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 language | English (US) |
|---|---|
| Pages (from-to) | 145-154 |
| Number of pages | 10 |
| Journal | Analytical Biochemistry |
| Volume | 210 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1993 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
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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 journal › Article
}
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 -