Validation of a sensitive radioimmunoassay to measure serum follicle-stimulating hormone in cattle: correlation with biological activity.

Abstract

Meaningful biological interpretation of the role of follicle-stimulating hormone (FSH) requires use of a validated radioimmunoassay (RIA) that closely estimates biologically active FSH, which was the objective of this work. Three FSH antibodies [NIDDK anti ovine FSH (oFSH); JAD anti oFSH; USDA anti bFSH] were screened against three tracer preparations [USDA oFSH-19-SIAFP-I2(USDA oFSH I2); LER1976a oFSH; USDA bFSH I2] in a RIA using USDA bFSH B1 or I2 as the assay standard. Sera obtained from three heifers at 4- to 8-h intervals for 5 days after injection of PGF2 alpha during the luteal phase were assayed for both FSH immunoactivity using each of the three optimized assay formats (NIDDK anti oFSH and JAD anti oFSH with USDA oFSH I2 as tracer; USDA anti bFSH with USDA bFSH I2 as tracer), and FSH bioactivity, using a rat Sertoli cell bioassay. Cross reactivity of bLH (NIH bLH B9) in all three assay formats was minimal (0.7, 0.9 and < 0.4% at 50% binding for the NIDDK, JAD and USDA antibodies, respectively). There was parallel displacement of tracer between bovine serum dilutions of 10 to 500 microliters and the two FSH standards. Correlations between JAD and USDA RIA data and bioassay results were not significant (P > or = 0.10), but were significant (r = 0.78; P = 0.0001) for the NIDDK RIA FSH and the bioactive FSH measurements. The assay sensitivity of the NIDDK RIA was 0.55 ng USDA bFSH B1 (0.013 ng USDA bFSH I2)/ml. The inter- and intra-assay CV were between 5.8 and 7.9 %. This RIA detected a pre-ovulatory FSH surge coincident with the LH surge, in all heifers studied. Furthermore, the emergence of each wave of follicle growth (up to day 12 of the cycle), was preceded by a transient increase (P < 0.02; days 0.5 to 1.5 and 8 to 10.5 of the cycle) in serum FSH, while LH concentrations remained unchanged. In conclusion, the RIA utilising NIDDK anti oFSH and USDA oFSH I2 as tracer provides a good estimate of bioactive FSH in cattle, and detects physiologically relevant increases in serum FSH related to emergency of each new wave of follicle growth.