| Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells. | |
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MedLine Citation:
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PMID: 17027850 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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We describe a method for the generation of high-affinity monoclonal antibodies, which combines the power of natural immune responses with in vitro panning, B cell culture, RT-PCR and expression of the recombinant product. B cells from immunised rabbits were incubated at approximately 1000-10,000 cells per well with solid phase antigen coated on the surface of 96-well ELISA plates. Extensive washing removed non-binding cells as well as those B cells, which bound with low affinity. Retained B cells were cultured for 7 days in the presence of activated rabbit splenocyte supernatant and irradiated EL-4-B5 mouse thymoma cells, to induce proliferation and secretion of immunoglobulin. Supernatants were screened to confirm the presence of specific antibody, before the cells were harvested en masse from individual positive wells. Single heavy- and light-chain variable region genes were recovered from individual wells by RT-PCR, critically without the need for isolation of single B cells. Paired VH and VL genes were subsequently expressed as recombinant antibodies and shown to retain the original activity and specificity of the B cell culture supernatants. The method has also been successfully applied to the generation of high-affinity antibodies to antigen expressed on the surface of target cells. |
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Authors:
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Daniel J Lightwood; Bruce Carrington; Alistair J Henry; Andrew J McKnight; Kenneth Crook; Karen Cromie; Alastair D G Lawson |
Publication Detail:
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Type: Journal Article Date: 2006-09-18 |
Journal Detail:
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Title: Journal of immunological methods Volume: 316 ISSN: 0022-1759 ISO Abbreviation: J. Immunol. Methods Publication Date: 2006 Oct |
Date Detail:
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Created Date: 2006-10-23 Completed Date: 2006-12-19 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 1305440 Medline TA: J Immunol Methods Country: Netherlands |
Other Details:
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Languages: eng Pagination: 133-43 Citation Subset: IM |
Affiliation:
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UCB Celltech, Antibody Centre of Excellence, UK. daniel.lightwood@celltech.ucb-group.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Antibodies, Monoclonal / biosynthesis*, genetics B-Lymphocytes / immunology* CHO Cells Clone Cells / immunology Cloning, Molecular Cricetinae Enzyme-Linked Immunosorbent Assay Immunoglobulin Heavy Chains / genetics, immunology Immunoglobulin Light Chains / genetics, immunology Immunoglobulin Variable Region / genetics, immunology Molecular Sequence Data OX40 Ligand / immunology* RNA / chemistry, genetics Rabbits Recombinant Proteins / genetics, immunology Reverse Transcriptase Polymerase Chain Reaction Surface Plasmon Resonance |
| Chemical | |
Reg. No./Substance:
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0/Antibodies, Monoclonal; 0/Immunoglobulin Heavy Chains; 0/Immunoglobulin Light Chains; 0/Immunoglobulin Variable Region; 0/OX40 Ligand; 0/Recombinant Proteins; 63231-63-0/RNA |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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