Figure 2.

(A) In the one-step analog method, labeled T4 is altered to prevent it from interacting with binding proteins. After equilibrium is achieved, the serum is removed and the occupancy of the antibody binding site is measured. The more the free T4, the less room there is for the labeled analog and the lower the signal. (B) In the labeled antibody method, free T4 competes with solid-phase T4 for a limited number of labeled antibody binding sites. The resulting solid-phase bound label is inversely proportional to the original free T4 concentration. (C) Indirect (tracer) equilibrium dialysis involves addition of radiolabeled T4 with free T4 calculated as the fraction of the total radiolabeled T4 that has crossed the dialysis membrane. (D) Direct equilibrium dialysis requires quantitative measurement of the free T4 present in the compartment from which the protein-bound T4 is excluded by radioimmunoassay or mass spectrometry. [Reproduced with permission from the editor of Ref. (6).]