| The incidence of cardiovascular diseases has increased during the last few decades, becoming a major cause of death in developed countries. Plasminogen activators including tissue-type plasminogen activator (t-PA), streptokinase (SK), urokinase (u-PA), and staphylokinase (SAK) are obvious choices for pharmaceutical treatment of blood clots. Therefore, accurate, rapid and cost effective assay methods for different plasminogen activators are of interest for production and clinical purposes as well as for standardization. Direct measurement methods are specific, but expensive. Indirect methods are less expensive and more versatile but they are difficult to standardize and require large amounts of reagents and sample. Recently, a thickness shear mode (TSM) acoustic sensor and a micro version of the rapid fibrin plate method (two-layer method) were used to develop a rapid and reliable method but with a high percentage of error.;This thesis is an investigation of an improved and reliable method for determination of performances and equivalences of plasminogen activators using a TSM sensor. To improve the two-layer method, the TSM sensor was redesigned to control the environmental conditions. To solve the diffusion and uniformity problems associated with the two-layer method, a new one-step method was developed. This method was optimized using a modified simplex algorithm. The first optimization was carried out for SK and the levels of thrombin, fibrinogen, transglutaminase, plasminogen and temperature were optimized to increase the precision of the method. In the optimization for t-PA, ratios of thrombin and plasminogen to fibrinogen were used as control variables to decrease the number of variables, and to make this method easily adjustable. Calibration experiments for t-PA, SK, u-PA, and SAK were carried out using the optimized method. Calibration curves for all activators showed the same power law pattern. However, the activators could be categorized into two groups, fibrin and nonfibrin specific, based on their mechanism and on the form of their calibration equations. The relative standard deviation percent of readings of the optimized method was 11-14% indicating an improvement in precision ever existing methods. As well, it has the potential to be considered as a reference method to measure and standardize the activity of different activators. |
