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

• Published in: Biochimica et biophysica acta Vol. 1850; no. 7; pp. 1397 - 1404
• Main Authors: Rossotti, Martín, Tabares, Sofía, Alfaya, Lucía, Leizagoyen, Carmen, Moron, Gabriel, González-Sapienza, Gualberto
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2015
• Subjects: Animals; antibodies; biotin; Biotin - immunology; biotinylation; bone marrow; Camelidae; Camelids, New World; CD11b Antigen - immunology; CD18 Antigens - immunology; Cell Line; cell membranes; Cell receptor; Cell Surface Display Techniques - methods; Cells, Cultured; dendritic cells; Dendritic Cells - immunology; Dendritic Cells - metabolism; drugs; Enzyme-Linked Immunosorbent Assay; epitopes; Flow Cytometry; Immunization - methods; Immunoprecipitation; In vivo biotinylation; Leukocyte Common Antigens - immunology; leukocytes; llamas; mice; Mice, Inbred BALB C; Nanobody; Peptides - immunology; Phage display; receptors; Receptors, Cell Surface - immunology; Reproducibility of Results; Single-Chain Antibodies - immunology; Single-Domain Antibodies - immunology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Flow cytometry; Nanobody; In vivo biotinylation; Phage display; Immunoprecipitation; Cell receptor
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.03.009

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, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. •Quantitative in vivo biotinylation of nanobodies in 96 culture blocks was optimized.•The biotin moiety allows high throughput FACS analysis of the VHH reactivity.•Pull-down experiments and MS identification are greatly facilitated.•On and off switching of the biotin addition allows binning of VHH epitopes on cells.