Clinical Research
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 15, 2003; 9(6): 1352-1355
Published online Jun 15, 2003. doi: 10.3748/wjg.v9.i6.1352
Construction and expression of a humanized M2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis
Xiao-Hua Jiang, Ren-Qian Zhong, Sheng-Qian Yu, Yin Hu, Weng-Weng Li, Xian-Tao Kong
Xiao-Hua Jiang, Department of Laboratory Medicine, 85 Hospital the Chinese PLA, Shanghai 200052, China
Ren-Qian Zhong, Weng-Weng Li, Xian-Tao Kong, Clinical Immunology Center of the Chinese PLA, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
Sheng-Qian Yu, Department of Nephrology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
Yin Hu, Department of Basic Science, Shanghai University of Engineering Science, Shanghai 200335, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr Xiao-Hua Jiang, Department of Laboratory Medicine, 85 Hospital of the Chinese PLA, Huashan Road, Shanghai 200052, China. jhlulu@citiz.net
Telephone: +86-21-62528805 Fax: +86-21-33110236
Received: July 18, 2002
Revised: August 1, 2002
Accepted: August 7, 2002
Published online: June 15, 2003
Abstract

AIM: To construct and express a humanized M2 autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC).

METHODS: cDNA fragments encoding M2-reactive epitopes of pyruvate dehydrogenase complex E2 (PDC-E2), branched chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) and 2-oxo-glutarate dehydrogenase complex E2 (OGDC-E2) 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-β-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---positive sera confirmed at Euroimmun Research Center (Germany). Using the purified BPO, M2 antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA.

RESULTS: The expressed BPO was observed with both antigenic reactivity and specificity of M2 autoantigens. The determination of M2 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, M2 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 M2 antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M2-antibody positive.

CONCLUSION: Compared 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.

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