Hook effect

Hook effect also called high-dose hook effect or the prozone effect, describes the paradoxical loss of signal that can occur in sandwich immunoassays in the presence of high concentrations of analyte ¹. The hook effect or the prozone effect is an immunologic phenomenon whereby the effectiveness of antibodies to form immune complexes is sometimes impaired when concentrations of an antibody or an antigen are very high. The formation of immune complexes stops increasing with greater concentrations and then decreases with extremely high concentrations, producing a hook shape on a graph of measurements. There are versions of the hook effect with excess antibodies and versions with excess antigens. An important practical relevance of the phenomenon is as a type of interference that plagues certain immunoassays and nephelometric assays, resulting in false negatives or inaccurately low results. Other common forms of interference include antibody interference, cross-reactivity and signal interference. The phenomenon is caused by very high concentrations of a particular analyte or antibody and is most prevalent in one-step (sandwich) immunoassays. Because of this, erroneously low results can be reported by the clinical laboratory. The hook effect is most commonly encountered with analytes that span a wide range of concentrations, such as those secreted by tumors including serum free light chains and prolactin ². Hook effect has also been reported in a number of ferritin immunoassays ³. Ferritin is an acute phase reactant and patients can have concentrations orders of magnitude above those commonly seen. High ferritin concentrations are especially common in macrophage activation syndrome and adult onset Stills disease ⁴. While rare, patients with these conditions can be encountered in tertiary care facilities such as ours. In these cases, erroneously reporting low ferritin concentrations can lead to inappropriate patient care.

Since the early 20th century, immunologists have noted that more is not always better: Increasing the amount of antibody in an antibody-antigen reaction could reduce, instead of increase, the amount of precipitating antibody-antigen complex ⁵. Similarly, mice receiving larger doses of anti-pneumococcus horse serum were not more, but less protected against pneumococcus infection ⁶. There was clearly a range of antibody concentrations above the optimum at which no effects or negative effects were obtained. This region of antibody concentrations was named the prozone, and the related observation the “prozone effect” ⁶ or after the shape of the complex formation curve the “high-dose hook effect” ⁷.

Over the following decades, the high-dose hook effect became better understood beyond its first application in immunology, and as a more general property of systems involving multivalent proteins. In 1997, Bray and Lay showed using simulations of various types of protein complexes that the prozone effect is a general phenomenon in biochemical complex formation, and occurs whenever one protein acts as a “linker” or “bridge” between parts of a complex ⁸.. This was corroborated using a mathematical model of an antibody with two antigen-binding sites by Bobrovnik ⁹ and in a DNA-binding experiment by Ha et al. ¹⁰.

The hook effect thus results from partially bound forms of the “linker” proteins competing with each other for binding partners, and as a consequence, there is a regime of concentrations where adding more linker protein will decrease the amount of fully formed complexes, rather than increase it (see Figure 2).

Clinical laboratories have sought to detect hook effect proactively through a variety of means. Some have instituted a separate orderable for when there is clinical suspicion hook effect might occur ¹¹. Others have employed neural learning networks to evaluate reaction curves and detect hook effect ¹². With regards to lateral flow assays, recent efforts have been reported that investigate reaction kinetics to reduce hook effect ¹³. Finally, laboratories have performed automatic re-analysis of ferritin concentrations that fall within the hook range on dilution ³.

Figure 1. Hook effect

Figure 2. High dose hook effect immunoassay

Footnote: Illustration of the hook effect of excess antigen and blocking antibodies on immunoassays. Binding of ligands A, B to a bivalent linker protein L. (a) Low linker concentration: availability of L limits the formation of total complexes (LAB, in colour). (b) Linker concentration on the order of ligand concentration: Formation of fully formed complex (LAB) reaches its maximum. (c) Concentration of linker L much higher than that of A or B: partially bound forms prevail, and formation of fully formed complex (LAB) goes down in absolute terms.

[Source ¹⁴ ]

Hook effect mechanism

Version with 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 agglutinate with the antigen to form immune complexes, then the test is interpreted as positive. However, if too many antibodies are present that can bind to the antigen, 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 ¹⁵.

Version with excess antigens

The hook 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 ¹⁶. 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 ¹⁷.

Examples include high levels of syphilis antibodies in HIV patients or high levels of cryptococcal antigen leading to false negative tests in undiluted samples ¹⁸. This phenomenon is also seen in serological tests for Brucellosis. The serological test is mainly seen in the precipitation reaction. 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 ¹⁹.

References

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