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 autoimmunity in individual patients and for studying the epidemiology of thyroid autoimmunity.

Thyroglobulin as an autoantigen: what can we learn about immunopathogenicity from the correlation of antigenic properties with protein structure?

“…Still, the sensitivity and specificity of the assay of anti‐hTg autoantibodies may have been underestimated, as they were more dependent on the method used than those of anti‐hTPO autoantibodies 12 . Anti‐hTg autoantibodies (≥ 1·0 U/ml) have also been detected in 10·4 ± 0·5% of 13 344 disease‐free subjects in the USA, 13 in agreement with previous estimates 14 . Their prevalence was higher in females and increased with age 13,15 , 16 and following viral infections 17 …”

Thyroid peroxidase and thyroglobulin autoantibodies in a large survey of populations with mild and moderate iodine deficiency

“…Many studies have shown a higher sensitivity and specificity of enzymelinked immunosorbent assays (ELISA) and RIA compared to assays based on passive haemagglutination in detecting thyroid autoimmunity. This has been found both when the diagnosis has been based on a combination of clinical and biochemical features (Laurberg & Pedersen, 1988;Laurberg et al, 1992) and when histopathological studies have indicated autoimmune thyroiditis with lymphocyte infiltration in the thyroid (Kasagi et al, 1996;Arai et al, 2000).…”

Correlation of Anti-Thyroid Peroxidase Antibodies (Anti-Tpo) With Thyroid Hormones in Local Population of Western Nepal.

“…TPO antibodies (TPO-Abs) appear to be involved in the tissue destructive processes associated with the hypothyroidism observed in Hashimoto's and atrophic thyroiditis [6]. Evaluation of the diagnostic value of an anti-TPO assay in patients with different types of thyroid disease and in controls showed increased anti-TPO antibodies in >90% patients with active autoimmune thyroiditis [7,8]. Measurement of this autoantibody is useful for settling the diagnostic quandary demonstrated by the evident inconsistency between thyroid function test parameters.…”