A SENSITIVE, NONISOTOPIC IMMUNOASSAY FOR PROGESTERONE USING THE AVIDIN-BIOTIN SYSTEM

A non-isotopic immunoassay for progesterone has been developed using s treptavidin-biotin system and time-resolved fluorometry. A biotinylate d progesterone derivative and a progesterone standard or sample was al lowed to react competitively with rabbit anti-progesterone in microtit er wells coated with anti-rabbit gamma-globulin (second antibody). A f luorescent product was then generated on the solid-phase by adding str eptavidin labeled with europium (Eu, a lantanide element). The fluores cent complex, (second antibody)-(anti-progesterone)-(biotinylated prog esterone derivative)-(Eu-labeled streptavidin) was quantified using a time-resolved fluorometer. This assay for progesterone was highly sens itive, precise and accurate. The lower detection limit was 5 pg/ml. Th e intra-assay coefficients of variation (CV) were 3.70-5.20% and the i nter-assay CV were 7.31-8.23%. The correlation with radioimmunoassay w as good (r = 0.965), This assay was used to measure the amount of prog esterone that accumulated in the culture medium of rat granulosa cells . The accumulation increased with the amount of insulin-like growth fa ctor-I added to the culture in the presence of follicle-stimulating ho rmone. The present assay with streptavidin-biotin system can be applie d to various tracers such as enzymes and fluorescent materials. This a pproach should also be widely applicable to antigens and haptens that can be labeled with biotin.