Murine Reagents and Analytical Tools to Enhance the Utility of Mouse Models for the Study of Human Thrombotic Disorders and Disease States.

The mechanisms that govern blood coagulation and fibrinolysis have been studied extensively by employing a variety of experimental techniques and in-vivo model systems. Murine models are a valuable resource to test new strategies in the areas of thrombosis and haemostasis. Because both normal and genetically altered mice are available, the murine models are ideal for this application. Murine models are in prevalent use and there exists a limited number of reagents available to assist the researcher. Development of murine reagents and analytical tools will enhance the utility of murine coagulation models by allowing researchers to acquire appropriate standards for characterization, validation, and screening of murine model systems. Recently, we have purified and characterized a group of nine mouse plasma proteins including the enzymatic forms of many of these proteins. A total of eight new polyclonal antibodies to murine antigens were generated including sheep a-mouse plasminogen and sheep anti-mouse factor X. Here we report on the development of two competitive based ELISA’s for the quantitation of mouse plasminogen and mouse factor X. The assay format utilizes purified antigen (plasminogen and factor X) standard. A standard curve is generated based upon the competition between fluid-phase antigen and an antigen-biotin conjugate for an immobilized affinity purified polyclonal antibody (sheep a-mouse plasminogen and sheep anti-mouse factor X). Solid-phase antibody/biotinyl-antigen complexes are detected using avidin-peroxidase. A colormetric signal, which is inversely proportional to the amount of antigen in the fluid-phase, is obtained following the addition of chromogenic substrate. Calibration curves are generated for an immobilized antibody ELISA by plotting the absorbance at 490 nm versus the concentration of non-biotin labeled antigen standard. Biotinylated-antigens were used at single concentrations of 50 ng/ml and 10 ng/ml for factor X and plasminogen respectively. In both cases, immobilized antibodies were adsorbed to microtiter plates at 5ug/ ml. Linearity of the factor X assay extends from approximately 10 ng/ml to 1000 ng/ml. The intra-assay coefficient of variation (CV) is ≤1.1 %, while the inter-assay CV is ≤5 %. Linearity of the plasminogen assay extends from approximately 0.1 μg/ml to 10 μg/ml. The intra-assay coefficient of variation (CV) is ≤2.0 %, while the inter-assay CV is ≤6.3 %. Preliminary studies have demonstrated the feasibility of these assays. With further testing and analysis these assays may prove useful to the researcher attempting to quantitate mouse plasma concentrations of these analytes.