Studies on the biological and immunological properties of human follitropin: profiles of two international reference preparations and of an aqueous extract of pituitary glands after electrofocusing

Abstract

Two international reference preparations, the First International Reference Preparation of Human Pituitary Gonadotropins (FSH [follitropin] and LH[luteinizing hormone]/ICSH [interstitial cell stimulating hormone]) for Bioassay (identified hereafter by its code no.: 69/104) and the First International Standard of Urinary FSH and LH (ICSH) for Bioassay (hereafter: hMG [human menopausal gonadotropin] 1st IS) and an aqueous extract of human pituitary glands (hPE) were fractionated in triplicate by isoelectric focusing on sucrose density gradient (Ampholine, pH range 2.5-10.0). The FSH activity was monitored in each fraction by an in vitro bioassay, a radioimmunoassay and a receptor binding assay. Unfractionated 69/104 was used in each assay system. Compared with the hPE, the activity profiles of the 69/104 and hMG reference preparations were spread over a significantly wider pH range; the biological activity eluted in the pH range of 3.5-5.0 was of the order of 90% for hPE, 72% for 69/104 and < 60% for hMG. Major variations in biological to immunological (B/I) and biological to receptor binding (B/R) activity ratios were observed in the individual electrofocusing fractions. The B/I ratios ranged from 0.8-2.2 (69/104), 1.0-5.7 (hMG) and 1.3-6.0 (hPE), respectively. The variation in the corresponding B/R ratios was: 0.5-1.7 (69/104), 0.58-4.0 (hMG) and 0.55-1.5 (hPE). When equal aliquots from each of the electrofocusing fractions were combined and this re-constituted pool was compared with the starting material, significant differences were observed in the B/I ratios: 1.63 instead of 1.0 (69/104), 2.58 vs. 2.34 (hMG) and 2.72 vs. 1.32 (hPE). There was no significant change in the mean B/R ratios before and after electrofocusing: 1.0 vs. 1.02 (69/104), 1.09 vs. 1.02 (hMG) and 1.05 vs. 1.04 (hPE). The mean recovery of biological activity in each of the 3 reconstituted pools of the 3 preparations was 97% for 69/104, 88% for hMG and 98% for hPE; the corresponding recoveries of receptor binding activities were 95, 94 and 98%, respectively. The mean recovery of immunological reactivity was 55% (69/104), 80% (hMG) and 47% (hPE), respectively. The reduction in the immunological reactivity of the 69/104 preparation without any apparent loss of biological or receptor binding activities following electrofocusing indicates that its immunoreactivity is unstable under mild experimental conditions, which do not influence its biological and receptor binding activities. Whereas this preparation might possibly be suitable as a standard for in vitro bioassays and receptor binding assays, it is unsuitable as a standard for the radioimmunoassay of hFSH.