Radioimmunoassays Specific for the Tertiary and Primary Structures of Thyroxine-Binding Globulin (TBG): Measurement of Denatured TBG in Serum*

Abstract

Antisera prepared by immunization of rabbits with human T₄-binding globulin (TBG) contained two popula- tions of antibodies: one directed against determinants of the native molecule, and the other directed against antigenic sites present only in denatured TBG. These two populations of antibodies were present in all nine antisera prepared in this or other laboratories that were tested. Exploiting this property of anti-TBG sera and using radioiodinated denatured TBG as a tracer, a RIA was developed which measures specifically denatured TBG in the presence of native TBG. The RIA for measuring denatured TBG used purified native TBG, which was denatured by reduction and pyridylethylation (RP-TBG) before labeling with ¹²⁵I. Native TBG was measured using the same antiserum, but the ¹²⁵I-labeled tracer was unmodified TBG. The sensitivity of the native TBG RIA was 0.25 ng purified native TBG. Equivalent amounts of native TBG in serum, desialylated TBG, and deglycosylated TBG produced superimposeable standard curves. The cross-reactivity with RP-TBG was less than 0.02%. The denatured TBG RIA had a sensitivity of 1 ng, and superimposeable curves were produced with equivalent concentrations of RP-TBG and heat-denatured native TBG. The cross-reactivity of 0.8% with native and degly-cosylated TBG was, at least in part, due to denatured TBG in the purified preparations. The specificity of the two RIAs is due to the existence of distinct and exclusive antigenic determinants i n native TBG and denatured TBG which are probably located on the surface of the tertiary structure and internally at the primary Structure of the molecule, respectively. Heat and acid pH treatments of serum produced a progressive loss in immu- noreactive native TBG, proportional to the loss of T₄-binding capacity. A reciprocal and quantitative increase in denatured TBG, as measured in the denatured TBG RIA, was found. T₄ partially protected the native TBG from denaturation. Denatured TBG was detected in sera from normal adults. The mean value was 6.05 ± 2.25 (±SD) μg/dl (n = 11). Similar values were found in 8 pregnant women, 5 men with familial partial TBG deficiency, and 15 hypothyroid 7 hepatic, and 8 renal failure patients. Significantly decreased concentrations were found in umbilical cord serum (2.43 ± 0.88 μg/dl; n = 12; P < 0.0001) and serum from neonates (2.89 ± 1.54 μg/dl; n = 7; P < 0.01). The mean value in thyrotoxic patients (3.36 ± 1.52 μg/dl; n = 14) was not significantly lower than normal. No denatured TBG was detected in sera from the 4 unrelated men with inherited complete TBG deficiency, nor could it be generated by heat treatment of these sera. Denatured TBG was not produced in vitro, since its concentration remained stable when whole blood was kept at 4 C or at room temperature for 48 h before the separation of serum or after repeated freezing and thawing. Denatured TBG is a normal constituent of serum, the concentration of which did not correlate with that of TBG. It is, however, absent in sera of subjects with inherited complete TBG deficiency. Whether it is a product of TBG degradation or a minor, poorly structured, component of TBG secreted by the liver remains a matter of speculation.