Isolation and characterization of two human monoclonal anti-phospholipid IgG from patients with autoimmune disease.
The antigenic specificity of anti-phospholipid antibodies (APA) is a matter of intensive investigation. To further characterize these antibodies, we attempted to isolate human monoclonal APA. B-cells of patients with at least one positive test for antibodies against cardiolipin, phosphatidylserine, beta2-glycoprotein I (beta2-GPI) or the lupus anti-coagulant were immortalized by transformation with Epstein-Barr virus and screened for production of specific IgG. Positive pools were fused with a heteromyeloma cell line and APA-secreting clones were isolated by standard procedures. Two monoclonal APA, HL-5B from a 51-year-old man with primary anti-phospholipid syndrome and recurrent cerebral microinfarctions, and RR-7F from a 48-year-old women with systemic lupus erythematosus but no evidence for thrombotic events were obtained. HL-5B is of the IgG2 subtype with lambda light chains, while RR-7F is IgG2 with kappa light chains. Both monoclonals show reactivity against cardiolipin and phosphatidylserine but lack reactivity against beta2-GPI or lupus anti-coagulant activity. To yield the same OD in the cardiolipin and phosphatidylserine ELISAs RR-7F must be used in an approximately 10-fold higher concentration than HL-5B, indicating a lower affinity towards these antigens. Interestingly, both mAPA can bind to cardiolipin in the absence of beta2-GPI. They do not cross-react with dsDNA but show reactivity against oxidized low-density lipoproteins. Analysis of the heavy chain mRNA of HL-5B and RR-7F showed that both are members of the VH3 family. While HL-5B shows extensive somatic mutations in the CDR1 and 2 regions, indicating that it was derived by a T cell-dependent antigen driven process, RR-7F is apparently germline encoded. The two monoclonal APA can be used as tools in further structural and functional analyses.
von Landenberg
C
C
Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, Germany.
Lackner
K J
KJ
von Landenberg
P
P
Lang
B
B
Schmitz
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D016736:Antiphospholipid Syndrome / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D002308:Cardiolipins / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D016682:Lupus Coagulation Inhibitor / Q000032:analysis; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010718:Phosphatidylserines / Q000276:immunology; D010743:Phospholipids / Q000276:immunology; D053482:beta 2-Glycoprotein I
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D002308:Cardiolipins; D006023:Glycoproteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D016682:Lupus Coagulation Inhibitor; D010718:Phosphatidylserines; D010743:Phospholipids; D053482:beta 2-Glycoprotein I