Agglutination – definition, reactions and applications

Agglutination definition

Agglutination is an antigen-antibody reaction in which a particulate antigen combines with its antibody in the presence of electrolytes at a specified temperature and pH resulting in the formation of visible clumping of particles. It occurs optimally when antigens and antibodies react in equivalent proportions. This reaction is analogous to the precipitation reaction in that antibodies act as a bridge to form a lattice network of antibodies and the cells that carry the antigen on their surface. Because cells are so much larger than a soluble antigen, the result is more visible when the cells aggregate into clumps.

When particulate antigens react with specific antibody, antigen-antibody complex forms visible clumping under optimum PH and temperature. Such a reaction is called agglutination. Antibodies that produce such reactions are called agglutinins.

What is agglutination?

Agglutination is the visible expression of the aggregation of antigens and antibodies. 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 the test is the observation of clumps resulting from that antigen-antibody complex formation. The quality of the result is determined by the time of incubation with the antibody source, amount and avidity of the antigen conjugated to the carrier, and conditions of the 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.

Agglutination differs from precipitation reaction in that since agglutination reaction takes place at the surface of the particle involved, the antigen must be exposed and be able to bind with the antibody to produce visible clumps. In agglutination reactions, serial dilutions of the 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 the antiserum is recorded as the reciprocal of the highest dilution that causes clumping. Since the cells have many antigenic determinants on their surface, the phenomenon of antibody excess is rarely encountered.

Prozone phenomenon

The condition of excess antibody, however, is called a prozone phenomenon. At a high concentration of antibody, the number of epitopes are outnumbered by antigen-binding sites. This results in the univalent binding of antigen by antibody rather than multivalently and thus, interferes in the crosslinking of antigen (Lattice formation).

Occasionally, antibodies are formed that react with the antigenic determinants of a cell but does not cause any agglutination. They inhibit the agglutination by the complete antibodies added subsequently. Such antibodies are called blocking antibodies. Anti-Rh antibodies and anti-brucella antibodies are few examples of such blocking antibodies.

Agglutination tests are easy to perform and in some cases are the most sensitive tests currently available. These tests have a wide range of applications in the clinical diagnosis of non- infectious immune disorders and infectious diseases. Agglutination reactions have a wide variety of applications in the detection of both antigens and antibodies in serum and other body fluids. They are very sensitive and the result of the test can be read visually with ease.

Applications of Agglutination Reactions

Cross-matching and grouping of blood.
Identification of Bacteria. E.g. Serotyping of Vibrio cholera, Serotyping of Salmonella Typhi and Paratyphi.
Serological diagnosis of various diseases. E.g Rapid plasma regains (RPR) test for Syphilis, Antistreptolysin O (ASO) test for rheumatic fever.
Detection of unknown antigen in various clinical specimens. E.g. detection of Vi antigen of Salmonella Typhi in the urine.