An enzyme immunoassay for determining total thyroxine in human serum using an ultramicroanalytical system

A simple, rapid competitive ultramicroElisa assay has been developed for the measurement of total thyroxine (T4) using only 10 microliters of serum. Our novel UME is based on fluorescence measurements of the hydrolytic product of 4-methyl-umbelliferyl-beta-D-galactopyranoside. T4-beta-Galactosidase conjugates and monoclonal antibodies were immobilized on polyvinyl plates, with sodium salicylate used as a blocking agent for thyroxine binding protein. The analytical steps were carried out using a semiautomated batch-assay system entitled "SUMA" (system for ultramicroanalysis). The T4 assay was completed in 2 h, with a measuring range of 24-386 nmol/L. The intra-assay coefficient of variation (CV) was 4.6-6.9% and the inter-assay C.V. 9.0-12.4% depending on the T4 concentrations. Percentage recovery ranged from 99.2-111%. Regression analysis showed a good correlation with an established radioimmunoassay (n = 121, r = 0.946, P less than 0.01).