The objective of this grant application is to develop an ultrasensitive, generally useful immunoassay technique based on amplification via clotting cascade. We have developed a method for measuring a factor X-activator isolated from Russell's viper venom (RVV-XA) at concentrations of less than 50 femtograms per ml (0.1 attomoles per sample). The technique, which we have named 'enzyme-linked coagulation assay' or ELCA, is based on the dection of thrombin generated from the clotting cascade using peroxidase- fibrinogen as a substrate. In this application we will use this highly sensitive enzyme assay for the development of an ultrasensitive ELISA assay, using RVV-XA coupled to antibodies. In this project, we will use these labeled antibodies to develop ultrasensitive 'sandwich' immunossays and apply them to measurement of antigens in ovarian cancer. We will first develop the method by preparing conjugates of RVV-XA with monoclonal antibodies to placental phosphatase, and by testing several protocols for 'sandwich' immunoassays with these reagents. This will yield a method which can discriminate the several allelic and cancer-associated variants of this enzyme, at concentration appropriate for serum, ascites and cyst fluids, and tumor tissues of ovarian cancer patients. This method will then be used to measure these antigens in ovarian cancer. Since we can measure variants of this enzyme at relevant concentrations in serum, this will be first detailed analysis of the change in concentrations of the two distinct isoenzymes of this enzyme during development of disease. We will also develop an immunoblot method using the ELISA-ELCA method, in order to identify specific isoenzymes after electrophoresis. Using the methodology developed for placental phosphatase, we will then analyze another group of antigens, i.e., those associated with ovarian cancer which appear to elicit an immune response in these patients (autologous ovarian tumor-associated antigens). These antigens will be identified using antibodies isolated from 'membrane fragments' of ovarian cancer ascites fluids, labeled with RVV-XA and used in an electrophoretic assay for these antigens. If these antigens are found to be tumor-specific, then attempts will be made to isolate them and prepare monoclonal antibodies to them.
| Durkee, K H; Roh, B H; Doellgast, G J (1993) Immunoaffinity chromatographic purification of Russell's viper venom factor X activator using elution in high concentrations of magnesium chloride. Protein Expr Purif 4:405-11 |
| Durkee, K H; Cheng, T M; Doellgast, G J (1990) Enzyme-linked coagulation assay: V. Amplified blotting assays using snake venom conjugates. Anal Biochem 184:375-80 |
