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Epitope-directed antibody selection by site-specific photocrosslinking

Sci Adv. 2020-04; 
Chen L, Zhu C, Guo H, Li R, Zhang L, Xing Z, Song Y, Zhang Z, Wang F, Liu X, Zhang Y, Ma RZ, Wang F.
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Proteins, Expression, Isolation and Analysis The cells were grown for an additional 30 hours at 30°C at 220 rpm overnight before harvesting by centrifugation at 6000g for 10 min. The cell pellet was lysed by sonication, and the resulting cell lysate was clarified by centrifugation at 13,000g for 30 min at 4°C. IL-1 or hC5a WT and mutants were purified on Ni-NTA resin (GenScript, L00250) following the manufacturer’s instructions. Get A Quote

摘要

Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human inter... More

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