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

Abstract

Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping of PDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five monoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fc), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial autoantigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2.

Figure 1

Antigen specificity of IgM and IgG secreting human monoclonals. Supernatants from human hybridoma were assayed for their reactivity against a panel of antigens by ELISA. The results were scored by measuring the optical densities of the individual wells. Note the high optical density (mean ± SEM) observed with PDC-E2 as antigen but not the other antigens, P < 0.01 Student’s t-test. ■ PDC-E2; ▨ histone; ds DNA; ▧ IgG (Fc); □ collagen; ▩ F protein; ▤ rBCKD-E2

Figure 2

Beef heart mitochondria were separated by SDS-PAGE, blotted and probed with antisera. Lanes 1, 2 and 3 correspond to the IgG monoclonals C6, C11 and G2 respectively. Lane 4 has been probed with a known positive PBC serum control. Lane 5 has been probed with a normal human serum control. Note that the human monoclonals in lanes 1, 2 and 3 react only to PDC-E2 and not to either protein X or BCKD-E2. In contrast, sera from the patient with PBC react to the 74 kDa PDC-E2, the 55 kDa protein X and the 52 kDa BCKD-E2. This pattern of unique reactivity to only the 74 kDa antigen with the human monoclonals was also found even with more concentrated preparations of antibody (data not shown).

Figure 3

Lysates of E. coli containing the full length human PDC-E2 were separated by SDS-PAGE, blotted and probed with various preparations of monoclonal C11. The 180 kDa band is the recombinant fusion protein. The monoclonal antibodies used were initially absorbed with E. coli crude lysates containing expression clones of various domains of PDC-E2. The monoclonal C11 was absorbed with the following: an irrelevant control plasmid (lane 1); the full length PDC-E2 (lane 2); amino acids 1–96 (lane 3); amino acid residues 91–227 (lane 4); amino acid residues 128–227 (lane 5); amino acid residues 136–227 (lane 6); amino acid residues 146–227 (lane 7); amino acid residues 160–227 (lane 8); amino acid residues 181–227 (lane 9); amino acid residues 229–401 (lane 10); amino acid residues 396–560 (lane 11). Note the removal of reactivity by the full-length PDC-E2, amino acid residues 91–227 (lane 4), 128–227 (lane 5) and 136–227 (lane 6). In contrast, control plasmid (lane 1) and clones containing amino acid residues 1–96 (lane 3), 146–227 (lane 7), 160–227 (lane 8), 181–227 (lane 9), 229–401 (lane 10) and 396–560 (lane 11) do not absorb reactivity. A similar pattern was seen with the other IgG monoclonal reagents.

Figure 4

A schematic representation of the reactivity of PBC sera and C6, C11 and G2 monoclonal antibodies against expression cDNA clones of human PDC-E2. Note the specific reactivity against the inner lipoyl domain and within amino acid residues 91–227, 128–227, 136–227 of the inner lipoyl domain. Other expression clones do not react. (+) Positive reactivity; (−) negative reactivity; (*) partial reactivity. Data compared with the control value of the full length PDC-E2 (Table 2) and for each monoclonal respectively.