INFLUENCE OF THE PURITY OF THE IODINATED TRACER ON THE SPECIFICITY OF THE RADIOIMMUNOASSAY OF HUMAN LUTEINIZING HORMONE

Publisher Website
Google Scholar
Abstract
Human luteinizing hormone (hLH), iodinated with 125I by use of a lactoperoxidase method, was fractionated by either cellulose adsorption, gel filtration or by the combination of these methods. The products of iodination were characterized by their in vitro biological LH activity and by their binding profiles with antisera to hLH, hLH.alpha. and hLH.beta. subunits. Several radioactive components were obtained after gel filtration with or without an initial cellulose adsorption step. One of these fractions was identified as biologically active hLH and another as the hLH.alpha. subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using 3 well defined and widely available antisera to hLH. The standard used was the hLH International Reference Preparation for immunoassay (68/40). Cross-reactivity was examined with several purified pituitary preparations, such as hFSH [human follicle stimulating hormone], HTSH [human thyroid stimulating hormone], hLH.alpha. and hLH.beta. subunit. A significantly higher cross-reactivity with hLH.alpha., hFSH and hTSH was obtained with the [125I]hLH.alpha. fraction as tracer than with biologically active [125I] hLH. In the radioimmunoassay [RIA] of hLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]hLH.alpha. tracer than with the biologically active [125I]hLH. The presence of [125I]hLH.alpha. in the [125I] hLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]hLH.alpha. should be separated from [125I]hLH prior to RIA. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration sysems) are inadequate for this purpose. An adequate separation can be achieved by the use of high resolution gel filtration systems.