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

Abstract

The equilibrium binding constants and the binding site concentrations of an immobilised rabbit antiserum towards progesterone-11α-hemisuccinate-bovine serum albumin were measured for different enzyme tracers. The enzyme tracers obtained by linking progesterone to horseradish peroxidase (HRP) through different spacer arm structures were: progesterone-11α-hemisuccinate-HRP (the homologous), progesterone-11α-hemiglutarate-HRP, progesterone-11α-hemiadipate-HRP, progesterone-11α-hemisebacate-HRP, progesterone-11α-hemisuccinamido-γ-aminobutyrate-HRP and progesterone-11α-hemisuccinamido-β-alaninate-HRP. If compared with the homologous enzyme tracer (provided with a five atom spacer arm and with an affinity of 1×10 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 which simulates the link between the carboxylic group of the progesterone derivative and the proteic lysine in the immunogen was inserted into 9 and 10 atom spacer arms in the very same position of the immunogen a sharp affinity increase was observed (1.2×10 9 M −1 for progesterone-11α-hemisuccinamido-β-alanilate-HRP and 1.5×10 9 M −1 for progesterone-11α-hemisuccinamido-γ-aminobutyrate-HRP). These results showed that in order to reach and exceed the homologous tracer affinity, a four or five atom spacer arm elongation must be coupled to a suitable amido group insertion. An identical trend was observed for the antibody binding site concentrations indicating the presence of two main classes of binding sites in the antiserum used.