Immunochemical detection by specific antibody to thrombin of prothrombin conformational changes upon adsorption to artificial surfaces

Abstract

Polyclonal antihuman alpha-thrombin antibodies produced in rabbits reacted minimally (less than 0.05%) in solution with human prothrombin. However, when prothrombin was adsorbed to artificial surfaces such as polyvinyl chloride (PVC), the cross-reactivity of surface-bound prothrombin with antibody IgG to thrombin (greater than 95% purity) was shown to be significantly enhanced. On PVC, the molar ratios of antibody IgG to thrombin/prothrombin approached the same level as that of antibody IgG to thrombin/thrombin when thrombin was adsorbed to the same material. The analyses of antigen-antibodies interaction, in solution with a direct binding assay by immune precipitation at high-speed centrifugation (160,000 g, 30 min), and on solid-phase PVC, were accomplished by use of double-labeling technique, i.e., 131I-thrombin (or 131I-prothrombin) and 125I-antibody IgG to thrombin. The results appear to suggest that prothrombin adsorption to PVC has resulted in some molecular conformational changes so that immunologically the adsorbed prothrombin resembles that of adsorbed thrombin on the same PVC surface.