Abstract
Intracellular single domain antibodies are recombinant proteins, comprising one variable region domain fragment, that bind specifically to intracellular molecules and can interfere with their particular functions within various cellular compartments. They are valuable tools in bioscience and potential macrodrugs in biotherapeutics; however, their application is still limited because of the difficulty and inefficiency of acquisition of functional intracellular antibodies. We describe here the new generation protocol for intracellular antibody capture to facilitate selection of functional single domains. This protocol uses a series of optimized single domain libraries, based on designed intracellular variable (VH or VL) region scaffolds, for direct in vivo isolation of single domains that bind to target proteins and interaction and for affinity maturation to develop sub-nM affinity antibody fragments. The method has advantages over other methods in that specific single domains are isolated directly within the reducing cellular environment and can be selected without in vitro antigen protein preparation.
In an accompanying methods paper, we describe a simple extension of the methodology to isolate subsets of IAC-captured single domains that interfere with protein–protein interactions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC (2005) Monoclonal antibody successes in the clinic. Nat Biotechnol 23:1073–1078
Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136
Lobato MN, Rabbitts TH (2003) Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol Med 9:390–396
Perez-Martinez D, Tanaka T, Rabbitts TH (2010) Intracellular antibodies and cancer: new technologies offer therapeutic opportunities. Bioessays 32:589–598
Visintin M, Tse E, Axelson H, Rabbitts TH, Cattaneo A (1999) Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc Natl Acad Sci U S A 96:11723–11728
Tse E, Lobato MN, Forster A, Tanaka T, Chung GTY, Rabbitts TH (2002) Intracellular antibody capture technology: application to selection of single chain Fv recognising the BCR-ABL oncogenic protein. J Mol Biol 317:85–94
Visintin M, Settanni G, Maritan A, Graziosi S, Marks JD, Cattaneo A (2002) The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies. J Mol Biol 317:73–83
Tanaka T, Rabbitts TH (2003) Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation. EMBO J 22:1025–1035
Tanaka T, Lobato MN, Rabbitts TH (2003) Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J Mol Biol 331:1109–1120
Tanaka T, Rabbitts TH (2008) Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds. J Mol Biol 376:749–757
Tanaka T, Rabbitts TH (2010) Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture. Nat Protoc 5:67–92
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246
Bartel PL, Chien C, Sternglanz R, Fields S (1993) Using the two-hybrid system to detect protein-protein interaction. In: Hartley DA (ed) Cellular interaction in development: a practical approach. IRL Press, Oxford, pp 153–179
Sambrook J, Russell PW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Engebrecht J, Brent R, Kaderbhai MA (1992) Escherichia coli, Plasmids, and Bacteriophages. In: Ausubel FM et al (eds) Short protocols in molecular biology, 3rd edn. Wiley, New York
Lundblad V (1992) Saccharomyces Cerevisiae. In: Ausubel FM et al (eds) Short protocols in molecular biology, 3rd edn. Wiley, New York
Tanaka T, Chung GTY, Forster A, Lobato MN, Rabbitts TH (2003) De novo production of diverse intracellular antibody libraries. Nucleic Acids Res 31:e23
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tanaka, T., Rabbitts, T.H. (2012). Intracellular Antibody Capture (IAC) Methods for Single Domain Antibodies. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-968-6_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-967-9
Online ISBN: 978-1-61779-968-6
eBook Packages: Springer Protocols