Improved radioimmunoassay for human TSH.
This study concerns the optimization of the human TSH (h-TSH) radioimmunoassay with special emphasis on reducing the heterogeneity of the 125I h-TSH tracer. Enzymatic iodination of h-TSH with glucose oxidase/lactoperoxidase was shown to be superior to either low or high dose chloramine-T procedures, producing a high specific activity reagent (70--150 microCi/microgram) with minimal evidence of damage. Tracer purification procedures not only affected initial immunoactivity but also storage stability and heterogeneity of the resulting 125I h-TSH. Tracers purified by combining concanavalin A-Sepharose adsorption and high resolution gel filtration (Sephadex G100), produced significantly lower (p less than 0.001) serum h-TSH measurements than were observed in less purified tracer materials. Concanavalin A-Sepharose adsorption yield of the 125I h-TSH iodination products closely correlated with the yield (r = 0.85, p less than 0.001) and immunoactivity (r = 0.90, p less than 0.001) of the tracer produced, thus making this an ideal method for initial tracer purification. Storage of tracer adsorbed to a solid support (concanavalin A-Sepharose) reduced technical manipulations without compromising tracer performance. Loss of specific activity was minimized by storage at -70 degrees C. The assay developed using these technical approaches showed a sensitivity limit of 0.005 +/- 0.001 (S.E.M.) microU/tube; 50% displacement at 0.18 +/- 0.08 (S.E.M.) microU/tube and complete delineation between euthyroid (n = 49, 2.44 +/- 0.18 (S.E.M.) mU/l, range 1.00--6.08) and hyperthyroid (n = 62, 0.34 +/- 0.02 (S.E.M.) mU/l, range 0.10--0.85), serum h-TSH levels.
D002850:Chromatography, Gel; D003208:Concanavalin A; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D011863:Radioimmunoassay / Q000379:methods; D013972:Thyrotropin / Q000097:blood*
D007457:Iodine Radioisotopes; D003208:Concanavalin A; D013972:Thyrotropin