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  • Title: The role of valency in the selection of anti-carbohydrate single-chain Fvs from phage display libraries.
    Author: MacKenzie R, To R.
    Journal: J Immunol Methods; 1998 Nov 01; 220(1-2):39-49. PubMed ID: 9839924.
    Abstract:
    Several strategies were investigated for phage display of anti-carbohydrate single-chain Fvs (scFvs) using the anti-Salmonella Se155-4 scFv as a model system. All were based on the knowledge that panning V(H) CDR libraries displayed in a standard pIII phagemid/helper phage format against immobilized multivalent carbohydrate antigens selects almost solely for mutants with higher yields of soluble product or scFvs that form dimers or higher oligomers even when the linker length is chosen to give monomeric molecules. Construction of scFv libraries, in a phagemid vector, with mutations that already provide higher yields and/or short linkers to promote dimeric display greatly reduced these undesired selection pressures. However. the panning of an error-prone library of the entire scFv in a short linker format yielded two mutants that existed partially in higher oligomeric forms, indicating that dimeric display did not entirely eliminate the selection pressure problem. In one mutant a Ser75Gly mutation led to the formation of greater amounts of dimeric, trimeric and tetrameric scFv and surface plasmon resonance analysis of these different forms gave quantitative data for the functional affinity of these different scFv forms. Finally, a phage vector was constructed and the original V(H) CDR library was transferred to this vector. This display format, in which scFv is displayed on all three to five copies of pIII, seemed to be superior in terms of selection on the basis of binding site affinity and as a display mode for scFvs with low intrinsic affinity. While the use of the phage vector did not lead to the isolation of higher affinity binders from the library employed here, it did almost entirely remove the undesired selection pressures in that there was selection for the wild-type sequence. It is suggested that the multivalency of display provided by phage vectors is preferable to any phagemid vector strategy for the de novo isolation of anti-carbohydrate antibodies from phage libraries.
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