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Phage Display Library Screening by Subtractive Selection

Creative Biolabs provides high-quality service in phage display antibody library construction and screening. In order to select antibodies with high affinity and specificity, we have developed a series of selection strategies. Among these strategies, subtractive selection allows highly conformation-specific selection for antibodies directed against functionally active, intact, multimeric epitopes on the cell surface molecules which can be tailored for diagnostic and therapeutic applications. Using this subtractive panning strategy, we can provide activation-specific single-chain antibodies for our customers within 4-6 weeks.

There is a high demand for conformation-specific antibodies in biological research, medical diagnosis as well as therapeutics. The traditional process of antibody generation does not allow targeting for conformation-specific antibodies since the immobilized targets on solid surfaces may properly display the desired epitopes. Creative Biolabs’ subtractive panning strategy could solve this problem by selecting conformation-specific antibodies against intact, functionally active, multimeric epitopes without alteration of structure.

Flow chart of the subtractive selection strategy (Eisenhardt et al. 2007) Fig. 1 Flow chart of the subtractive selection strategy (Eisenhardt et al. 2007).

Our subtractive strategy is based on an initial depletion step against non-target epitopes followed by a selection step for the target epitope, therefore allows a highly specific selection for activation-specific conformations (Fig. 1). Each panning round starts with a depletion step, in which phages are primarily incubated with non-activated cells. Phages which bind to the non-activated cells are centrifuged down with the cells and discarded. The supernatant, containing the phages that are not binding to the non-activated cells will be transferred to the selection round activated cells expressing receptors in their activated conformation, and the supernatant will be co-incubated. Cells are centrifuged down with the binding phages, and the supernatant is discarded. Phages, which bind to the activated cells in the selection step are binding to the pelleted cells, and then, it will be eluted and transferred to the next panning round. In this way, the antibodies targeting the active cell receptors could be identified.

Creative Biolabs’ scientists have extensive experience in phage display library screening based on subtractive selection strategy. It is highly effective in reducing the number of nonspecific clones and simplifying the washing process, and finally reducing the lots of potential ligands. This strategy generates antibodies with high specificity by changing selection matrix during panning rounds. In addition, it also generates antibodies with high affinity by changing elution conditions and using competitive elution.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

  1. Eisenhardt, S.U.; et al. Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc. 2007, 2(12):3063-73.



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