Monovalent binding of autoantibodies to beta2-glycoprotein I, detected using surface plasmon resonance at low antigen density.
The precise mechanism of interaction between autoantibodies and beta2-glycoprotein I (beta2GPI) and the experimental conditions to be used for their detection are still under debate. Until now, these interactions have been studied under static conditions. We have investigated the interactions of purified IgG from 25 lupus anticoagulant-positive patients with immobilized beta2GPI under flow conditions by real-time analysis based on surface plasmon resonance technology. Sensor chips were coated with purified human beta2GPI coupled to dextran via amino groups at low densities (1.4, 1.8 or 2. 4 ng beta2GPI/mm2). Four patients' IgG displayed efficient binding and had the highest so-called antiphospholipid IgG levels by enzyme-linked immunosorbent assay (ELISA) and the highest absorbance values in an anti- beta2GPI ELISA at a beta2GPI density reported to be around 12 ng/mm2. Binding of antibodies to the beta2GPI sensor chips proved to be dependent upon the IgG concentration and beta2GPI density and was inhibited by a rabbit antibody against beta2GPI. Similar association and dissociation profiles were observed for the four efficient binders. The fast rate of dissociation limited the binding of autoantibodies to beta2GPI and was highly suggestive of a monovalent association, confirmed by binding of Fab fragments under similar experimental conditions. In conclusion, monovalent binding of low-affinity antibodies to beta2GPI immobilized at a density as low as 1.8 ng/mm2 could be detected using surface plasmon resonance.
Regnault
V
V
UMR CNRS 7563, Laboratoire d'Hématologie, Faculté de Médecine, Hémostase-Hématologie et Service de Médecine H du CHU, Nancy, France.
De Maistre
E
E
Wahl
D
D
Lecompte
T
T
D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000378:metabolism*; D016736:Antiphospholipid Syndrome / Q000175:diagnosis*; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D020349:Surface Plasmon Resonance; D053482:beta 2-Glycoprotein I
D017152:Antibodies, Antiphospholipid; D006023:Glycoproteins; D007106:Immune Sera; D007074:Immunoglobulin G; D053482:beta 2-Glycoprotein I