Improved radioimmunoassay for human TSH

Abstract

This study concerns the optimization of the human TSH (h-TSH) radio-immunoassay 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 μCi/μg) 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 < 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 < 0.001) and immunoactivity ( r = 0.90, p < 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°C. The assay developed using these technical approaches showed a sensitivity limit of 0.005 ± 0.001 (S.E.M.) μU/tube; 50% displacement at 0.18 ± 0.08 (S.E.M.) μU/tube and complete delineation between euthyroid ( n = 49 2.44 ± 0.18 (S.E.M.) mU/1, range 1.00–6.08) and hyperthyroid.( n = 62 0.34 ± 0.02 (S.E.M.) mU/1, range 0.10–0.85), serum h-TSH levels.