An ELISA method for the detection of autoantibodies to adrenal cortex

An enzyme-linked immunosorbent assay (ELISA) is described for autoantibodies to adrenal cortex. Microsomes were prepared from fresh human adrenal glands, and microtitre ELISA plates were incubated at 4 degrees C overnight with 25 micrograms antigen/ml, the optimal concentration for the system. Optimal dilution of patient's serum was 1/500. Peroxidase-labelled anti-human IgG and IgM sera were used in separate tests and o-phenylenediamine and H2O2 served as substrate. Intra-assay variance of optical density units was 4.5%, and inter-assay variance was negligible when antigen preparations from 2 different adrenal glands were compared. All sera positive or negative at first test gave the same qualitative result in a second. Non-organ-specific binding of sera containing mitochondrial or ribosomal antibodies was eliminated by a blocking ELISA system where the antigen-coated plates were incubated with test sera, and in a second step, peroxidase-labelled IgG from an adrenal antibody-positive control serum was added. In this test, optimal antigen concentration for coating of plates was 6.25 micrograms/ml and optimal serum dilution 1/50. The ELISA proved more sensitive and reproducible than indirect immunofluorescence. Adrenal antibodies detected by ELISA were usually of IgG class alone and only 1 of the 30 positives also contained IgM specificity. 30 out of 38 sera (79%) from patients with 'idiopathic' Addison's disease were positive whereas immunofluorescence revealed only 23 (61%) at first testing and another 4 positives when sera were tested on different adrenal glands. The ELISA described is useful for both scientific work and clinical diagnosis of autoimmune adrenalitis.