Specificities and characteristics of mitochondrial protein antigens assessed by enzyme linked immunosorbent assay for anti-mitochondrial antibodies. Relationship to immunofluorescent and precipitating antibodies.

M-A, M-B and M-C are autoantibodies to mitochondrial proteins frequently found in primary biliary cirrhosis. To study the characteristics and specificities and to isolate antigens reacting with the autoantibodies a sensitive assay (ELISA) was established. Using this technique a significantly elevated level of antibodies was detected in patients with primary biliary cirrhosis (mean OD 460.2, s.d. 145.8) compared to normals (133.7 +/- 49.7). The assay correlated well with the indirect immunofluorescence test for detecting anti-mitochondrial antibodies and the mitochondrial fluorescence could be abolished by absorption of autoantibodies with the mitochondrial fraction. All three types of antibodies could be detected by ELISA; however, sera containing the combination of two or more antibodies yielded higher ELISA values. The ELISA confirmed that the M-A antigen is trypsin and acid (pH 3.0) sensitive but DNAase and RNAase resistant while the M-B antigen is DNAase and trypsin sensitive. The antigens were enriched in the supernatant isolated from the mitochondrial fraction centrifuged at 1,800 g for 60 min and 2% polyethylene glycol precipitates of the mitochondrial fraction. The antigens were found phosphate buffer soluble and therefore could also be enriched by phosphate buffer extraction of the mitochondrial proteins. Thus, ELISA described here provided a sensitive method in the assessment of characteristics and purification of autoantigens related to mitochondrial antibodies.