Ag-AB
ANTIGEN-ANTIBODY REACTIONS AND SELECTED TESTS
TEACHING OBJECTIVES
To describe the nature of Ag-Ab reactions
To compare and contrast antibody affinity and avidity
To delineate the basis for antibody specificity and cross reactivity
To discuss the principles of commonly used tests for antigen/antibody reaction
Introduction
Antigen-Antibody (Ag-Ab) Interaction is a biochemical reaction between antibodies and specific antigens to form an immune complex
The antigenic determinant or epitope of antigens is recognized by the variable region of the Antibody
Initiates a series of immunological responses to act against the respective antigens for their removal or destruction.
Antigen-Antibody Interaction
Antigen
Any foreign substances that enters the body and elicit or react with inmmune response.
Directly elicit an immune response (immunogen) or require the help of some other molecules (carrier proteins) to do so (hapten).
Can be proteins, peptides, lipids, or, polysaccharides
Antibody (Ab)
Component produced by the immune system in response to antigens.
Produced by plasma B-cells
Antigen binding site is called paratope.
Types: IgG, IgA, IgM, IgE, IgD
Antigen-Antibody Interaction
Interactions between antigens and antibodies are known as antigen-antibody reactions
The reactions are highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens.
Antibodies recognize molecular shapes (epitopes) on antigens.
Highly specific reaction
Non-covalent interaction (Van der Waals forces, Ionic bonds, Hydrogen bonds, Hydrophobic interactions )
The strength of antigen-antibody interaction is expressed in terms of avidity and affinity.
Affinity
Affinity is the strength of the reaction between a single antigenic determinant (epitope) and an antibody’s antigen binding site (Fab).
Avidity
A measure of the overall strength of an antibody-antigen complex.
Avidity is influenced by the affinity of the antibody for epitope, the valence of both the antibody and the antigen, and structural arrangement of epitopes and paratopes.
Specificity
The ability of an individual antibody combining site to react with only one antigenic determinant.
The ability of a population of antibody molecules to react with only one antigen.
There is a high degree of specificity in antigen-antibody reactions.
Antibodies can distinguish differences in:
The primary structure of an antigen
Isomeric forms of an antigen
Secondary and tertiary structure of an antigen
Cross reactivity
The ability of an individual antibody combining site to react with more than one antigenic determinant. OR
The ability of a population of antibody molecules to react with more than one antigen.
Cross-reactions arise because the cross-reacting antigen shares an epitope in common with the immunizing antigen.
It has an epitope which is structurally similar to one on the immunizing antigen (multi-specificity).
Cross reactivity
Factors Affecting Measurement of Ag/Ab-reactions
The ease with which one can detect antigen-antibody reactions will depend on a number of factors.
1. Affinity
The higher the affinity of the antibody for the antigen, the more stable will be the interaction.
The ease with which one can detect the interaction is enhanced.
2. Avidity
Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.
3. Antigen to antibody ratio
The ratio between the antigen and antibody influences the detection of antigen-antibody complexes
Prozone: is a possible cause of a false-negative antigen-antibody reaction caused by excessive amount of antibody
Postzone: refers to the excess of antigen resulting in no lattice formation in an agglutination reaction
Equivalence zone: a variable ratio of antigen and antibody that results in precipitation in which there is no unbound antibody or antigen.
4. 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 the antigen by the antibody.
If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.
Types of Antigen-Antibody Interaction
Ag-Ab reactions are basically of two types:
In Vivo (Occurring in natural conditions): It includes the general antibody-mediated immune response in the body against any antigen.
Agglutination, Precipitation, Complement fixation, Neutralization, Ab Dependent Cell-Mediated Toxicity, Opsonization
In Vitro (Done in artificial conditions): It includes a series of serological tests performed in laboratories to detect antigens or antibodies for many diseases.
Agglutination, Precipitation, Complement fixation, Neutralization, ELISA, Radioimmunoassay (RIA), Immunofluorescence, Western Blotting
Precipitation Reaction
The reaction between soluble (small) antigens and soluble antibodies forms an insoluble precipitate.
The proportion of Ag and Ab in the reaction must be equivalent for the reaction to occur.
Antibodies involved are called precipitins
Types: In solution (Flocculation and Ring Test)
In Agar (Immunodiffusion)
In Agar with an electric field (Immunoelectrophoresis)
Agglutination Reaction
The reaction between insoluble (large) antigens and soluble antibodies leads to agglutination.
Formation of visible clumps occurs.
Occurs on the surface of the particle involved.
Antibodies involved are called agglutinins.
Types: Direct/Active (Slide, Tube, Heterophile agglutination test and, Coombs’ test,)
Passive (Latex, Haemagglutination and, Coagglutination test)
Applications of agglutination tests
Haemagglutination
It is a passive agglutination reaction that involves RBCs as carrier particles.
RBCs of sheep, humans, chicks, etc. are used
Used in Blood Typing
In the detection of parasitic infections and viral diseases such as influenza, mumps, and measles.
Coomb's Test (Antiglobulin Test)
Direct Coomb's Test
When antibodies bind to erythrocytes, they do not always result in agglutination.
This can result from the antigen/antibody ratio being in antigen excess or antibody excess or in some cases electrical charges on the red blood cells preventing the effective cross linking of the cells.
These antibodies that bind to but do not cause agglutination of red blood cells are referred to as incomplete antibodies.
Direct Coomb's Test
To detect the presence of non-agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the antibody coating the red cells.
This anti-immunoglobulin can now cross-link the red blood cells and result in agglutination.
Indirect Coomb's Test
Complement Fixation
It is the process of binding or fixing the serum complement proteins with the Ag-Ab complex which further initiates a series of immune responses against the antigen.
It is used in the detection of any specific antigen or antibody in the patient’s serum.
Reagents: Antigen, Antibody, Complement, and, Indicator System
Common complement fixation test is Wasserman’s Test: For detection of Syphilis
Other Ag-Ab Reaction Tests
Enzyme-Linked Immunosorbent Assay (ELISA)
Sensitive techniques for the detection of the presence of antigen or antibody and quantification as well in case of clinical diagnosis of many diseases
Enzymes are used for labeling
Immunofluorescence
It is a type of immunoassay technique in which fluorescent dyes are used for the visualization of Ag-Ab reactions.
Detects surface antigens or antibodies
Neutralization: Virus Neutralization Tests, Toxin Neutralization Tests
Applications of Antigen-Antibody Interaction
The most common use is the determination of blood groups i.e. blood typing.
Rapid diagnosis test kits used for pregnancy, malaria, dengue, etc. are based on this principle.
Serological ascertainment of exposure to infectious agents.
Quantification of drugs, hormones, viral antigens, etc.
Detection of presence or absence of proteins in serum.
To study the characteristics of different immunodeficiency diseases.
To perform confirmatory tests for infections such as Western Blotting for HIV infection.
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