The objective of this research is to develop specific methods for the detection and quantitative measurement of P210bcr-abl in cells, tissues and body fluids from Philadelphia chromosome- positive patients with chronic myelogeneous leukemia. The intent is to develop sensitive assays to detect low levels of the marker protein, P210bcr-abl, in patients with residual disease. A radioimmune assay (RIA) will be developed as a rapid methods for specifically detecting P210bcr-abl. Three different anti-peptide antibodies have been developed which can detect the P210-bcr-abl protein. One antibody detects the bcr portion of the protein whereas the other two detect the abl portion of the protein. These antibodies, and others to be produced, will be used in a double antibody immunometric assay. The strategy is to attach an antibody detecting one segment of the chimeric protein (e.g. bcr encoded N-terminal sequences) to a bead which allows the binding of P210bcr-abl. A second antibody (anti-abl), tagged with an isotope or fluorescent label, is used that is capable of binding the more C-terminal abl part of the protein. The amount of double antibody complex will be determined by measuring the amount of tagged antibody specifically bound to the immunobead; this number will allow estimation of the amount of P210 in the sample being analyzed. This assay will be used to monitor levels of P210 in various tissues and fluids of patients and normal individuals. In addition monoclonal antibodies to peptide sequences predicted from the two bcr-abl junctions of P210bcr-abl will be prepared and used to develop a competitive RIA. This approach will provide a specific test to detect P210 in Philadelphia-positive cases of CML. A second assay based on immunoblotting methods will be developed to verify and confirm the results of the RIA; such an assay has potential as the method of choice for detecting P210 in a reliable and specific manner. The immunoblot assay will use both anit-bcr and anti-abl peptide antibodies under conditions where each antibody can be competed with its respective peptide immunogen to allow assessment of the specificity of the blot detection. A third assay will be developed which will allow detection of the tyrosine kinase associated with P210 and related proteins. The normal c-abl product (P145), although having a tyrosine kinase activity, does not score positive in our anti-abl kinase assay. Thus, a specific is very feasible.
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| Li, W J; Smith, L A; Kabat, K G et al. (1989) Differential effects of tumor promoters on P210bcr-abl expression. Hematol Pathol 3:113-23 |
