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Schofield DJ Pope AR Clementel V Buckell J Chapple SDj Clarke KF Conquer JS Crofts AM Crowther SR Dyson MR Flack G Griffin GJ Hooks Y Howat WJ Kolb-Kokocinski A Kunze S Martin CD Maslen GL Mitchell JN O'Sullivan M Perera RL Roake W Shadbolt SP Vincent KJ Warford A Wilson WE Xie J Young JL McCafferty J 《Genome biology》2007,8(11):R254-18
We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation. 相似文献
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A. D. Blackader Frank G. Pedley H. E. Macdermot J. H. Elliott R. R. Struthers D. B. Mendel C. R. Bourne J. A. Nutter H. M. Young C. K. P. Henry R. V. B. Shier R. B. Malcolm A. W. Young Percy G. Bell H. H. Burnham J. Beattie Norman Brown L. R. Hess F. W. Harvey Harold Orr J. H. Elliott E. S. Mills John Hunter L. J. Austin Madge T. Macklin 《CMAJ》1929,21(5):632-644
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Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways. 相似文献
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