What is an Antigen (Ag)?
(Anti= against; gen=thing that produces or causes)
Any foreign substances that when entering our body sometimes self-elicit a series of immune responses and are precisely called immunogens. Whereas some of them don’t directly elicit an immune response but require the help of some other molecules (carrier proteins) to do so and are called hapten. The immunogens and hapten are collectively called antigens.
– They can be proteins, peptides, lipids or, polysaccharides.
– Antibody binding site is called an epitope.
– Abbreviation: “Ag”
What is an Antibody (Ab)?
An antibody is simply the component produced by the immune system in response to antigens. So basically antigens are the generator of antibodies. They interact with each other to induce an immune response.
– Also called immunoglobulins (Ig)
– Produced by plasma B-cells
– Antigen binding site is called paratope.
– Types: IgG, IgA, IgM, IgE, IgD (Pneumonics: “GAMED”)
What is an Antigen-Antibody (Ag-Ab) Interaction?
It is a biochemical reaction between antibodies and specific antigens when they come closer to a distance of several nanometers. It is the binding of paratopes of antibodies to specific antigens on their epitopes that initiates a series of immunological responses to act against the respective antigens for their removal or destruction.
Antigen + Antibody ⇄ Ag-Ab complex → Immune Response
According to the law of mass action:
Ka = Rate of the association constant
Kd = Rate of the dissociation constant
Keq = Equivalence constant
At equilibrium, the ratio of the concentration of product i.e. [Ag-Ab complex], and reactants i.e. [Antigen]-[Antibody] are constant and called equivalence constant.
Antigen-Antibody (Ag-Ab) Interaction Properties
– Highly specific reaction
– Occurs in an observable manner
– Non-covalent interaction ( Van der Waals forces, Ionic bonds, Hydrogen bonds, Hydrophobic interactions )
– No denaturation of antibodies and antigens
– Affinity: It is the strength with which one antigen binds on a single antigen-binding site on an antibody.
– Avidity: It is a broader term than affinity. It is a measure of the overall strength of the Ag-Ab complex. It depends on:
- The affinity of the antibody
- Valency(no. of binding sites) of antibody and antigen
- And the structural arrangement of epitopes and paratopes.
– Cross-Reactivity: It refers to the ability of an antibody to bind to similar epitopes of different antigens.
Stages of Antigen-Antibody (Ag-Ab) Interaction
1. Primary Stage: It is the initial interaction between antigens and antibodies.
– Without any visible effects
2. Secondary Stage: It is the irreversible interaction between antigens and antibodies.
– With visible effects
Factors Affecting Antigen-Antibody Interaction
Many factors affect Ag-Ab reactions. Some of the common factors are:
- Temperature: It depends on the chemical nature of epitopes, paratopes and, bonds involved. Eg. Hydrogens bonds are stable at low temperatures and hydrophobic bonds are stable at high temperatures.
- pH: Optimal pH range is 6.5 to 8.5. Extreme pH values change the conformation of antibodies and inhibit the reaction.
- Ionic strength: The effect of ionic is important in blood group serology. Here the reaction is significantly influenced by sodium and chloride ions. In normal saline solution, Na+ and Cl− cluster around the complex and partially neutralize charges, potentially interfering with antibody binding to antigen. This could be problematic when low-affinity antibodies are used.
- Enzyme Treatment: Many proteolytic enzymes are used to enhance the Ag-Ab reactions. Some of the commonly used ones are papain, ficin, bromelin.
- Concentrations of Ag and Ab: Increase in the concentration of antigen and antibody enhances the reaction.
- No. of antigen-binding sites: More the no. of antigen-binding sites on the antibody, the more is the chances of interaction. For eg., IgM is pentamer and hence has 10 binding sites whereas IgG is monomer hence has only 2 binding sites so IgM will bind more efficiently with antigens.
- Structural arrangement: If the structure of epitope and paratope is such that they could fit well as lock and key then it enhances the interaction between antigen and antibody.
Types of Antigen-Antibody Interaction
Ag-Ab reactions are basically of two types:
1. In Vivo (Occurring in natural condition): It includes the general antibody-mediated immune response occurring in our body against any antigen.
2. In Vitro (Done in artificial condition): It includes a series of serological tests performed in laboratories to detect antigens or antibodies in case of many diseases.
In Vivo Reactions
- Complement fixation
- Ab Dependent Cell-Mediated Toxicity
In vitro Reactions
- Complement fixation
- Radioimmunoassay (RIA)
- Western Blotting
- Immunochromatograhy (ICT)
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.
– In the presence of electrolytes
– At specific pH and Temperature
– Antibodies involved are called precipitins
– Types: In solution (Flocculation and Ring Test)
In Agar (Immunodiffusion)
In Agar with an electric field (Immunoelectrophoresis)
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)
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.
Note: In the case of sensitivity agglutination reaction is more sensitive than precipitation reaction.
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
Enzyme-Linked Immunosorbent Assay (ELISA)
It is one of the sensitive techniques for the detection of the presence of antigen or antibody and quantification as well in case of clinical diagnosis of many diseases such as AIDS, Ebola, Pernicious anemia, different parasitic infections, etc. Enzymes are used for labeling.
– Detects free antigens or antibodies.
– Reagents: Coating buffer, Blocking buffer, Wash Buffer, Substrates, Sample and, Assay Diluents.
– Types: Direct, Indirect and, Sandwich ELISA.
When Ag-Ab interactions result in the rupture or lysis of RBC, it is called hemolysis which results in the release of Haemoglobin. It can be catalyzed by enzymes called hemolysins. It is the demonstrable endpoint of some reactions.
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.
– Fluorescent dyes such are fluorescein isothiocyanate and lissamine rhodamine used.
– Types: Direct and Indirect.
In neutralization, the biological effects of viruses and toxins are neutralized by homologous antibodies called neutralizing antibodies.
Types:- Virus Neutralization Tests (Eg. Viral hemagglutination inhibition test)
– Toxin Neutralization Tests (Eg. Schick test, antistreptolysin O test, etc.)
It is a type of immunoassay in which radioisotopes are used for labeling the antigen or antibody to detect the formation of the Ag-Ab complex.
– Can determine very small quantities of Ag and Ab in the serum.
– Used for the quantification of hormones, drugs and, viral antigens.
The first step in the Ag-Ab interactions involves the formation of the Ag-Ab complex and it is called sensitization. It is not observable and is observed only after agglutination of the formed complex using different reagents. IgG antibodies can sensitize red cells with the corresponding antigens hence are called sensitizing antibodies.
It is called so because of its similarity to Southern Blotting.
– Protein separation is done by electrophoresis.
– Used in the detection of proteins.
– Confirmatory test in the diagnosis of HIV.
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 detection as well as detection of several diseases such as malaria, dengue, etc. are based on this principle. They require very little time for the tests.
- 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.
Limitations of Antigen-Antibody Interaction
- Require higher expertise and equipment which are not mostly available in some developing or underdeveloped countries. Hence these techniques are not used in those places mostly.
- RDTs do not reliably detect low-density parasitemia (≤200 parasites/µL).
- In different cases of antibody detections, its hard to differentiate between early and late infections as the antibodies remain in our blood for a long time even after the cure of infection.
References and Sources
- Goldsby R.A., Kindt T.J., Osborne B.A., (1999) Kuby Immunology, 4th edition, W.H.Freeman & Co Ltd., pg. 137- 160
- Parija S.C., (2009), Textbook of Microbiology and Immunology, 2nd edition, Elsevier, a division of Reed Elsevier India Private Limited, pg. 94-115
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