title: Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.
abstract: OBJECTIVE
  To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE).
  
  
  METHODS
  A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations.
  
  
  CONCLUSIONS
  Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.
authors.affiliation: MCP Hahnemann University, Philadelphia, Pennsylvania, USA.
authors.forename: S N
authors.initials: SN
authors.lastname: Seal
authors.forename: R M
authors.initials: RM
authors.lastname: Hoet
authors.forename: J M
authors.initials: JM
authors.lastname: Raats
authors.forename: M Z
authors.initials: MZ
authors.lastname: Radic
mesh_terms: D000328:Adult; D000974:Antibodies, Antinuclear / Q000235:genetics; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000276:immunology; D004247:DNA / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D019151:Peptide Library; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000974:Antibodies, Antinuclear; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger; D004247:DNA; D001120:Arginine