Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Leonidas G. Bachas, Mark E. Meyerhoff

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.

Original languageEnglish (US)
Pages (from-to)223-238
Number of pages16
JournalAnalytical Biochemistry
Volume156
Issue number1
DOIs
StatePublished - Jul 1986
Externally publishedYes

Keywords

  • binding assays
  • bridging group recognition
  • enzyme conjugate substitution
  • enzyme labels
  • immunochemical methods

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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