Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells.

摘要: Abstract Background Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, therapeutic applications. While preparation against purified antigens is straightforward, generation nanobodies difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform throughput identification receptor based on use biotin handle. Methods Using biotin-acceptor peptide tag, in vivo biotinylation 96 well culture blocks was optimized allowing parallel analysis by flow cytometry ELISA, direct pull-down/MS target identification. Results The potential this strategy demonstrated selection characterization panels Mac-1 (CD11b/CD18), MHC II mouse Ly-5 leukocyte common antigen (CD45) receptors, from VHH library obtained llama immunized with bone marrow derived dendritic cells. By off switching addition biotin, method also allowed epitope binning selected Nbs directly Conclusions This streamlines potent antigens, constitute ready-to-use biotinylated reagents. General significance will accelerate discovery which comprise largest group drug analytical targets.