Effect of homologous and heterologous spacer arms of progesterone - horse radish peroxidase conjugates on the equilibrium constants for an immobilised anti-progesterone antiserum

Citation
G. Giraudi et al., Effect of homologous and heterologous spacer arms of progesterone - horse radish peroxidase conjugates on the equilibrium constants for an immobilised anti-progesterone antiserum, ANALYT CHIM, 417(1), 2000, pp. 95-100
Citations number
6
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
ANALYTICA CHIMICA ACTA
ISSN journal
00032670 → ACNP
Volume
417
Issue
1
Year of publication
2000
Pages
95 - 100
Database
ISI
SICI code
0003-2670(20000717)417:1<95:EOHAHS>2.0.ZU;2-4
Abstract
The equilibrium binding constants and the binding site concentrations of an immobilised rabbit antiserum towards progesterone-11 alpha-hemisuccinate-b ovine serum albumin were measured for different enzyme tracers. The enzyme tracers obtained by linking progesterone to horseradish peroxidase (HRP) th rough different spacer arm structures were: progesterone-11 alpha-hemisucci nate-HRP (the homologous), progesterone-11 alpha-hemiglutarate-HRP, progest erone-11 alpha-hemiadipate-HRP, progesterone-11 alpha-hemisebacate-HRP, pro gesterone-11 alpha-hemisuccinamido-gamma-aminobutyrate-HRP and progesterone -11 alpha-hemisuccinamido-beta-alaninate-HRP. If compared with the homologo us enzyme tracer (provided with a five atom spacer arm and with an affinity of 1x10(9) M-1) the affinity of enzyme tracers with spacer arms of at most six atoms showed a 10-fold decrease whereas about a two-fold decrease was observed with spacer arms of seven and nine atoms. When an amido group whic h simulates the link between the carboxylic group of the progesterone deriv ative and the proteic lysine in the immunogen was inserted into 9 and 10 at om spacer arms in the very same position of the immunogen a sharp affinity increase was observed (1.2x10(9) M-1 for progesterone-11 alpha-hemisuccinam ido-beta-alanilate-HRP and 1.5x10(9) M-1 for progesterone-11 alpha-hemisucc inamido-gamma-aminobutyrate-HRP) These results showed that in order to reac h and exceed the homologous tracer affinity, a four or five atom spacer arm elongation must be coupled to a suitable amido group insertion. An identic al trend was observed for the antibody binding site concentrations indicati ng the presence of two main classes of binding sites in the antiserum used. (C) 2000 Elsevier Science B.V. All rights reserved.