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- W2578581621 abstract "Analyses of biological samples today include protein microarray, a high-throughput technology that can be used for protein expression profiling, targeting disease related biomarkers. Affinity microarray utilises scFv antibodies, fixed in an ordered pattern on a solid support. Albeit high biocompatibility and sensitivity, this technique causes randomised antibody orientation, which could negatively influence the reactivity of the arrayed antibodies. The photo-reactive unnatural amino acid p-benzoyl-L-phenylalanine (pBpa), can be incorporated into a scFv structure using site-directed mutagenesis and a specific RNA (tRNA)/aminoacyl-tRNA synthetase pair. Through a technology known as dock ‘n’ flash, pBpa can be covalently coupled to cyclic carbohydrate β-cyclodextrin both in-solution and on a coated surface upon excitation to 350- 360 nm wavelength light. The technique enables predictable scFv orientation and thereby exposed binding sites, a property desirable in the development of a new microarray based platform. This master’s thesis project explored the possibility to incorporate pBpa into 10 scFv antibodies of five specificities through site-directed mutagenesis. It aimed to evaluate the antibody functionality and on-chip performance. Due to obstacles during scFv+/pBpa+ transformation, along with challenging antibody production, the project was not complete in respect to its originally planned protein evaluations. In was concluded that TOP10 competent E. coli are not appropriate hosting scFv+/pBpa+ transformation. Instead it was found that Rosetta-gami 2 and NovaBlue E. coli are suitable scFv+/pBpa+ transformation hosts, and that NovaBlue can partake in mutant scFv antibody production. These new data hopefully contribute to a possible future development of a new high-performance microarray immobilization technique. (Less)" @default.
- W2578581621 created "2017-01-26" @default.
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- W2578581621 date "2016-01-01" @default.
- W2578581621 modified "2023-09-27" @default.
- W2578581621 title "Generation and molecular characterization of recombinant scFv antibodies, microarray adapted by molecular design" @default.
- W2578581621 hasPublicationYear "2016" @default.
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