Measurement of anti-thyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay

Abstract

Anti-thyroglobulin IgG in human serum was measured by a novel and sensitive immune complex transfer enzyme immunoassay. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between human anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. The polystyrene ball was washed to eliminate most nonspecific IgG in test serum, and the complex was eluted from the polystyrene ball, to which nonspecific IgG had been adsorbed, with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay was 1,000 to 3,000-fold more sensitive than the conventional enzyme immunoassay, in which a human thyroglobulin-coated polystyrene ball was incubated with serum containing human anti-thyroglobulin IgG and, after washing, with rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in about 10% of healthy subjects and in all patients with Graves' disease and chronic thyroiditis. The principle of the present method may make it possible to sensitively measure other autoantibodies including anti-thyroid peroxidase and anti-thyrotropin receptor antibodies to aid diagnosis of thyroid diseases.