Detection of anti-branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 antibody in primary biliary cirrhosis by ELISA using recombinant fusionprotein

Anti-MZ of anti-mitochondrial antibody (AMA) is a serological marker of pri mary biliary cirrhosis (PBC), Anti-pyruvate dehydrogenase complex-E2 (anti- PDC-E2) is recognized as the most frequently occurring anti-M2, and a routi ne laboratory test for this antibody has already been established, However, it is also known that there are patients with PBC who are negative for ant i-PDC-E2, For the serological diagnosis of these patients, immunoblotting f or anti-Mas is indicated, However, the technique currently utilized is too laborious to allow testing of a large number of samples, In this study, me have developed an enzyme-linked immunosorbent assay (ELISA) using a recombi nant fusion protein in order to evaluate anti-branched chain Zero-acid dehy drogenase complex-E2 (anti-BCOADC-E2), another frequently occurring anti-M2 in PBC patients. KB cell lines (CCL 17) mere utilized as source material, and BCOADC-E2 cDNA (971 bp) including the lipoic acid binding domain was am plified by polymerase chain reaction. The amplified region was subcloned in to pEX-3 vectors and expressed, and the resulting fusion protein (beta-gala ctosidase/beta COADC-E2) was utilized as antigen for an ELISA, We ascertain ed the specificity of this antigen by inhibition tests with ELISA and immun oblotting. We defined the cut-off optical density (OD) value as the mean 3 SD (0.146) of sera from 60 normal controls. Anti-BCOADC-E2 could not be d etected with this assay in sera from normal controls and from patients with autoimmune hepatitis and chronic viral hepatitis, Anti-BCOADC-E2 was detec ted in 119 of 210 sera (56.7%) from patients with PBC, In addition, anti-BC OADC-E2 was detected in 48 of 99 (48.5%) sera from PBC patients who mere ne gative for anti-PDC-E2, sere, we have succeeded in developing a new ELISA f or detecting anti-BCOADC-E2. This system is antigen-specific and easily per formed. This assay should allow routine testing of a large number of serum samples, and should become especially useful for the serodiagnosis of anti- PDC-E2-negative PBC patients.