Purification of Anti-Thyroglobulin IgG from Human Serum

Abstract

The high prevalence of subjects in the general population and among patients with thyroid diseases who are positive for serum anti-thyroglobulin antibodies, together with the inconsistency of hybridoma techniques to obtain large amounts of anti-thyroglobulin antibodies, prompted us to prepare anti-thyroglobulin antibodies from human serum. An anti-thyroglobulin antibody positive serum was absorbed with CNBr-activated sepharose 4B conjugated to thyroglobulin and anti-thyroglobulin antibodies eluted by acid pH (3.0), basic pH (10.7) and detergent (SDS 3 g/l or 7 g/l). Preliminary experiments carried out with rabbit anti-human thyroglobulin allowed us to purify an Ig-fraction that retained its anti-thyroglobulin activity by immunoprecipitation and tanned red cell haemagglutination. When subjected to agarose gel electrophoresis and Silver stain, this fraction has the mobility of rabbit Ig. Further experiments were carried out using Hashimoto's serum (tanned red cell haemagglutination 1:40620). pH 3.0 was found to give the best yield of stable antibodies. An IgG fraction was eluted at a concentration of 10±1.2 mg/l of serum. This fraction has the same electrophoretic mobility as human IgG and is close to pure human anti-thyroglobulin antibody. A dose-response curve was built up at a concentration range between 0.016 mg–2 mg of IgG anti-thyroglobulin antibody per litre. The slope of the curve parallels that of a dilution curve of the whole serum, suggesting that the purified antibodies are representative of the whole antibody population. In conclusion, we provide a method for preparing considerable amounts of highly purified anti-thyroglobulin antibodies from human serum for application in clinical medicine and basic research, and in particular which provides a standard for measuring anti-human thyroglobulin serum antibodies by weight rather than the presently used standard expressed in U/ml. Furt h e rmore, this reagent could act as an ideal immuno-vector for the diagnosis and therapy of differentiated thyroid carcinoma.