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dc.contributor.advisor Gunn, Michael D en_US
dc.contributor.author Lipes, BD
dc.contributor.author Chen, YH
dc.contributor.author Ma, H
dc.contributor.author Staats, HF
dc.contributor.author Kenan, DJ
dc.contributor.author Gunn, MD
dc.coverage.spatial England
dc.date.accessioned 2009-01-02T16:24:50Z
dc.date.issued 2008-05-30
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/18455737
dc.identifier S0022-2836(08)00405-1
dc.identifier.citation J Mol Biol, 2008, 379 (2), pp. 261 - 272
dc.identifier.uri http://hdl.handle.net/10161/903
dc.description Dissertation en_US
dc.description.abstract The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.
dc.format.extent 261 - 272
dc.format.mimetype application/pdf
dc.language eng
dc.language.iso en_US
dc.relation.ispartof J Mol Biol
dc.relation.isversionof 10.1016/j.jmb.2008.03.072
dc.subject Animals
dc.subject Antibody Affinity
dc.subject Antibody Specificity
dc.subject Biological Assay
dc.subject Cell Line
dc.subject Drosophila melanogaster
dc.subject Humans
dc.subject Immunoglobulin Fragments
dc.subject Immunoglobulin Variable Region
dc.subject Mice
dc.subject Mice, Inbred BALB C
dc.subject Mice, Inbred C57BL
dc.subject Peptide Library
dc.subject Receptors, Cell Surface
dc.subject Recombinant Proteins
dc.subject Toll-Like Receptor 2
dc.title An entirely cell-based system to generate single-chain antibodies against cell surface receptors.
dc.type Journal Article
dc.department Immunology en_US
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/18455737
pubs.issue 2
pubs.organisational-group /Duke
pubs.organisational-group /Duke/School of Medicine
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments/Immunology
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Medicine
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Medicine/Medicine, Cardiology
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Pathology
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers
pubs.organisational-group /Duke/School of Medicine/Institutes and Centers/Duke Human Vaccine Institute
pubs.publication-status Published
pubs.volume 379
dc.identifier.eissn 1089-8638

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