Detection of novel non-M2-related antimitochondrial antibodies in patients with anti-M2 negative primary biliary cirrhosis

Abstract

Objective: In 95% of patients with primary biliary cirrhosis (PBC) antimitochondrial antibodies (AMA) can be detected reacting with at least one of the five components of the M2-antigen identified as 2-oxo-acid-dehydrogenase complex (OADC). However, among our PBC-sera 15-20% are anti-M2 negative by ELISA and Western blotting (WB) but show in the immunofluorescence test (IFT) the typical AMA-staining. Aim of the present study was, to characterize the target antigen(s) of these non-M2-related AMA.

Patients and methods: Analyzed were sera from 27 patients with clinically and histologically proven PBC being AMA positive by IFT but anti-M2 negative by ELISA and WB. They were tested by WB against various 100.000 g supernatants obtained after sonication of mitochondria from rat liver, bovine heart and pig kidney. These were further separated by isopycnic sucrose density centrifugation using different sucrose density fractions.

Results: Fourteen of the 27 AMA positive/anti-M2-negative sera (52%) reacted in the WB with a 60kDa- and 8 (29%) with an 80kDa-protein, both present in the 100.000g supernatant from bovine heart mitochondria accumulating at sucrose densities 1.14-1.16. An identity of these determinants with any of the M2-related antigens could be excluded. In the 60kDa band components of the mitochondrial enzymes F1F0-ATPase, ubiquinone cytochrome C reductase and acyl CoA dehydrogenase were detected by MALDI-TOF analysis; the 80kDa protein could not be further characterized.

Conclusions: AMA positive/anti-M2 negative PBC sera contain antibodies to further mitochondrial antigens at 60 and 80kDa which do not correspond to any of the M2-determinants. Those antibodies can be detected to a lesser extent in sera from patients with classical anti-M2 positive PBC but not in patients with other hepatic and non-hepatic disorders.