Immunogenetic analysis of a panel of monoclonal IgG and IgM anti-PDC-E2/X antibodies derived from patients with primary biliary cirrhosis.
OBJECTIVE
Autoantibodies with specificity for the E2 component of the pyruvate dehydrogenase complex (PDC-E2) are commonly present in primary biliary cirrhosis. The aim of this study was to generate and characterise human anti-PDC-E2 monoclonal antibodies and analyse immunoglobulin gene usage and mutation for clues to pathogenesis.


METHODS
Peripheral B-lymphocytes from two patients with primary biliary cirrhosis were used to generate heterohybridomas secreting PDC-E2 specific monoclonal antibodies. The antibodies were characterised by ELISA, immunoblotting, indirect immunofluorescence and enzyme inhibition techniques, and their encoding immunoglobulin genes were amplified, cloned and sequenced.


RESULTS
Four IgGlambda and one IgMlambda monoclonal antibodies specific for PDC-E2 were generated: all gave bands at 74 kD and 52 kD on PDC immunoblots, two clones were specific for the lipoylated inner lipoyl domain, and all inhibited target enzyme function. Sequence analysis suggested unrestricted VH gene usage, but a strong preference for lambda light chains. The extent of somatic mutation was high (3-20%), with evidence for antigen selection in 3/5 VH sequences.


CONCLUSIONS
These monoclonal antibodies closely resemble the hallmark autoantibodies of primary biliary cirrhosis. Their specificities demonstrate true cross reactivity between an epitope on PDC-E2 and Protein X, and the existence of a subset of B cells that recognise only the lipoylated form of the antigen. The pattern of immunoglobulin gene mutations suggests an antigen-driven selection of high affinity IgG autoantibodies, supporting a possible role for exogenous antigen in the pathogenesis of primary biliary cirrhosis.
Thomson
R K
RK
Tenovus Laboratory, and University Medicine, Southampton University Hospitals, UK. rkt@soton.ac.uk
Davis
Z
Z
Palmer
J M
JM
Arthur
M J
MJ
Yeaman
S J
SJ
Chapman
C J
CJ
Spellerberg
M B
MB
Stevenson
F K
FK
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D005260:Female; D005815:Genetic Code; D006801:Humans; D015151:Immunoblotting; D007125:Immunogenetics; D007074:Immunoglobulin G / Q000032:analysis* / Q000235:genetics; D007075:Immunoglobulin M / Q000032:analysis* / Q000235:genetics; D008105:Liver Cirrhosis, Biliary / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009154:Mutation; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011768:Pyruvate Dehydrogenase Complex; D051047:Dihydrolipoyllysine-Residue Acetyltransferase