Defined analyte-enzyme conjugates as signal generators in immunoassays

Se Hwan Paek, Leonidas G. Bachas, Willfried Schramm

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

We investigated the synthesis of progesterone-horseradish peroxidase (P-HRP) conjugates and products purified by affinity chromatography. The obtained preparatious were characterized with an immobilized monoclonal antibody in solid-phase immunoassays. Three homogeneous P-HRP conjugates were isolated. Two preparations were identified to contain a single progesterone ligand on the enzyme molecule. A third preparation contained two progesterone ligands. We postulate that conjugation can occur at two different positions on the enzyme, and that the different microenvironment of the protein structure surrounding the ligand contributes to different binding constants of the conjugates with immunoglobulin. By comparing the effective binding constants derived from affinity chromatography and from Scatchard analysis, we have demonstrated that the divalent conjugate binds to antibody immobilized on planar surfaces only by a single attachment due to steric restriction. Dose-response curves for progesterone using the isolated P-HRP conjugates have been investigated and compared.

Original languageEnglish (US)
Pages (from-to)145-154
Number of pages10
JournalAnalytical Biochemistry
Volume210
Issue number1
DOIs
StatePublished - Jan 1 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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