An enzyme immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum

Abstract

An enzyme-linked sandwich immunoassay using human thyroglobulin conjugated with β- d-galactosidase and silicone rods coated with human thyroglobulin was developed for the measurement of circulating anti-thyroglobulin autoantibody. The volume of serum needed for the assay was as little as 5 μ1. The sensitivity of the assay was approximately 7 × 10 −15 mol/tube of antithyroglobulin immunoglobulin G corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by (1) parallelism of the standard curve with dilution of sample sera of patients with thyroid diseases, and (2) non-detectability of anti-thyroglobulin in autoantibody in the sera of normal subjects. The precision of the assay was proven by (1) sufficient recovery of antithyroglobulin immunoglobulin G added to serum, and (2) coefficients of variance within and between assays were 6.5 to 10.3% and 4.9 to 14.1%, respectively. No effect of thyroglobulin on the present assay was observed when the ratio of the amount of thyroglobulin to that of anti-thyroglobulin immunoglobulin G was lower than 1:10. Furthermore, a significant correlation was observed between anti-thyroglobulin autoantibody concentrations measured by our enzyme immunoassay and those by tanned red cell hemagglutination ( r = 0.78), and between those by the enzyme immunoassay and those by radioimmunoassay ( r = 0.80).