Defined Analyte-Enzyme Conjugates as Signal Generators in Immunoassays
Paek, Se-Hwan; Bachas, Leonidas G.; Schramm, Willfried
Paek S. H., , Bachas L. G., , Schramm W., (1993/04)."Defined Analyte-Enzyme Conjugates as Signal Generators in Immunoassays." Analytical Biochemistry 210(1): 145-154. <http://hdl.handle.net/2027.42/30861>
AbstractWe investigated the synthesis of progesterone-horseradish peroxidase (P-HRP) conjugates and products purified by affinity chromatography. The obtained preparations 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.
MetadataShow full item record
Accessibility: If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.