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 ...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 Ez (PDCE2), 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. coliM15 (pREP4) for expression, which was induced by isopropylthio-β-Dgalactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M2-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 M2antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M2-antibody positive.
CONCLUSION: Compared with the routine immunofluorescenoe 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.展开更多
文摘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 Ez (PDCE2), 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. coliM15 (pREP4) for expression, which was induced by isopropylthio-β-Dgalactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M2-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 M2antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M2-antibody positive.
CONCLUSION: Compared with the routine immunofluorescenoe 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.
