ANTIGEN-ANTIBODY REACTIONS

Antigens and antibodies combine with each other specifically and in an observable manner. In the body, they form the basis of antibody mediated immunity in infectious diseases. In laboratory, they help in diagnosis of infections, in epidemiological surveys, in the identification of infectious agents.

Antigen – antibody reactions in vitro are known as serological reactions. Stages of Ag – Ab reactions Primary stage: Initial interaction between Ag & Ab – invisible Rapid, occurs at low temperatures. Reaction is reversible. Ag & Ab is bound to each other by weak Van der Waal’s forces, Ionic bonds & Hydrogen bonding.

Secondary stage: It is an irreversible interaction with visible effects. Demonstrable events – Precipitation Agglutination Lysis of cells Killing of live antigens Neutralization of toxins Complement fixation Immobilization of motile organisms Enhancement of phagocytosis.

GENERAL FEATURES OF Ag – Ab REACTIONS The reaction is specific. Entire molecules react and not the fragments There is no denaturation of the antigen or antibody during the reaction. The combination occurs at the surface. The combination is firm but reversible. The firmness is influenced by the affinity & avidity of the reaction.

Affinity – refers to the intensity of attraction between the antigen & antibody molecules. It is the function of closeness of fit between the epitope & antigen binding region of its Ab. Avidity – strength of the bond after the formation Ag-Ab complexes. It is a better indicator of the strength of interactions in real biological systems. 6. Both Ags & Abs participate in the formation of agglutinates or precipitates. 7. Antigens & antibodies can combine in varying proportions. Both Ags & Abs are multivalent.

Types of Antigen – Antibody Reactions Precipitation reaction Agglutination reaction Neutralization reaction Complement fixation test Immobilization test Opsonisation Immunofluorescence Radioimmuno assay Enzyme immunoassay

PRECIPITATION REACTION PRINCIPLE: When a soluble Ag combines with its Ab in the presence of electrolytes (NaCl) at a suitable temperature & pH, the Ag-Ab complex forms an insoluble precipitate. Precipitation can take place in liquid media or in gels such as agar, agarose or polyacrylamide.

ZONE PHENOMENON The amount of precipitate formed is greatly influenced by the relative proportions of Ags & Abs. If increasing quantities of Ags are added to the same amount of antiserum in different tubes, precipitation is found to occur most rapidly & abundantly in the middle tubes. Preceding tubes – Ab excess (Prozone) Middle tubes – Ag & Ab in equivalent proportions (Zone of equivalence) Later tubes – Ag excess (Post zone)

Mechanism of precipitation Marrack (1934) proposed the lattice hypothesis – mechanism of precipitation The multivalent antigens combine with bivalent Abs in varying proportions, depending on the Ag – Ab ratio on the reacting mixture. Precipitation results when a large lattice is formed consisting of alternating Ag & Ab.

Marrack’s hypothesis Marrack’s hypothesis

Applications of Precipitation reaction It can be carried out as either a quantitative or qualitative test. Sensitive for the detection of Ags. Types of precipitation reactions: Ring test Slide test Tube test Immunodiffusion Electroimmunodiffusion

IMMUNODIFFUSION (precipitation in gel) Advantages of immunodiffusion: Reaction is visible as a distinct band of precipitation. Stable, can be stained for preservation. Indicates identity, cross reactions, non identity between different Ags.

Single diffusion in one dimension (Oudin procedure) Ab is incorporated in agar gel in a test tube & Ag solution is layered over it. Ag diffuses downward through the agar gel – forming a line of precipitation.

Single diffusion in two dimension (Radial Immunodiffusion) In this method antiserum solution containing antibody is incorporated in a agar gel on a slide. Wells are cut and antigen is applied in the gel. Then Ab present in the gel reacts with the Ag which diffuses out of the well. Precipitation rings are formed around the wells. The diameter of the ring is directly proportional to the concentration of antigen.

Double diffusion in one dimension (Oakley-Fulthorpe procedure) Ab is incorporated in agar gel Above which is placed a column of plain agar. The Ag is layered over it. The Ag & Ab move towards each other through the intervening column of plain agar & form the precipitate.

4. Double diffusion in two dimensions (Ouchterlony procedure) Helps to compare different antisera & antigens directly. Agar gel is poured on a slide & wells are cut . Antiserum – central well Different Ags in the surrounding wells. 20

Reaction of identity Lack of relatedness Partial identity Fig 2 – lack of relatedness Partial identity 21

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