Agglutination is aggregation of particulate matter (antigen) which occurs when it combines with specific antibody. It was first observed in 1896 by Gruber and Durham when serum antibody was found to react with bacterial cells.
It is an antigen–antibody reaction in which a particulate antigen combines with its antibody in presence of electrolytes at a specified temperature and pH resulting in formation of visible clumps.
When particulate antigens reacts with specific antibody, antigen antibody complex forms visible clumping under optimum PH and temperature. Such reaction is called agglutination and is visible expression of aggregation of antigens and antibodies. Antibodies that produce such reactions are called agglutinins and particulate antigens that are aggregated are termed agglutinogens.
Agglutination differs from precipitation reaction in that since agglutination reaction takes place at surface of particle involved, antigen must be exposed and be able to bind with the antibody to produce visible clumps.
This reaction occurs optimally when antigens and antibodies react in equivalent proportions. It is analogous to precipitation reaction in that antibodies act as a bridge to form a lattice network of cells that carry antigen on their surface and antibodies. As cells are larger than a soluble antigen, the result is more visible when cells aggregate into clumps.
Agglutination reactions apply to particulate test antigens that have been conjugated to a carrier. The carrier could be artificial (such as latex or charcoal particles) or biological (such as red blood cells). These conjugated particles are reacted with patient serum presumably containing antibodies. The endpoint of test is observation of clumps resulting from that antigen-antibody complex formation.
The quality of result is determined by the time of incubation with antibody source, amount and avidity of antigen conjugated to the carrier, and conditions of test environment (e.g., pH and protein concentration).
Various methods of agglutination are used in diagnostic immunology and these include latex agglutination, flocculation tests, direct bacterial agglutination, and hemagglutination.
In this reactions, serial dilutions of antibody solution are made and a constant amount of particulate antigen is added to serially diluted antibody solutions. After several hours of incubation at 37°C, clumping is recorded by visual inspection. The titer of antiserum is recorded as reciprocal of highest dilution that causes clumping. Since cells have many antigenic determinants on their surface, the phenomenon of antibody excess is rarely encountered.
The condition of excess antibody, however, is called prozone phenomenon. At high concentration of antibody, number of epitopes are outnumbered by antigen binding sites. This result in univalent binding of antigen by antibody rather than multivalent and thus, interfere in crosslinking of antigen (Lattice formation).
Occasionally, antibodies are formed that react with antigenic determinants of a cell but does not cause any agglutination. They inhibit agglutination by complete antibodies added subsequently. Such antibodies are called blocking antibodies. Anti-Rh and Anti-Brucella antibodies are few examples of such blocking antibodies.
Agglutination tests are easy to perform and in some cases most sensitive tests currently available. These tests have a wide range of applications in the clinical diagnosis of non- infectious immune disorders and infectious disease. Agglutination reactions have wide variety of applications in detection of both antigens and antibodies in serum and other body fluids.
Agglutination reactions can be broadly divided into two groups:
Applications of Agglutination Reactions:
- Serological diagnosis of various diseases. E.g. Antistreptolysin O (ASO) test for rheumatic fever, Rapid plasma regain (RPR) test for Syphilis.
- Identification of Bacteria. E.g. Serotyping of Vibrio cholera, Serotyping of Salmonella Typhi and Paratyphi.
- Cross matching and Blood grouping.
- Detection of unknown antigen in clinical specimens. E.g. detection of Vi antigen of Salmonella Typhi in urine.
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