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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3187779?pdf=render |
| _version_ | 1818859209019621376 |
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| author | Fortunato Ferrara Pawel Listwan Geoffrey S Waldo Andrew R M Bradbury |
| author_facet | Fortunato Ferrara Pawel Listwan Geoffrey S Waldo Andrew R M Bradbury |
| author_sort | Fortunato Ferrara |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-19T09:08:32Z |
| format | Article |
| id | doaj.art-0c042e9ff1714f0ebaefad9539444eb6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-19T09:08:32Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-0c042e9ff1714f0ebaefad9539444eb62022-12-21T20:28:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-01610e2572710.1371/journal.pone.0025727Fluorescent labeling of antibody fragments using split GFP.Fortunato FerraraPawel ListwanGeoffrey S WaldoAndrew R M BradburyAntibody 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.http://europepmc.org/articles/PMC3187779?pdf=render |
| spellingShingle | Fortunato Ferrara Pawel Listwan Geoffrey S Waldo Andrew R M Bradbury Fluorescent labeling of antibody fragments using split GFP. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC3187779?pdf=render |
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