Radioimmunoassay of Estradiol-17β Without Chromatography

Several radioimmunoassays for the estimation of estradiol-17β in serum have been reported. For the generation of antisera, the majority of these methods have used steroids conjugated to a carrier protein through sites on the A or D rings. The resulting antisera have shown considerable cross reactivity with related steroids necessitating the use of various chromatographic separation steps prior to the actual radioimmunoassay. The use of haptens in which the carrier protein was conjugated to a steroid molecule through carbon 6 on the B ring (1,2,3) has resulted in the production of highly specific antisera. Conjugation of the protein molecule to other positions which leave the A and D rings available for recognition by antibody should also result in the generation of antibodies with enhanced specificity. In this report, an antiserum was prepared to a conjugate of an 11β-hemisuccinate derivative of estradiol-17β to bovine serum albumin. The resulting antiserum appeared to be specific for 17β-estradiol and was sensitive to substitutions on the A, B and D rings. With the use-of this antiserum, it has been possible to develop a sensitive, accurate and specific radioimmunoassay for the estimation of estradiol-17β in sera from humans. This antiserum was sufficiently specific to allow the use of a simple benzene extract of serum without subsequent chromatography. The use of iodinated E2-β-11α-TME as labeled tracer further simplified the procedure by allowing the use of gamma ray spectrometry, rather than liquid scintillation counting with its attendant expense and inconvenience.