title: Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells.
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, 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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents.
  
  
  CONCLUSIONS
  This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay.
authors.forename: Martín
authors.initials: M
authors.lastname: Rossotti
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay.
authors.forename: Sofía
authors.initials: S
authors.lastname: Tabares
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay.
authors.forename: Lucía
authors.initials: L
authors.lastname: Alfaya
authors.affiliation: Parque Lecoq, IMM, Montevideo, Uruguay.
authors.forename: Carmen
authors.initials: C
authors.lastname: Leizagoyen
authors.affiliation: Centro de Investigación en Bioquímica Clínica e Inmunología, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Moron
authors.affiliation: Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay. Electronic address: ggonzal@fq.edu.uy.
authors.forename: Gualberto
authors.initials: G
authors.lastname: González-Sapienza
mesh_terms: D000818:Animals; D001710:Biotin / Q000276:immunology; D039481:CD11b Antigen / Q000276:immunology; D018821:CD18 Antigens / Q000276:immunology; D002161:Camelids, New World; D002460:Cell Line; D061505:Cell Surface Display Techniques / Q000379:methods; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007114:Immunization / Q000379:methods; D017493:Leukocyte Common Antigens / Q000276:immunology; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000276:immunology; D011956:Receptors, Cell Surface / Q000276:immunology*; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D039481:CD11b Antigen; D018821:CD18 Antigens; D010455:Peptides; D011956:Receptors, Cell Surface; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies; D001710:Biotin; D017493:Leukocyte Common Antigens
keywords: Cell receptor; Flow cytometry; Immunoprecipitation; In vivo biotinylation; Nanobody; Phage display
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