Abstract
A double antibody radioimmunoassay (RIA) for the measurement of bullfrog basic gonadotropin (GTH) is described. The antiserum was raised against a highly purified bullfrog basic GTH. Bullfrog basic GTH-IV (pI=9.33) was used for radioiodination and as a reference standard. The dissociation constant of the binding of the antibody to bullfrog basic GTH-IV was 8.28×10-12M. The standard curve was obtained with 0.1-25.6ng/ml of bullfrog basic GTH-IV. When bullfrog sera were assayed by this RIA system, the intra-assay coefficient of variation and inter-assay coefficient of variation were 4.45% and 5.6%, respectively. The accuracy of the assay was shown as the recovery of bullfrog basic GTH-IV added to the pooled bullfrog sera. With a range of 0.1-12.8ng/ml, the recovery was 104.78±11.91%(Mean±S. D.). When bullfrog pituitary extract was fractionated by isoelectric focusing (IEF) and assayed for gonadotropin by this RIA system and also by radioreceptor assay systems employing Xenopus or Anolis testicular homogenates, the RIA positive peaks agreed well with those obtained with Xenopus RRA, indicating that the present RIA system measures LH-like gonadotropin. The inhibition curves of pituitary preparations from bullfrog, brown frogs and dark-colored frog were almost parallel to the standard curve, while those of other anuran and urodele species were not. This RIA system was highly sensitive, but allowed only a narrow application, because of species-specificity.
