Introduction to Immunology and Serology Diagnostic Techniques

1 .Key Term:

- Immunology: is both the study of the human 

immune system and the field of medicine that treats

diseases of the immune system.

- Serology: is the branch of science concerned with

serum to measure either antigens or antibodies in sera.

- Immunodiagnostic: is a medical diagnostic based on

highly specific interaction between an antibody and an

antigen. The antibody is used to detect the presence of

the antigen.

-Antibodies: also known as immunoglobulins (Igs).

They are a group of serum proteins (globulins),

secreted in a “soluble” form by B lymphocyte in

response to antigenic stimuli. These antibodies bind

with the stimulating antigen and inactivating it.

- Antigen-binding site: Hypervariable region of an

antibody molecule that is the location where binding a

specific antigen takes place.

- Epitope: A part of an antigen, also as an antigen

determinate, that bind with a specific antibody .

2 .Structure of Immunoglobulin:

The immunoglobulin molecules has two distinct regions,

one of which (Fab) contains an antigen binding site that

bind to an antigen, whereas the other (Fc) contain

receptor that interact with a complement or phagocytes.

3.Immunoglobulins: Ag-Ab reactions:

3-1. Natural of Ab Reactions:

A. Lack and Key concept

Antigen-antibody binding sites are located within the

hypervariable segments of the variable region (VL and VH)

on Fab segment of an antibody molecule. Any change in the

hypervariable regions of an antibody may alter its specificity .

The concept of specificity or “exact fit” of the two molecules

has been compared to a “lock and key fit” where the “lock”

refers to the antigen – site (antibody) and the “key” to the

epitope on an antigen.

B. Non-covalent Bonds

The bonds that hold the antigen in the antibody

combining site are all non-covalent in natural. These

include hydrogen bonds, electrostatic bonds. Multiple

bonding between the Ag and the Ab ensures that the

Ag will be bound tightly to the Ab.

C. Reversible

Since Ag-Ab reaction occur via non-covalent bonds

they are by their nature reversible.

3.2 Affinity and Avidity

1. Affinity:

The term refers to an intrinsic forces of attraction or

association between an antibody (antigen-binding site)

and one epitope on corresponding antigen (univalent

antigen).

It is the sum of the attractive and repulsive forces

operating between the antigenic determinant and the

combining site of the antibody.

2 .Avidity:

When the antigen consist of several repeating and

identical epitopes (multivalent antigen), the avidity

between an antigen and an antibody is the sum of the

affinities involved (i.e., affinity between antibodies and

multivalent antigens, known as avidity)

Affinity refer to the strength between a single antigenic

determinate and an individual antibody combining site

whereas avidity refer to overall strength of binding

between multivalent Ag’s and Ab’s.

3.3 Specificity and Cross Reactivity

A. Specificity

Specificity refers to the ability of an individual antibody

combining site to react with only one antigenic

determinant or ability of population of antibody

molecules to react with only one antigen.

B. Cross reactivity

Cross reactivity refers to ability of an individual antibody

combining site to react with more than one antigenic

determinant or ability of population of antibody

molecules to react with more than one antigen.

Cross reactivity arise because:

- The cross reacting antigen shares an epitope in

common with antigen.

- Because it has an epitope which is structurally similar

to one antigen.

4 .Factors Affecting Measurement of Antigen – Antibody

Reactions:

The only way that one knows that an antigen-antibody

reaction has occurred is to have some means of directly

or indirectly detecting the complexes formed between

the antigen and antibody. The ease with one can detecte

antigen-antibody reaction will depend on a number of

factors.

A. Affinity

The higher affinity of the antibody for the antigen, the

more stable will be the interaction is enhanced.

B. Avidity

Reaction between multivalent antigens and multivalent

antibodies are more stable and those easer to detect.

The binding between an antibody and a multivalent

antigen is much stronger (has stronger avidity) than

binding between an antibody and single epitope.

C. Ag-Ab ratio

The ratio between the antigen and antibody influences

the detection of Ag/Ab complexes because the size of

the complexes formed in related to the concentration of

the antigen and antibody. This is depicted in this figure .

Ag excessAb excessEquivalence – Lattice formation

D. Physical form of the antigen

The physical form of the antigen influences how one

detects its reaction with an antibody. If the antigen is a

particulate, one generally looks for agglutination of

antigen by the antibody. If the antigen is soluble one

generally looks for the precipitation of the antigen after

the production of large insoluble Ag/Ab complexes .

5 .Formation of Monoclonal and Polyclonal Antibodies:

5.1 Monoclonal antibodies:

Identical antibodies with unique specificity made by

single B cell.

5.1 Polyclonal antibodies:

Different antibodies with different specificity made by

different clone of B cell.

5.3 Researchers make monoclonal and antibody by:

* Injecting specific antigen into a host animal (typically a

mouse).

* Isolating antibody-producing cells (B cells) from the

spleen of the mouse.

* Fusing these B cells with a specific type of tumor cell

that grows easily in culture and produces antibodies.

* Isolating successful hybridomas (fused cells) that

produce antibodies specific for the antigen of interest.

Quality Assurance In Serology And Immunology Laboratory

1 .Key Term

* Quality Assurance means a comprehensive process used

by the laboratory to prevent and control errors that may

occur at any interval from the time a test is ordered until it is

reported.

* Quality Control Program, means those quality control,

requirements established for clinical laboratories a provided

in applicable federal law and regulations.

* The purpose of quality assurance is to prevent as many

errors as possible and to detect those that do occur and the

procedures used to recognize and minimize them.

2.1 Pre-analytical Factors:

The term “Pre-analytical Factors” include all steps that a

test sample must undergo until the actual test is carried

out . They included:

2.1.1age depended variation:

age depended changes of changes of concentration or

activities occur in a number of hematological and

immunological analytes. Hence laboratory results should

be interpreted keeping in a view the age of the patient

e.g. titer of ASO different from neonate and adult.

2.1.2 Incorrect specimen identification or mislabeling:

An incomplete specimen identification will obviously give

wrong information despite all the precautions taken for a

proper analytical procedure.

2.1.3 Prolonged transportation and storage:

prolonged storage prior to processing of the specimen

can affect the results especially since many organisms

do not survive for long unless they are kept in an

environment which is rapidly changing.

2.1.4 Selection of appropriate sample or collection the

right specimen:

An appropriate sample should be collected in an

appropriate way, collection the right specimen is of

critical importance.

2.1.5 Selection of right test method:

Selection of right test method is of paramount

importance.

2.1.6 Sending the sample to the right laboratory:

The sample for analysis of particular analyte should be

send to the right laboratory undertaking that particular

investigation. A laboratory not doing a test and yet

receiving the samples for analysis will cause inordinate

delay as well as a decline in the quality of the sample .

2.1.7 Centrifugation:

Hematolysed blood specimens are not suitable for

serological studies. It is always advisable to avoid

factors which cause hemolysis such as centrifuge blood

sample at high speed before clotting.

2.2 Analytical Controls:

Analytical Quality controls depend upon the following:

2.2.1 Equipment reliability.

2.2.2 Reagent stability, integrity and efficiency.

2.2.3 Adequate calibration.

2.2.4 Procedure reliability using standard operating manuals.

2.2.5 Specificity, precision and accuracy of the test of the high

order.

2.2.6 Proficiency personnel and continuous updating of their

Knowledge.

2.2.7 Selection of the right technique of proven efficacy for

which reliable reagents are available .

2.3 Post-analytical controls:

1. after the proper analytical process is over, it is

important to record the findings on the right

requisition slip in a clear way.

2. Any possible remarks on the result obtained

should be entered, as well as the normal range of

the results.

3 .The significance of the result obtained should

be highlighted wherever required.

4. There should be frequent dialog between

laboratory personnel and the physicians for

appropriate use of the facilities and right

interpretation of results obtained in the laboratory.

Done by : Dr. Nada