ANTIGEN-ANTIBODY REACTIONS
Introduction:
ANTIGEN
Antigen is defined as foreign substance capable of inducing a specific immune response, which is also called immunogen.
Antibody
Is a protein produced from plasma cell consisting of two iden-tical heavy chains & light chains that recognizes a particular epitope on an antigen and reacts with it in an observable manner
Antigens and antibodies combine with each other specifically in an observable manner. The reactions between antigens and antibodies are useful in many ways. 1) They form the basis of antibody mediated immunity against infectious diseases. 2)They cause tissue injury in some type of hypersensitivity reactions and autoimmune diseases. 3) In the laboratory, they help (a) in the diagnosis of infections, (b) in epidemio-logical surveys, (c) in the identification of infectious agents and nonin-fectious agents such as enzymes. In general these reactions can be used for the identification of either antigens or antibodies. Antigen-Antibody reactions in vitro are known as serological reactions.
The reaction between antigen and antibody occurs in three stages.
1.Primary stage
2.Secondary stage
3. Tertiary stage
Primary stage:
The primary stage is the initial reaction between the two, without any visible effects. This reaction is rapid, occurs even at low tempera-tures and obeys the general law of physical chemistry and thermody-namics. The reaction is reversible, the combination between antigen and antibody molecules being effected by the weaker intramolecular forces such as vander waals forces, ionic bonds and hydrogen bonding rather than by the firmer covalent bonding. The primary reaction can be detected by estimating free and bound antigen or antibody sepa-rately in the reaction mixture.
Secondary stage:
The secondary stage leads to demonstrable events such as pre-cipitation, agglutination, lysis of cells, killing of live antigens, neutral-ization reactions, complement fixation and enhancement of phagocy-tosis.
Thus the antigen causing Agglutination was called agglutinin that causing precipitation precipitin.Thus the antibody causing agglutination was called Agglutinogen, that causing precipitin precipitinogen.
Tertiary stage:
Some antigen-antibody reactions occurring in vivo initiate chain reactions that lead to neutralization or destruction of injurious antigens, or to tissue damage. These are the tertiary reactions and include hu-moral immunity against infectious disease as well as clinical allergy and other immunological diseases.
Antigen-Antibody reactions have the following general characteristics:
1. The reaction is specific. An antigen combines only with its homolo-gous antibody and vice versa. The specificity, however is not ab-solute and cross reaction may occur due to antigenic similarity and relatedness.
2. Entire molecules react and not fragments.
3. There is no denaturation of the antigen or the antibody during the reaction.
4. The combination occurs at the surface.
5. The combination is firm but reversible. The firmness of the union is influenced by the Affinity and the Avidity of the reaction. Affinity refers to the intensity if interaction between the Antigen and Anti-body. Avidity is the strength of the bond after the formation of the complexes.
6. Both antigens and antibodies participate in the formation of agglu-tinates or precipitates.
7. Antigens and Antibodies can combine in varying proportion
Many methods are available for the measurement of antigen and antibody participating in the reactions.
Measurement may be in terms of mass or more commonly as units or titer.
The antibody titer of a serum is the highest dilution of the serum which gives an observable reaction with antigen in the particular reac-tion
The titer of a serum is influenced by the nature and the type and conditions of the test
There are two important parameters of serological tests. They are sensitivity and specificity. Sensitivity refers to the ability of the test to detect even very minute quantity of the antigen or antibody.
Specificity refers to the ability of the test to detect reactions be-tween homologous antigens and antibodies only.
1. Precipitation
2. Agglutination
3. Neutralisation
When a particulate antigen is mixed with its antibody in the pres-ence of electrolytes at a suitable temperature and pH, the particles are clumped together or agglutinated.
Agglutination is more sensitive than precipitation for the detec-tion of antibodies. The same principles govern agglutination and pre-cipitation. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions. The zone phenomoenon may be seen when either an antibody or antigen is in excess.
Incomplete or monovalent antibodies do not cause agglutina-tion, though they combine with the antigen. They may act as ‘blocking antibodies’, inhibiting agglutination by the complete antibody added subsequently.
1. Slide Agglutination
2. Tube Agglutination
3. Passive Agglutination
4. Hemagglutination
When a drop of the appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in the drop of a saline on a slide or tile, agglutination takes place. A positive result is indicated by the clumping together of the particles and the clearing of the drop. Mix-ing the antigen and the antiserum with a loop or gently rocking the slide facilitates the reaction. Depending on the titer of the serum, agglutina-tion may occur instantly or within seconds. Clumping occurring after a minute may be due to drying of the fluid and should be disregarded.
It is essential to have on the same slide a control consisting of the antigen suspension in saline, without the antiserum, to ensure that the antigen is not agglutinable. Agglutination is usually visible to the naked eye but may sometimes require confirmation under the microscope. Slide agglutination is a routine procedure for the identification of many bacterial isolates from clinical specimens. It is also the method used for blood grouping and cross matching.
This is a standard quantitative method for the measurement of antibodies. When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilution of an antiserum in test tubes, the agglutination titer of the serum can be estimated. Tube agglutination is routinely employed for the serological diagnosis of typhoid, brucello-sis and rickettsial fever. Widal agglutination test is done for typhoid, Brucella agglutination test is done for Brucellosis and Weil Felix test is done for rickettsial infections
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