- Meeting abstract
Characterization of binding interactions in solution between β-2-glycoprotein I and monoclonal antibodies
Arthritis Research & Therapy volume 4, Article number: 91 (2002)
The circulating so-called antiphospholipid antibodies associated with autoimmune thrombophilia appear in most cases to be directed against phospholipid-binding proteins, chiefly apolipoprotein H (β-2-glycoprotein I, β2gpI) or prothrombin. Accordingly, the determination of antibodies reacting with β2gpI in solid-phase immunoassays is useful in the diagnostic workup of thrombophilic patients. These antibodies are characterized by significant binding only when β2gpI is immobilized on "high binding", chemically activated, or phospholipid-coated surfaces. It is not yet known, however, if the binding of antibodies is a result of a specific conformation that β2gpI attains when immobilized on a surface or simply a reflection of a sufficiently high epitope density. We are establishing solution binding assays based on capillary electrophoresis to measure antibody-β2gpI interactions without immobilizing any of the interacting molecules. Here we describe the optimization of analysis conditions in uncoated and coated capillaries for native and succinylated human β2gpI. Monoclonal anti-β2gpI and the humanized monoclonal antibodies proposed as standards for solid-phase assays were used as model ligand systems and we demonstrate the measurement and quantitation of binding. The influence of changes in buffer conditions such as ionic strength, pH, and of buffer additives including heparin - a known β2gpI ligand - is easily assessed by this approach and minute amounts of biological material are consumed (nanoliter injection volumes). The set-up is well suited for determining weak affinity interactions and will be helpful in providing answers to the questions about solution affinities of patient antibodies for native β2gpI and thus for understanding the molecular pathology behind the thrombophilic effect of anti-β2gpI autoantibodies.
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Heegaard, N., Schou, C., Kogutowska, E. et al. Characterization of binding interactions in solution between β-2-glycoprotein I and monoclonal antibodies. Arthritis Res Ther 4 (Suppl 1), 91 (2002). https://doi.org/10.1186/ar538