The influence of heterology, enzyme label and assay conditions on the sensitivity of microtitre plate enzymeimmunoassays for progesterone in milk.

Seven antisera raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (P11-HS) were used in microtitre plate enzymeimmunoassays (EIAs) for progesterone to identify improvements in sensitivity achievable by using various heterologous labels. EIAs using beta-galactosidase linked to P11-HS, 11 alpha-hydroxyprogesterone 11-hemimaleate (P11-HM), 11 alpha-hydroxyprogesterone 11-glucuronide (P11-Glu) or progesterone 3-(o-carboxymethyl) oxime (P3-CMO) were compared. Loss of sensitivity through bridge recognition was least evident using the P11-Glu derivative. The same seven antisera were used to evaluate assay sensitivity using beta-galactosidase, alkaline phosphatase, penicillinase and peroxidase linked to P11-HS or P11-Glu as label. Consistent improvements were achieved with the heterologous assays in the order penicillinase greater than alkaline phosphatase/peroxidase greater than beta-galactosidase: with penicillinase, sensitivity generally exceeded that of RIA. These data provide evidence for the general efficacy of the combination 11 alpha-hemisuccinate (immunogen bridge) and 11 alpha-glucuronide (label bridge) in reducing bridge recognition. EIA performed at 4 degrees C provided greater sensitivity than at ambient temperature (21 degrees C) or 40 degrees C, however, ambient temperature incubation provided a practical compromise. Equilibrium was not achieved under any of the conditions investigated.