Immunisation techniques. AntibodyImmunisation techniques. Antibody(polyclonal and monoclonal)(polyclonal and monoclonal)

production, purification, labelling.production, purification, labelling.Hybridoma technology,Hybridoma technology,

fermentationfermentation

Department of Immunology and BiotechnologyUniversity of Pécs

Isotype

Idiotype

Structure of an antibody

Antibody and antigenic determinant

Antibody and antigenic determinant (epitope)

Types of antigenic determinants

A complex antigen generates polyclonal immuneresponse

Monoclonal antibodies:antibodies with the same specificity,produced by a cell line derived from only one activatedB cell,able to recognise only one epitpe

Polyclonal antibodies:mixture of antibodies with different specificities,product of more B cell clones,recognise different epitopes of an antigen

Y Y

Y

Ag

Y YY

Ag

Immunization

Selection of the adequate animal:• Monoclonal antibodies – mouse or rat• Policlonal antibodies – rabbit, sheep, goat

Antigen:• hapten (5-10 kD >) – carrier• native or modified molecules, cells• Administration: intramuscular, subcutaneous

or intracutan

Immunization

Adjuvants: delay the absorption of the antigen and non-specifically stimulate the immune system (activation ofmacrophages) resulting in increased antibody secretion.

Examples:• Incomplet adjuvants: contain mineral oils (incomplete Freund)• Complete Freund: heat-inactivated Mycobacterium tuberculosis

suspended in mineral oil• Aluminium-hydroxid gel• Muramyl-dipeptide (Mycobacterium tbc.)• Bacterial vaccines (e.g. Bordetella pertussis) co-administration

with immunogens can increase the antibody production againstthese immunogens

• Liposome

Immunization

Purification of polyclonal antibodies

Antibodies are extracted from the sera ofimmunized animals.

• IgM• precipitation of Ig fraction with chemical

methods: based on molecular weight andsolubility

• „euglobulin precipitation”• Further purification with gel chromatography• Density gradient centrifugation

•• IgG:IgG:•• Ion-exchange chromatographyIon-exchange chromatography•• Affinity chromatography (Staph.Affinity chromatography (Staph.

aureus protein A, Streptococcus proteinaureus protein A, Streptococcus proteinG)G)

Purification of polyclonal antibodies

• IgA, D, E: multiple step methods IgA: after Zn precipitation; ion-exchange chromatography

and gel filtration IgE: affinity chromatography

Immunosorbent methods:Antigen specific isolation on the surface of Sepharose,

cellulose or polyacrylamid gels

Purification of polyclonal antibodies

Antibody testing

Testing of titer and specificity should be donein the same system where the antibody will be applied

e.g.: ELISA flow cytometry immunohistochemistry

Hybridoma technique,production of monoclonal antibody

With the hybridoma technique we are able to selecta specifically activated (e.g. antibody production),but limited life span immune cell and keep it in vitro(proliferation).

The basis of the technique is that we fuse in vitro anactivated immune cell with a B-cell/plasma cell-derived tumor cell line.

Plasma cell

Antibody productionImmortality

Immortality and antibodyproduction

Myeloma

Hybridoma

Hybridoma production

Hybridoma production

1. Cell fusion: spleen cell (from immunized animal) + non-secretingmouse myeloma (Sp-2/0-Ag14) + PEG

2. Selection: Selection of the desired hybrides (spleen cell + myeloma)

• Sp-2 cells are deficient for HGPRT and TK enzymes (purineand pyrimidine synthesis), but spleen cells have them, so bythe use of a selective medium (HAT) we can select the spleencell – Sp2 hybrids

Pathways of DNA synthesis

Selection of the antibody producing hybrid cultures, testing(ELISA)

• Cloning:dilution of the antibody producing cells so that on a96 well plate statistically less than 1 cell is seeded in awell. Testing antibody production of monoclones.

Antibody produced by a single clone of cells:MONOCLONAL ANTIBODY

Hybridoma production

Monoclone

4. Mass antibody production:(1) production of hybridoma supernatant in vitro;(2) administration of hybridoma cells ip. in mice (ascites);(3) fermentor (artificial mouse).

Hybridoma production

Applications of monoclonal antibodies

•• Preparative methodsPreparative methods–– Protein purificationProtein purification

•• Analytical methods (Diagnostic purposes,Analytical methods (Diagnostic purposes,research)research)–– Identification and isolation of lymphocyte subgroupsIdentification and isolation of lymphocyte subgroups

and clonesand clones–– TTumor detection and imagingumor detection and imaging–– Routine laboratory techniques- serologyRoutine laboratory techniques- serology

•• TherapyTherapy–– TumorTumor–– Autoimmune diseasesAutoimmune diseases–– Chronic inflammationChronic inflammation

1g gold ~ € 201g mAb ~ € 800If you have the monoclone

Conjugation

Fluroescent dye – e.g.: FITCEnzyme – e.g.: HRPOBiotin

Enzyme conjugates

Enzyme converts substrates thatmanifests in colour reaction.

HRPO,ALP

Immunohistochemical slides

Fluorescent dye conjugates

FITC,PE

Imunofluorescence

PE

FITC

FITC

Flow cytometry

Toxin conjugates

Ricin

A toxin conjugated to an antibody binding tothe surface antigen of the target cell will beinternalized by the target cell. Then the toxinis released into the cytoplasm and exerts toxiceffect (e.g. Ricin can block the proteinsynthesis of the cell).

Radioisotope conjugates

• γ ray emission, low dose Diagnostic purposes – radioactive imagining Diagnosis of tumors and micrometastases

• α, β ray emission, high doseTreatment of tumors

Bispecific antibodies

TUMOR

Effector cell• NK• Tc• Macrophage

A bispecific antibody is created by chemical crossbinding of the Fab fragments of two different Igs.

Effector cell specific Fab part:• CD3 Tc• CD64 (FcγR1) high affinity FcγR; Mφ • CD89 FcαR; Mφ , granulocytes

Tumor specific Fab part

Therapeutic monoclonal antibodies registered by FDAName Specificity Type Application Year

Orthoclone OKT3 CD3 Murine Graft rejection 1986

ReoPro GpIIb/gpIIa Chimeric Cardiovascular diseases 1994

Rituxan(mabthera) CD20 Chimeric Non-Hodgkin lymphoma 1997

Zenapax CD25 Humanised Graft rejection 1997

Remicade TNFα Chimeric Crohn’s disease, rheumatoid arthritis 1998

Simulect CD25 Chimeric Graft rejection 1998

Synagis RSV Humanised Respiratoric synctitium virus infection 1998

Herceptin Her-2 Humanised Metastatic breast carcinoma 1998

Mylotarg CD33 Humanised Acute Myelogenous Leukemia (AML) 2000

Campath CD52 Humanised B cell chronic lymphoid leukemia(B-CLL) 2001

Zevalin CD20 Murine B cell non-Hodgkin-lymphoma 2002

Erbitux EGFR Chimeic Colorectal carcinoma (EGFR+ tumors?) 2004

Avastin VEGF Humanised Colorectal carcinoma 2004

Tysabri α4β1/7 integrin Humanised Multiple sclerosis 2004

Murine antibodies

The first therapeutic monoclonal antibody registeredby FDA.

OKT3 – mouse monoclonal anti-CD3 (1986).

This therapy is quite effective but its repeated use isaccompanied with severe immunological side effects:

HAMA (human anti-mouse-antibodies)

The constant part of an Ig is conserved, but there are some differencesbetween human and mouse Igs.

HAMA can be detected after 8-12 days of treatment, the peakconcentration is after 25-30 days.

Chimeric antibodiesThe Fv region of the chosen monoclonal antibodygene is fused to Fc part of a human Ig gene.

Approximately 75 of a chimera Ig % is of humanorigin.

The specificity of the antibody is similar to that of theoriginal mouse antibody.

The in vivo half life and effector functions of the Igare similar to those of the original human antibody.

HACA (human anti-chimeric-antibodies)

Less immunogeneicity, but sometimes HACA can be detected.

Humanised antibodies

The genes of the CDR regions of a mousemonoclonal antibody are implanted into the genesof human antibody.

More than 90% of the antibody is of human origin.

The specificity of the humanized antibody is similarto that of the original mouse antibody.

The in vivo half life and effector functions of thehumanized antibody are similar to those of theoriginal human antibody.

US Food and Drug Administration nomenclature of therapeutic antibodies

Chimeric mAb Fully human mAb Murine mAb Humanised mAb

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Monoclonal antibodyChimeric antibodyHumanised antibodyImmunomodulatory antibodyAntitumor antibodyCardiovascular antibodyHumanMurine The Lancet Volume 369, Issue 9573, 12 May 2007-18 May 2007, Pages 1641-1657