the spe cimens from mouse fusions. Those found positive by ELISA also showed no binding even when they were grown to stationary phase (i.e when the cells were allowed to grow until the nutrients exhausted and the cells started to die) when the antibody concentration expected to be max­imum in the supernatant.

In rat fusions the phase separation was also accomplished using donkey anti-rat serum neat and diluted 1:5. Screening the supernatants for both hPTH(l-34) and bPTH(l-84) antibody at least three times did not result in any positive clones and the maximum binding observed was negligible around 2%. Table 3.7 gives a summary of the findings from assay of products from mouse and rat fusions.

3.7.3.5. Characterization of monoclonal antibody

The ELISA method is more sensitive than immunodiffusion, How­ever, the crossreactivity of the different antibody sometimes results in

a high background. The results using ELISA of four clones of mouse monoclonal were shown in figure 3.12 The immunoglobulin class and subclass of the other clones were shown in table 3.8. In mouse immu­noglobulin typing experiments,predpitin lines were visible after 24 hours incubation at room temperature.

The class and subclass typing of rat monoclonal antibodies are shown in table 3.9, and their typing results from ELISA are shown in figure 3.13. In rat immunoglobulin typing assay, the precipitin lines were visible after 48 hours incubation at room temperature.

MOUSE AND RAT MONOCLONAL ANTIBODIES

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Table 3.8.Ig class and subclass of mouse monoclonal antibody clones

Clone name Ig class and subclass4/PTH/NS1 IgGl4/PTH/NS2 IgGl4/PTH/NS3 IgG2b4/PTH/NS4 IgGl4/PTH/NS5 IgG2a5/PTH/SP1 IgG2b5/PTH/SP2 IgGl5/PTH/SP3 IgGl5/PTH/SP4 IgG2b5/PTH/SP5 IgG2b5/PTH/SP6 IgG2a5/PTH/SP7 IgG2a

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 115 - CHAPTER 3

Table 3.9Ig class and subclass of rat monoclonal antibody clones

Clone name Ig class and subclassR/PTH/6 IgMR/PTH/9 IgMR/PTH/14 IgMR/PTH/22

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3.7.3.6. Purification of monoclonal antibody

The elution profile of mouse antibody from each column has been shown in figure 3.14, 3.15, 3.16. The finding from SDS-PAGE analysis of the purified antibody from protein A column are shown in plate 3.1. The results of the purification of rat monoclonal antibodies using MONO Q anion exchange column and gel filtration are given in Figures 3.17 and 3.18 and the SDS-PAGE analysis data in plate 3.2.

3.8. DISCUSSION AND CONCLUSION

3.8.1. Mouse monoclonal antibodies

Balb/c mice have been commonly used in the production of monoclonal antibodies,although the immunization schedules seem to vary a great deal among workers in the field. A common route of immunization is the intrapertoneal injection; other routes have been used less frequently, only after having been shown to produce a better response.

The immunization schedule followed in this project was to investi­gate whether the response of Balb/c mice to hPTH(l-34) is affected by the route of immunization, and also the form of antigen free and conjugate. It is known that the conjugation enhances the immune response and yields a high affinity antibody. However, Nussbaum investigated the conjugation of hPTH(l-34) to a carrier protein and found no difference in response between the two forms (Nussbaum et al., 1985). Van de Walls (Van De Walle et al., 1983) also used Balb/c in the development of monoclonal antibody to PTH. The mice responded, and antibody was of the IgG.

In the present project the doses were a lot lower than what pre­vious workers used. The mice were injected with lower doses in order to enhance the selection of more specific response and higher affinity antibodies.

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- 1 2 2 - CHAPTER 3

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- 123 - CHAPTER 3

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MOUSE AND RAT MONOCLONAL ANTIBODIES

- 124 - CHAPTER 3

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MOUSE AND RAT MONOCLONAL ANTIBODIES

- 1 2 5 - CHAPTER 3

The results show that hPTh(l-34) has to be conjugated in order to eli­cit detectable antibody response in Balb/c mice. The route of immunization seems to be not particularly important.

3.8.2. Rat Monoclonal Antibodies

The species difference in regard to immune response is a well known phenomenon, as is the individual differences between members of the same species. The rats immunized with the free hPTH amino- terminal (1-34) fragment, showed a response that tended to be related to the route of immunization. They only responded when immunized through the intra-muscular route. This is illustrated again when rats immunized intra-dermally produced no response until after they were re- immunized intra-muscularly. In this latter group some animals might have developed tolerance, one rat responded when re-immunized intra-muscularly, whereas the remaining three did not respond. They had been exposed to the antigen before, and suppressor T cells might have been activated to prevent the B cell response.

The mouse fusion technique is principally that used by Milstein in 1975 with the improvements made to it over the last 10 to 13 years. The specific efficiency is notably low, and the underlying reason might be associated with any step or steps in the procedure. The secondary in-vitro immunization evidently improved the specific efficiency, and the clones stabilized were originally derived from secondary in-vitro stimulation.

The monoclonal antibodies were all of the IgG class, an indi­cation that primary stimulation of the B lymphocytes may have pre­ceded the in-vitro stimulation.

MOUSE AND RAT MONOCLONAL ANTIBODIES

- 126 - CHAPTER 4

CHAPTER 4

GOAT POLYCLONAL AND MONOCLONAL ANTIBODIES TO hPTH(l-34)

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 127 - CHAPTER 4

4.1. INTRODUCTION

Antisera to PTH have been raised in various animal species, including guinea pigs (Berson et al., 1963), chicken (Reiss and Canterbury 1968, Chase and Slatopolsky 1974, and Vieira et al., 1986), goat (Fisher et al., 1974, Manning et al., 1977) and sheep (Wood et al., 1980, Mallette et al., 1981). The tendency for immunological recogni­tion of certain regions of PTH amongst different species certainly does exist.

The choice of animal for immunization is usually determined by the cost of the animal, cost of its maintenance, the longevity and possibly the species if there is clear cut evidence that certain species respond better than others to the peptide hormone region selected. In general almost all species will respond and some antibody will be detected in the serum of the animal. However, the amount of serum obtained at each bleed, the titre and the affinity of the antibody are all of importance to the use of the antibody and the choise of animal especially because the bleeds tend to differ from each other markedly. The titre and affinity of the antibody will determine the type of assay in which it can be used. Although most assays favour the use of high affinity antibodies, in some assays even an antibody with a moderate or low affinity can be used provided a large amount of the antibody is available.

Large animals are usually preferred because of the long life expectancy and the large amount of serum available. On the other hand antibodies of high affinity and titre are usually obtained from small animals like rooster and guinea pig. The large amount of blood obtainable from the large animals also can be used in the pro­duction of monoclonal antibodies. In small animals where no myeloma cell lines is available the only source of B-lymphocytes is the spleen; this limits the use of the same animal in the production of both mono- and polyclonal antibodies. Large animals in the other hand are more suited for the dual purpose, as large amounts of blood can be taken

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 128 - CHAPTER 4

and the development of cell lines suitable for fusion have been found to be possible (Groves et al., 1987)

Since the introduction of monoclonal antibodies in 1975 (Kohlerand Milstein 1975), the interspecific cell hybridization has beenincreasingly used in attempts to produce monoclonal antibodies fromspecies for which myeloma cell lines are not readily available. Fusionbetween rat spleen cells and mouse myeloma cell line has been used toproduce monoclonal antibodies to mouse antigens (Trowbridge1978). Human monoclonal antibodies were also produced using thesame approach in the early stages (Scwaber and Rosen 1978). However,

beenthe extent to which the human modelhas*studied, the inves­tigation of stable human cell lines and examinationof the literature devoted to this subject, indicate it may bedifficult to develop myeloma cell lines from any less

studied species.

Nonetheless, with the modern developments in biotechnology involving the production of proteins in-vitro, and the development of an in-vitro large scale production systems for monoclonal antibodies, the use of cross-species fusion and the subsequent production of monoclonal antibodies in-vitro look- feasible.

Early interspecific fusions of rat and human with mouse myeloma cell lines, prompted large scale use of this approach in the pro­duction of monoclonal antibodies from other species. Immortalization of sheep cell for the production of monoclonal antibodies was first attempted in 1981 (Tucker et al., 1981). This was done by fusing the ovine lymph node or spleen cells with mouse myeloma cell lines.Loss of immunoglobulin production occurred despite the good growth of hybridoma in culture. Later, a technique used in human fusion to increase the efficiency of fusion and stabilize the resulting hybridoma (Ostberg and Pursch 1983, Teng et al., 1983) was adapted successfully with other species (Tucker et al., 1984, Anderson et al., 1986, Groves et al., 1987, Tucker et al., 1987, Groves et al.,

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 129- CHAPTER 4

1988). The use of interspecific hybridoma after their sensitization to aminopterin as fusion partners overcomes some of the problems of interspecific fusion, not least the stability of cell lines pro­duced. This approach have been used to produce human, chimpanzee (Van Meurs and Jonker 1986), cattle (Groves et al., 1987) and sheep aiitibodies. The use of a second and third generation of refusion have also been used in the production of bovine antibodies (Tucker et al., 1987). All fusion reported to date depend on the incorporation into one cell of gene of two different species (i.e mouse and sheep) but not three.

4.2. MATERIALS AND METHODS

4.2.1. Conjugation of hPTH( 1-34)

hPTH(l-34) from Sigma was conjugated to Key Holy Lim­pet hemocyanin using the glutaraldehyde method as described in chapter 3. hPTH(l-34); 52 ug was dissolved in 1 ml of sterile water,added to 9 ug of Key Holy Limpt hemocyanin after activation with 2.5% glutaraldehyde. The solution was mixed for few seconds and then mixed with non ulcerative adjuvant in the ratio of 1:1.

4.2.2. Immunization

A goat was immunized with hPTH(l-34), in the free form. After two months a booster injection was given. Two further booster injections were given with two months interval between them. The conjugated hPTH(l-34) was then used for boosting to enhance the antibody titre. The last boost was 50 ug hPTH(l-34) given in the free form. Table 4.1 describes the immunization schedule and materials used.

4.2.3. Monitoring of serum antibody

The goat was bled 9-10 days after each immunization and every two weeks thereafter. The blood collected from the jugular vein was kept at 4°C overnight,then the blood was centrifuged in a Beckman J6B

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 130- CHAPTER 4

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- 131 - CHAPTER 4

refrigerated centrifuge at 3000 rpm for 30 minutes at 4 °c . The serum was decanted and sodium azide was added to it to a concentration of 0.1%. The serum was stored at 4°C . The bleeds were assayed using both RIA and ELISA as described in chapter 2.

4.3. CHARACTERIZATION OF ANTIBODY

4.3.1. Specificity

The goat serum was assessed for the specificity for hPTH(l-34) using the following peptides:

RH1 (hPTH(9-15)), RH2 (hPTH( 18-24)), RH3 (hPTH(24-28)), RH4 (hPTH(28-34)) all synthesised in the Chemistry department,University of Surrey. hPTH(39-84), hPTH(l-84) obtained from Markiting Asocis- tion. LH, GH, TSH, Prolactin and hCG all supplied by the National Institute of Biological studies. The bleeds tested were G8/19187 (the first bleed after the 2 nd immunization) and G8/11687 (the first bleed after the 4th immunization).

In RIA a standard curve was constructed, and different concentrations Ipg/ml-lOOug/ml of the four small N-terminal peptides,the C-terminal(39-84) and lug/m l-lpg/m l of the whole molecule hPTH(l-84) were allowed to react with the antibody for 3 days. The labelled hPTH(l-34) was then added and the tubes were incubated for a further 3 days. The antigen antibody complex was precipitated with second antibody and PEG 6000, and the pellet counted for 100 seconds in multigamma counter.

ELISA PVC plates were coated with lOOug/ml of the four small peptides RH1, RH2, RH3, RH4 and the C-terminal(39-84), and with 2ug/ml of the whole molecule(l-84) and hPTH(l-34), and the plates incubated at 4°C overnight. After washing and blocking of the free sites, the serum, diluted to give absorbance of 0.75 at 490 nm, was added to the plates and the incubation continued overnight at 4° C .

The plates were washed again and the bound antibody was visualized using HRPOase second antibody and the substrate.

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 132- CHAPTER 4

Other unrelated peptides were also tested using the same ELISA and RIA methods. In ELISA a concentrations between 0.5 and 2 ug/ml were adsorbed on PVC ELISA plates and the antibody allowed to react to them. The bound antibody was revealed with enzyme labelled second antibody and substrate.

4.3.2. Affinity constant

The two bleeds chosen for cross reactivity were also used for affinity constant determination. The scatchard plots were constructed from the results of standard curves, where the concentration of labelled hPTH(l-34) added per tube was also calculated. A computer program has been used for plotting these results and calculating the various constants.

4.3.3. Displacement and standard curves:

The displacement of bound hPTH(l-34) was assayed using ELISA and RIA. In ELISA the bound hPTH(l-34) was displaced using the same solid phase hPTH(l-34). The antiserum was diluted serially and 200 ul of each dilution was incubated with different concentrations of hPTH(l-34) overnight at 4°C . Then 200 ul of the mixture was transferred to a PVC plate coated with 2 ug/ml hPTHl-34) and the plate incubated overnight at 4° C . After washing the bound antibody was visualized using HRPOase labelled second antibody and substrate.

In liquid phase RIA the serum was serially diluted and 100 ul of each dilution incubated with different concentrations of hPTH(l-34) and incubated overnight at 4° C . The next day the labelled hPTH(l-34) ,10000 cpm in 100 ul, was added to all tubes. The incuba­tion continued overnight at 4°C . The bound antigen antibody was pre­cipitated using second antibody and PEG 6000 and the pellet counted using multigamma counter.

The standard curve was constructed using liquid phase RIA. The titre was chosen from the antiserum dilution curve and the assay was set up as follows:

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 133 - CHAPTER 4

To duplicate LP3 tubes 100 ul of anti-hPTH(l-34) serum diluted 1:1200 (at which 50% binding of } 25I -hPTH(l-34)is obtained ) was added followed by 100 ul of dilution buffer. Standard hPTH(l-34) was diluted to give a concentration between lpg/m l and lug/m l and 100 ul of each concentration was added to the appropriate tubes. The tubes were mixed and incubated at 4°C overnight. The labelled hPTH(l-34), 10000 cpm in 100 ul, was added to all tubes, the tubes mixed and incubated overnight at 4°c . The bound antigen-antibody was precipi­tated with second antibody and PEG 6000. The pellet was counted in multigamma counter.

4.4. GOAT MONOCLONAL ANTIBODY

4.4.1. Myeloma cell line

The myeloma cell line was developed by Mr B.Morris and his co-workers in the Biochemistry Department University of Surrey. The murine-ovine myeloma cell line was selected from fusion of NS1 with ovine B lymphocytes. This cell line was grown in 8 azaguanine and developed for fusion with ovine B lymphocytes.

One vial of cells was thawed out quickly in a 37° C water bath, the cells were washed in RPMI 10 % FCS twice and resuspended in 10 ml of RPMI 10 % FCS, and plated in 20 ml tissue culture flask. The cells were maintained at log phase growth for at least a week before fusion.

4.4.2. Blood collection

The goat was immunized with 100 ug of unconjugated hPTH(l- 34) in incomplete adjuvent, and boosted with 50ug of unconjugated hPTH(l-34) as described above. Three days after boosting, 200 ml of blood was collected into 10 ml heparin vacutainer tubes, the tubes were mixed thoroughly with the anticoagulant, and the mixing con­tinued until the blood was ready to be used.

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 134 - CHAPTER 4

4.4.3. Lymphocyte separation

The blood was pooled in two 200 ml dragons, and diluted 1:1 v /v with sterile phosphate buffered saline pH 7.4 (Flow laboratory). Then the lymphocytes were separated by density gradient centrifugation on lymphopaque (Nyegard,UK). The lymphocytes were washed twice with Hank’s balanced salt solution, and repelleted by centrifugation. The pellet was resuspended in RPMI 10 % FCS. The lymphocytes were counted using trypan blue.

4.4.4. Fusion and plating out

Preparation of mouse feeder layer: the spleen of unimmunized mouse was taken aseptically, and processed as described in chapter 3.

Preparation of Myeloma cell line: the myeloma cell line was grown in a log phase. The day before fusion the cells were counted using trypan blue, and they were seeded at 5x 105 cells/ml. On the day of fusion, the cells were counted again. The required volume was centri­fuged, and the cells were resuspended in fresh medium and kept in the incubator until needed.

4.4.5. Goat-Heteromyeloma fusion

The goat lymphocytes were divided in two parts. One half was fused with NS1 mouse myeloma, and the other half fused with ovine- murine heteromyeloma.

The goat lymphocytes were prepared as mentioned above. They were counted, and centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended in the heteromyeloma medium, and mixed giving a ratio of 2:1 for lymphocytes to heteromyeloma cells. The mixture was pelleted by centrifugation at 1000 rpm for 5 minutes. The cells were drained of any solution using a Pasteur pipette. The edge of the tube was knocked on the bench to release the cells. An aliquot of pre­warmed 50 % PEG 1500 (0.8 ml) was added slowly to the cells over 1 minute. The cells were left standing without disturbance for another minute, then diluted slowly with 10 ml of serum free RPMI

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 135- CHAPTER 4

over 6 minutes. The mixture was left to stand on the bench for another 10 minutes without disturbance. The cells were pelleted by centrifugation at 1000 rpm for 5 minutes, and resuspended in 200 ml of RPMI 10 % FCS containing 2X HAT. The cells were plated out in eight 24-well tissue culture plates, 1ml per well. Feeder layer(lm l) was added to each well and the plates were incubated at 37° C in humidified atmosphere containing 5% C 02 .

4.4.6. Goat-Mouse NS1 myeloma fusion

The mouse NS1 myeloma cell line was prepared as described in chapter 3. On the day of fusion the cells were counted, then pelleted and resuspended in fresh medium. The goat lymphocytes were prepared and counted as mentioned above. The lymphocytes were mixed with NS1 in the ratio of 5:1 lymphocytes to NSl,and pelleted by centrifugation. The cells, drained of any liquid, were released by knocking the edge of the tube on the bench. The fusion was carried out as for goat-heteromyeloma. The cells were seeded in eight 24 well tissue culture plates, and incubated at 37°C in humidified atmo­sphere containing 5% C 02 .

4.4.7. Screening

The plates were screened primarily under the microscope. Those which showed positive growth were subjected to further assays to select for the specific antibody producers. As for rat and mouse fusions before, ELISA and RIA assay systems were used. The methods were the same as that described in section 2.2.0 and 2.3.0. ELISA and, RIA liquid phase for the hPTH(l-34) and RIA liquid phase for bPTH(l-84) were used to screen for antibody production.

For ELISA, donkey anti goat IgG was supplied by Guildhay Antisera Ltd and was used at 1:8000 dilution. Antigen and antibody incubations were all carried out overnight.

In RIA for hPTH( 1-34), three assays were run together, one included neat donkey anti goat serum , the second donkey anti goat

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 136 - CHAPTER 4

serum diluted 1:10 and in the third type dextran ~ coated charcoal was used for separation.

In liquid phase RIA for bPTH(l-84), two assays were run together, the first with donkey anti goat serum diluted 1:10, and the second using dextran coated charcoal.

The cells were screened three times, and the positive wells in more than one assay in the first screening were cloned. Those wells which showed a positive growth were assayed for anti-hPTH(l-34) and anti-bPTH( 1-84) specific antibodies. The positives were expanded and cloned using limiting dilution method.

4.4.8. Cloning

The cells were cloned using the limiting dilution method. Each positive well was cloned in two 96 well tissue culture plates. Three different concentrations of cells were used, 50 cells/ml, 10 cells/ml and 5 cells/ml. The 5 cells/ml concentration was cloned and plated in one 96 well tissue culture plate, the other two concentrations were plated each in half plate. In the wells the cells were mixed with the mouse feeder layer,and the plates were incubated at 37° C in humidified atmo­sphere with 5% CO 2 • The plates were inspected for growth after seven days. Those containing one clone were tested for specific antibody secretion.

4.5. RESULTS

4.5.1. Serum polyclonal antibody

The bleeds from the goat were tested for hPTH(l-34) antibody using both liquid phase RIA, and ELISA. As expected, after each boost the titre of the bleeds increased to a maximum, then decreased. How­ever, the first bleed after each boost gave the highest titre and, then either the titre sustained for a few weeks and started to drop down, or went down immediately. The titre after the first boost was 1/1200, and in all other bleeds the titre never went higher than that.Figure 4.1 shows a typical change of titre after the first boost.

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 137 - CHAPTER 4

Figure 4.2 shows the titre of all bleeds. Figure 4.3 shows the dilution curve using ELISA.

The standard curve and results from displacement studies are shown in figure 4.4 and 4.5 respectively. There is a small effect of prolonged incubation upon antiserum dilution curve,as can be seen in figure 4.6.

4.5.2. Characterization of antibody

Specificity : The specificity of the two best bleeds were tested. The bleeds were chosen according to the titre.

Different fragments of hPTH(l-34) as well as other unrelated peptides were used. The small fragments within the N-terminal sequence were used to map the antigenic determinant. While the whole molecule (1-84) and the C-terminal (39-84)PTH along with other unrelated peptides were used to test the specificity of the anti­body.

The cross-reactivity of goat anti-hPTH(l-34) antibody has been tested using ELISA and RIA. Figure 4.7 shows the cross-reactivity of fragments within the N-terminal fragment (1-34), C-terminal (39- 84) and the whole molecule using ELISA. The binding of antibody to h P T H (l-34) has been taken as 100% binding, while other fragments binding calculated as a percentage of that of hPTH(l-34). The results of RIA were shown in figure 4.8 a,b and c. It can be seen that the cross-reactivity with the whole molecule is almost 100% in RIA, while only 10% in ELISA. It seems that the antibody will not differentiate between the whole molecule and the N-terminal (1-34) in RIA, but once the whole molecule is adsorbed to solid phase support (ELISA plates) the antibody will bind to it minimally.

The cross-reactivity with other peptides was minimal in both ELISA and RIA. The cross-reactivity was 0.1-1 percent in ELISA and less than 0.5% in RIA.

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bleeds used previously for cross reactivity were determined on the basis of scatchard plot. From the RIA results the affinity constant was found to be 5x 1091/mol for both the bleeds.

4.5.3. Monoclonal antibody

The volume of goat blood used (200 ml) yielded 2xl08 lympho­cytes, of which one half was fused with ovine-murine heteromyeloma, and the other half fused with mouse NS1 myeloma cell line. The screening started after 12 days, primary microscopic screening revealed 100 % growth with a mean of 5 clones in each well of the goat heteromyeloma, and 100 % growth with a mean of 3 clones in each well in goat-mouse fusion.

After the microscopic screening the media changed, and after three days screening for specific antibody to hPTH(l-34) and bPTH(l- 84) was carried out using liquid phase RIA and ELISA.

In RIA screening the goat-heteromyeloma resulted in five clones positive to hPTH(l-34). The wells were assayed three times and in each the cells were found to secrete antibody to hPTH(l-34). The antibody was detectable by all three methods used to precipitate the antibody- antigen complex (neat donkey anti-goat, donkey anti-goat diluted 1:10 and dextran coated charcoal). The results are shown in figure 4.9.

The antibody did not cross-react with bPTH(l-84), when the same antigen was used as radiolabelled ( ,125/ -bPTH(l-84)). The use of both donkey anti-goat and dextran coated charcoal to precipitate the antigen-antibody complex showed no binding. The results of both fusions are summarised in table 4.2.

It was noted that the goat-heteromyeloma hybridoma clones were bigger and grew faster than the goat-mouse hybridoma clones. In general, the clones were much smaller than mouse-mouse clones.

The media was changed three days before screening, and the supernatant taken was used in all assays. All the positive clones stoped secreting the antibody six days after the first screening.

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4.6. DISCUSSION AND CONCLUSION

4.6.1. Polyclonal antibody

The goat was commonly used in the production of polyclonal antibody to hPTH. It is considered to be a good responders and recom­mended for use in PTH antibodies (Mallete 1983). In this study the immunization regimen adopted proved to be effective; the goat responded to the free fragment only and as low as 100 ug, used in the first immunization, was enough to elicit a good response. The specificity of the antibody however confirms the recent claim of an antigenic determinant within the 18-25 amino-acid sequence of this fragment (Veiere et al., 1987). Hence it is not surprising that the antibody cross-reacts 100% with the whole molecule (1-84), but shows no binding to 39-84 amino- acid sequence. The discrepancy between ELISA and RIA cross reactivity results might be arrtibuted to the different principle underlying each technique. The presentation of the antigen is completely different in these two assay systems. While the antibody binds hPTH(l-84) in solution where allantigenic determinants are available, in ELISA the adsorption to PVC plate restrict the determinants available for binding. Other peptides unrelated to PTH did not bind the antibody in either ELISA or liquid phase RIA.

4.6.2. Monoclonal antibody

Many species have been used in cross-species fusion. This is the first report of goat-mouse fusion, and an attempt to use goat in cross­species heteromyeloma fusion.

All other cross-species fusions reported before were found to lose quickly their non-myeloma chromosomes; the goat-mouse fusion in the present study is no exception. The fusion was successful, and 100% growth was achieved. However, specific clones producing anti­body to hPTH(l-34) has not been detected. This is not surprising since even in the mouse and rat fusions where compatible cells are used

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 152- CHAPTER 4

this can happen. Moreover, it is encouraging in that improvements in the specific efficiency of fusion is easier to contend with than fusion efficiency. These cell lines can also be used as heteomyeloma in future fusions for the stabilization of cell lines.

The loss of foreign chromosomes noted in all cross-species fusion and the production of stable clones was shown to be achieved by the use of heteromeyloma as fusion partners (Tucker et al., 1987, Tucker et al., 1985, Groves et al., 1988, Groves et al., 1987, Teng et al., 1985). Moreover, the use of primary, secondary and tertiary hetro-hybridoma (heteromyeloma) was also found to be possible (Tucker et al., 1987). However, so far the possibility of heteromyeloma of two species to be crossed with a third one has not been tried. In this study a hetero­myeloma of ovine-mouse was fused with goat blood lymphocytes. The resulting hybridomas demonstrated the production of specific antibody to hPTH(l-34). The cell lines also showed a very good specificity in that they did not appear to recognise the same fragment adsorbed to PVC ELISA plate nor to bPTH(l-84) in liquid phase RIA.

GOAT POLY- AND MONOCLONAL ANTIBODIES

- 153 - CHAPTER 5

CHAPTER 5

APPLICATION OF MON OCLONAL ANTIBODIES

APPLICATION OF MONOCLONALS

- 154- CHAPTER 5

5.1. INTRODUCTION

In contrast to polyclonal antibodies, monoclonal antibodies offer selectivity of a finer degree, in conjunction with the homogeniety and availability in large quantities. This allows easier standardization of immunoassay procedures and their in the laboratory on a wider scale. Furthermore, the monoclonal antibody also made it possible to expand the applications of antibodies using various combination of their pro­perties, and the design of novel assay systems. A summary of these advantages is given in table 5.1.

The clinical, biological and other uses of monoclonal antibodies have been extensively reviewed (Foster 1982, Hubbard 1983, Davis and Rao 1984, James et al., 1984). In this study the antibodies to hPTH(l- 34) were intended for use in immunoassay and immunocytochemical investigation.

5.2. IMMUNOCYTO CHEMISTRY

5.2.1. M aterials

Affinity purified donkey anti-mouse HRPOase and Alkaline phosphatase-labelled IgG was obtained from Guildhay antisera Ltd. Affinity purified sheep anti-mouse alkaline phosphatase labelled IgG was supplied by Nordic. Naphthol AS:B1 phosphoric acid and Fast Red TR were supplied by Sigma. 3,3’-Diaminobenzidine tetrahydro- chloride (DAB) was obtained from BDH.A11 other chemicals were of analytical grade.

APPLICATION OF MONOCLONAL ANTIBODIES

- 155 - CHAPTER 5

Table 5.1comparison of monoclonal and polyclonal antibodies properties

Property Polyclonal antibody Monoclonal antibodyConcentration of immunoglobulin

lOmg/ml 0.1-10mg/ml

Concentration of desired antibody

0.1-1.0mg/ml 0.1-1.0mg/ml

Reproducibility variable from batch to batch

absolute

Cross-reactivity common rare,unless the same antigenic determinant present in other antigens

Supply limited for each animal and each bleed

unlimited

Properties spectrum of all immunoglobulins with behavior varies, as it is spec­trum of all immu­noglobulins

one class and subclass,with pre­selected properties by screening

Cost relatively cheap expensive

APPLICATION OF MONOCLONAL ANTIBODIES

- 156 - CHAPTER 5

5.2.2. Buffers

Veronal acetate buffer, pH 9.2: 0.9715 g of sodium acetate (trihydrate), 1.4715 g of sodium barbitone and 2.5 ml of 0.1M HC1 were dissolved and made up to 250 ml with decarbonated distilled water.

Tris-HCl buffer pH 7.6: Tris(hydroxymethyl)aminomethane 9.1g, sodium chloride 25.5 g were dissolved in 750 ml of distilled water and mixed thoroughly. The pH was adjusted to 7.6 using 1.0M HC1 and the volume completed to 1000 ml.

Citrate acetate buffer pH 5.0: 5.3 g of citric acid was dissolved in 500 ml of distilled water, and 12.0 g of ammonium acetate was dis­solved in 3 litres of distilled water. The citric acid solution was added slowly to the ammonium acitate solution until the pH was 5.0.

Peroxidase blocking solution: 120 ml of hydrogen peroxide 30% v /v was added slowely to 200 ml of methanol. The solution was used within a week, and not more than twice.

Alkaline Phosphatase Substrate solution: Naphthol AS:B1phosphoric acid (sodium salt), 5mg, was dissolved in 3 drops of dimethylformamide (BDH), and 5mg Fast Red TR was dissolved in 10ml veronal buffer pH 9.2. The naphthol solution was added to theFast Red solution, mixed, and filtered. This was usually prepared just before use.

Substrate solution for Horseradish peroxidase: 125 mg of DAB were dissolved in 340 ml of citrate acetate buffer pH 5.0. The DAB dissolved in small volume of buffer first, then the rest of the buffer volume added, and the solution was sonicated. This reagent was prepared just before use each time.

5.2.3. Tissue samples

Formalin-fixed tissue sections of human parathyroid glandadenoma and/or carcinoma, chief cell carcinoma, were obtained during surgery or as an autopsy samples. These were supplied ready for use

APPLICATION OF MONOCLONAL ANTIBODIES

- 1 5 7 - CHAPTER 5

(Courtesy of Mr Peter Jenkins, Pathology, Royal Surrey County Hospital)

5.2.4. Procedure for Alkaline phosphatase staining

The tissues were dewaxed by taking them through xylene and different concentrations of alcohol and finally to water. The tissues were then incubated in 20% acetic acid to destroy any endogenous alkaline phosphatase activity. Then they were rinsed in PBS pH 7.4, the excess buffer was wiped and 200 ul of PBS containing 0.1% normal serum was spread on the tissue to block the non specific binding of the antibody. The slides were incubated for 1 hour at room temperature in humidified box to reduce the evaporation.

The slides were washed with PBS, and 200 ul of the first antibody diluted in PBS-2% BSA were spread on the slides, and the slides were incubated at 4°C overnight in a humidified box.

The slides were then washed three times 10 minutes each with PBS- Tween, 200 ul of alkaline phosphatase labelled IgG diluted in PBS- 2% BSA was added to each slide and, incubated for 90 minutes at room temperature, they were washed again with PBS-Tween as before.

The substrate solution, 1 ml, was added to each slide, and the slides were incubated at room temperature for 45-60 minutes.The tis­sues were washed with distilled water, tap water, then counter­stained with Mayer’s haemalum for 4 minutes. The tissues were washed in running tap water until the colour was blue. Finally, the slides were mounted in glycerin jelly and left to dry. They were inves­tigated using light microscope with 25 or 40 X lens.

5.2.5. Procedure for Horseradish peroxidase staining

The tissues were dewaxed as before, and the endogenous peroxidase was blocked by incubating the slides for 10 minutes in the blocking solution.

APPLICATION OF MONOCLONAL ANTIBODIES

- 158 - CHAPTER 5

After blocking the slides were soaked in running tap water for another 10 minutes, the excess water was removed, and the free sites were blocked with 1% normal serum in Tris-HCl pH 7.6 for 1 hour at room temperature.

The excess liquid was removed, 200 ul of the the first anti­body (anti-hPTH(l-34) antibody) was added, and the slides incubated at 4°C overnight in a humidified box.

The slides were washed three times, 10 minutes each. Tris- HC1 buffer containing 0.5% Tween 20, and 200 ul of the second antibody (HPROase labelled antibody) was added to each slide, and incubated for 60 minutes at room temperature.

After washing three times with Tris-HCl-Tween as above, the slides were incubated with the substrate for 3-5 minutes depending upon the visual appearance of brown colour, the reaction stopped by immersing the slides in distilled water.

The tissue sections were placed in Elhrich’ acid heamatoxylin (supplied by R A Lamb Ltd) for 5 minutes. They were gently washed in running tap water for 5 minutes, followed by differentiation in acid alcohol (1% v /v hydrochloric acid in 70% v /v alcohol) with gentle agitation for approximately 5 seconds.

The sections were again rinsed in running tap water for 5 minutes and examined microscopically to ensure sufficient differentiation. They were rinsed in tap water for 10 minutes , and then progressively dehydrated by passing through 70% alcohol, 100% alcohol twice for 30 seconds and finally cleared in xylene twice for 30 seconds and 5 minutes respectively.

The sections were mounted using DPX (BDH chemicals Ltd) and a coverslip, taking them straight from xylene.

5.3. DOUBLE SANDWICH ELISA

APPLICATION OF MONOCLONAL ANTIBODIES

- 159 - CHAPTER 5

5.3.1. Materials

Polystyrene plates were supplied by Dynatech, Donkey ant- mouse and anti-goat HRPOase IgG labelled were obtained from Guil- dhay Antisera, Guildford Surrey.

5.3.2. Purification of goat anti-PTH antibody

5.3.2.1. Caprylic acid and ammonium sulphate precipitation method

The method was essentially that adapted by McKinney and Par­kinson (McKinney and Parkinson 1987). The serum was diluted with acetate buffer 1:5 and the pH adjusted to 4.5. Caprylic acid was added slowly to the concentration of 25ul/ml of diluted sample while the solution was mixed. The solution was mixed for further 30 minutes at room temperature, and the mixture centrifuged at 3000 rpm in Beckman J6 refrigerated centrifuge for 15 minutes. The super­natant was decanted, and cooled down by putting it on ice for 10 minutes. The immunoglobulins were then precipitated using 45% ammonium sulphate. Ammonium sulphate was added to the supernatant (277mg/ml); the solution was stirred for 30 minutes and centrifuged at 3000 rpm for 15 minutes in J6 Beckman refrigerated cen­trifuge . The pellet was resuspended in PBS and dialysed overnight against 2 litres of PBS. The dialysate was kept at 4°C .

5.3.2«2. Anion exchange method

An DEAE anion exchange cartridge was made available by Guil- dhay Antisera Ltd. The column was equilibrated by running 50 ml of phosphate buffer pH 6.4. 1 ml of serum was added to 9 ml of phos­phate buffer pH 6.4, and run through the column. The serum was washed down using the same buffer and, 5 ml fractions were collected. The column was washed with acetic acid, and regenerated again by washing it with phosphate buffer pH 8.8.

The protein content of the fractions was measured at 280 nm on Cecil spectrophotometer, and the protein concentration was calculated

APPLICATION OF MONOCLONAL ANTIBODIES

- 160- CHAPTER 5

(Hudson and Hay 1980).

5.3.3. Purification of mouse monoclonal antibody

Mouse monoclonal antibodies were purified using Protein A as mentioned in chapter 3. The protein concentration was calculated as above.

5.3.4. Conjugation of monoclonal antibody w ith HRPOase enzyme

The mouse monoclonal antibody was labelled with peroxidase according to Beyzavi et al ( 1987).

Buffers

Ethanolamine buffer pH 8.0: 5ml ethanolamine (BDH) was diluted to 50 ml with distilled water, 5ml glycine buffer (0.05M) was added and the pH was adjusted to 8.0 with 1M Hcl. The volume was made up to 100 ml with distilled water.

5.3.5. Method

HRPOase (10 mg) was dissolved in 2ml distilled water, and 0.4ml of 0.1M sodium periodate was added to the enzyme solution and mixed at room temperature for 30 minutes. The solution was then dialysed against ImM sodium acetate buffer pH 4.4 overnight at 4°C .

Affinity purified monoclonal antibody (5 mg) was dialysed against 0.1 M carbonate/bicarbonate buffer pH 9.5 overnight at 4°C .

The enzyme and antibody solutions were combined together and mixed for 2 hours at room temperature. Ethanolamine buffer pH 8.0 (2 ml) was added and the solution was left standing for another hour before it was dialysed against two litres of PBS buffer pH 7.5. The labelled antibody was used without purification.

5.3.5.1. Double sandwich ELISA procedures

Titration of both monoclonal and polyclonal antibodies were car­ried out, by coating the rows of ELISA plate with different concentra­tions of one of them and, then incubating with hPTH. The second

APPLICATION OF MONOCLONAL ANTIBODIES

- 161 - CHAPTER 5

antibody was added at different concentrations in the columns of ELISA plate. The same procedure was used for both the antibodies. When the concentrations of the different antibodies were determined, the sensitivity was assessed using different concentrations of hPTH(l-34).

Both antibodies were tried as bottom layer and top layer in the assay. The general procedure is as follows:

The antibody was diluted in carbonate-bicarbonate buffer, pH 9.6, to concentrations of 20,10,5,2 and 1 ug/ml, 200 ul of solution was added to the appropriate wells and the plate incubated overnight at 4°C . After washing the plates with PBS, the free sites were blocked by incubating 200 ul of PBS-BSA in each well for 1 hour at 37° C .

hPTH( 1-34),200 ul,2 ug/ml was added to all wells, and the incu­bation continued for another 2 hours at 37°C . The plate was washed with PBS-Tween 20, anti-PTH antibody, diluted in PBS-BSA-Tween 20 to 20,10,5,2 and 1 ug/ml, was added to the corresponding wells and the plate incubated overnight at 4°C .

The plate was washed with PBS-BSA-Tween 20, and 200 ul of second antibody HRPOase labelled was added to all wells and incu­bated for 2 hours at 37° C . After washing, 150 ul of substrate was added to each well. After incubation for 30 minutes at 37° C . 50 ul of 2M sulphuric acid was added to stop the reaction and the plate read at 490 nm in automatic ELISA reader.

5.4. RESULTS

5.4.1. Immunocytochemistry

Monoclonal antibodies were titrated with HRPOase and ALP labelled anti-mouse IgG. The optimum dilution for each monoclonal antibody was established, as well as for the HRPOase and ALP anti­mouse IgG. The optimum dilutions are described in table 5.2.

The antibody were screened first using HRPOase - labelled donkey anti-mouse IgG. The tissue sections were blocked and the antibody was

APPLICATION OF MONOCLONAL ANTIBODIES

- 162- CHAPTER 5

allowed to react to the tissue, the bound antibody then revealed using HRPOase -labelled antibody and DAB. The positive was dark brown colour, while the negative shows no brown colour in the tissue. The results of HRPOase system are shown in plates 5.1 - 5.8.

The results obtained using HRPOase system consistently gave high background staining. This made the distinction between the positive and negatie staining very difficult. The chromogen used (DAB) occa­sionally was found to give inconsistant results. The staining was light in all of the antibodies tested, and changing the HRPOase dilution always led to increased background staining. However, with careful microscopical examination positive signs could be seen.

The same tissue sections were used with ALP labelled anti-mouse antibody. The titres were determined as previously described with HRPOase and they are given in table 5.2.

The use of ALP labelled anti-mouse was found to give better results than HRPOase labelled antknouse antibody, the background was low and the positive was clearly visible as the red colour is very much recognizable. All the antibodies tested were found to be positive; some were weak and the colour was not very strong. The results were consistent unlike the experience with HRPOase where a great deal of variation was seen between different batchs of DAB (chromogen). The outcome of the experiments with ALP-labelled anti-mouse anti­body are shown in plates 5.9 - 5.14.

5.4.2. Double sandwich ELISA

The goat anti-hPTH(l-34) antiserum was purified using precipi­tation and anion exchange resin. The precipitation method yielded a relatively pure material as judged by SDS-PAGE analysis shown in plate 5.15. However, the antibody comprised only 10% of the total

APPLICATION OF MONOCLONAL ANTIBODIES

- 163 - CHAPTER 5

Plate 5.1: The binding of monoclonal antibody 4/PTH/NS1 to chief cell carcinoma tissue . Using HRPOase labelled an ti­body.

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Plate 5.2: The binding of monoclonal antibody 4/PTH/NS2 to chief cell carcinoma tissue . Using HRPOase labelled antibody.

APPLICATION OF MONOCLONAL ANTIBODIES

^

164 - CHAPTER 5

Plate 5.3: The binding of monoclonal antibody 4/PTH/NS3 to chief cell carcinoma tissue . Using HRPOase labelled an ti­body.

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K m F nPlate 5.4: The binding of monoclonal antibody 5/PTH/SP1 to chief cell carcinoma tissue . Using HRPOase labelled an ti­body.

APPLICATION OF MONOCLONAL ANTIBODIES

- 165 CHAPTER 5

Plate 5.5: The binding of monoclonal antibody 5/PTH/SP2 to chief cell carcinoma tissue . Using HRPOase labelled an ti­body.

Plate 5.6: The binding of monoclonal antibody 5/PTH/SP3 to chief cell carcinoma tissue . Using HRPOase labelled an ti­body.

APPLICATION OF MONOCLONAL ANTIBODIES

- 166 - CHAPTER 5

Plate 5.7: The binding of monoclonal antibody 5/PTH/SP7 to chief cell carcinoma tissue.Using HRPOase labelled an ti­body.

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Plate 5.9: The binding of monoclonal antibody5/PTH /SP4 to chief cell carcinoma tissue.Using ALP labelled antibody.

Plate 5.10: The binding of monoclonal antibody5/PTH/SP2 to chief cell carcinoma tissue.Using ALP labelled antibody.

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Plate 5.11: The binding of monoclonal antibody5/PTH/SP4 to chief cell carcinoma tissue.Using ALP labelled antibody.

Plate 5.12: The binding of monoclonal antibody4/PTH/NS6 to chief cell carcinoma tissue.Using ALP labelled antibody.

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CHAPTER 5

APPLICATION OF MONOCLONAL ANTIBODIES

immunoglobulins precipitated and the use of acid and ammonium sul­phate caused some destruction of antibody.

In anion exchange chromatographis purification, the antibody eluted in one peak collected in three tubes, and the tubes were kept separate. The protein was monitored at 280 nm and the concentration was calculated accordingly. The elution of the antibody from the anion exchange column is shown in figure 5.1. Fraction number 3 was found to contain more protein and gave a higher binding to hPTH(l- 34). In indirect ELISA, therefore, it was used in all studies.

5.4.3. Assay

Chequerboard ELISA of both goat anti-hPTH(l-34) polyclonal and mouse monoclonal antibodies were carried out using hPTH(l-34) only. Different concentrations were used to assess the sensitivity of the assay.

The monoclonal antibodies were tested as a solid phase first to assess their capability to bind to hPTH(l-34). This was considered to be the appropriate use of these low affinity antibodies, especially when the material is available in large amount and also can be produced relatively easily. The results from three different monoclonals are shown in figures 5.2 - 5.4. The binding of hPTH(l-34) was time dependent ,as an increase in the binding was observed when the anti­body was incubated with hPTH(l-34) for up to 48 hours as shown in figure 5.5. There were also no difference in the binding value between the antibodies when tested in a standard curve (figure 5.6).

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results are shown in figures 5.7 - 5.9.

Mouse monoclonal was labelled using the periodate method, and used in the assay instead of anti-mouse labelled antibody. The titration of the labelled monoclonal was shown in figure 5.10. The use of HRPOase labelled monoclonal antibody in hPTH(l-34) ELISA standard curve was shown in figure 5.11. It is clear that the sensitivity of the assay using the enzyme labelled monoclonal antibody is low. The addi­tion of more concentrated labelled monoclonal antibody only increases the NSB rather than increasing the sensitivity. There seems to be a cross-rectivity between the goat and mouse antibodies. This can be seen in the difference between the blank and the negative controls used. When the wellswere not coated with the antibody the reading was low, whereas with antibody coating in the absence of antigen the reading was high. It is also confirmed when simply goat and mouse antibodies were mixed and incubated in plastic tube before the addition to an antigen coated plate, no binding was observed i.e no effective mono­clonal antibody left to bind the hPTH(l-34) on the plate.

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5.5. DISCUSSION AND CONCLUSION

The immunocytochemical studies demonstrate the binding of antibody to the native hormone(s). It is important that they recog­nize the native hormone in the tissue or serum if they are to be used in clinical studies. It is the first study to demonstrate the binding of monoclonal antibodies raised against synthetic hPTH to parathy­roid tissue. The binding of these monoclonal antibodies to parathyroid tissue containing hPTH, might be useful in localizing tumors secreting hPTH in tissues other than the parathyroid gland, or in localizing and possibly estimating the size of adenomas in parathyroid glands.

The double sandwich ELISA developed for the measurement of hPTH(l-34) demonstrates the analytical potential of the mono­clonal and polyclonal antibody produced. From the studies carried out using both the monoclonal and polyclonal antibodies,it is apparent that the affinity of the monoclonal antibodies is not suitable for detecting very low concentration of hPTH(l-34). Therefore, this assay cannot be used in clinical assessment of patient samples since the nor­mal range is far lower than that possible with this assay. The use of high non- isotopic label may improve the sensitivity.

The use of monoclonal antibody as a solid phase (capture antibody) was found to be inferior to that of polyclonal antibody. This could be related to the specificity and the possible alteration of binding site of the monoclonal antibodies. Other explanation might be the singular specificity of monoclonal antibody with probably one or near that number of antigenic determinant units per antigen molecule. Hence the limited signal intensity observed. Indeed the sensi­tivity of immunoassays and the affinity of antibodies was shown to be enhanced by the mixing of monoclonal antibodies (Elhrich and Moyle 1983, Elhrich et al., 1982, Moyle et al., 1983).

The labelling of the monoclonal antibody did not increase the sensitivity of the assay. Also the increase in concentration of the labelled monoclonal antibody did not improve the assay in this

APPLICATION OF MONOCLONAL ANTIBODIES

- 1 8 6 - CHAPTER 5

respect contrary to the: report of Wada (Wada et al., 1982). The use of low concentration of labelled antibodies, on the other hand was sug­gested to be better in constructing a calibration curve (Tshikawa et al., 1982). In the former study, the dilution of labelled monoclonal antibody showed very little difference to the sensitivity, ivity.

In double site ELISA, the use of monoclonal antibody as a top layer proved to be superior to their use as solid phase (capture anti­body). This could be related to the fine specificity of monoclonal anti­body, and the possible alteration of the binding site of monoclonal antibody upon adsorption can not be precluded.

APPLICATION OF MONOCLONAL ANTIBODIES

- 187 - CHAPTER 6

CHAPTER 6

DISCUSSION AND CONCLUSIONS

DISCUSSION AND CONCLUSIONS

- 188 - CHAPTER 6

Both radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) were chosen for screening for monoclonal antibodies to hPTH(l-34) , because they comply with the essential features of the screening assays mentioned in chapter 2 i.e fast, reliable and can handle large numbers of samples. It was expected that the combined use of these two fundamentally different techniques (Ekins 1981) would allow the selection of antibodies with different characteristics (Miller et al., 1986). Both assays were found to be very suitable for evaluating the polyclonal antisera as well as for screening the monoclonal antibo­dies in tissue culture supernatants. The liquid phase RIA was used for the screening of monoclonal antibodies in all fusions. However, in prac­tice the liquid phase assay was not the most valuable in detecting monoclonal antibodies. Different methods were used to enhance the pre­cipitation of antigen-antibody complexes. The second antibody and PEG method was found to be the best, because it gave the lowest non­specific binding in comparison with the dextran coated charcoal (Zanelli et al., 1983 ). The liquid phase RIA was found to be particularly useful in screening for goat monoclonal antibodies specific to hPTH(l-34). This is the first occasion in which goat monoclonal antibodies have been produced and detected.

The iodination of hPTH(l-34) is difficult and the most commonly used methods often result in substantial damage to the hormone (Teitelbaum 1983) and yield a less immuno-active peptide. In this study a method using iodogen (1,3,4,6-tetrachloro-3a,6a- diphenylglycoluil) was developed. Although iodogen method has been used for the iodination of other peptides (Schrader and O’Malley 1981), this is the first report of it being used for hPTH. This iodogen method was found to be superior to the chloramine T method in iodinating hPTH(l-34). The incorpoation of iodine into hPTH(l-34) was higher as indicated by the elution profile of the labelled hormone from sephadex G15 column. The labelled hormone from the iodogen method resulted in higher binding than that obtained from the chloramine T method as indicated by the antiserum dilution curve. Results from this study also

DISCUSSION AND CONCLUSIONS

- 1 8 9 - CHAPTER 6

show that 1.5 mCi sodium iodide used in many published protocols is unnecessary (Zanelli et al., 1980), and that only one third is required for iodination. This can be seen from the elution profile of the .125I

-hPTH( 1-34) from the G15 column, where the labelled peak using iodogen gave twice the number of counts per second than that of chloramine T. Moreover, less damage to the peptide occured in the iodogen method; this was evident since all iodinated fractions from the iodogen method were useable in the assay, whilst only one fraction with high binding was seen after chloramine T iodination.

ELISA provided an effective, fast, reliable method which could handle a large number of samples with ease. The application of ELISA procedure B (using antigen non-coated wells as blanks) was successful in avoiding the detection of false positives. This criteria has been adapted by many workers for the same reason (Cheng 1988, Kenna et al., 1985). Blocking agents are not all effective in preventing non­specific binding to the wells of ELISA plates (Kenna et al., 1985). While gelatin and bovine serum albumin were found to have the same effect in blocking the free sites of the wells of ELISA plates, the use of casein in our hands blocked even the binding of the antibody. The detergent Tween 20 was found to prevent the binding of rat antibody to solid phase antigen. Interestingly, this is a widely used detergent in ELISA. It is normally used for washing and in antibody dilution buffers. However, recently it has been shown both to enhance (Cheng and Yap 1988, cheng 1988) and decrease (Gharavi and Lockshin 1988) the binding of human anticardiolipin antibodies. It seems from our results that this effect may depend on the antibody characteristics. It is noteworthy that the binding of mouse and goat antibodies is not affected.

Only four published papers describe attempts to produce mono­clonal antibodies to hPTH (Nussbaum et al., 1981, Van De Walle et al., 1983, Nussbaum et al., 1985, Vieira and Neer 1987). In most studies bovine parathyroid hormone (bPTH) was used as immunogen and screening is for antibodies that cross-react with hPTH. There are

DISCUSSION AND CONCLUSIONS

- 190 - CHAPTER 6

significant differences in amino-acid sequence between hPTH and bPTH (Arnaud 1983), and these have been shown to affect the antibody characteristics (Mallete 1983, Zanelli et al., 1983a). It has been shown that the use of hPTH in the production of polyclonal antibody resulted in more specific antibodies to hPTH (Mallette 1983, Vieira et al., 1987). Furthermore, with the availability of synthetic hPTH and fragments, although expensive, their use as immunogens may lead to a more specific antibody.

We report the successful production of monoclonal antibody to hPTH(l-34) using the synthetic N-terminal hPTH(l-34) sequence as immunogen. The results of this study represent a good basis for the immunization and production of rodent monoclonal antibodies to hPTH(l-34). The mouse system has many advantages and is generally preferred for the production of monoclonal antibodies (Goding 1986, Campbell 1984). With the exception of our research work, all mono­clonal antibodies produced against PTH so far are of mouse origin. However, Balb/c mice were found to be good responders when immun­ized with bPTH, and have been used successfully in the production of monoclonal antibodies (Van De Walle et al., 1983). In our hands Balb/c mice were found to respond when immunized with hPTH(l-34) conju­gated to a carrier protein, contrary to previous reports that Balb/c mice are poor responders to hPTH(l-34) (Nussbaum et al., 1985), in which the authors attributed the poor response to genetic control. Results from our study, of the route of immunization and the form of antigen illustrate that Balb/c mice do respond to hPTH(l-34) and their response depends on the form of antigen. The free peptide was found to be non-immunogenic but produced a response when conjugated to a car­rier protein. The route of immunization in the mouse has not been investigated thoroughly using the conjugated hPTH(l-34). The intra- peritoneal route of immunization was used in the mouse and proved successful for the conjugate, but not the free hPTH(l-34). The intramuscular and intradermal routes of immunization produced no response when the free peptide was used as immunogen. The high cost

DISCUSSION AND CONCLUSIONS

- 191 - CHAPTER 6

of the antigen prevented a full investigation of immunogenicity and the response.

A novel in vitro immunization was used in the production of monoclonal antibodies to hPTH(l-34). This method has proved success­ful in producing monoclonal antibodies against calmodulin when the in vivo immunization had failed (Pardue et al., 1983) and has been used to increase the number of positive clones in fusion (Siraganinan et al., 1983). In this study, the method has resulted in the successful produc­tion of monoclonal antibodies to hPTH(l-34), when the use of the in vivo immunized spleen cells has failed. Moreover, all the antibodies produced were of IgG class which probably indicates a secondary response. Eleven cell lines were established as positive to hPTH(l-34) using ELISA. However, none showed any binding to radio-labelled hPTH(l-34) in liquid phase RIA. This was probably because of the specificity of the antibodies and/or the possible structural alteration in the antigen during iodination. Indeed, Nussbaum investigated several other molecules in which amino acid substitution was made specially to aid the iodination of hPTH(l-34), but despite such manipulation good antigen-antibody binding was not achieved (Nussbaum et al., 1981).

In immunocytochemical studies using the mouse monoclonal anti­bodies to hPTH(l-34), one antibody gave very strong positive binding to chief cells. All hybridoma lines tested were found to bind with the tissue antigen(s), though some rather weakly. Thus their binding to the natural hormone was demonstrated. It is expected that these cell lines will be of significant value in such studies, and they could used in the clinical detection and/or localization of PTH tumors. The mouse antibo­dies were unfortunately found to be of low affinity which makes them unsuitable for hPTH(l-34) immunoassay purposes. This is because the assay is very demanding in terms of antibody affinity, especially when the level of circulating hPTH(l-34) is markedly low in comparison with other forms of the hormone which circulate in 10-15 fold higher concentration.

DISCUSSION AND CONCLUSIONS

- 192- CHAPTER 6

Rats are not often used for the production of monoclonal antibo­dies. In most publications the use of rats was reserved for the produc­tion of monoclonal antibodies to mouse antigens. The rat spleen cells are usually fused with mouse myeloma cell lines, but very rarely with rat myeloma cell lines. This could be as a result of the difficulties encountered in growing the parent myeloma line or equally the result­ing hybrids. Here we report our experience with the rat Y3 myeloma cell line, and the first successful cross-species rat monoclonal antibodies to hPTH(l-34). Investigation of the immunogenicity was carried out in a similar way to that for mice. The route of immunization was investi­gated first using the free hPTH(l-34). Surprisingly, the results show substantial differences between mice and rats. Rats responded to immunization with the free fragment (1-34), but only when immun­ized intramuscularly. Intraperitoneal and intradermal immunization routes proved completely unsuccessful. The use of the free synthetic fragment was preferred, because it has a unique tertiary structure in solution quite distinct from the whole molecule, while conjugation to a carrier protein carries the risk of changing this conformation (Atassi 1986). In contrast to mice in which conjugation was necessary, rats responded very well to the free peptide.

Rat-rat fusion has certain advantages. It is probably that the difficulties of maintaining the rat myeloma cells have been the major reason underlying the poor use of rat-rat fusions in monoclonal anti­body technology. In this study Y3 rat myeloma cell line was used as fusion partner. Even though different conditions were used for cell maintenance, fusion experiments conducted with this cell line were not successful. Rat-mouse cross-species fusion, on the other hand, looked very promising. These fusions resulted in six positive cell lines, all of which produced IgM class antibodies. The production of IgM in such a situation was somewhat surprising. It is possible that only a primary immune response was generated in the responding rats.

The goat polyclonal antibody binds both the whole molecule hPTH(l-84) and hPTH(l-34). The cross-reactivity study with PTH

DISCUSSION AND CONCLUSIONS

- 193 - CHAPTER 6

peptides suggests that the antibody binds to the C-terminal end of the hPTH(l-34), the same region recently identified as antigenic region in the hPTH(l-34) (Vieira and Neer 1987). This antibody might be useful in setting up immunoradiometric or immunochemiluminometric assays similar to those described before (Brown et al., 1987, Nussbaum et al., 1987, Brown et al., 1988). The goat responded well to hPTH(l-34) and allowed the production of a novel heteromyeloma by cross- species fusion. This differs from the normal heteromyeloma fusion in the sense that genetic contribution from three different species is involved; a mouse- sheep heteromyeloma was fused with goat peripheral blood lymphocytes. The outcome of this was a hetero-hybridoma, generated for the first time, which produce goat monoclonal antibodies with good specificity to hPTH(l-34). The fusion efficiency was found to be very high (100%). Unfortunately these hetero-hybridomas lost their ability to secrete antibody 10 days after the screening assays gave positive results.

FUTURE WORK

This study provides a substantial amount of valuable background material for the raising of monoclonal antibodies to hPTH-related pep­tides. On the basis of the findings presented here, the following research work would be extremely valuable: (a) Affinity purification of the monoclonal antibodies described above and labelling, for example, with radio-label, for use in immunoassay, (b) Examination of the fusion efficiency of Y3 rat myeloma cell line and optimization of rat-rat fusions; continuation of rat-mouse work particulary in the light of the good primary response of the rat; testing of primary in vitro immuniza­tion with a view to incorporating it into the routine immunization pro­tocol, (c) Development of goat-mouse non-secretor hybridomas for the use as fusion partners; the use of second and third generation hybrido­mas in particular as fusion partners is required for the production of stable and continuously-secreting hetero-hybridomas, (d) Further stu­dies of the use of fragments of hPTH for raising monoclonal antibodies

DISCUSSION AND CONCLUSIONS

- 1 9 4 - CHAPTER 6

to hPTH, thereby opening up new areas of research on the structure and functions of parathyroid hormone.

DISCUSSION AND CONCLUSIONS

REFERENCES

- 196 -

[A]

Abbott, S. R., Nissenson, R. A., Teitelbaum, A. R., Clark, O. H., and Arnaud, C. D.(1980). Biologically active parathyroid hormone in human hyperparathyroid serum: Assay and Characterization. T rans.

A ssoc. A m . P h ysicians, 93: 192

Abelson, H. T., and Rabstein, L. S.,(1970). Lymphosarcoma: Virus- induced thymic-independent disease in mice. Cancer Res., 30: 2213.

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HYBRIDOMAVolume 6, Number 4, 1987Mary Ann Liebert, Inc., Publishers

Monoclonal Antibodies to Human Parathyroid Hormone (1-34) and

Their Use in the Immunocytochemical Detection of Parathyroid Tumours

I.A. MOHAMED, R. HUBBARD, E. AH-SING, and J. CHAKRABORTYDepartment of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH, England

A B ST R A C T

Monoclonal antibodies against the b iologically a ctiv e N -term inal fragm ent of human parathyroid hormone, hPTH (1-34), w ere produced. The procedure included the use of novel secondary im m unization in vitro of mouse spleen ce ll cu ltures. D issociated spleen ce lls from primary im m unized B alb /c m ice, w ere cultured for five days in the presence of thym ocyte conditioned media (TCM) and synthetic hPTH (1-34). Contrary to previous findings by other workers, in our hands B alb /c m ice responded w ell. Follow ing im m unization the spleen ce lls w ere fused with NS1 m yelom a ce lls and cultured for eleven days before screening for antibody. Using an enzym e linked im munosorbent assay (ELISA) a number of positive clon es w ere d etected .

P ositive c e lls -w e r e cloned by lim iting dilution and fifte e n sp ec ific m onoclonal hybridomas w ere produced. The immunoglobulin c lass of the d ifferen t m onoclonal antibodies was found to be IgG l. The im m unocytochem ical reaction was tested with ch ie f ce ll carcinom a tissue and found to be clearly positive.

IN TRO DUC TIO N

Parathyroid hormone (PTH) is an 84 am ino-acid single polypeptide chain, with a m olecular w eight of 9300 D altons. PTH serves an im portant role in the regulation o f extracellu lar calcium lev e l, which is m ediated by its e f fe c ts on kidney, bone and gut. Plasm a contains a heterogeneous m ixture of PTH fragm ents (1,2) largely o f carboxy-term inal fragm ents o f PTH, and only 2-25% in tact PTH (3). While the main form of the hormone secreted by the parathyroid glands is the in tact PTH (4), m etabolism by the liver and perhaps the kidneys, represents the final activation of this hormone thus contributing to the presence of these PTH -related peptides in circulation .

It is generally accep ted that the N -term inal fragm ent of PTH contains the structural requirem ents for the physiological a c tiv ity (5), and to date the C -term inal fragm ent has not been assigned any hormonal function.

381

It appears from published reports that serum N -term inal PTH leve ls are useful in discrim inating betw een healthy subjects and hyperparathyroid p atients (6 ,7 ,8 ,3 ,9 ,10) in venous ca th eteriza tion studies for loca liza tion of parathyroid tissue (11), ' hypercalcaem ic secondary hyperparathyroidism , chronic renal failure, and in the investigation of the pharm acokinetics of exogenously adm inistered hPTH (1-34) (12,13). H ow ever, from the view point of reliab ility and sen sitiv ity the current N -term inal PTH im m unoassays based on polyclonal antibodies are far from adequate for this purpose (14). We, therefore, undertook in the present study, the developm ent of m onoclonal antibodies to h-PTH (1-34) which m ight offer an im provem ent in som e of the laboratory investigations o f calcium -related disorders.

M ATERIALS A N D M ETHODS

Im m unization and C ell Fusion:B alb /c m ice were im m unized with hPTH (1-34) conjugated with bovine

serum albumin, and then im m unized with unconjugated hPTH (1-34) for two more doses within four w eeks. N ovel secondary in vitro im m unization of spleen ce lls was carried out by a m ethod sim ilar to that reported previously (15). A fter five days of in vitro im m unization, the spleen ce lls w ere fused with NS1 m yelom a ce lls in the ratio of 5:1 using polyethylene g lycol, M.W. 1500, (16). The ce lls w ere plated out in four 24 w ell tissue culture p lates, containing feeder layer ce lls in two tim es the normal concentration of hypoxanthine, aminopterin. and thym idine (HAT). The p lates were incubated at 37 C in a hum idified atm osphere of 5% CCL for eleven days. Those w ells which showed positive growth when exam ined under the m icroscope w ere screened using indirect ELISA.

ELISA:Figure 1 shows the indirect ELISA assay which has been adapted for

screening the culture supernatants for the presence of the desired antibodies. A 200 yl aliquot of hPTH (1-34) solution, at a concentration of 1 yg /m l in phosphate buffer saline (PBS), pH 7.4 was added to each w ell of a 96-w ell polyvinylchloride p late. A fter incubation overnight the p late was washed three tim es w ith PBS pH 7.4 containing 0.1% gelatin and 0.05% tw een 20. The sam ple or standard was added to the p late, incubated at 37°C for two hours and washed as before. The next step was the addition of peroxidase-labelled donkey anti-m ouse second antibody to each w ell, incubation at 37°C for another tw o hours, follow ed by m easurem ent o f the colour developed at 490 nm thirty m inutes after the addition of orthophenylene diam ine (OPD) as substrate for peroxidase.

D eterm ination of Immunoglobulin C lasses and Subclasses:The m onoclonal antibodies w ere allowed to react with immunoglobulin

class sp ec ific anti-sera (Serotec) in im m unodiffusion tes ts .These antibody characterisations w ere further confirm ed using double

antibody sandwich ELISA. P olystyrene m icrotitre p lates w ere coated with donkey anti-m ouse im munoglobulins (Guildhay, Guildford, U.K.) and the supernatants allowed to bind to it. The sheep c la ss-sp ec ific an ti-sera were then diluted 1:1000 and applied to the p late.

Donkey anti-sheep antibody labelled w ith horseradish peroxidase (Guildhay) was added and allowed to react for one hour at 37°C . The p late was washed and the enzym e visualised using an orthophenylene diamine (OPD) reaction .

Im m unocytochem istry:P araffin waxed ch ie f ce ll carcinom a tissue s lices 6-8 ym w ere dewaxed

and the endogenous alkaline phosphatase blocked, the free s ite s w ere also

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Step 1

S t e p 2 PTH1 - 3 4

PT H1 - 3 4

S t e p 4PTH1-34

FIGURE 1 ., Indirect ELISA o f Human Parathyroid Hormone (1-34) S p ec ificM onoclonal A ntibodies.

Step‘1 - A dsorbtion of ly g /m l antigen to polyvinylchloridep late overnight at 4 C.

Step 2 - Addition of cu ltu re supernatant antibody andincubation for 2 hours at 37 C.

Step 3 - The addition of enzym e labelled donkey an ti-m ouseantibody and incubation for 2 hours at 37 C.

Step 4 - The addition o f en zym e substrate, orthophenylenediam ine (OPD) and incubation for 30 m inutes at 37 C. Sulphuric acid addition to stop the reaction .

blocked using 1% non-immune sera. Supernatant (200 yl) from growing ce lls was spread over the tissue and incubated at 4°C overnight. The tissues w ere washed using phosphate buffered saline and 200 yl of alkaline phosphatase conjugated antibody was added. Incubation was continued for a further one hour at room tem perature. A fter washing, the colour was visualised using naphthol AS:BI and fast red.

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RESULTS

Sera from m ice im m unized with hPTH (1-34), w ere used to se le c t the conditions for the ELISA system . A chequerboard ELISA for positive mouse serum is shown in Figure 2. From the screening te s ts for m onoclonal antibody against hPTH (1-34), a number of positive w ells w ere d etected . The ce lls w ere cloned tw ice using a lim iting dilution m ethod. A to ta l of f ifteen ce ll lines w ere identified as positive and the corresponding hydridomas stored in liquid nitrogen.

The im munoglobulin class and subclass w ere found to be IgGl by im m unodiffusion and confirm ed using ELISA. The resu lts are given in Figure3.

In im m unocytochem istry, one clone was tested and found to give a positive result with tissue s lices from a ch ie f c e ll carcinom a as shown in Figure 4.

2 -.00

1 .8o

1 . 4 0

1 . 2 0

l.oo

.80

. 6 0 J

. 4 0 -B -

. 2 0

.00

. 5 0 1 . 5 0 2 . 5 01.00 2 .00.00

Antigen coating concentration pg/ml

FIGURE 2., T itration o f B alb/c m ice antiserum to human parathyroid :hormone (1-34) using d ifferen t antigen concentrations for coating of the plate.

A ntiserum dilution - 1:1,000 ( • ----- • ) , 1:2,000 (■ ■ ),1:4,000 (▼-----▼ ), 1:8,000 (♦ -----* ), 1:16,000 (□------□ ),1:32,000 (A.------ A ) , 1:64,000 (O------O)

IgGl IgG2a IgG3 Igfl

I qo C l a s s & S u b c l a s s

FIG U R E 3., Typing o f fo u r M o n o c lo n a l a n t ib o d ie s using doub le a n t ib o d y sa n d w ic h ELISA.

1 /3 /6 ( m ) , 1 /2 /6 ( @ ) , 2 /2 /6 ( « ) , C 8 ( * ) .

f

:J L

' Jk V**- * «# • v ' 4- U AUiir * -

. - ; ..

4:

#

I

vy '^ C > ;■ ^ »**<

•ttt T#*1 4#'

FIG U R E 4., (a) T h e b ind ing o f M o n o c lo n a l a n t ib o d y to hP T H in s e c t i o n sf ro m p a r a t h y r o id c h ie f c e l l c a r c in o m a t i s su e , c o u n te r s t a i n e d w i th h a e m a l u m (x 400).

385

F IG U R E 4., (b) T h e s a m e c o n d i t i o n s as in (a) e x c e p t t h a t insulinM o n o c lo n a l a n t ib o d ie s w e r e used as a n e g a t iv e c o n t r o l (x 400).

DISCUSSION

S p e c i f i c a n t ib o d y to hP T H (1-34) is n e e d e d b e c a u s e th is f r a g m e n t is b io lo g ic a l ly a c t i v e , an d i t s m e a s u r e m e n t is o f g r e a t i m p o r t a n c e . T he a v a i l a b i l i t y o f m o n o c lo n a l a n t ib o d y to th is f r a g m e n t should o v e r c o m e so m e of t h e i n h e r e n t p r o b l e m s o f th e a s sa y , a n d m a y a l low th e m e a s u r e m e n t of th is f r a g m e n t in n o r m a l in d iv id u a ls w h ic h is n o t p o ss ib le w i th th e e x is t in g p o ly c lo n a l a n t ib o d y . T h e a n t ib o d ie s r e p o r t e d h e r e p r o d u c e d by i m m u n iz a t io n m v i t r o m a y , t h e r e f o r e , h a v e an i m p a c t on th e in v e s t ig a t io n o f p a r a t h y r o id d i s o r d e r s by im p ro v in g th e hP T H (1-34) a s sa y .

B a lb / c m ic e h a v e b e e n r e p o r t e d to be ve ry p oor r e s p o n d e r s to hP T H (1- 34) (17). In c o n t r a s t , w e fo u n d t h a t th is s t r a in o f m ic e r e s p o n d e d w ell to h - P T H (1-34). F u r t h e r m o r e , t h e im m u n o g lo b u l in c la s s in ou r p r e p a r a t i o n s w e r e o f t h e IgGl t y p e , w h e r e a s N u s sb a u m (17) fo u n d th e m all to be IgM. In t h e c a s e o f b ov ine P T H im m u n o g e n , B a lb / c m ic e w e r e r e sp o n s iv e w hen used f o r th e p r o d u c t io n o f m o n o c lo n a l a n t ib o d ie s (18). M o s t of t h e s e a n t ib o d ie s w e r e r e p o r t e d to be IgG c la s s w i th only one b e in g IgM.

In c o n c lu s io n , w e h a v e show n t h a t B a lb / c m o u se sp le e n c e l l s re sp o n d w el l to h u m a n P T H (1-34) and c a n be used fo r m o n o c lo n a l a n t ib o d y p r o d u c t io n . T h e ro le o f jn v i t r o im m u n i z a t io n in thrs r e s p e c t c a n n o t be d i s c e r n e d a t th is s t a g e o f our s tu d y . F u r t h e r w o rk on th e c h a r a c t e r i s a t i o n of t h e s e a n t ib o d ie s and t h e i r a p p l i c a t io n s is in p ro g re s s .

REFERENCES

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2. S i lv e rm a n , R . an d Y a lo w , R .S . J . C l in . I n v e s t . , 32, 1958-1971 (1973).

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3. Seger, G .Y. Ehdocrine control of bone and calcium m etabolism . Eds. D .Y. Chan, J.T. P o tts Jr. and T. Fujita. E lsevier Science Publishers. Am sterdam 1984.

4. Habener, J .F ., P ow ell, D. and Murray, T.M., e t al., Proc. N atl. Acad. Sci.j USA 68, 2986-2991 (1971);

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9. P o tts , J.T . Jr., Seger, G.V. and Endres, D.B. Los A ngeles; N icolas Institu te, 1-9 (1983).

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11. Dunlop, D .A .B ., Papapoulous, S.E., Lodge, R.W., Falton, A .J ., Kendall, B.E. and O'Roirdan, J.L.H. Br. J. R adiol., 53, 183-191 (1980).

12. K ent, G .N ., Loveridge, N ., R eeve, J. and Z anelli, J.M. C linical S cien ce , 68, 171-177 (1985).

13. Z anelli, J.M. and R afferty , B. J. Immunoassay 1, 289-308 (1980).

14. Vierira, J.G .H ., O liveira, H .A .D ., M aciel, R .M .D., M esquita, G.H. and Russo, E.M.K. J. Im munoassay., 7 (1&2), 57-72 (1986).

15. Pardue, R .L., Brady, R .C ., Perry, G.W. and Dedman, J .R . J. C ell B iol., 96, 1149-1154 (1983).

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17. Nussbaum, S .R ., Lin, C .S., P o tts , J.T. Jr., R osenthal, A.S. and R osenblatt, M. Meth. E nzym ol., 109, 625-638 (1985).

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Address reprint requests to; R. HubbardDepartment of BiochemistryUniversity of SurreyGuildfordSurrey GU2 5XHEngland

Received for publication April 6, 1987.Accepted for publication April 22, 1987.

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