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-mu-l 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 < 0.01).