Murine Reagents and Analytical Tools

描述（由申请人提供）：通过采用各种实验技术和体内模型系统，已对控制血液凝固和纤维蛋白溶解的机制进行了广泛研究。最近，基因靶向（敲除小鼠）和转基因技术已被用于创建小鼠品系，其表型代表人类血栓性疾病和疾病状态。与此同时，在新药开发过程中通常使用鼠凝血模型。在开发新的促凝血、抗凝和溶凝药物的过程中，需要进行基础研发研究以及安全性、有效性和药代动力学研究。因为正常和基因改变的小鼠都是可用的，所以鼠模型是该应用的理想模型。基于鼠模型被普遍使用并且存在有限数量的试剂可用于辅助研究人员的事实，开发用于该研究领域的商业试剂组是必要的。该项目旨在通过产生一系列可用于人类系统的试剂和分析工具来提高小鼠凝血模型的实用性。长期目标是生产一系列可销售的鼠源性试剂，包括：a）用于血浆重建实验和测定标准化的纯化的鼠凝血因子; B）用于Western印迹、免疫组织化学、免疫沉淀、流式细胞术和ELISA应用的鼠凝血因子的抗体;和c）用于凝血酶原、酶、辅因子、抑制剂、活化肽和酶/抑制剂复合物的ELISA测定。在I期支持期间，我们纯化并表征了一组9种小鼠血浆蛋白，包括凝血酶原和因子X的酶形式。总共产生了七种新的针对鼠抗原的多克隆抗体。我们还证明了两种基于竞争的ELISA最终测量因子X和纤溶酶原的鼠血浆浓度的可行性。这些试剂目前由HTI销售，可供购买。我们正试图获得第二阶段的资金，通过增加我们不断增长的可用鼠产品清单，进一步推进第一阶段取得的进展。我们计划纯化额外的止血蛋白，产生额外的多克隆和单克隆抗体，并确定和开发临床上重要的免疫测定与这些试剂。

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.