Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

Abstract

Adrenocortical autoantibodies (ACA), present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF) on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA) technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13) and granulomatous (N = 9). ACA-PLPA were detected in 10/22 (45%) patients: 9/13 (69%) with the idiopathic form and 1/9 (11%) with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%): 10/13 (77%) with the idiopathic form and 1/9 (11%) with the granulomatous form. Twelve of the 13 idiopathic addisonians (92%) were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time). However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.

Autoantibodies against recombinant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17alpha-hydroxylase in Addison's disease and autoimmune polyendocrine syndrome type III

“…Recently, a 51 kDa antigen from the placenta and the granulosa cells has been shown to react with sera from the majority of AD patients positive for StCA-IIF (31). Although the ACA-IIF titers found in these patients were not in the lower range (1:16 and 1:32), the possibility of a false positive result cannot be excluded, since this is a semiquantitative method influenced by the presence of background staining, lipemia and quality of the adrenal cortex tissue (23). Among AD patients positive for SCCAb and/or 17OHAb, discrepant results between StCA-IIF and SCCAb and/or 17OHAb could also be due to the differences in the sensitivity and/or specificity of the assays.…”

“…One possible explanation for the presence of adrenal autoantibodies in one granulomatous AD patient is the presentation to the immune system of adrenal autoantigens resulting from inflammatory destruction of the adrenal cortex and subsequent autoantibody production in a genetically predisposed subject. Alternatively, one cannot rule out the possibility that a granulomatous adrenalitis supervened in a patient with an already ongoing autoimmune process (23).…”

“…ACA were detected by a classical IIF technique, using thin cryostatic sections of normal bovine adrenal glands as the source of antigen (23). ACA-IIF titers were determined by testing serial twofold dilutions of the serum until reaching the end point.…”

Doença de addison de etiologia auto-imune

“…Também podem fazer parte desta sín-drome a distrofia do ectoderma, o hipogonadismo hipergonadotrófico, o vitíligo, a anemia perniciosa, a alopecia, as síndromes de má absorção e a hepatite crônica ativa (15,18). Doenças tiroidianas autoimunes estão raramente presentes (11%) e, embora o diabetes mellitus do tipo I também seja pouco freqüente (4% a 12%), auto-anticorpos anti-GAD 65 podem ser demonstrados em 41% dos pacientes, mesmo na ausência de diabetes mellitus clinicamente manifesto (13,15,19). A SPA do tipo I é extremamente rara, exceto na Finlândia, onde sua freqüência estimada é de aproximadamente 1/25.000 habitantes.…”

“…Além da IFI, outras técnicas podem ser utilizadas para a detecção dos ACA, entre elas: a ELISA (63), o radioimunoensaio (64) e a técnica da proteína-A imunoperoxidase (PAIP) (65). Esta última, além de apresentar sensibilidade e especificidade comparáveis à IFI, tem as vantagens de dispensar o uso do microscópio de imunofluorescência e de permitir o estoque das lâmi-nas por tempo prolongado (65).…”