A new solid-phase immunoradiometric assay for anti-thyroglobulin autoantibody

A newly developed sensitive and quantitative immunoradiometric assay (IRMA) for anti-thyroglobulin (anti-Tg) autoantibody is described. Serum samples to be tested are added to wells of polyvinyl microtiter plates coated with human thyroglobulin. After removal of the unbound material, anti-Tg antibody is determined by adding purified 125I-anti-human immunoglobulin G antibody. Using 1.0 microliter of serum anti-Tg antibody was detected in 81.2% of patients with Hashimoto's thyroiditis or idiopathic myxedema (n = 32), in 46.4% of those with Graves' disease (n = 28), in 11.9% of subjects with other thyroid disorders (n = 42) and in 4.2% of normal controls (n = 71). Similar percentages of positive tests were observed by passive hemagglutination (PH) and a good correlation was found between the antibody levels determined by the two techniques. Using larger amounts of serum (100 microliters) detectable anti-Tg antibody by IRMA was found in the majority of patients with thyroid autoimmune disorders who had negative PH tests. Quantitative measurements of anti-Tg antibody by IRMA could be obtained by using purified anti-Tg antibody as standard reference. The minimum detectable amount of anti-Tg antibody was 0.5 ng. The present method is proposed as a simple and convenient technique for quantitative measurement of any antibody, using wells coated with the appropriate antigen

A new solid-phase immunoradiometric assay for antithyroid microsomal antibody

A new sensitive, quantitative, and specific immunoradiometric assay (IRMA) for antithyroid microsomal (anti-M) antibody has been developed. Samples to be tested are incubated within wells of polyvinyl microtiter plates coated with solubilized thyroid microsomal antigen. After removal of unbound material, anti-M antibody is detected by adding purified [125I]antihuman immunoglobulin G (IgG) antibody. Using 1.0 microliter serum, anti-M antibody was found by IRMA in all of the patients with Hashimoto's thyroiditis or idiopathic myxedema (n = 19), in 86% of those with Graves' disease (n = 42), in 10.9% of subjects with other nonautoimmune thyroid disorders (n = 37), and in 8.4% of normal controls (n = 71). A good correlation was found with the results obtained in anti-M antibody tests by passive hemagglutination. Using larger volumes of serum (up to 100 microliters), anti-M antibody detectable by IRMA was found in some patients with Graves' disease and negative passive hemagglutination tests. Quantitative measurements of anti-M antibody by IRMA could be performed using a standard IgG preparation containing high levels of anti-M antibody. The minimal detectable amount ranged between 1-2 ng IgG, corresponding to a sensitivity 15-30 times greater than that of the competitive binding radioassay. We suggest that the present IRMA may be proposed as a general technique for the detection of different organ-specific autoantibodies

A solid-phase immunoradiometric assay for gastric parietal cell antibodies

We recently described a solid phase immunoradiometric assay (IRMA) for anti-thyroglobulin and anti-thyroid microsomal antibodies. In the present study a similar IRMA for gastric parietal cell antibodies (PCA) has been developed. Samples to be tested are incubated within wells of polyvinyl microtitre plates coated with solubilized gastric microsomal antigen. After removal of unbound material, PCA is detected by adding purified 125I-anti-human IgG antibody. A good correlation was found with the results of PCA assays obtained by indirect immunofluorescence. In contrast, negative PCA by IRMA were consistently obtained in sera containing high titres of several other organ specific and non-organ specific autoantibodies. PCA determinations by IRMA were than carried out in a series of normal controls and patients with autoimmune or non-autoimmune thyroid disorders. Positive results were obtained in three of 70 (4.3%) apparently normal subjects, in 16 of 87 (18.4%) patients with Hashimoto's thyroiditis, in 10 of 48 (20.8%) with idiopathic myxoedema, in 25 of 95 (25.6%) with Graves' disease and in five of 64 (7.8%) with other non-autoimmune thyroid disorders. Preliminary results showed that quantitative measurements of PCA by IRMA could be performed using a serum containing high levels of PCA as standard reference. In conclusion, PCA may be easily and specifically detected using the same IRMA procedure previously developed for anti-thyroid antibodies. We therefore suggest that the present IRMA may be proposed as a general technique for the detection of different organ specific autoantibodies

Surface markers and function of circulating thyroid autoantibody-producing cells.

The in vitro synthesis of antithyroglobulin (anti-Tg) and antithyroid microsomal (anti-M) autoantibodies by peripheral blood mononuclear cells (MNC) from patients with autoimmune thyroid diseases was investigated using sensitive immunoradiometric assays. Cultures were carried out in the presence or in the absence of pokeweed mitogen (PWM). Thyroid autoantibodies were undetectable in supernatants of MNC cultures from 9 normal subjects. Supernatants of MNC cultured without PWM had detectable levels of anti-Tg and anti-M in 5 (19.3%) and in 2 (7.7%) of 26 patients with autoimmune thyroid diseases, respectively. In the presence of PWM, a marked increment in the antibody concentrations occurred in all but 1 of these cultures, and the number of positive cultures increased to 13 (50.1%) for anti-Tg and to 15 (57.7%) for anti-M. Studies of MNC fractions depleted of T lymphocytes (non-T cells) were carried out on selected patients showing antibody synthesis only after PWM stimulation. Autoantibody production was not found with non-T cells, but the effect of the mitogen was restored by readdition of T cells. Irradiation (1000 rad) of T cells before coculturing significantly enhanced autoantibody production. With this model no significant functional difference was found between autologous and allogenic T cells from thyroid autoimmune disease patients or from normal subjects. The cells involved in PWM-driven thyroid autoantibody synthesis, as defined by depletion studies, were lymphocytes bearing DR antigens and surface immunoglobulin G (IgG) without detectable surface immunoglobulin M (IgM). Depletion from MNC suspensions of Tg-binding cells abolished PWM-stimulated anti-Tg production, but did not alter the synthesis of anti-M. Further studies were carried out on MNC from a single patient with Hashimoto's thyroiditis, whose non-T cells consistently produced large amounts of anti-M and total IgG in the absence of PWM. The addition of PWM to these unfractionated MNC slightly increased the production of anti-M, but inhibited antibody synthesis after depletion of T lymphocytes. Interestingly, the addition of autologous T lymphocytes to non-T cells inhibited the spontaneous synthesis of anti-M. These data indicate that in vitro synthesis of anti-Tg and anti-M by MNC may be frequently induced by stimulation with PWM in patients with thyroid autoimmune disorders. PWM-stimulated synthesis of thyroid autoantibodies appears to be T-cell dependent and modulated by radiosensitive T lymphocytes. The cells responsible for PWM-dependent thyroid autoantibody synthesis are B lymphocytes with surface membrane IgG and have receptors specific for the autoantigen