Isolation, characterization and sequence analysis of five IgG monoclonal anti-beta 2-glycoprotein-1 and anti-prothrombin antigen-binding fragments generated by phage display

J Immunol. 1999 Oct 15;163(8):4604-11.

Abstract

We have isolated five monoclonal IgG anti-beta 2-glycoprotein-1 (anti-beta 2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta 2GP-1-enriched mAbs (B14, B22, B27) reacted with beta 2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with beta 2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for beta 2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between beta 2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to beta 2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta 2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta 2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aged
  • Amino Acid Sequence
  • Antibodies, Antiphospholipid / chemistry
  • Antibodies, Antiphospholipid / genetics
  • Antibodies, Antiphospholipid / isolation & purification
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / metabolism
  • Bacteriophages / chemistry*
  • Bacteriophages / genetics
  • Bacteriophages / immunology
  • Base Sequence
  • Binding Sites, Antibody
  • Binding, Competitive
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • Glycoproteins / immunology*
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / isolation & purification
  • Immunoglobulin Fab Fragments / metabolism*
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / genetics
  • Immunoglobulin G / isolation & purification*
  • Immunoglobulin G / metabolism
  • Male
  • Molecular Sequence Data
  • Prothrombin / immunology*
  • Prothrombin / metabolism
  • Sequence Analysis, DNA
  • beta 2-Glycoprotein I

Substances

  • Antibodies, Antiphospholipid
  • Antibodies, Monoclonal
  • Glycoproteins
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G
  • beta 2-Glycoprotein I
  • Prothrombin