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Article: Selection of antibodies for intracellular function using a two-hybrid in vivo system

TitleSelection of antibodies for intracellular function using a two-hybrid in vivo system
Authors
Issue Date1999
PublisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.org
Citation
Proceedings Of The National Academy Of Sciences Of The United States Of America, 1999, v. 96 n. 21, p. 11723-11728 How to Cite?
AbstractExpression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single- chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two- hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.
Persistent Identifierhttp://hdl.handle.net/10722/162281
ISSN
2015 Impact Factor: 9.423
2015 SCImago Journal Rankings: 6.883
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorVisintin, Men_US
dc.contributor.authorTse, Een_US
dc.contributor.authorAxelson, Hen_US
dc.contributor.authorRabbitts, THen_US
dc.contributor.authorCattaneo, Aen_US
dc.date.accessioned2012-09-05T05:18:37Z-
dc.date.available2012-09-05T05:18:37Z-
dc.date.issued1999en_US
dc.identifier.citationProceedings Of The National Academy Of Sciences Of The United States Of America, 1999, v. 96 n. 21, p. 11723-11728en_US
dc.identifier.issn0027-8424en_US
dc.identifier.urihttp://hdl.handle.net/10722/162281-
dc.description.abstractExpression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single- chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two- hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.en_US
dc.languageengen_US
dc.publisherNational Academy of Sciences. The Journal's web site is located at http://www.pnas.orgen_US
dc.relation.ispartofProceedings of the National Academy of Sciences of the United States of Americaen_US
dc.subject.meshAnimalsen_US
dc.subject.meshAntibody Affinityen_US
dc.subject.meshAntigen-Antibody Reactionsen_US
dc.subject.meshBlotting, Westernen_US
dc.subject.meshCho Cellsen_US
dc.subject.meshChloramphenicol O-Acetyltransferaseen_US
dc.subject.meshCricetinaeen_US
dc.subject.meshDisulfides - Metabolismen_US
dc.subject.meshGenes, Reporteren_US
dc.subject.meshImmunoglobulin Fragments - Genetics - Isolation & Purificationen_US
dc.subject.meshModels, Immunologicalen_US
dc.subject.meshSaccharomyces Cerevisiae - Immunologyen_US
dc.subject.meshTransfectionen_US
dc.subject.meshTransformation, Geneticen_US
dc.subject.meshTwo-Hybrid System Techniquesen_US
dc.titleSelection of antibodies for intracellular function using a two-hybrid in vivo systemen_US
dc.typeArticleen_US
dc.identifier.emailTse, E:ewctse@hku.hken_US
dc.identifier.authorityTse, E=rp00471en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1073/pnas.96.21.11723en_US
dc.identifier.pmid10518517-
dc.identifier.scopuseid_2-s2.0-0032716364en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0032716364&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume96en_US
dc.identifier.issue21en_US
dc.identifier.spage11723en_US
dc.identifier.epage11728en_US
dc.identifier.isiWOS:000083166800010-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridVisintin, M=7004492062en_US
dc.identifier.scopusauthoridTse, E=7005019454en_US
dc.identifier.scopusauthoridAxelson, H=7004201331en_US
dc.identifier.scopusauthoridRabbitts, TH=7103136845en_US
dc.identifier.scopusauthoridCattaneo, A=15829214500en_US

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