Murine Reagents and Analytical Tools

DESCRIPTION (provided by applicant): The mechanisms that govern blood coagulation and fibrinolysis have been studied extensively by employing a variety of experimental techniques and in-vivo model systems. Recently gene targeting (knockout mice) and transgenic techniques have been employed to create strains of mice whose phenotypes represent human thrombotic disorders and disease states. In conjunction with this, murine coagulation models are routinely used in the process of new drug development. During the course of developing new pro-coagulant, anti-coagulant and clot dissolving drugs, basic R&D studies as well as safety, efficacy, and pharmacokinetic studies are required. Because both normal and genetically altered mice are available, the murine models are ideal for this application. Based on the fact that murine models are in prevalent use and there exist a limited number of reagents available to assist the researcher, it is imperative to develop a commercial group of reagents for use in this field of study. This project was designed to enhance the utility of mouse coagulation models by generating a battery of reagents and analytical tools comparable to what is available for application to the human system. The long-term objective is the production of a marketable line of murine derived reagents that include: a) purified murine coagulation factors for plasma reconstitution experiments and assay standardization; b) antibodies to murine coagulation factors for Western blot, immunohistochemistry, immunoprecipitation, flow cytometry and ELISA applications; and, c) ELISA assays for the zymogens, enzymes, cofactors, inhibitors, activation peptides and enzyme/inhibitor complexes of coagulation. During phase I support we purified and characterized a group of nine mouse plasma proteins including the enzymatic forms of both prothrombin and factor X. A total of seven new polyclonal antibodies to murine antigens were generated. We also demonstrated the feasibility of two competitive based ELISA's to ultimately measure murine plasma concentrations of both factor X and plasminogen. These reagents are currently being marketed by HTI and are available for purchase. We are attempting to secure phase II funding to further the advances made in phase I by adding to our growing list of available murine products. We plan to purify additional hemostatic proteins, generate additional polyclonal and monoclonal antibodies, and identify and develop clinically important immunoassays with these reagents.