Detection of Antimitochondrial Autoantibodies in Immunofluorescent Ama–Negative Patients With Primary Biliary Cirrhosis Using Recombinant Autoantigens

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Abstract
Antimitochondrial antibodies (AMA) are the serologic hallmark of primary biliary cirrhosis (PBC). However, depending on the clinical laboratory, from 5% to 17% of PBC patients are consistently AMA–negative, using native mitochondrial antigens and a variety of conventional assays including immunofluorescence (IMF) and enzyme–linked immunosorbent assay (ELISA). The major immunoreactive mitochondrial autoantigens are the E2 members of the 2–oxo–acid dehydrogenase complex family, including pyruvate dehydrogenase complex–E2 (PDC–E2), branched chain 2–oxo acid dehydrogenase complex–E2 (BCOADC–E2), and oxo–glutarate dehydrogenase complex–E2 (OGDC–E2); cDNAs of these proteins have now been cloned, sequenced, and their B–cell epitopes defined. In the present study, we cloned cDNAs encoding these proteins from human, not bovine, sources, and expressed the recombinant proteins in a newly developed ELISA that employs a unique Escherichia coli buffer, and compared the data with previous assays using both AMA–positive and –negative patients. Using this new assay and our criteria for positive as an optical density (OD) greater than 10 SD above the mean of control sera, the AMA–positive rate of 191 PBC sera was 94% (179 of 191) compared with 84% (161 of 191) by IMF. None of the 316 control sera were reactive. Using our recombinant assays, we focused attention on the 30 IMF–AMA-negative patients. Twenty–two of 30 (73%) of these patients were positive using this new ELISA. The group of 30 IMF–AMA-negative/ELISA–positive patients did not differ significantly from a comparable population of IMF–AMA-positive patients with respect to age, sex distribution, liver function tests, elevation of serum IgM, or pathologic stage.

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