Changes in binding affinity of a monoclonal antibody to a platelet binding domain of fibrinogen adsorbed to biomaterials

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Abstract
Previously, we found that when fibrinogen-coated polyurethanes resided in a buffer for a period of time (the 'residence time') platelet adhesion to these materials decreased. Other changes in adsorbed fibrinogen such as decreases in polyclonal antibody binding and SDS elutability supported the conclusion that fibrinogen undergoes postadsorptive conformational changes. Subsequently we measured the binding of monoclonal antibodies to the three putative platelet binding sites on fibrinogen, using a single mid-range concentration of antibody. We found that binding of a monoclonal antibody to the platelet binding site at the C-terminus of the gamma chain of fibrinogen changed little with residence time, while binding of monoclonal antibodies to the other two putative binding sites on fibrinogen either increased with residence time (RGDF at Aα 95-98), or first increased and then decreased with residence time (RGDS at Aα 572-575). In the current study, we measured antibody binding affinity, Ka, by measuring antibody binding at a series of antibody concentrations. This is a more sensitive method for detecting changes in adsorbed fibrinogen than measuring antibody binding from a single antibody concentration. The Ka was determined for two antibodies, M 1 (4A5), which binds to a platelet binding domain of fibrinogen (y 402-411) and R 1 (155 B1616), which binds to residues 87-100 of the Aα chain (containing an RGDF site). A summary of the results for the M1 antibody are as follows. The Ka was higher for M1 binding to fibrinogen adsorbed to Immulon I® than to Biomer®, Biospan® or poly(ethylene terephthalate), suggesting that fibrinogen adsorbed to Immulon I® is more platelet adhesive than fibrinogen adsorbed to the other polymers. On Biospan®, the Ka decreased from 2.8 x 109 to 1.0 x 109 M-1 after a 24 h 37°C residence time, which correlated with the decrease in platelet adhesiveness of adsorbed fibrinogen observed previously under these conditions. The change in Ka was greater when adsorbed fibrinogen was kept under denaturing conditions. For example, the Ka decreased from 2.8 x 109 to 0.8 x 109 M-1 after a 1 h 70°C residence time whereas it remained approximately the same, 2.9 x 109 M-1, after a 24 h 0°C residence time.

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