ELCA ASSAY OF CLINICALLY IMPORTANT CLOTTING FACTORS

A NEW TECHNIQUE HAS BEEN DEVELOPED TO ASSAY CLOTTING FACTORS: SOLID-PHASE FIBRINOGEN AND LABELLED FIBRINOGEN TOGETHER ARE USED AS A SUBSTRATE TO DETECT THROMBIN GENERA- TED FROM THE CLOTTING CASCADE. WHEN THROMBIN CLEAVES BOTH FORMS OF FIBRINOGEN TO FIBRIN, THE LABELLED FIBRIN IN SOLU- TION ASSOCIATES WITH THE SOLID PHASE. THIS TECHNIQUE, WHICHHAS BEEN NAMED "ENZYME-LINKED COAGULATION ASSAY" OR ELCA, ISMORE SENSITIVE THAN EXISTING METHODOLOGIES AND CAN CONVENIENTLY BE MODIFIED TO MEASURE AN CLOTTING FACTOR OF INTEREST. THIS TECHNIQUE WAS DEVELOPED USING MICROTITER PLATES COATED WITH FIBRINOGEN AS THE SOLID PHASE, AND PEROXIDASE-FIBRINOGEN IN SOLUTION AS THE LABELLED FIBRINOGEN. BECAUSE PIPETTING EQUIPMENT AND PLATE READERS DESIGNED FOR ELISA ASSAYS IN MICROTITER PLATES CAN BE USED IN THIS ASSAY, SPECIAL EQUIPMENT DOES NOT HAVE TO BE DESIGNED FOR PERFORMING THIS ASSAY. THE OBJECTIVE OF THIS PROJECT IS TO DEVELOP KITS CONTAINING APPROPRIATE MIXTURES OF REAGENTS FOR ASSAY OF ANTITHROMBIN III, PROTEIN C, AND FACTORS II, X, VIII, AND IX. THE ASSAY WILL BE DESIGNED USING A SINGLE FORMAT FOR DILUTION OF PLASMAS AND ADDITION OF REAGENTS, AND SOFTWARE WILL BE DEVELOPED FOR ANALYSIS OF DATA TRANSFERRED DIRECTLY TO A MICROCOMPUTER FROM THE PLATE READER. THIS TECHNIQUE, WHEN DEVELOPED IN KIT FORM FOR THE ASSAY OF SPECIFIC FACTORS, SHOULD IMPROVE THE DIAGNOSIS OF ANUMBER OF DISEASES FOR WHICH CLOTTING FACTOR ASSAY IS AN IMPORTANT COMPONENT IN DISEASE MANAGEMENT.