INFLUENCE OF THE PURITY OF THE IODINATED TRACER ON THE SPECIFICITY OF THE RADIOIMMUNOASSAY OF HUMAN FOLLICLE-STIMULATING HORMONE

Abstract

The objective of this study was to establish whether [125I]hFSH [human follicle stimulating hormone] subunits are formed during iodination of hFSH by a mild (lactoperoxidase) technique, and if so, which fractionation procedure provides the best separation of [125I]hFSH from its subunits. In addition, the effect of 125I subunits on the specificity of hFSH radioimmunoassay [RIA] technique was examined. The products of iodination of hFSH were fractionated by a number of techniques consisting of low and high resolution gel filtration procedures and adsorption on either cellulose or Concanavalin [Con] A coupled to Sepharose. The efficacy of the adsorption methods was assessed by a subsequent high resolution gel filtration procedure (Ultrogel AcA 54). Radioreceptor and RIA methods were used to monitor the profiles of hFSH, hFSH.alpha. and hFSH.beta. subunits and to assess the recoveries of radioreceptor-active hFSH obtained by the different procedures. Iodination of a highly purified hFSH preparation invariably resulted in the formation of both [125I]hFSH and [125I]hFSH subunits. The proportion of radioactivity associated with the 125I subunit peak to that of the [125I]hFSH peak was 10% when the iodination products after low resolution gel filtration were fractionated by a high resolution gel filtration procedure. The corresponding amount of subunits obtained by adsorption on cellulose and Con A was 71% and 6%, respectively. In comparison with low resolution gel filtration there was a partial separation of [125I]hFSH from [125I]hFSH subunits on Con A while no separation was effected on cellulose. 125I subunits were probably formed during fractionation on cellulose. The recoveries of hFSH activity following fractionation on cellulose and Con A were significantly lower (27% and 65%) than those obtained by either low or high resolution gel filtration techniques. A marked reduction in the specificity of the RIA of hFSH was found when [125I]hFSH subunits rather than [125I]hFSH were used as tracer in the assay. The presence of 125I subunits in [125I]hFSH preparations used as tracer in the RIA of hFSH can diminish the specificity of the assay. The routine employment of a high resolution gel filtration procedure, which provides a complete separation of 125I subunits from the intact hormone, is suitable for the elimination of this source of assay invalidity while the other fractionation procedures tested (cellulose adsorption or Concanavalin A) are not.