Martín Rossotti1, Sofía Tabares1, Lucía Alfaya1, Carmen Leizagoyen2, Gabriel Moron3, Gualberto González-Sapienza4. 1. Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay. 2. Parque Lecoq, IMM, Montevideo, Uruguay. 3. Centro de Investigación en Bioquímica Clínica e Inmunología, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina. 4. Cátedra de Inmunología, DEPBIO, Facultad de Química, Instituto de Higiene, UDELAR, Montevideo, Uruguay. Electronic address: ggonzal@fq.edu.uy.
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. GENERAL SIGNIFICANCE: This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
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 mouseLy-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. GENERAL SIGNIFICANCE: This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
Authors: K E Conrath; M Lauwereys; M Galleni; A Matagne; J M Frère; J Kinne; L Wyns; S Muyldermans Journal: Antimicrob Agents Chemother Date: 2001-10 Impact factor: 5.191
Authors: Martin A Rossotti; Andrés González-Techera; Julio Guarnaschelli; Lucia Yim; Ximena Camacho; Marcelo Fernández; Pablo Cabral; Carmen Leizagoyen; José A Chabalgoity; Gualberto González-Sapienza Journal: MAbs Date: 2015 Impact factor: 5.857
Authors: Sofia Tabares-da Rosa; Linda A Wogulis; Mark D Wogulis; Gualberto González-Sapienza; David K Wilson Journal: J Mol Recognit Date: 2018-07-23 Impact factor: 2.137
Authors: Martin A Rossotti; Henk van Faassen; Anh T Tran; Joey Sheff; Jagdeep K Sandhu; Diana Duque; Melissa Hewitt; Xiaoxue Wen; Jegarubee Bavananthasivam; Saina Beitari; Kevin Matte; Geneviève Laroche; Patrick M Giguère; Christian Gervais; Matthew Stuible; Julie Guimond; Sylvie Perret; Greg Hussack; Marc-André Langlois; Yves Durocher; Jamshid Tanha Journal: Commun Biol Date: 2022-09-09