Agglutination Tests: Types, Principle, Uses

When the specific antibodies (agglutinins) bind to surface antigens of bacteria, virus, or any antigens immobilized in particulate matter (such as latex particle) and cause the formation of visible clumps, such test is called agglutination test.

Agglutination test can be performed in:

• Surface of glass slides: Rapid reading is required because evaporation affects
• Test tubes: More sensitive because of longer incubation

Reagents available commercially.

Agglutination test can be performed for the:

• Identification of an organism with known antibodies
• Identification of serum antibodies with known antigens.

Bacterial agglutination test

1. Measure the antibody produced by the host against bacterial agglutinins
2. Best performance when used in sterile physiologic saline

Uses: Disease diagnosis, most appropriate test when bacterial agent is difficult to cultivate in vitro. 

Some examples of such diseases are:

1. Tetanus: Clostridium tetani
2. Yersiniosis: Yersinia pestis
3. Leptospirosis: Leptospira spp.
4. Brucellosis: Brucella spp
5. Tularemia: Francisella tularensis

Slide Agglutination test

• Use of Antisera (Ab) to identify Salmonella and Shigella by causing agglutination of the organisms
• Diagnostic purpose (hospital laboratories): Antisera directed against the cell wall O antigens of Salmonella and Shigella
• Epidemiologic purpose (public health laboratories): Antisera against flagellar H antigen and capsular Vi antigen of salmonella.

Particle agglutination test

1. Agglutination of an artificial carrier particle with a specific antigen bound to its surface
   1. Size of the carrier enhances the visibility of agglutination.
2. Examples include
   1. Latex particles: Latex agglutination test
   2. Treated Red blood cells i.e. hemagglutination
   3. Whole bacterial cells.
3. Reaction is dependent on
   1. Amount and avidity of antigen bound to carrier
   2. Time of the incubation with specimen
   3. Environment of interaction (pH, protein concentration etc)

Hemagglutination

1. Treated animal RBC is used as a carrier of antigen
2. Passive hemagglutination: Antigens that are being bound by antibodies are not the antigen of RBC but are passively bound antigens.
3. Examples
   1. Microhemagglutination test for Syphilis (MHA-TP)
   2. Hemagglutination treponemal test for Syphilis (HATTS)
   3. Passive hemagglutination tests for antibody to extracellular antigen of Streptococci
   4. Rubella indirect hemagglutination test
   5. Hemagglutination Inhibition Test (HAI) for Avian Influenza
   6. Quantitative Micro Hemagglutination Test (HA)

Latex agglutination test for Ag detection

1. Latex beads coated with a specific antibody are agglutinated in the presence of homologous antigens (bacteria).
2. Used to determine the presence of capsular antigen of
   1. H. influenzae
   2. N. meningitidis: rapid detection of meningococcal capsular polysaccharides in CSF.
   3. Streptococcus spp.
   4. C. neoformans

Latex agglutination test for Ab detection

1. Latex particles coated  with specific antigen
2. Commercially available for the accurate and sensitive detection of antibodies to
   1. Cytomegalovirus
   2. Rubella virus
   3. Varicella-zoster virus
   4. Heterophile antibody of infectious mononucleosis
   5. Teichoic acid antibodies of Staphylococci
   6. Antistreptcoccal antibodies
   7. Mycoplasma antibodies, etc.

Coagglutination (COAG)

Specific antibody is bound to the surface protein A of staphylococci (Cowan type 1 strain of Staphylococcus aureus). Soluble microbial antigen in the specimen is mixed with the COAG reagent, resulting in the agglutination of the staphylococcal cells.

Antibody COAG Reagent + Antigen in specimen = Staphylococcal cells (Agglutinated)

References

1. Levinson. (2010). Review of Medical Microbiology and Immunology. McGraw-Hill.
2. Attar, Z. J., Chance, M. L., el-Safi, S., Carney, J., Azazy, A., El-Hadi, M., Dourado, C., & Hommel, M. (2001). Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta tropica, 78(1), 11–16. https://doi.org/10.1016/s0001-706x(00)00155-8 
3. Johnson, J., Duffy, K., New, L., Holliman, R. E., Chessum, B. S., & Fleck, D. G. (1989). Direct agglutination test and other assays for measuring antibodies to Toxoplasma gondii. Journal of clinical pathology, 42(5), 536–541. https://doi.org/10.1136/jcp.42.5.536
4. Waltman, D. (2017). Developments in detection strategies for salmonella enteritidis in layer hen flocks. Producing Safe Eggs, 161–185. https://doi.org/10.1016/b978-0-12-802582-6.00009-4