Thrombus detection remains a challenge to the physician who must manage certain life-threatening problems such as coronary artery thrombi or pulmonary emboli neither of which can be reliably diagnosed. The long term objective of the proposed research is the use of fibrin specific antibodies to localize intravascular deposits of fibrin, a constitutive protein network in thrombi. During the current grant period, a novel immunochemical strategy to obtain fibrin specific antibodies has been validated: a synthetic peptide, chosen to mimic the amino terminus of the Beta-chain in human fibrin, has been used to derive three monoclonal anti(human)fibrins, 64C5, 55D10, and 59D8.
The aims of the present grant proposal (Y04-Y06) are to 1) develop suitable animal models for a quantitative evaluation of Mab-64C5, -55D10 and -59D8 as thrombus detecting agents and 2) prepare different antifibrin antibodies as alternative thrombus detecting agents. Both chicken and rabbits will be used to develop models suitable for detection of experimentally induced thrombi using radiolabeled monoclonal antifibrins. If the results are promising, considerations unique to in vivo scintigraphic imaging will be investigated; ie. choice of ideal radioisotope (eg. 99mTc or 111In), non-denaturing labelling with radioisotope, and biodistribution studies. As a part of this objective, we intend to characterize the manner in which 64C5, 55D10, and 59D8 recognize fibrin ad its circulating degradation products. The likehood of immunocrossreactivity with degradation products and circulating fibrin monomers is the basis for the second objective, the search for alternative antifibrin antibodies. We will synthesize fibrin-unique peptides for us as antigens: the amino terminal heptapeptide of the Alpha-chain in human fibrin, the Factor XIIIa mediated crosslink site in Gamma-chain dimers (2 peptides), and the amino terminal heptapeptides exposed upon plasmic cleavage of fibrin Beta-chains. These peptides will then be used as antigens to immunize rabbits and mice with the aim of obtaining antibodies which bind to human fibrin but not fibrinogen.
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