Separation of antibody-bound and free human follicle-stimulating and luteinizing hormones by ethanol-ammonium acetate.

Abstract

ABSTRACT The use of 66% ethanol containing 6.6% ammonium acetate provided a simple and economical method for the separation of antibody-bound and free 131I-labelled hormones in incubation mixtures of low protein concentrations used in the radioimmunoassay of human pituitary FSH and LH. The accuracy of the procedure was confirmed by similar results obtained in the evaluation of radiation damage to the tracer, binding of the tracer to the antibody and the separation of the antibody-bound and free 131I-labelled hormones by the use of ethanol-ammonium acetate solution and chromato-electrophoresis. The sensitivity of the measurement of the hormones was 10 pg with a precision of ± 5%; thus allowing the determination of hormone concentrations in as little as 20 to 50 μl of the plasma samples. There was a 98.5 to 102% recovery of the hormones added to the plasma samples of pre-determined hormone concentrations. The use of highly purified antigens for radioisotopic labelling and standard as well as purified antisera provided specific measurements of FSH and LH in the plasma. Various dilutions of a post-menopausal plasma sample yielded responses parallel to those obtained for the doseresponse curves of the FSH and LH standards by logit-log plots, thus confirming the validity of the assay. The mean plasma FSH and LH levels in normal men, children and in women during the luteal phase were 2.7 and 2.6, 1.1 and 1.2, and 1.8 and 2 ng/ml, respectively, whereas the plasma of hypophysectomized subjects showed trace to detectable levels of FSH and LH.