SpringerProtocols: Abstract: Identification of the Specificity of Isolated Phage Display Single-Chain Antibodies Using Yeast Two-Hybrid Screens

13. Identification of the Specificity of Isolated Phage Display Single-Chain Antibodies Using Yeast Two-Hybrid Screens

By: Nicolaj Rasmussen², Henrik J. Ditzel²

Abstract

Full Text

Download PDF (255K)

A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens.

Affiliation(s): (2) Medical Biotechnology Center, Institute of Medical Biology, University of Southern Denmark, Winsloewparken 25, 3, DK-5000 Odense, Denmark

Book Title: Antibody Phage Display: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 562 | Pub. Date: Mar-01-2009 | Page Range: 165-176 | DOI: 10.1007/978-1-60327-302-2_13

Subject: Microbiology

Key Words: Phage display - scFv - Yeast two-hybrid - Antigen identification

References

Download References

1.	Ditzel, H. J., Binley, J. M., Moore, J. P., Sodroski, J., Sullivan, N., Sawyer, L. S. W.,Hendry, R. M., Yang, W. P., Barbas, C. F., and Burton, D. R. (1995) Neutralizing recombinant human antibodies to a conformational V2- and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure. J. Immunol. 154, 893–906.

2.	Clark, M. A., Hawkins, N. J., Papaioannou, A., Fiddes, R. J., and Ward, R. L. (1997) Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library. Clin. Exp. Immunol. 109, 166–174.

3.	Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., and Winter, G. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597.

4.	Jensen, K. B., Jensen, O. N., Ravn, P., Clark, B. F., and Kristensen, P. (2003) Identification of keratinocyte-specific markers using phage display and mass spectrometry. Mol. Cell. Proteomics 2, 61–69.

5.	Jakobsen, C. G., Rasmussen, N., Laenkholm, A. V., and Ditzel, H. J. (2007) Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78. Cancer Res. 67, 9507–9517.

6.	Cai, X., and Garen, A. (1995) Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc. Natl. Acad. Sci. USA 92, 6537–6641.

7.	Coronella, J. A., Spier, C., Welch, M., Trevor, K. T., Stopeck, A. T., Villar, H., and Hersh, E. M. (2002) Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast. J. Immunol. 169, 1829–1836.

8.	Hansen, M. H., Nielsen, H., and Ditzel, H. J. (2001) The tumor-infiltrating B cell response in medullary breast cancer is oligoclonal and directed against the autoantigen actin exposed on the surface of apoptotic cancer cells. Proc. Natl. Acad. Sci. USA 98, 12659–12664.

9.	Robert, R., Jacobin-Valat, M. J., Daret, D., Miraux, S., Nurden, A. T., Franconi, J. M., and Clofent-Sanchez, G. (2006) Identification of human scFvs targeting atherosclerotic lesions: selection by single round in vivo phage display. J. Biol. Chem. 281, 40135–40143.

10.	Fields, S., and Sternglanz, R. (1994) The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10, 286–292.

11.	Young, K. H. (1998) Yeast two-hybrid: so many interactions, (in) so little time. Biol. Reprod. 58, 302–311.

12.	Chardin, P., Camonis, J. H., Gale, N. W., Vanaelst, L., Schlessinger, J., Wigler, M. H., and Barsagi, D. (1993) Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260, 1338–1343.

13.	Mizuno, K., Kitamura, A., and Sasaki, T. (2003) Rabring7, a novel Rab7 target protein with a RING finger motif. Mol. Biol. Cell 14, 3741–3752.

14.	Rosenberger, G., Gal, A., and Kutsche, K. (2005) AlphaPIX associates with calpain 4, the small subunit of calpain, and has a dual role in integrin-mediated cell spreading. J. Biol. Chem. 280, 6879–6889.

Comments (Loading...)

Post comment/View all comments