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• W2045132978 startingPage "93" @default.
• W2045132978 abstract "Affibody molecules specific for human TNF‐α (tumour necrosis factor‐α) were selected by phage‐display technology from a library based on the 58‐residue Protein A‐derived Z domain. TNF‐α is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF‐α‐blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine‐free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli ‐produced and IMAC (immobilized‐metal‐ion affinity chromatography)‐purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF‐α. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF‐α. For Z TNF‐α:185 , subnanomolar affinity ( K D =0.1–0.5 nM) for human TNF‐α was demonstrated, as well as significant binding to TNF‐α from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF‐α receptor, since this interaction could be efficiently blocked by the Z TNF‐α:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF‐α binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody‐based reagents for the diagnosis of inflammation is discussed." @default.
• W2045132978 created "2016-06-24" @default.
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• W2045132978 date "2009-10-01" @default.
• W2045132978 modified "2023-10-17" @default.
• W2045132978 title "Generation of tumour-necrosis-factor-α-specific affibody molecules capable of blocking receptor binding<i>in vitro</i>" @default.
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• W2045132978 doi "https://doi.org/10.1042/ba20090085" @default.
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