Current methods for in vivo thrombus detection either lack specificity or pose additional health risks. With the tools of molecular immunology and syntheitic peptide chemistry, we plan to prepare a clinically useful, diagnostic antibody that possesses specificity for human fibrin (the structural protein in thrombi) and provides sufficient sensitivity for in vivo thrombus detection by gamma scintigraphy. Our strategy will emphasize the differences between fibrin and its soluble precursor, fibrinogen, and will focus on ways to select fibrinspecific antibodies. Synthetic peptides will be designed to mimic fibrinunique epitopes and used to elicit antibodies that will bind human fibrin in the presence of fibrinogen. This approach was validated during the previous grant period when we used a synthetic peptide corresponding to the N terminus of the human fibrin beta-chain to elicit monoclonal antibodies that bound to fibrin monomer in the presence of fibrinogen. The in vivo specificity of one monoclonal antibody (64C5) was confirmed in experiments using chicken, rabbit and dog models. In the research proposed here, we focus on the design and testing of various fibrin-like peptides corresponding to thrombin cleavage sites and Factor XIIIa cross-link sites, and describe plans for an improved screening strategy that uses fibrin miniclots rather than fibrin monomers as antigens.
Our specific aims i nclude the in vivo testing of antibody 5D7 as a gamma scintigraphic agent for detecting experimental thrombi; the preparation of monoclonal antibodies that avidly bind to clotted fibrin; the synthesis and refinement of cross-link antigens related to gamma-chain cross-link sites; and the structural and immunochemical study of the alpha-chain cross-link site in order to identify a new fibrin-unique epitope. Three specific benefits will result from these experiments: First, they will provide a detailed immunochemical analysis of unique epitopes on the fibrin molecule. They will also suggest general methods for differentiating any protein from its precursor. Second, they will provide reagents that can tag thrombi in vivo. Third, they will lay the foundation upon which a sensitive and specific noninvasive test for life-threatening thromboembolism can be built.
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