Enzyme immunoassay for thyroxine and triiodothyronine in human serum, with use of a covalent chromatographic separation method.

A practicable enzyme immunoassay for measurement of thyroxine and triiodothyronine in serum was developed, involving a separation method based on the thiol-disulfide interchange reaction. Serum samples at alkaline pH were incubated for 1 h with antibodies and antigens labeled with beta-D-galactosidase. Each reaction mixture was then passed through a 0.1-mL column containing (anti-IgG) antibody immobilized on Sepharose 4B, to which the anti-IgG antibodies were coupled by means of a disulfide bond (3 g of IgG fraction per liter). The column was washed and the bound form of the label then was eluted from the column with a buffer containing dithiothreitol (25 mmol/L) by splitting the disulfide bonds between the anti-IgG antibody molecules and Sepharose matrix. From the enzyme activity in the eluate, concentrations of thyroxine or triiodothyronine in serum could be determined. Values obtained by this method and those obtained by a radioimmunoassay correlated well (thyroxine, r = 0.97, slope = 1.1 y-intercept = -0.94 microgram/L, n = 70; triiodothyronine, r = 0.98, slope = 0.91, y-intercept = 0.067 microgram/L, n = 77.