Influence of the purity of the iodinated tracer on the specificity of the radioimmunoassay of human luteinizing hormone.
Human luteinizing hormone (HLH) iodinated with 125I by the 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, HLHalpha and HLHbeta 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 HLHalpha subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using three 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, HTSH, HLHalpha and HLHbeta subunit. A significantly higher cross-reactivity with HLHalpha, HFSH and HTSH was obtained with the [125I]HLHalpha fraction as tracer than with biologically active [125I]HLH. Furthermore, in the radioimmunoassay of HLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]HLHalpha tracer than with the biologically active [125I]HLH. It is concluded that the presence of [125I]HLHalpha in the [125I]HLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]HLHalpha should be separated from [125I]HLH prior to radioimmunoassay. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration systems) are inadequate for this purpose. However, an adequate separation can be achieved by the use of high resolution gel filtration systems.
D001681:Biological Assay; D002850:Chromatography, Gel; D005640:Follicle Stimulating Hormone / Q000032:analysis; D006801:Humans; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007986:Luteinizing Hormone / Q000032:analysis* / Q000302:isolation & purification; D011863:Radioimmunoassay / Q000379:methods*
D007106:Immune Sera; D007457:Iodine Radioisotopes; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone