Characterization and epitope mapping of human monoclonal antibodies to PDC-E2, the immunodominant autoantigen of primary biliary cirrhosis

Leung, Patrick S.C.; Krams, Sheri M.; Muñoz, Santiago J.; Surh, Charles P.; Ansari, Aftab A.; Kenny, Thomas P.; Robbins, Dick L.; Fung, John J.; Starzl, Thomas E.; Maddrey, Willis C.; Coppel, Ross L.; Gershwin, M. Eric

doi:10.1016/0896-8411(92)90187-u

Cited by 24 publications

(9 citation statements)

References 46 publications

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“…Thus, these Fabs have similar specificities to those found in human PBC sera and in human monoclonal antibodies (41) and should allow identification of the VH and VL gene usage of these autoantibodies. Comparison of V gene sequences from human monoclonal antibodies and combinatorial autoantibodies with corresponding germ-line V genes will allow determination of the role of somatic mutation in production ofthese high-affinity Fabs.…”

Section: Discussionmentioning

confidence: 99%

“…This is similar to the binding profile offive human monoclonal antibodies derived by fusion of a regional lymph node from a PBC patient with a mouse-human heterohybrid cell line. All monoclonal antibodies were specific for PDC-E2 and showed minimal or no detectable binding to protein X (41).…”

Section: Discussionmentioning

confidence: 99%

“…The concentrated Fabs were resuspended in nonreducing SDS/PAGE sample buffer and electrophoresed by SDS/PAGE followed by immunoblotting as described (37,38 Specificity Determination. Antigen specificity was determined by ELISA as described (41). Microtiter plates were coated overnight at 4°C with recombinant human PDC-E2 containing the inner lipoyl domain (37), porcine PDC (Sigma), recombinant BCKD-E2 (42,43), histone-(Worthington), calf thymus double-stranded DNA (Sigma), and bovine serum albumin (Sigma) at 10 ,ug/ml in bicarbonate buffer (pH 9.5).…”

Section: Methodsmentioning

confidence: 99%

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Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

Cha

Leung

Gershwin

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTmRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the A phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC-dihydrolipoamide acetyltansferase, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) -were isolated, appearing at a frequency of 0.01% in the combinatorial immunoglobulin library. These Fab clones recognize human PDC-E2 with high affinity (Ka = 10-710-9 M-1). Using both immunoblotting and ELISA, LC1-LC6 showed little cross-reactivity to any of the other autoantigens commonly recognized by PBC sera or to other antigens commonly recognized by PBC sera or to other antigens such as histone, calf thymus DNA, and bovine serum albumin. The Fab monoclonal antibodies show a typical anti-mitochondrial staining patterm in HEp-2 cells but react strongly with the luminal aspect ofbiliary epithelial cells ofpatients with PBC. Our results demonstrate that a recombinant combinatorial immunoglobulin library can be used to isolate high-affinity Fabs against a specific autoantigen. Such reagents wfll facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

Cha

Leung

Gershwin

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous studies on identification of the T cell and AMA epitopes have focused on the major domains of the PDC-E2 as well as the requirement of LA for epitope recognition. [24][25][26][27][28][29][30][31][32] The consensus of these studies is that the immunodominant epitope is located within the ILD. We report herein on the characterization of 17 mAbs against the major autoantigen of PBC, PDC-E2, with particular emphasis on fine mapping of the epitopes that are recognized.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: The molecule versus the mimic

Migliaccio

2001

Hepatology

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The 2-oxo-acid dehydrogenase complexes and, in particular, the E2 component of the pyruvate dehydrogenase complex (PDC) are the target of antimitochondrial antibodies (AMA). More than 95% of primary biliary cirrhosis (PBC) patients have detectable levels of autoantibodies to PDC-E2 and in general these react with a region of the molecule that contains the prosthetic group lipoic acid (LA). LA is vital to the function of the enzyme, although there is conflicting evidence as to whether its presence is required for PDC-E2 recognition by AMA. Some, but not all, monoclonal antibodies (mAbs) to PDC-E2 produce an intense staining pattern at the apical surface of bile duct epithelial cells (BEC) in patients with PBC, and it has been argued that the molecule at the apical surface of PBC bile duct cells is a modified form of PDC-E2 or a cross-reactive molecule, acting as a molecular mimic. Herein, we characterize the epitopes recognized by 4 anti-PDC-E2 mAbs that give apical staining patterns (3 mouse and 1 human). In particular, by using a combination of recombinant antigens, competitive inhibition assays, and a unique peptide-on-bead assay, we determined that these apically staining mAbs recognize 3 or 4 distinct epitopes on PDC-E2. More importantly, this suggests that a portion spanning the entire inner lipoyl domain of PDC-E2 can be found at the BEC apical surface. In addition, competition assays with patient sera and a PDC-E2-specific mAb showed significant epitope overlap with only 1 of the 3 mouse mAbs and showed a differential response to the peptide bound to Primary biliary cirrhosis (PBC) is a destructive autoimmune disease of intrahepatic bile ducts characterized by inflammation of the portal triads, fibrosis, and the presence of antimitochondrial antibodies (AMA). 1,2 The major autoantigens recognized by AMA are related members of the 2-oxoacid dehydrogenase complexes including the pyruvate dehydrogenase complex (PDC-E2), the branched chain ketoacid dehydrogenase complex-E2, and the 2-oxo-glutarate dehydrogenase complex-E2. It has been hypothesized that a molecular mimic of the PDC-E2 molecule is expressed at high levels in the apical region of biliary epithelial cells (BEC) in PBC. [3][4][5] We have previously described a series of 8 monoclonal antibodies (mAbs) against the mitochondrial antigens of PBC that produce this disease-specific staining pattern. 6,7 Essentially, when probed with these disease-specific mAbs, small bile ducts in tissue from patients with PBC, but not controls, show intense staining at the apical surface of the cells lining the lumen. This pattern is in addition to the normal cytoplasmic pattern seen with mAbs against mitochondrial proteins. The fact that such apical staining is seen only with these select mAbs but not all mAbs that react with PDC-E2 has led to the hypothesis that a molecule(s) cross-reactive with PDC-E2 is located at the apical surface of PBC BEC. 3,4 Extensive efforts have been made to elucidate the AMA epitopes and link them to pathology. The consensus is that altho...

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“…The enzyme complex includes pyruvate dehydrogenase complex (PDC), the branched chain a-ketoacid dehydrogenase complex (BCKD), and the oxoglutarate dehydrogenase complex (OGDC), E1, E2 and E3 (Yeaman, 1989). Among these molecular targets, AMA are most commonly directed to the E2 component of PDC (Leung et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Oral Tolerance and Pyruvate Dehydrogenase in Patients with Primary Biliary Cirrhosis

Suzuki

Water

Gershwin

et al. 2002

Journal of Immunology Research

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Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the immunological destruction of intralobular bile ducts and serum anti-mitochondrial antibodies (AMA). Based upon previous work of oral tolerance and autoimmunity, we hypothesized that feeding the mitochondrial autoantigens of PBC would alter the clinical course and the level of antimitochondrial antibodies. The bovine pyruvate dehydrogenase complex (PDC) was purified and 5 mg fed in gelatin capsules to 6 patients with early stage PBC for 6 months. Antimitochondrial antibodies and liver biochemistries were measured at every 3 months for 12 months. The clinical trial was completed for all patients except for 1 who showed deterioration of pre-existing skin rash during treatment, which disappeared within 2 weeks after treatment was discontinued. However, after 1 year, neither the titers of AMAs nor liver biochemistries were significantly changed by this treatment. This is the first trial to test the efficacy of oral tolerance induction in PBC. However, the data, which limited in scope, did not demonstrate efficacy and further highlights the difficulties in showing continuing evidence of tolerance induction in autoimmunity.

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Characterization and epitope mapping of human monoclonal antibodies to PDC-E2, the immunodominant autoantigen of primary biliary cirrhosis

Cited by 24 publications

References 46 publications

Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis.

Heterogeneous response of antimitochondrial autoantibodies and bile duct apical staining monoclonal antibodies to pyruvate dehydrogenase complex E2: The molecule versus the mimic

Oral Tolerance and Pyruvate Dehydrogenase in Patients with Primary Biliary Cirrhosis

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