Defined analyte-enzyme conjugates as signal generators in immunoassays

S. H. Paek, Leonidas G Bachas, W. Schramm

Research output: Contribution to journalArticle

26 Citations (Scopus)

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 - 1993
Externally publishedYes

Fingerprint

Signal generators
Immunoassay
Progesterone
Enzymes
Horseradish Peroxidase
Immobilized Antibodies
Affinity chromatography
Ligands
Affinity Chromatography
Immunoglobulins
Monoclonal Antibodies
Molecules
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Defined analyte-enzyme conjugates as signal generators in immunoassays. / Paek, S. H.; Bachas, Leonidas G; Schramm, W.

In: Analytical Biochemistry, Vol. 210, No. 1, 1993, p. 145-154.

Research output: Contribution to journalArticle

@article{8df56e08986840558dbdc2cd49a663f8,
title = "Defined analyte-enzyme conjugates as signal generators in immunoassays",
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.",
author = "Paek, {S. H.} and Bachas, {Leonidas G} and W. Schramm",
year = "1993",
doi = "10.1006/abio.1993.1165",
language = "English (US)",
volume = "210",
pages = "145--154",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Defined analyte-enzyme conjugates as signal generators in immunoassays

AU - Paek, S. H.

AU - Bachas, Leonidas G

AU - Schramm, W.

PY - 1993

Y1 - 1993

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0027173198&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027173198&partnerID=8YFLogxK

U2 - 10.1006/abio.1993.1165

DO - 10.1006/abio.1993.1165

M3 - Article

C2 - 8489010

AN - SCOPUS:0027173198

VL - 210

SP - 145

EP - 154

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -