CHARACTERIZATION OF A RAPID AND SENSITIVE ENZYME-IMMUNOASSAY (EIA) FOR PROGESTERONE APPLIED TO CONDITIONED CELL-CULTURE MEDIA

Progesterone accumulation in conditioned media is a frequently employe d endpoint for in vitro cell culture of steroidogenic cells. Although radioimmunoassay (RIA) has been the predominant method for measurement of progesterone, a number of nonradiometric immunoassays have been de scribed but they have not been applied to conditioned media. Here, we report the characterization of a microtitre plate enzyme immunoassay ( EIA) for determination of progesterone in conditioned media. The EIA h as a sensitivity of 0.3 pg per well with intraassay and interassay coe fficients of variation of 7.3 and 10.2%, respectively. The specificity of the EIA is no different than that of a comparable RIA showing cros sreactivities of less than 0.1% for other steroids except 5alpha-pregn an-3,20-dione (47%) and 11alpha-hydroxyprogesterone (18%). Progesteron e levels from conditioned cell culture media of either rat or human gr anulosa cell cultures measured by both EIA and RIA were in close agree ment (r = 0.96) and serial dilutions of culture samples in the EIA wer e parallel to those of the standards. Also, extraction of culture medi a prior to EIA was found not to be necessary. Thus, this EIA is a high ly sensitive and specific assay that provides a rapid, simple, inexpen sive, and non-radiometric alternative to radioimmunoassay for measurem ent of progesterone in conditioned cell culture media.