Abstract: The present invention provides methods for the simultaneous measurement of triiodothyronine (T.sub.3) and thyroxine (T.sub.4) in biological fluids such as serum by direct equilibrium dialysis and immunoassay. Specifically, the method comprises dialyzing the serum sample to equilibrium in a physiological buffer system so that the free T.sub.3 and the free T.sub.4 are separated from T.sub.3 and T.sub.4 bound to serum proteins. The method further comprises combining a measured quantity of the dialyzed serum sample having free T.sub.3 and free T.sub.4 with reagents comprising a measured quantity of T.sub.3 labelled with a detectable marker and a measured quantity of T.sub.4 labelled with a detectable marker; an anti-T.sub.3 antibody of sufficient specificity and in sufficient quantity to bind a measurable quantity of the free T.sub.3, and an anti-T.sub.4 antibody of sufficient specificity and in sufficient quantity to bind a measurable quantity of the free T.sub.4.

Abstract: This invention relates to a highly accurate, rapid and simple estimation of thyroxine (T.sub.4) directly from blood serum and also relates to the accurate measurement of triiodo-L-thyronine (T.sub.3) directly from blood serum. More specifically, the invention relates to a rapid, specific and reliable radioimmunoassay (RIA) technique for measurement of both T.sub.4 and T.sub.3 in unextracted serum. The method requires very small amounts of serum, e.g., 25 microliters (.mu.l) to measure T.sub.4 concentration in nearly all specimens representing clinical states of eu-, hypo- and hyperthyroidism, and 250 .mu.l to measure T.sub.3 concentrations in specimens representing most clinical states.