Isolation, characterization and sequence analysis of five IgG monoclonal anti-beta 2-glycoprotein-1 and anti-prothrombin antigen-binding fragments generated by phage display.
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.
Chukwuocha
R U
RU
Department of Medicine, Division of Rheumatology, University of California, Los Angeles 90095, USA. rchukwu@ucla.edu
Hsiao
E T
ET
Shaw
P
P
Witztum
J L
JL
Chen
P P
PP
D000368:Aged; D000595:Amino Acid Sequence; D017152:Antibodies, Antiphospholipid / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001435:Bacteriophages / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000276:immunology* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D053482:beta 2-Glycoprotein I
D017152:Antibodies, Antiphospholipid; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D053482:beta 2-Glycoprotein I; D011516:Prothrombin