ULTRASENSITIVE IMMUNOASSAYS FOR CLOTTING FACTORS

WE SEEK TO DEVELOP AN ULTRASENSITIVE IMMUNOASSAY FOR PEPTIDES WHICH ARE RELEASED DURING THE ACTIVATION OF CLOTTING FACTORS. UNTIL NOW, THE ONLY ACTIVATION PEPTIDE WHICH HAS RECEIVED WIDESPREAD ACCEPTANCE FOR CLINICAL DIAGNOSIS IS THE FIBRINOPEPTIDE A RELEASED WHEN FIBRINOGEN IS CONVERTED TO FIBRIN BY THROMBIN. OTHER ACTIVATION PEPTIDES SHOULD BE RELEASED EARLIER IN THE PROCESS OF THROMBOSIS, AND MEASUREMENT OF THESE COULD BE OF VALUE IN EVALUATING THE 'THROMBOTIC STATE". WE WILL ATTEMPT TO DESIGN SYSTEMS FOR EVALUATING THE UTILITY OF THESE OTHER PEPTIDES (E. G. PEPTIDES OF II, X, IX, XI). THEY WILL FIRST APPLY A NEW IMMUNOASSAY AMPLIFICATION SYSTEM BASED ON PREPARING SNAKE VENOM CONJUGATES OF PEPTIDES (SPECIFICALLY RUSSELL'S VIPER VENOM FACTOR X ACTIVATOR). THE CONJUGATES WILL BE MEASURED USING A NEW ULTRASENSITIVE COAGULATION ASSAY BASED IN MICROTITER PLATES (ENZYME-LINKED COAGULATION ASSAY OR ELCA, THE CORE TECHNOLOGY OF ELCATECH). DURING THE PHASE I EFFORT, THEY WILL PREPARE CONJUGATES OF FIBRINOPEPTIDE A AND F1.2 OF PROTHROMBIN WITH RBB-XA, AND DEVELOP COMPETITIVE ASSAYS USING THESE CONJUGATES. VARIOUS ASSAY PROTOCOLS WILL BE TESTED TO OBTAIN OPTIMAL SENSITIVITY FOR COMPETITIVE ASSAYS FOR THESE PEPTIDES, AND THESE ASSAYS WILL BE COMPARED WITH STANDARD RADIOIMMUNOASSAY (RIA) PROTOCOLS. ONCE THE MOST SENSITIVE PROTOCOL FOR MEASUREMENT OF PEPTIDES IS IDENTIFIED, THEY USE THIS PROTOCOL FOR MEASUREMENT OF THESE PEPTIDES IN PLASMA SAMPLES, AGAIN USING STANDARD RIA FOR COMPARISON. THIS PROJECT WILL EXTEND THE NUMBER OF APPLICATIONS FOR THIS BASIC TECHNOLOGY IN THE AREA OF THROMBOSIS RESEARCH, RESULTING IN A SINGLE TECHNOLOGY WHICH IS UNIVERSALLY APPLICABLE TO MEASUREMENT OF RELEVANT COAGULATION PARAMETERS.