Insulin autoantibodies in Graves' disease--before and after carbimazole therapy.

Insulin autoantibodies (IAA) are well documented in patients with insulin-dependent diabetes (IDDM) prior to the administration of insulin and in patients with reactive hypoglycaemia--the insulin autoimmune syndrome (IAS). It has been suggested that IAA can be induced by the administration of drugs containing sulphydryl groups, such as carbimazole, and they have been frequently described in Graves' disease. An alternative explanation is the clustering of autoantibodies in autoimmune disease. We studied 39 patients (37 females, two males, age range 14 to 61 years; mean 33.8 years) with proven Graves' disease and no previous treatment with carbimazole. Fifteen of the 39 patients had a family history of other autoimmune diseases. IAA and thyroid autoantibodies were assayed at diagnosis and monthly thereafter while on treatment with carbimazole, for up to 6 months. IAA were measured using a direct-binding solid-phase ELISA and specificity was confirmed by absorption studies using insulin covalently coupled to Sepharose beads. At diagnosis 33 of the 39 patients (85%) were positive for thyroid microsomal antibodies, 13 (33%) were positive for thyroglobulin antibodies, and 4 (10%) were positive for IAA. All IAA-positive patients had microsomal antibodies at diagnosis, and two had thyroglobulin antibodies in addition. After 4 months on carbimazole, the frequency of thyroid microsomal autoantibodies was unchanged (83%), while that of anti-thyroglobulin antibodies had fallen (8.6%). All four IAA-positive patients remained positive, and studies of binding to human, porcine and bovine insulin demonstrated that one serum, initially human insulin specific, later became cross-reactive with all three. We conclude that low titres of IAA are found in Graves' disease, and are associated with the presence of autoimmunity rather than the carbimazole. Symptomatic hypoglycaemia, however, is rare in Caucasian patients.

Insulin autoantibody polymorphisms with greater discrimination for diabetes in humans.

Insulin autoantibodies, like islet cell antibodies, are found not only in the sera of newly diagnosed Type 1 (insulin-dependent) diabetic patients and their relatives, but also in patients with other autoimmunities who do not develop diabetes. Insulin autoantibodies are oligo/monoclonal and frequently binding-site restricted. As determinant selection is genetically determined, we questioned whether certain polymorphisms of insulin autoantibodies, identified by their binding site on the insulin molecule, could better discriminate for Type 1 diabetes, which is also HLA determined. First, we raised monoclonal antibodies to human insulin by classic fusion methods in order to determine the range of antibody polymorphism, and identified five distinct types by their binding profiles to a panel of insulin variants, using an enzyme-linked immunosorbent assay. Two of these polymorphisms, type A and type B, were subsequently found in insulin autoantibody positive human sera using the same panel of insulin variants, and successfully distinguished diabetes-related from diabetes-unrelated individuals. Thus, the type B polymorphism was responsible for binding in 60% of 41 insulin autoantibody positive individuals with polyautoimmune disease but no personal or family history of diabetes (diabetes unrelated), but in only 2% of a group which comprised 17 newly-diagnosed insulin autoantibody positive Type 1 diabetic patients, 19 insulin autoantibody positive discordant twins of Type 1 diabetes and six insulin autoantibody positive healthy siblings of Type 1 diabetic patients (diabetes related) (p less than 0.01). Isolation of the type A polymorphism alone reduced the proportion of false negatives in the insulin autoantibody test for diabetes relatedness from 49% to 20% without diminishing its specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved ELISA for thyroid microsomal auto-antibodies. Comparison with haemagglutination and immunofluorescent techniques.

Thyroid microsomal antibodies (TMA) and thyroglobulin antibodies (TGA) are strongly associated with auto-immune thyroid disease. TMA and TGA have been mostly detected by means of either immunofluorescence (IF), tanned red cell haemagglutination (TRCH), or radio-immunoassay (RIA) until the recent development of the enzyme-linked immunosorbent assay (ELISA). The ELISA has not been as extensively used in TMA detection as in the assay for TGA. The RIA method, though more sensitive, is technically and materially very demanding while the TRCH and IF are simpler to perform but are less sensitive. The main problem with the ELISA for the detection of TMA appears to be the interference of TGA present in some test sera reacting with thyroglobulin present as a contaminant in the thyroid microsomal preparation. In this study, we compared the TMA results from an ELISA system designed to eliminate TGA interference with those of IF and TRCH. The ELISA system in which TGA interference was eliminated without concurrent inhibition of the test reaction that gives rise to false negatives was more sensitive than either IF or TRCH. A significant number of samples was falsely reported as negative by both TRCH and IF. The correlation of the degree of positivity between TRCH and ELISA was moderate and was higher than that between ELISA and IF, though both were highly significant. The ELISA technique for TMA detection described here is a more efficient, more sensitive and also more cost-effective system than either TRCH or IF, both of which should now be replaced by ELISA, provided steps are taken to avoid false positives and false negatives.

On the relationship between islet cell antibodies and insulin autoantibodies in patients at risk from insulin dependent diabetes.

The frequencies of islet cell antibodies (ICA) and insulin autoantibodies (IAA) were studied in three clinically well defined groups, using an aprotinin sensitised indirect immunofluorescence assay for ICA and a direct binding solid ELISA for IAA, and the association of these two serological markers for insulin dependent diabetes analysed. Frequency of ICA was 10.7% in siblings of diabetics, 15.5% in discordant identical twins and 65.9% in newly diagnosed diabetic patients. Frequency of IAA was 7.1% in siblings, 46.7% in discordant twins and 38.6% in newly diagnosed diabetic patients. No correlation was demonstrated between the two autoantibodies in the siblings. In the newly diagnosed diabetic patients there were sera positive or negative for both, but 22 (50%) of the sera showed dissociation between the two antibodies. The studies of twins showed that IAA and ICA fluctuated independently with time, and demonstrate the inappropriateness of seeking such an association in cross-sectional surveys. An association could not be demonstrated in this group even if data from multiple samples taken at different points in time were pooled, scoring an individual as positive if at any time their sera had been positive for the corresponding antibody. Thus our data showed no correlation between ICA and IAA in any of the groups studied.

Problems associated with the presence of immunofluorescent heterophile antibody in sera submitted for autoantibody screening.

A small proportion of sera submitted for routine immunofluorescent antibody screening contains heterophile antibodies. The reaction patterns produced by the heterophile antibody may be confused with or mask specific autoantibody patterns, leading to false positive or false negative autoantibody results. In this study we report the development of a method in which fresh sheep red cells are used to absorb heterophile antibody from sera without affecting specific autoantibody which may be present. This technique was used on a panel of 142 sera known to contain heterophile antibody but not initially reported as containing specific autoantibodies by immunofluorescence. After absorption with sheep red cells the heterophile antibody was completely removed from the sera under test and 27 (19%) specimens were shown to contain previously undetected autoantibodies. Only 6 (4%) of the sera, however, contained autoantibodies at a titre which would be reported as a positive result on routine screening. These results suggest that there may be a significant number of sera submitted for routine autoantibody screening which contain autoantibodies that are masked by the presence of heterophile antibody. Selective use of the absorption technique offers a simple solution to this problem.

The incidence of occult thyroid disease associated with thyroid antibodies identified on routine autoantibody screening.

The presence of thyroid microsomal and/or thyroglobulin antibodies has been recorded over a 2 year period of 15 000 consecutive autoimmune profile request. Where there had been no initial clinical suspicion of thyroid diseases, 332 requests showed positive thyroid antibodies, and of these 63 (19%) had abnormal in vitro thyroid function tests (TFT). No differences were observed between the abnormal and normal groups with respect to the presence of different autoantibodies or to the age and sex distributions. Of these subjects with clinically unsuspected hypothyroidism but with abnormal TFTs, 29% were commenced on thyroxine therapy and experienced a symptomatic improvement, 25% remain well on no therapy and 9% continue on no treatment but with symptoms possibly attributable to hypothyroidism. 3% became clinically hypothyroid during a follow-up period of 2 years. 5% died of unrelated causes and there was inadequate follow-up information on the remainder. This study provides further confirmation that when thyroid antibodies, and in particular thyroid microsomal antibody, are found unexpectedly, a significant proportion of patients will have biochemical evidence of hypothyroidism and may benefit from appropriate treatment.

Failure to demonstrate any interaction between polyclonal rheumatoid factors and IgG antinuclear antibodies or other IgG autoantibodies.

The autoantibody profile results from 750 randomly selected patients with rheumatoid factor (RF) and/or antinuclear antibody (ANA) positive tests were retrospectively analysed in order to discover if there was any evidence to show that the presence of polyclonal rheumatoid factors affects the incidence and titre of IgG-ANA in these patients. The incidence of IgG-ANA in the RF-positive group (34.4%) was significantly greater than that found in the RF-negative groups (19.6%) (P < 0.001), and there was no significant difference between the mean IgG-ANA titres of the two groups (P = 0.987). The possibility that the presence of IgM-RF in serum might be responsible for the inhibition of IgG-ANA was examined by treating the sera of selected patients who were RF-positive but IgG-ANA-negative with the dissociating agent D-penicillamine (DP). After treatment, IgG-ANA could not be detected in any of the sera. Similar studies were carried out to ascertain if there was a masking effect by RF on two other IgG autoantibodies, anti-keratin antibody (AKA) and gastric parietal cell antibody (GPCA). There was no evidence from these studies that the presence of RF affects the incidence of either AKA or GPCA. We conclude from these results that the presence of RF is not a significant factor controlling the incidence of either IgG-ANA or other IgG autoantibodies and that the routine treatment of RF-containing serum with a dissociating agent before testing for autoantibodies would be unnecessary.