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Steroid 21-Hydroxylase Autoantibodies: Measurements with a New Immunoprecipitation Assay¹

FIRS Laboratories, RSR Ltd. (H.T., M.S.P., M.P., J.F.S., J.S., S.C., L.P., T.A., B.R.S., J.F.), Llanishen, Cardiff, United Kingdom CF4 5DU; the Institute of Semeiotica Medica, University of Padua (C.B., M.V.), Padua, Italy; and the Department of Medicine, University of Wales College of Medicine (H.T., M.S.P., J.S., S.C., T.A., B.R.S., J.F.), Cardiff, United Kingdom CF4 4XN

Address all correspondence and requests for reprints to: Dr. Bernard Rees Smith, FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom CF4 5DU.

Autoantibodies (Abs) to steroid 21-hydroxylase (21-OH) are a major component of adrenal cortex Abs and are characteristic of autoimmune Addison’s disease. We have developed a new method for measuring Abs to 21-OH based on ¹²⁵I-labeled recombinant human 21-OH produced in yeast. With this assay, 21-OH Abs were detected in 43 of 60 (72%) sera from patients with isolated Addison’s disease, 11 of 12 (92%) autoimmune polyglandular syndrome type I sera, 27 of 27 (100%) autoimmune polyglandular syndrome type II sera, and 24 of 30 (80%) sera from patients who were positive for adrenal cortex antibodies by immunofluorescence but had no overt Addison’s disease. 21-OH Abs were found by ¹²⁵I assay in 4 of 150 (2.7%) sera from patients with insulin-dependent diabetes mellitus, 1 of 77 (1.3%) Graves’ sera, 1 of 67 (1.5%) Hashimoto’s sera, and 6 of 243 (2.5%) sera from healthy blood donors. 21-OH Abs were not detected in 9 sera from patients with Addison’s disease due to tuberculosis, 32 sera from patients with noninsulin-dependent diabetes mellitus, 35 sera from patients with myasthenia gravis, or 17 sera from patients with premature ovarian failure. There was good agreement between the ¹²⁵I-labeled 21-OH assay and an assay based on ³⁵S-labeled 21-OH produced in an in vitro transcription/translation system (r = 0.86; n = 129; P < 0.001). In the case of sera from patients with Addison’s disease, insulin-dependent diabetes mellitus, Graves’ disease, and Hashimoto’s disease and from healthy blood donors that were low positive in the ¹²⁵I assay, neutralization studies with unlabeled 21-OH confirmed the presence of specific 21-OH Abs.

Overall, the 21-OH Ab assay based on ¹²⁵I-labeled 21-OH showed good sensitivity, precision, and disease group specificity. This, combined with a simple assay protocol and the convenience of ¹²⁵I handling and counting, make it attractive for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of inheritance of adrenal autoimmunity. In addition, studies of the effect of treatment or possible preventative measures on 21-OH Ab levels in individuals without overt adrenal failure may suggest ways of delaying the onset of autoimmune Addison’s disease.

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