Sensitive enzyme-linked immunosorbent assay for measurement of autoantibodies to human thyroid peroxidase

Abstract

The development of a sensitive assay for detection of autoantibodies against one of the major thyroid antigens, thyroid peroxidase (TPO), is described. TPO was purified from human thyroid tissue by: (1) isolation of thyroid microsomes using homogenization and differential centrifugation, (2) solubilization of membrane proteins by Zwittergent 3–14, and (3) anion exchange liquid chromatography on a FPLC Mono Q column. Autoantibodies against TPO (TPO-Ab) were measured using an enzyme-linked immunosorbent assay (elisa) with serum samples diluted 1:100. Standards containing 70, 7, 0.7, 0.02 and 0 U ml⁻¹ TPO-Ab were employed (reference standard code 66/387 NIBSC, London, UK). The detection limit was 0.02 U ml⁻¹ corresponding to 2 U ml⁻¹ in undiluted serum. The inter- and intra-assay coefficients of variation were 8.6% and 5.3%. In 109 healthy control subjects TPO-Ab was found in 9 (8.3%), while 43 (97.7%) out of 44 patients with newly diagnosed untreated Graves' disease had detectable TPO-Ab in serum. All of 16 patients with newly diagnosed spontaneously developing primary hypothyroidism had circulating TPO-Ab (range 16–7000 U ml⁻¹). The new assay is a valuable tool for evaluation of thyroid auto-immunity in individual patients and for studying the epidemiology of thyroid autoimmunity.

Key Words:

• autoimmunity
• hyperthyroidism
• hypothyroidism
• Graves' disease
• thyroid disease