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Phage Display Library Screening by Capture Lift

Creative Biolabs provides high-quality service in phage display antibody library construction and screening. In order to discover antibodies reactive with unidentified cell surface antigens, the capture lift assay, which is a modified plaque lift assay is used. The capture lift strategy has greater sensitivity in the discovery of higher-affinity antibodies (Abs) that recognize unique epitopes and novel Abs to rare antigen (Ag) present in complex mixtures.

Cloning and expression of functional antibody fragments in bacteria and on the surface of filamentous phage have revolutionized the processes of antibody discovery and engineering. However, the enrichment of the highest affinity Fabs displaying may result in slow dissociation rates. Also, a large number of unique clones displaying low or intermediate affinities to diverse epitopes may be lost in the enrichment steps. An optimal screening system should permit the rapid characterization of all clones present in the library. Phage-expressed libraries of Fabs can be screened by filter lifts probed with Ag avoiding prior enrichment or selection steps. The capture lift assay is modified from the filter-lift assay. Phage-expressed soluble Fabs are selectively captured on the nitrocellulose, significantly enhancing the signal of the assay. The screening procedure of this strategy is as follows (Fig. 1). Firstly, a nitrocellulose filter is incubated with a capture reagent, and then the remaining binding sites on the filter are blocked. A bacterial lawn is infected with the Ab-phage library and is overlaid with the pretreated nitrocellulose filter. The filter is removed and probed with target molecules. At last, isolate the clones of interest from the agar plate aligned with the filter.

Flow chart of the capture-lift screening procedure (Watkins 2002).
Fig.1 Flow chart of the capture-lift screening procedure (Watkins 2002).

Using capture lift strategy, we can simultaneously analyze thousands of antibody clones. More importantly, it can be used with crude detergent-solubilized cell extracts. Therefore, it is very useful to discover Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Capture lift screening does not require protein fixation steps which may alter antigenicity or expose intracellular epitopes. Finally, because plaques arise from infection by a single phage, every clone present in the library can be screened, permitting the identification and isolation of rare, non-abundant Fab species.

Featured Advantages


Creative Biolabs develops this capture lift strategy to screen high-affinity Abs and Abs to rare Ags present in complex mixtures. This highly sensitive approach allows the targeting of less abundant antigens and the detection of weaker antibody-antigen interactions.

Creative Biolabs’ scientists have extensive experience in phage display library screening based on capture lift. The increased sensitivity and normalization of the amount of Fab bound on the nitrocellulose make capture lift assay a great method for discovery and engineering of phage-expressed Fabs.

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

References

  1. Watkins, J. D. Screening of phage-expressed antibody libraries by capture lift. Methods Mol Biol. 2002, 178:187-93.
  2. Watkins, J.D.; et al. Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries. Anal Biochem. 1998, 256(2):169-77.



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