Reactivity of an immobilized anti-progesterone antiserum with homologous and heterologous progesterone–horseradish peroxidase conjugates

Abstract

The binding and selectivity features of an immobilised anti-progesterone antiserum were studied by the use of four different enzyme tracers: progesterone 11α-hemisuccinate–horseradish peroxidase (P-11α-HS–HRP), progesterone 11α-carboxymethyl ether–horseradish peroxidase (P-11α-CME–HRP), progesterone 11β-carboxymethyl ether–horseradish peroxidase (P-11β-CME–HRP) and progesterone 3-(O-carboxymethyl)oxime–horseradish peroxidase (P-3-CMO–HRP). The antiserum–tracer affinities generally showed a remarkable reduction in respect to the affinity of the analyte because of the steric hindrance of the enzyme and, among the four tracers, the higher affinity value was evaluated for the P-11α-HS–HRP (homologous to the immunogen molecule). The concentration of antibody binding sites interacting with the tracers showed the presence of different classes of antibodies able to react with variable affinity with tracers and analyte, as confirmed by the cross-reactivity values measured towards different progesterone derivatives. The assays performed with the tracers showed that an increase of sensitivity can be obtained using enzyme tracers provided with heterologous structure features with respect to the immunogen molecule.