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

Autores
Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Victor Gabriel; González Sapienza, Gualberto
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Rossotti, Martín. Universidad de la República. Facultad de Química; Uruguay
Fil: Tabares, Sofía. Universidad de la República. Facultad de Química; Uruguay
Fil: Alfaya, Lucía. Universidad de la República. Facultad de Química; Uruguay
Fil: Leizagoyen, Carmen. No especifíca;
Fil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina
Fil: González Sapienza, Gualberto. Universidad de la República. Facultad de Química; Uruguay
Materia
CELL RECEPTOR
FLOW CYTOMETRY
IMMUNOPRECIPITATION
IN VIVO BIOTINYLATION
NANOBODY
PHAGE DISPLAY
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/185731
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/185731
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cellsRossotti, MartínTabares, SofíaAlfaya, LucíaLeizagoyen, CarmenMoron, Victor GabrielGonzález Sapienza, GualbertoCELL RECEPTORFLOW CYTOMETRYIMMUNOPRECIPITATIONIN VIVO BIOTINYLATIONNANOBODYPHAGE DISPLAYhttps://purl.org/becyt/ford/3.1https://purl.org/becyt/ford/3Background 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.Fil: Rossotti, Martín. Universidad de la República. Facultad de Química; UruguayFil: Tabares, Sofía. Universidad de la República. Facultad de Química; UruguayFil: Alfaya, Lucía. Universidad de la República. Facultad de Química; UruguayFil: Leizagoyen, Carmen. No especifíca;Fil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República. Facultad de Química; UruguayElsevier Science2015-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/185731Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Victor Gabriel; et al.; Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells; Elsevier Science; Biochimica et Biophysica Acta - General Subjects; 1850; 7; 7-2015; 1397-14040304-41651872-8006CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbagen.2015.03.009info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:31:43Zoai:ri.conicet.gov.ar:11336/185731instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:31:44.154CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
title Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
spellingShingle Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
Rossotti, Martín
CELL RECEPTOR
FLOW CYTOMETRY
IMMUNOPRECIPITATION
IN VIVO BIOTINYLATION
NANOBODY
PHAGE DISPLAY
title_short Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
title_full Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
title_fullStr Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
title_full_unstemmed Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
title_sort Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
dc.creator.none.fl_str_mv Rossotti, Martín
Tabares, Sofía
Alfaya, Lucía
Leizagoyen, Carmen
Moron, Victor Gabriel
González Sapienza, Gualberto
author Rossotti, Martín
author_facet Rossotti, Martín
Tabares, Sofía
Alfaya, Lucía
Leizagoyen, Carmen
Moron, Victor Gabriel
González Sapienza, Gualberto
author_role author
author2 Tabares, Sofía
Alfaya, Lucía
Leizagoyen, Carmen
Moron, Victor Gabriel
González Sapienza, Gualberto
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv CELL RECEPTOR
FLOW CYTOMETRY
IMMUNOPRECIPITATION
IN VIVO BIOTINYLATION
NANOBODY
PHAGE DISPLAY
topic CELL RECEPTOR
FLOW CYTOMETRY
IMMUNOPRECIPITATION
IN VIVO BIOTINYLATION
NANOBODY
PHAGE DISPLAY
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.1
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv 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.
Fil: Rossotti, Martín. Universidad de la República. Facultad de Química; Uruguay
Fil: Tabares, Sofía. Universidad de la República. Facultad de Química; Uruguay
Fil: Alfaya, Lucía. Universidad de la República. Facultad de Química; Uruguay
Fil: Leizagoyen, Carmen. No especifíca;
Fil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina
Fil: González Sapienza, Gualberto. Universidad de la República. Facultad de Química; Uruguay
description 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.
publishDate 2015
dc.date.none.fl_str_mv 2015-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/185731
Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Victor Gabriel; et al.; Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells; Elsevier Science; Biochimica et Biophysica Acta - General Subjects; 1850; 7; 7-2015; 1397-1404
0304-4165
1872-8006
CONICET Digital
CONICET
url http://hdl.handle.net/11336/185731
identifier_str_mv Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Victor Gabriel; et al.; Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells; Elsevier Science; Biochimica et Biophysica Acta - General Subjects; 1850; 7; 7-2015; 1397-1404
0304-4165
1872-8006
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbagen.2015.03.009
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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