In 1975 we developed a LH bioassay term RICT (rat interstitial cell testosterone) for measurement of circulating LH, 5-fold more sensitive than conventional radioimmunoassay. We have now developed a simplified and rapid method of comparable sensitivity for the measurement of LH/hCG in plasma, tissue extracts or incubation media. This bioassay uses microtiter plates and the product is, measured by an enzyme-linked immunoassay procedure using transfer solid phase. The procedure can be caried out in less than five hours (versus greater than 24 hr. for RICT) with minimal reagent preparation. The principle and steps presented can be used also for the assay of pituitary and hypothalamic hormones or any protein hormone that can stimulate the release of a cell product which can be measured by the present approach (ie. steroid, cyclic nucleotide, gonadotropin). We used RICT assay to assess biological LH activity secreted in response to endogenous and low dose exogenous GnRH, pulses in normal men. The absence of non-specific plasma effects in the LH bioassay was demonstrated by the finding of undetectable levels of LH bioactivity despite low but measurable immunoactivity in 10 hypogonadotropic men. In normal men exogenous low dose (10 mu g) i.v. GnRH administration resulted in preferential release of bioactive LH, with a consequent significant increase in the median plasma bio- to immunoactive LH ratio. This pattern mimicked that of endogenous LH pulsatility. The preferential increase in bioactive plasma LH in response to exogenous or endogenous GnRH might reflect the smaller initial distribution volume and slower metabolic clearance rate of bio versus immuno LH. We have demonstrated a sustained inhibitory actions on bio and immunoactive LH of a potent GnRH antagonist (N-acetyl-D-pCLPhel,2-D-Trp3-D-Alal0GnRH10) in postmenopausal women, and shown that the GnRH antagonist binds avidly to serum proteins and has a prolonged plasma residence time. This may explain the observed extended duration of the antagonist action in vivo.
