High diagnostic accuracy for idiopathic Addison's disease with a sensitive radiobinding assay for autoantibodies against recombinant human 21-hydroxylase.

Abstract

Autoantibodies against 21-hydroxylase (P450c21) are common in idiopathic autoimmune Addison's disease. In the present work, we have developed a sensitive radiobinding assay using in vitro translated recombinant human 35S-P450c21. Levels of P450c21 antibodies (P450c21-Ab) were expressed as a relative index (P450c21 index) using a P450c21-Ab positive Addisonian serum and two antibody-negative healthy sera as positive and negative standards in healthy individuals. The upper level of normal was the mean + 3 SD. Positivity for P450c21-Ab was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated 35S-P450c21. In 38 Addisonian patients, P450c21-Ab were found in 24/28 (86%) idiopathic, 0/5 post-tuberculosis, 0/3 adrenoleukodystrophy, and 0/2 post-adrenalectomy sera. Among healthy individuals, 1/70 (1.4%) were positive. The P450c21 index, as an estimate of P450c21-Ab levels, correlated inversely with the duration of idiopathic Addison's disease (r = -0.527; P = 0.007): 16/16 (100%) positive in patients with less than 20 yr and 8/12 (67%) positive in patients with more than 20 yr disease duration. The availability of this simple and sensitive radiobinding assay to evaluate levels of P450c21-Ab will permit large clinical studies as well as screening subjects at risk. In addition, the general population can now be screened to evaluate the predictive value of P450c21-Ab for Addison's disease.

Adrenal and ovarian autoimmunity

“…Recently, the laboratory diagnosis of AAD has been enriched by use of specific recombinant autoantigens. Two different immunoprecipitation assays for the detection of autoantibodies to 21-OH (21-OH Abs) have been developed, one based on 35 S-labeled 21-OH produced in an in vitro transcriptiontranslation system (56,70), the other based on 125 Ilabeled recombinant 21-OH produced in yeast (59). Immunoprecipitation assays based on 35 S-21-OH are highly specific and sensitive with a good reproducibility but there are limitations associated with the use of DNA and 35 S-labeled material.…”

Analysis of extended human leukocyte antigen haplotype association with Addison's disease in three populations

“…Steroidogenic P450 cytochrome enzymes have been identified as target antigens of the autoimmune response (2,3) and circulating antibodies against 21-hydroxylase are a sensitive diagnostic marker of the disease with a tendency to decrease in percentage when adrenal gland destruction progresses (4). The disease is often found as a part of polyendocrinopathy syndromes combining it with other organ-specific autoimmune diseases, thyroid disease and type 1 diabetes (T1D) being the most frequent associates (5).…”

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

“…On the other hand, the different binding ability of protein A and of rabbit antisera for human IgG subclasses may explain, in part, the discrepancies between the two assays. The use of antigenspecific immunoprecipitation assays for autoantibodies against P450c21 (32,33) or P450c17 and P450scc (19) could be instrumental in the understanding of the mechanisms responsible for the discordance between ACA-PLPA and ACA-IIF observed in some patients with AD. Recently, Colls et al (34), using an immunoprecipitation assay, detected autoantibodies against P450c21 (P450c21-Ab) in 66% of patients with autoimmune AD and observed a significant positive correlation between the immunoprecipitation results and those obtained with the IIF.…”