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Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture

Abstract

Single-domain intracellular antibodies are antibody variable segments that bind to specific target proteins inside cells. These antigen-binding variable regions can interfere with protein function or perturb protein–protein interactions and can be used as tools for research, especially functional genomics and proteomics and interfering with the protein interactome. This protocol (Intracellular Antibody Capture, IAC3) describes the isolation of functional variable heavy (VH) or variable light (VL) segments from diverse libraries. The protocol comprises four principle steps: validation of a bait antigen; initial screening in yeast of a single domain library; generation of sub-libraries after the initial screen; and finally confirmation of the positive clones interaction with antigen in yeast and mammalian cells. Each library (initial, second and third) screening takes upto 2 weeks and the overall procedure <3 months. This third generation IAC method has many advantages, including isolation of single domain intracellular antibodies with high affinity and specificity by direct in vivo genetic selection.

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Figure 1: Schematic representation of various antibody formats.
Figure 2: Flow chart of the third generation intracellular antibody capture (IAC) technology.
Figure 3: Initial single domain antibody libraries.
Figure 4: Maps of plasmid vectors used for yeast intracellular antibody capture (IAC) screening.
Figure 5: Assembly PCR to generate the second and third single domain sub-libraries with randomized complementarity determining regions (CDRs).
Figure 6: Maps of plasmid vectors for the mammalian luciferase reporter assays.
Figure 7: Representative western blot to detect LexA–Ag fusion protein expression in bait strains.
Figure 8: Example of auto-activation test of L40 yeast bait strains expressing LexA–antigen fusion protein.
Figure 9: Confirmation of yeast iDab clones isolated from library screening.
Figure 10: Example of PCR amplification of DNA fragments for sub-library construction and confirmation of single-domain VP16 plasmids by restriction enzymes digestion.

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Acknowledgements

This protocol was developed from work funded by the National Foundation for Cancer Research, Medical Research Council (UK), the Leukaemia Research Fund (UK) and the University of Leeds. We would like to thank Drs. Hanif Ali and Andrew Dixon for their comments and editing of the detailed protocol.

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T.T. and T.H.R. wrote the manuscript.

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Correspondence to Terence H Rabbitts.

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Tanaka, T., Rabbitts, T. Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture. Nat Protoc 5, 67–92 (2010). https://doi.org/10.1038/nprot.2009.199

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