Cross-Reactivity of Rabbit Anti-Bovine Prothrombin/Thrombin IgGs With Bovine Factor V/Va-Related Antigens

The purpose of this study was to determine whether rabbit anti-bovine prothrombin/thrombin immunoglobulin Gs (IgGs) would cross-react with bovine factor V/Va-related antigens. Bovine prothrombin, crude thrombin, as well as 2 purified versions of thrombin, that is, thrombin 4A (the previous version of Thrombin-JMI marketed prior to 2008) and 4B (the currently marketed version of Thrombin-JMI), were administrated to individual groups of rabbits on days 0, 21, 42, 91, 123, and 151 using standard immunologic methods. Blood was drawn from each rabbit on days 30, 50, 105, 137, and 165 and the pooled antisera from individual groups were purified to obtain the IgGs using protein G affinity columns. By probing bovine factor V/Va samples, the possible cross-reactivity of each IgG collected at different time points (from day 30 to day 165) was explored using Western blotting techniques. The results indicated that rabbit anti-bovine prothrombin and crude thrombin IgGs could cross-react strongly with bovine factor V/Va in an immunization time-dependent manner. However, antibodies generated in thrombin 4A-treated rabbits presented much weaker cross-reactivity with bovine factor V/Va. Furthermore, no cross-reactivity with bovine factor V/Va-related antigens was observed when the anti-bovine thrombin 4B IgG collected at any time point was used. The results suggest that thrombin 4B preparation contains the least bovine factor V/Va contaminants among the bovine prothrombin/thrombin preparations studied and the amount of bovine factor V/Va contaminants in bovine thrombin 4B is too small to elicit the generation of antibodies against bovine factor V/Va in rabbits.