Sensitive Human Thyrotropin Immunoradiometric Assay Set Up by the Identification and Minimization of Nonspecific Bindings

Abstract

An immunoradiometric assay (IRMA) of human thyrotropin (hTSH), based on magnetic solid phase separation, was studied especially in terms of its nonspecific bindings (B0) which were identified as a product of the interaction between an altered form of radioiodinated anti-hTSH monoclonal antibody (125l-mAB) and the uncoupled magnetizable cellulose particle (matrix). Preincubation with the same matrix, solid phase saturation with milk proteins, tracer storage at 4°C and serum addition during incubation were found to be particularly effective in preventing their formation. These findings were used to reproducibly decrease nonspecific bindings to values < 0.1% (or < 70 cpm), thus increasing the signal-to-noise ratio (B60/B0) up to values of 300-500. This way hTSH radioassays were obtained with functional sensitivities of about 0.05 mlU/L and analytical sensitivities of the order of 0.02 mlU/L. Such sensitivities, and, more importantly, a general improvement in assay performance, were obtained in a highly reproducible manner and all over the useful tracer life.