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Related Experiment Videos

Multiple epitope interactions in the two-step sandwich immunoassay.

S A Fernando1, G S Wilson

  • 1Department of Chemistry, University of Kansas, Lawrence 66045.

Journal of Immunological Methods
|July 6, 1992
PubMed
Summary

The hook effect in sandwich immunoassays occurs when labeled antibodies bind to multiple epitopes on an analyte. This study demonstrates how analyte characteristics influence this hook effect, particularly with repeating epitopes.

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Area of Science:

  • Biochemistry
  • Immunology
  • Analytical Chemistry

Background:

  • The hook effect is a phenomenon observed in sandwich immunoassays, leading to a false-negative result at high analyte concentrations.
  • Understanding the hook effect is crucial for accurate immunoassay development and interpretation.

Purpose of the Study:

  • To investigate the hook effect in two-step sandwich immunoassays.
  • To elucidate the role of analyte characteristics and epitope interactions in causing the hook effect.
  • To examine the influence of analyte concentration and liquid-phase antibody on the hook effect.

Main Methods:

  • Experimental and theoretical investigation of the hook effect in two-step sandwich immunoassays.
  • Utilized three different analytes: biosynthetic human growth hormone (hGH), dimeric hGH (D-hGH), and ferritin.

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  • Employed monoclonal antibodies targeting specific epitopes on the analytes.

    • Main Results:

    • The sandwich immunoassay for hGH, with distinct epitopes, showed no hook effect.
    • Dimeric hGH (D-hGH), possessing repeating epitopes, demonstrated a hook effect when labeled antibodies interacted with multiple epitopes.
    • Ferritin also exhibited multiple epitope interactions, contributing to the hook effect.

    Conclusions:

    • The hook effect in two-step sandwich immunoassays is caused by the desorption of bound analyte.
    • Analyte characteristics, specifically the presence of multiple or repeating epitopes, are key factors influencing the hook effect.
    • Conformational changes in the analyte after labeled antibody binding to several epitopes lead to desorption and the hook effect.