Monovalent binding of autoantibodies to beta(2)-glycoprotein I, detected using surface plasmon resonance at low antigen density

The precise mechanism of interaction between autoantibodies and beta(2)-gly coprotein I (beta(2)GPI) and the experimental conditions to be used for the ir detection are still under debate. Until now, these interactions have bee n studied under static conditions. We have investigated the interactions of purified IgG from 25 lupus anticoagulant-positive patients with immobilize d beta(2)GPI under flow conditions by real-time analysis based on surface p lasmon resonance technology. Sensor chips were coated with purified human b eta(2)GPI coupled to dextran via amino groups at low densities (1.4, 1.8 or 2.4 ng beta(2)GPI/mm(2)). Four patients' IgG displayed efficient binding a nd had the highest so-called antiphospholipid IgG levels by enzyme-linked i mmunosorbent assay (ELISA) and the highest absorbance values in an anti- be ta(2)GPI ELISA at a beta(2)GPI density reported to be around 12 ng/mm(2). B inding of antibodies to the beta(2)GPI sensor chips proved to be dependent upon the IgG concentration and beta(2)GPI density and was inhibited by a ra bbit antibody against beta(2)GPI. Similar association and dissociation prof iles were observed for the four efficient binders. The fast rate of dissoci ation limited the binding of autoantibodies to beta(2)GPI and was highly su ggestive of a monovalent association, confirmed by binding of Fab fragments under similar experimental conditions. In conclusion, monovalent binding o f low-affinity antibodies to beta(2)GPI immobilized at a density as low as 1.8 ng/mm(2) could be detected using surface plasmon resonance.