Isolation of human anti-branched chain alpha-oxo acid dehydrogenase-E2 recombinant antibodies by Ig repertoire cloning in idiopathic dilated cardiomyopathy.
The generation of human monoclonal autoantibodies is critical for understanding humoral immune response in autoimmunity. In this study, Ig gene repertoire cloning was performed from a regional lymph node of a patient with idiopathic dilated cardiomyopathy (IDCM), and the resulting combinatorial IgG library was screened with bovine branched chain alpha-oxo acid dehydrogenase-E-2 (BCOADC-E2), one of the autoantigens in IDCM. After three rounds of affinity selection, we isolated three human recombinant IgG Fab molecules, named BC1, BC2 and BC3, that specifically react with BCOADC-E2 by ELISA. Interestingly, BC2 showed weak cross-reactivity to pyruvate dehydrogenase complex-E2 (PDC-E2), another mitochondrial autoantigen found in primary biliary cirrhosis (PBC), and their kappa light chain genes have 95% homology with a light chain of the human anti-DNA antibody. Although the exact pathogenic effect of anti-BCOADC-E2 autoantibodies is still unknown in IDCM, the potential binding specificity and limited light chain gene usage of our recombinant IgG molecules may shed light on the initial mechanism as to how autoantibodies start developing in IDCM.
Lee
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Division of Food & Biotechnology, College of Agriculture & Life Sciences, Kangwon National University, Chunchon, Korea.
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Cha
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D000217:Acyltransferases / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002311:Cardiomyopathy, Dilated / Q000235:genetics / Q000276:immunology*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000276:immunology*; D002417:Cattle; D003001:Cloning, Molecular; D015723:Gene Library; D005803:Genes, Immunoglobulin / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D000217:Acyltransferases; C033588:dihydrolipoamide acyltransferase