Further Studies on the Radioimmunoassay of Gonadotropin-Releasing Hormone: Effect of Radioiodination, Antiserum and Unextracted Serum on Levels of Immunoreactivity in Serum1

Abstract

Studies were undertaken to determine the effect of method of radioiodination, antisera and sera on levels of endogenous gonadotropin-releasing hormone (GnRH)-like activity in serum as quantified by radioimmunoassay. GnRH was radioiodinated by the chloramine T or lactoperoxidase procedure and purified on columns of Sephadex G-25 (0.8 .times. 20 cm, eluted with 0.01 N acetic acid) or on columns of QAE Sephadex G-25-120, an anion exchange resin (0.6 .times. 25 cm, eluted with 0.1 M borate buffer, pH 9.2, containing 0.1% gelatin). Unlabeled GnRH, monoiodo-GnRH and diiodo-GnRH were separated on the QAE Sephadex columns. Levels of immunoassayable GnRH-like activity in sera of ewes were lower (P < 0.05) when antiserum R-42 (high affinity) was used for quantification than when antiserum R-31 (low affinity) was used. Furthermore, levels of immunoassayable GnRH-like activity in sera were lower when monoiodo-GnRH from the lactoperoxidase radioiodination was used for quantification than when other radioiodinated preparations were used with either the high affinity or low affinity antiserum. Incubations of GnRH with sera and subsequent fractionation on 0.8 .times. 20 cm columns of Sephadex G-25 indicated that macromolecular components of serum bind radioiodinated GnRH. This was particularly evident after radioiodination of GnRH by the chloramine-T procedure. Binding of [3H]GnRH or unlabeled GnRH to macromolecular components of sera could not be demonstrated. To determine if serum was capable of destroying the immunological activity of GnRH, 1.0 ng unlabeled GnRH was incubated at 0, 22 or 37.degree. C from 0-240 min with 1.0 ml aliquots of pools of sera obtained from cattle, dogs, horses, humans, rats and sheep. The disappearance of GnRH was determined by radioimmunoassay. The half-disappearance times for GnRH incubated in sera at 37.degree. C ranged from less than 15 min in sera from horses to approximately 240 min in sera from humans and sheep. Intermediate half-disappearance times were noted for GnRH in sera of cattle, dogs and rats. The half-disappearance time of GnRH in serum from all species increased dramatically at lower temperatures. Unless procedures to eliminate binding of radioiodinated GnRH to macromolecular components of serum and to eliminate destruction of immunological activity of GnRH are used, actual levels of GnRH in the unknown will be overestimated.