Enzyme-Linked Immunosorbent Assay of Progesterone in Serum Using Penicillinase as Label

An enzyme-linked immunosorbent assay (ELISA) for progesterone measurement in serum or plasma samples using penicillinase as label enzyme is reported. A C3 and C11 derivatives of progesterone were prepared and conjugated to bovin serum albumin (BSA). Polyclonal antibody against these two immunogens were prepared in New Zealand white rabbits. Purified Ig fractions of antibodies were immobilized onto the wells of microtiter plates. Progesterone 3-(O-carboxymethyl ) oxime was linked to penicillinase and used as tracer (enzyme-conjugate). The standard assay completed within three hours had a low limit of detection, from 5 pg/well (50 pg/ml) covering up to 1 ng/well (10 ng/ml). For the first time in this assay the color development in case of penicillinase as enzyme label was measured in the wells directly. Recoveries were measured to be within the range of 95-98%. Percent Coefficient of variations (CV%) obtained between and within runs of several assays were 3% and 7% respectively. When compared between values of progesterone measured by radioimmunoassay (RIA) and present ELISA method, a correlation value of r = 0.9 was obtained. Freeze-dried progesterone-enzyme conjugate was found stable for at least three months at 4° C without using any other preservative.