Cargando…

Fluorescent Labeling of Antibody Fragments Using Split GFP

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chai...

Descripción completa

Detalles Bibliográficos
Autores principales: Ferrara, Fortunato, Listwan, Pawel, Waldo, Geoffrey S., Bradbury, Andrew R. M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187779/
https://www.ncbi.nlm.nih.gov/pubmed/21998685
http://dx.doi.org/10.1371/journal.pone.0025727
_version_ 1782213349782585344
author Ferrara, Fortunato
Listwan, Pawel
Waldo, Geoffrey S.
Bradbury, Andrew R. M.
author_facet Ferrara, Fortunato
Listwan, Pawel
Waldo, Geoffrey S.
Bradbury, Andrew R. M.
author_sort Ferrara, Fortunato
collection PubMed
description Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.
format Online
Article
Text
id pubmed-3187779
institution National Center for Biotechnology Information
language English
publishDate 2011
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-31877792011-10-13 Fluorescent Labeling of Antibody Fragments Using Split GFP Ferrara, Fortunato Listwan, Pawel Waldo, Geoffrey S. Bradbury, Andrew R. M. PLoS One Research Article Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems. Public Library of Science 2011-10-05 /pmc/articles/PMC3187779/ /pubmed/21998685 http://dx.doi.org/10.1371/journal.pone.0025727 Text en Ferrara et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ferrara, Fortunato
Listwan, Pawel
Waldo, Geoffrey S.
Bradbury, Andrew R. M.
Fluorescent Labeling of Antibody Fragments Using Split GFP
title Fluorescent Labeling of Antibody Fragments Using Split GFP
title_full Fluorescent Labeling of Antibody Fragments Using Split GFP
title_fullStr Fluorescent Labeling of Antibody Fragments Using Split GFP
title_full_unstemmed Fluorescent Labeling of Antibody Fragments Using Split GFP
title_short Fluorescent Labeling of Antibody Fragments Using Split GFP
title_sort fluorescent labeling of antibody fragments using split gfp
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187779/
https://www.ncbi.nlm.nih.gov/pubmed/21998685
http://dx.doi.org/10.1371/journal.pone.0025727
work_keys_str_mv AT ferrarafortunato fluorescentlabelingofantibodyfragmentsusingsplitgfp
AT listwanpawel fluorescentlabelingofantibodyfragmentsusingsplitgfp
AT waldogeoffreys fluorescentlabelingofantibodyfragmentsusingsplitgfp
AT bradburyandrewrm fluorescentlabelingofantibodyfragmentsusingsplitgfp