Agglutination

 “Agglutinin” is an obsolete term for an antibody capable of causing cells to come together in macroscopic clumps. Agglutination (clumping) of bacteria by serum was perhaps the earliest observed manifestation of in vitro immune reactions. The phenomenon is due to crosslinking of cells through the reaction of multivalent antibody molecules with cell-surface antigens. The quantity of antibody necessary for crosslinking is generally very small, yet the cell masses involved in agglutination generate a response visible to the naked eye. Because of these characteristics, this oldest of techniques remains one of the most sensitive and widely used types of immunoassay.

Agglutination immunoassays can employ cells or synthetic particles. The two broad categories of cell-based agglutination assay are termed “direct” and “passive”. In the direct assay, antibodies cause clumping of microbes or blood cells by reaction with surface antigens endogenous to those cells. Typically, the immune status of serum in a test sample will be assessed, based on the highest dilution that will still agglutinate target cells. Direct agglutination assays are widely used to diagnose infectious disease by the presence of specific antibodies in acute or convalescent serum. The “monospot” assay for infectious mononucleosis is an example of this application (1). In passive agglutination immunoassays, the target antigen is exogenous, and is coated covalently or by adsorbtion onto the cells used for the assay. Test samples are then assayed as for the direct agglutination method. Many variants of direct and passive agglutination are in use for research or diagnostic procedures. For example, the test for Rh blood type is a two-step method in which antibodies in the test sample react with endogenous Rh antigen on erythrocytes, then addition of anti-human IgG induces agglutination (2).

Availability of uniformly sized latex particles allowed development of a chemically defined substrate on which to attach antigen or antibody (3). The role of the particles was precisely analogous to that of cells, in that their aggregation in response to antibody–antigen crosslinking generated an optically detectable signal. The advantage of inert particles was in their stability, compared to cells, and in interassay reproducibility. Measurement of agglutination was at first by turbidometry. Development of laser light-scattering techniques have greatly improved the sensitivity of detection (4) and have led to development of automated instruments for quantitation of agglutination reactions.

References

1. C. L. Lee, I. Davidsohn, and R. Slaby (1968) Am. J. Clin. Pathol. 49, 3–11. 

2. R. R. A. Coombs, A. E. Mourant, and R. R. Race (1945) Br. J. Exp. Pathol. 26, 255–266. 

3. J. M. Singer and C. M. Plotz (1956) Am. J. Med. 21, 888–892. 

4. V. Schulthess, R. J. Cohen, N. Sakato, and G. B. Benedek (1974) Immunochemistry 13, 955–962.

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