Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis.

aimTo construct and express a humanized M(2) autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC).

methodscDNA fragments encoding M(2)-reactive epitopes of pyruvate dehydrogenase complex E(2) (PDC-E(2)), branched chain 2-oxo-acid dehydrogenase complex E(2) (BCOADC-E(2)) and 2-oxo-glutarate dehydrogenase complex E(2) (OGDC-E(2)) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into the plasmid vector pQE-30 and then transferred into E. coli M15 (pREP4) for expression, which was induced by isopropylthio-beta-D-galactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M(2)-positive sera confirmed at Euroimmun Research Center (Germany). Using the purified BPO, M(2) antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA.

resultsThe expressed BPO was observed with both antigenic reactivity and specificity of M(2) autoantigens. The determination of M(2) antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay. With the newly established method, M(2) antibodies were found in 100 % (20/20) of patients with PBC. Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M(2) antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M(2)-antibody positive.

conclusionCompared with the routine immunofluorescence assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.