Detection of anti-branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 antibody in primary biliary cirrhosis by ELISA using recombinant fusion protein.
Anti-M2 of anti-mitochondrial antibody (AMA) is a serological marker of primary biliary cirrhosis (PBC). Anti-pyruvate dehydrogenase complex-E2 (anti-PDC-E2) is recognized as the most frequently occurring anti-M2, and a routine laboratory test for this antibody has already been established. However, it is also known that there are patients with PBC who are negative for anti-PDC-E2. For the serological diagnosis of these patients, immunoblotting for anti-M2s is indicated. However, the technique currently utilized is too laborious to allow testing of a large number of samples. In this study, we have developed an enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion protein in order to evaluate anti-branched chain 2-oxo-acid dehydrogenase complex-E2 (anti-BCOADC-E2), another frequently occurring anti-M2 in PBC patients. KB cell lines (CCL 17) were utilized as source material, and BCOADC-E2 cDNA (971 bp) including the lipoic acid binding domain was amplified by polymerase chain reaction. The amplified region was subcloned into pEX-3 vectors and expressed, and the resulting fusion protein (beta-galactosidase/BCOADC-E2) was utilized as antigen for an ELISA. We ascertained the specificity of this antigen by inhibition tests with ELISA and immunoblotting. 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 detected with this assay in sera from normal controls and from patients with autoimmune hepatitis and chronic viral hepatitis. Anti-BCOADC-E2 was detected in 119 of 210 sera (56.7%) from patients with PBC. In addition, anti-BCOADC-E2 was detected in 48 of 99 (48.5%) sera from PBC patients who were negative for anti-PDC-E2. Here, we have succeeded in developing a new ELISA for detecting anti-BCOADC-E2. This system is antigen-specific and easily performed. 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.
Miyakawa
H
H
Fourth Department of Internal Medicine, Teikyo University School of Medicine, Kanagawa, Japan.
Abe
K
K
Kitazawa
E
E
Kikuchi
K
K
Fujikawa
H
H
Matsushita
M
M
Kawaguchi
N
N
Kako
M
M
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007658:Ketone Oxidoreductases / Q000276:immunology*; D008105:Liver Cirrhosis, Biliary / Q000175:diagnosis / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008930:Mitochondria, Liver / Q000276:immunology; D009097:Multienzyme Complexes / Q000276:immunology*; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)