Antigen-Antibody Reaction Based Tests: Introduction, Types with Descripti

Antigen-Antibody Reaction Based Tests: Introduction, Types with Description and Application

Introduction of Antigen-Antibody Reaction

Antigen-antibody reaction ( interaction between antigen and antibody) is reversible, occurs at the surface and there is no denaturation of antigen (Ag) and antibody(Ab) during the reaction. Richard J. Goldberg gave the first correct description of the antigen-antibody reaction at the University of Wisconsin in 1952 and it came to be known as “Goldberg’s theory” (of Ag-Ab reaction). This reaction is highly specific, and an antigen reacts only with antibodies produced by itself or with closely related antigens. The antibody recognizes molecular shapes (epitope, an antigenic determinant) on the antigen. The affinity of the antibody for the antigen is one of the most important features in determining antibody efficacy in vivo. The attachment between antigen and antibody includes various non-covalent interactions between epitope and variable region (VH/VL) domain of an antibody. Chemical bonds responsible for the antigen-antibody reaction are electrostatic bonds, hydrogen bonding. hydrophobic interactions and Van der Waals bonds. Factors affecting Ag-Ab reactions are temperature, pH, and Ionic strength. The strength of antigen-antibody interactions is determined by the following terms-

Affinity: It is the combined strength of total on-covalent interactions between the single antigen-binding site of antibody and single epitope is the affinity of the antibody for that epitope.

Low-affinity antibody: It binds antigen weakly and dissociates readily whereas high-affinity Ab binds antigen tightly and remains bound longer.

Avidity: It is the strength of multiple interactions between multivalent antibody and antigen and it is better to measure the binding capacity of antibody than affinity. High avidity can compensate for low affinity.

Cross-reactivity: Antibodies elicited by one antigen can cross-react with unrelated antigens if they share identical epitopes or have similar chemical properties.

Types of Antigen-Antibody Reaction

Precipitation
Flocculation
Agglutination
Complement Fixation Test
Neutralization Tests
Radio – Immunoassay (RIA)
Enzyme Immuno Assay (EIA)
Immunochromatography
Immunoelectrophoresis
Immuno Blotting technique
Immunofluorescence

Precipitation reaction

When soluble antigen combines with its antibody in the presence of electrolyte forms insoluble (visible) precipitates. This type of antigen-antibody reaction is more sensitive for antigen detection. e.g. Elek’s test (diphtheria toxin), Biken test (to detect enterotoxigenic E. coli)

The antigen can be detectable both qualitatively as well as quantitatively.

It is more useful for antigen detection to as least as 1µg.

Flocculation

It is a special type of precipitation where precipitates remain suspended instead of sedimentation. e.g. VDRL, Kahn test (a tube flocculation test for syphilis), RPR

Agglutination

Antigen-antibody reaction in which antibody combines with a particulate antigen such reaction called agglutination. It is more sensitive than precipitation for antibody detection. It is further subcategorized into the following types-

Slide agglutination
Tube agglutination
Indirect passive haemagglutination
Reverse passive haem-agglutination
Antiglobulin ( Coomb’s) test

Slide agglutination:-serotyping e.g. blood grouping ( ABO and Rh typing) and cross match ( major and minor)
Tube agglutination: Widal test, Weill- Felix reaction, Paul Bunnel test.
Indirect passive haemagglutination:

Where antibodies are coated on RBC
Turkey red cells are often used as carrier particles because they are nucleated and sediment rapidly.
Red cells coated with antigens are called sensitized cells e.g. TPHA

Reverse passive haem-agglutination:- The technique utilizes stabilized red cells coated with specific viral antibodies.
Antiglobulin ( Coomb’s) test: It is applicable for the detection anti-Rh Ab and incomplete antibody of brucellosis. It is of two types, direct Coomb test (DCT) and indirect Coomb test (ICT). DCT is applicable to assist diagnose the cause of RBC destruction (hemolytic anemia); investigating a blood transfusion reaction; to diagnose hemolytic disease of the newborn (HDN). ICT uses to determine whether there are antibodies to the Rh factor in the mother’s blood in a case of hemolytic disease of the newborn (HDN).

Complement fixation test

Complement fixation test brief form is CFT. Complement is a nonspecific protein found in our normal serum. This CFT was extensively used for the diagnosis of syphilis caused by Treponema pallidum, a serological test called Wassermann test or Wassermann reaction named after the introducer surname bacteriologist August Paul von Wassermann in 1909. The whole complement system is made up of nine components i.e. complement one ( C1)to C9 Complement proteins are heat-labile and are destroyed by heating at 56°C for 30 minutes. Complement binds to antigen and antibody ( Ag-Ab) complex. When the antigen is an RBC it causes lysis of red blood cells ( RBCs). It has the ability to screen against a large number of viral and bacterial infections at the same time.

Neutralization Tests

When antitoxin combines with its corresponding toxin, neutralization occurs. Few examples of these tests are the Schick test (diphtheria) and the Dick test ( Streptococcus pyogenes).

Radio – Immunoassay (RIA)

Major analytes up to picogram (10 –¹²g) quantities
The most common label used is radio Isotopes and measured by gamma-spectrometer.
Used for quantification of hormones, drugs, tumor marker, viral antigen, etc.

Enzyme Immuno Assay (EIA)

It measures enzymes labeled antigen, antibody and it may be homogenous or heterogeneous. Major types of heterogenous EIA is enzymes linked ImmunoSorbent Assay (ELISA)

ELISA ( enzymes linked ImmunoSorbent Assay)

This technique involves the use of an enzymes system and an immunosorbent ( an absorbing material specific for one component of reaction, antibody, or antigen). The absorbing material could be either agarose or a solid matrix ( microwell, membrane).

Enzyme used-

Horseradish peroxidase
alkaline phosphates

Substrate used-

O- Phenyl diamine dihydrochloride (OPDDH)- for peroxidase
P- nitrophenyl phosphate (PNP) -for alkaline phosphatase

Modification of ELISA

Indirect ELISA:- detect antibody → Antigen is coated on the plate surface.
Direct ELISA / Sandwich ELISA: detect antigen→ Antibody is coated on the plate surface.

Immunochromatography

Immunochromatography is an association between chromatography and immunoassay ( antigen-antibody reaction). This technology is widely applicable in laboratory medicines for the rapid detection of antigens (various microbes) and antibodies. Depending on the test, either antigen (for detection of antibody) or antibody (for detection of antigen) are immobilized on a nitrocellulose membrane in discreet spots in a cartridge device. Following antibody or antigen capture and a flow-through wash step, sequential addition of an enzyme-labeled conjugate and substrate results in the appearance of a colored reaction product in the form of bands or spots directly on the membrane. e.g. rapid/ quick/ spot tests of HIV, HCV antibody tests, HBsAg test

Immuno Blotting technique

Southern blotting – detect DNA
Northern blotting – detect RNA
Western blotting – defection protein ( confirmatory test of HIV)
Eastern blotting- to analyze protein post-translational modifications (PTMs)

Immunoelectrophoresis

Immunoelectrophoresis is a combination of immuno-diffusion and electrophoresis. It is a special type of precipitation that occurs in agar under an electric field. Initially, an antigen mixture is separated by electrophoresis and then tested by double immuno-diffusion.

Immunofluorescence

Immunofluorescence also called cell imaging technique is a combination of Immuno and fluorescence and this assay is used primarily on biological samples and is classically defined as a procedure to detect antigens in cells using antibodies. The specificity of the antibody to its antigen is the base for immunofluorescence.

Application of Antigen-Antibody Reaction

These Ag-Ab reaction-based tests are widely used in laboratory techniques for the serological tests of blood compatibility and various pathogenic infections.

Tests based on precipitation principle are Elek’s test (diphtheria toxin), Biken test (to detect enterotoxigenic E. coli)
Flocculation based tests are VDRL, Kahn test (a tube flocculation test for syphilis), RPR
Assay belonging to agglutination are blood grouping, X-match, Widal test, Weill- Felix reaction, Paul Bunnel test, TPHA, Coomb’s tests ( direct and indirect)
Complement fixation test (CFT) uses to screen a large number of viral and bacterial infections at the same time.
Neutralization tests are Schick (diphtheria) and Dick test ( Streptococcus pyogenes).
RIA uses for the quantification of hormones, drugs, tumor markers, and viral antigens.
ELISA is widely applicable to detect antigens and antibodies of various microorganisms.
Immunochromatographic test- This test is widely adopted as the quick or spot or screening test for laboratory diagnosis of various diseases etiological agents as well as the conditions and they are-

Detection of toxins
Pregnancy tests- detection of human chorionic gonadotropin (hCG)
Diagnosis of parasitic infections-Malaria rapid test, ImmunoCardSTAT for Cryptosporidium/Giardia
Diagnosis of bacterial infections-ImmunoCard Mycoplasma, which detects Mycoplasma pneumoniae specific IgM in serum samples, ImmunoCardSTAT SpSA for H. pylori antigens in stool, Vibrio cholerae O1 and O139 from stool specimens, Binax NOW Streptococcus pneumoniae antigen card – Streptococcus pneumoniae antigen detection in CSF or in urine
Diagnosis of viral Infections-Antigen detection e.g. Binax NOW RSV, Remel Xpect RSV, ImmunoCardSTAT! For Rotavirus, Influenza A/B (BinaxNOW Influenza A & B), Dengue NS1, SARS-CoV-2 antigen, HBsAg test. Antibodies detection e.g. detection of HIV-1 and HIV-2 antibodies (Tridot), Dengue IgM and IgG, SARS-CoV-2 IgM, and IgG

9.Immunoelectrophoresis is used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum, M-proteins in serum and urine.

10. Immuno Blotting technique uses to detect DNA, RNA, protein, and PTMs.

11. Immunofluorescence is a type of immunohistochemistry method which utilizes fluorophores to visualize various cellular antigens.

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