Murine Reagents and Analytical Tools

描述(由申请人提供)：通过使用各种实验技术和体内模型系统，已经广泛地研究了控制血液凝固和纤溶的机制。最近，基因打靶(基因敲除小鼠)和转基因技术被用来创造表型代表人类血栓形成障碍和疾病状态的小鼠品系。与此相结合，小鼠凝血模型在新药开发过程中也被常规使用。在开发新的促凝剂、抗凝剂和溶解血栓药物的过程中，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。由于既有正常小鼠，也有基因改变的小鼠，小鼠模型是这种应用的理想选择。基于这样一个事实，即小鼠模型被广泛使用，而可用于辅助研究人员的试剂数量有限，开发用于这一研究领域的商业化试剂群是当务之急。该项目旨在通过产生一系列试剂和分析工具来增强小鼠凝血模型的实用性，这些试剂和分析工具可与可用于人类系统的试剂和分析工具相媲美。长期目标是生产一系列商品化的小鼠衍生试剂，包括：a)用于血浆重建实验和测定标准化的纯化小鼠凝血因子；b)用于免疫印迹、免疫组织化学、免疫沉淀、流式细胞术和ELISA应用的小鼠凝血因子抗体；以及c)用于凝血酶原、酶、辅因子、抑制物、激活肽和酶/抑制物复合体的ELISA法。在I期支持期间，我们纯化并鉴定了一组9种小鼠血浆蛋白，包括凝血酶原和凝血因子X的酶形式。总共产生了7种新的抗鼠抗原的多克隆抗体。我们还证明了两种基于竞争的酶联免疫吸附试验最终测定小鼠血浆中凝血因子X和纤溶酶原浓度的可行性。这些试剂目前正由HTI销售，可供购买。我们正试图获得第二阶段的资金，以通过增加我们日益增长的可用小鼠产品清单来进一步推进第一阶段取得的进展。我们计划提纯更多的止血蛋白，产生更多的多克隆和单克隆抗体，并利用这些试剂鉴定和开发具有临床重要性的免疫分析方法。

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.