A method for the preparation of human thyroxine-binding globulin; its importance in the establishment of an accurate and specific radioimmunoassay

Pure thyroxine-binding globulin (TBG) is required in radioimmunoassay to prepare monospecific antisera, [125I]TBG and as primary standard. Homogeneous TBG was prepared by a three-stage affinity chromatography procedure; it could not be dissociated into subunits and its molecular weight by SDS polyacrylamide gel electrophoresis was 59 000. The amino acid composition was in agreement with two earlier reports. The secondary structure determined by circular dichroism in the far U.V. showed it to contain 24% each of alpha-helix and beta-pleated sheet. Serum TBG was determined by a 24-h radioimmunoassay using polyethyleneglycol to separate bound and free TBG. Serum TBG (mg/l, mean +/- S.D.) was: normal men 15.3 +/- 2.11 (n = 34), normal women 18.4 +/- 2.72 (n = 32) (P less than 0.005), in women on oral oestrogens 24.0 +/- 5.0 (n = 23), in normal pregnancy 38.6 +/- 3.0 (n =37), in cord blood 21.7 +/- 3.5 (n = 25) (P less than 0.001) and in euthyroid subjects aged over 60 years 17.8 +/- 4.5 (P n.s.). In women with thyroid disease TBG was reduced in hyperthyroidism: 15.5 +/- 2.5 (n = 28) and elevated in hypothyroidism: 21.0 +/- 4.0 (n = 25). Wider use in TBG-assay of non-denatured TBG of proven composition and structure should decrease the scatter in reference ranges and improve its value as a routine thyroid function test as both a primary measurement and as the T4 : TBG ratio.