Identifying novel fibrin-binding antibodies
Brown, Wendy E.
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Atherosclerosis causes coronary heart disease, the leading cause of death in America today. Fibrin plays an important role in blood coagulation, especially in connection to atherosclerosis. Given the subtle and progressive nature of the disease, current diagnostic techniques are severely limited and rely on conjecture to locate plaque lesions. These shortcomings present the opportunity for the development of more specific technology. To that end, excess fibrin deposition may be exploited as a characteristic of atherosclerosis. We propose to develop an in-vivo fibrin-based targeting system. The first step was to select a targeting scheme that will preferentially bind to fibrin. Therefore, the Tomlinson I scFv phagemid library was used to select antibody fragments that preferentially bound a fibrin clot substrate. Three rounds of biopanning assays were performed on a fibrin clot in order to generate an enriched phage population that exhibits preferential binding to fibrin. Gel electrophoresis and DNA sequencing was used to analyze PCR samples after each screen to ensure the maintenance of the genetic insert encoding for the antibody linked to the phage coat protein and variability in the five diversity sites on the variable light and heavy chains of the displayed antibodies. After three rounds of selection, an enriched antibody population with maintained genetic diversity that exhibits preferential binding to fibrin was obtained. This is a crucial step in the identification and characterization of single antibody clones with desired binding characteristics. Future studies will involve ELISAs to identify the antibodies that most strongly bind to fibrin, SPR and immunolabeling to characterize antibody binding. It is the hope that this study and those following will progress toward the development a fibrin-based targeting system for minimally-invasive imaging modalities or delivery of therapeutics to treat atherosclerotic patients who do not exhibit symptoms of plaque formation.