Hook effect

Mechanism and in vitro importance

Prozone – excess antibodies

In an agglutination test, a person's serum (which contains antibodies) is added to a test tube, which contains a particular antigen. If the antibodies interact with the antigen to form immune complexes, called agglutination, then the test is interpreted as positive. However, if too many antibodies that can bind to the antigen are present, then the antigenic sites are coated by antibodies, and few or no antibodies directed toward the pathogen are able to bind more than one antigenic particle. Since the antibodies do not bridge between antigens, no agglutination occurs. Because no agglutination occurs, the test is interpreted as negative. In this case, the result is a false negative. The range of relatively high antibody concentrations within which no reaction occurs is called the prozone.

Postzone – excess antigens

The effect can also occur because of antigen excess, when both the capture and detection antibodies become saturated by the high analyte concentration. In this case, no sandwich can be formed by the capturing antibody, the antigen and the detection antibody. In this case, free antigen is in competition with captured antigen for detection antibody binding. Sequential addition of antigen and antibody, paired with stringent washing, can prevent the effect, as can increasing the relative concentration of antibody to antigen, thereby mediating the effect.

Examples include high levels of syphilis antibodies in HIV patients or high levels of cryptococcal antigen leading to false negative tests in undiluted samples. This phenomenon is also seen in serological tests for Brucellosis. It may be seen in precipitation reactions. The antibody that fails to react is known as the blocking antibody and prevents the precipitating antibody from binding to the antigens. Thus the proper precipitation reaction does not take place. However, when the serum is diluted, the blocking antibody is as well and its concentration decreases enough for the proper precipitation reaction to occur.

In vivo observations

Lewis Thomas described in his memoir a physiologic experiment of 1941 in which he observed the prozone effect in vivo: immunity in rabbits to meningococcus, which was robust, unexpectedly decreased when immunization was used to induce a heightened antibody response. In other words, getting the rabbits' bodies to produce more antibodies against this bacterium had the counterproductive effect of decreasing their immunity to it. From the viewpoint of an overly simplistic notion of the antibody/antigen relationship, this seems paradoxical, although it is clearly logical from a viewpoint duly informed by present-day molecular biology. Thomas was interested in pursuing this physiologic research further, and remained so for decades afterward, but his career took him in other directions and he was not aware of anyone having pursued it by the time of his memoir. One kind of relevance that he hypothesized for this in vivo blocking antibody concept was as a driver of human susceptibility to certain infectious diseases. In the decades since, the concept has also been found to have clinical relevance in allergen immunotherapy, where blocking antibodies can interfere with other antibodies involved in hypersensitivity and thus improve allergy treatment.

References

References

1. (2012-03-23). "Advances in Immunoassay Technology". InTech.
2. (1989-03-01). "Reply". Gut.
3. (2015-02-01). "Antigen excess in modern immunoassays: To anticipate on the unexpected". Autoimmunity Reviews.
4. Miller, James. (April 2004). "Interference in immunoassays: avoiding erroneous results". CLI.
5. (2009-11-30). "Assessment of the prozone effect in malaria rapid diagnostic tests". Malaria Journal.
6. (2004-05-01). "Interferences in Immunoassay". The Clinical Biochemist Reviews.
7. (November 1993). "Prozone phenomenon in secondary syphilis. Has its time arrived?". Arch. Intern. Med..
8. (September 1980). "False-negative cryptococcal antigen test". JAMA.
9. (2001-02-01). "Utility of commonly used commercial human chorionic gonadotropin immunoassays in the diagnosis and management of trophoblastic diseases". Clinical Chemistry.
10. Thomas, Lewis. (1983). "The Youngest Science: Notes of a Medicine-Watcher".
11. (2006). "IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking". J Clin Invest.