Determination of thyroxine binding globulin (TBG) in human serum by fluorescence excitation transfer immunoassay.
Human thyroxine binding globulin (TBG) was purified to near homogeneity by chromatography on thyroxine-substituted Sepharose 4B, DEAE-Sephadex A-50, Sephadex G-150 and n-decylamine-substituted Sepharose 4B. TBG was quantitated by homogeneous fluorescence excitation transfer immunoassay; the preparation of dye-protein conjugates is described. The fluorescence of fluorescein-TBG is quenched when an immune complex is formed with tetramethylrhodamine-antibody. In the presence of unlabeled TBG, the relative fluorescence intensity is increased and can be related quantitatively to the concentration of competing antigen. The described assay for TBG has a range corresponding to serum concentrations of 5--75 microgram/ml; the correlation with radioimmunoassay is 0.96. The coefficients of variation for replicate determinations of single samples of serum with low, normal and elevated TBG concentrations were 3.5%, 2.7% and 2.6% respectively; between-assay variations were 8.8%, 8.6% and 2.6% respectively.
D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G; D013975:Thyroxine-Binding Proteins / Q000302:isolation & purification*
D007074:Immunoglobulin G; D013975:Thyroxine-Binding Proteins