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