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

Sanghoon Cha, Patrick S Leung, M. Eric Gershwin, Mark P. Fletcher, Aftab A. Ansari, Ross L. Coppel

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the λ phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC - dihydrolipoamide acetyltransferase, 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 = 10-7-10-9M-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 pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients 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 will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.

Original languageEnglish (US)
Pages (from-to)2527-2531
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number6
StatePublished - Mar 15 1993

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Dihydrolipoyllysine-Residue Acetyltransferase
Biliary Liver Cirrhosis
Autoantigens
Autoantibodies
Immunoglobulins
Antigens
Clone Cells
Immunoglobulin Genes
Bovine Serum Albumin
Serum
Immunoblotting
Bacteriophages
Histones
Autoimmune Diseases
Immunoglobulin G
Lymph Nodes
Epithelial Cells
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Staining and Labeling

Keywords

  • Autoimmunity
  • Combinatorial immunoglobulin antibodies
  • Primary biliary cirrhosis
  • Pyruvate dehydrogenase complex-E2

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Combinatorial autoantibodies to dihydrolipoamide acetyltransferase, the major autoantigen of primary biliary cirrhosis. / Cha, Sanghoon; Leung, Patrick S; Gershwin, M. Eric; Fletcher, Mark P.; Ansari, Aftab A.; Coppel, Ross L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 6, 15.03.1993, p. 2527-2531.

Research output: Contribution to journalArticle

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AB - mRNA from a regional lymph node of a patient with primary biliary cirrhosis (PBC) was used to construct a combinatorial immunoglobulin library in the λ phage vector system. Six human monoclonal IgG Fab clones (LC1-LC6) specific for the major autoantigen of PBC - dihydrolipoamide acetyltransferase, 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 = 10-7-10-9M-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 pattern in HEp-2 cells but react strongly with the luminal aspect of biliary epithelial cells of patients 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 will facilitate the analysis of immunoglobulin gene structure, idiotype, and antigen-antibody interactions in autoimmune disease.

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