Abstract
Despite the availability of antibody libraries for the selection of receptor molecules, the large number of established and well-characterized hybridoma lines still represent a useful source for recombinant antibody genes. This protocol describes the PCR amplification, cloning, and a small-scale expression test for the generation of scFv fragments from hybridoma cell lines. Particular emphasis was placed on frequently observed problems and pitfalls of this method.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Huston, J. S., Levinson, D., Mudgett-Hunter, M., et al. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879–5883.
Bird, R. E., Hardman, K. D., Jacobson, J. W., et al. (1988) Single-chain antigen-binding proteins. Science 242, 423–426.
Whitlow, M. and Filpula, D. (1991) Single-chain Fv proteins and their fusion proteins. Methods Companion Methods Enzymol. 2, 97–105.
Orlandi, R., Güssow, D. H., Jones, P. T., and Winter, G. (1989), Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 3833–3837.
Ørum, H., Andersen, P. S., Øster, A., et al. (1993) Efficient method for constructing comprehensive murine Fab antibody libraries displayed on phage. Nucleic Acids Res. 21, 4491–4498.
Dübel, S., Breitling, F., Fuchs, P., et al. (1994) Isolation of IgG antibody Fv-DNA from various mouse and rat bybridoma cell lines using the polymerase chain reaction with a simple set of primers. J. Immunol. Methods. 175, 89–95.
Chen, Y. W., Word, C. J., Jones, S., Uhr, J. W., Tucker, P. W., and Vitetta, E. S. (1986) Double isotype production by a neoplastic B cell line. I. Cellular and biochemical characterization of a variant of BCL that expresses and secretes both IgM and IgG1. J. Exp. Med. 164, 548.
Shimizu, A., Nussenzweig, M. C., Han, H., Sanchez, M., and Honjo, T. (1991) Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene. J. Exp. Med. 173, 1385.
Pauza, M. E., Rehmann, J. A., and LeBien, T. W. (1993) Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains. J. Exp. Med. 178, 139.
Welschof, M., Terness, P., Kolbinger, F., et al. (1995) Amino acid sequence based PCR primers for the amplification of human heavy and light chain immunoglobulin variable region genes. J. Immunol. Methods 179, 203.
De Haard, H. and Kazemier, B. (1995) The effect of mutations in the primer encoded FR1 and FR4 regions of VH and VK on the reactivity of scFv-s. Hum. Antibod. Hybridomas 6.
Krauss, J., Arndt, M. A. E., Martin, A. C. R., Liu, H., and Rybak, S. (2002) Specificity grafting of human antibody frameworks selected from a phage display library, in Proceedings of the 13th International IBC Conference on Antibody Engineering, IBC, Westborough, MA.
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.
Breitling, F., Dübel, S., Seehaus, T., Klewinghaus, I., and Little, M. (1991) A surface expression vector for antibody screening. Gene 104, 147–153.
Dübel, S., Breitling, F., Fuchs, P., Braunagel, M., Klewinghaus, I., and Little, M. (1993) A family of vectors for surface display and production of antibodies. Gene 128, 97–101.
Dörsam, H., Braunagel, M., Kleist, C., Moynet, D., and Welschof, M. (1997) Screening of phage displayed libraries, in Molecular Diagnosis of Infectious Diseases, Methods in Molecular Medicine, vol. 13 (Reischl, U., ed.), Humana, Totowa, NJ, pp. 595–610.
Li, J. Y., Sugimura, K., Boado, R. J., et al. (1999) Genetically engineered brain drug delivery vectors: cloning, expression, and in vivo application of an anti-transferrin receptor single chain antibody-streptavidin fusion gene and protein. Protein Eng. 12, 787–796.
Plückthun, A. and Pack, P. (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology 3, 83–105.
Kipriyanov, S. M., Moldenhauer, G., Martin, A. C., Kupriyanova, O. A., and Little, M. (1997) Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity. Protein Eng. 10, 445–453.
Leung, S. O., Goldenberg, D. M., Dion, A. S., et al. (1995) Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2. Mol. Immunol. 32, 1413–1427.
Co, M. S., Scheinberg, D. A., Avdalovic, N. M., et al. (1993) Genetically engineered deglycosylation of the variable domain increases the affinity of an anti-CD33 monoclonal antibody. Mol. Immunol. 30, 1361–1367.
Nishimura, E., Mochizuki, K., Kato, M., et al. (1999) Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle. Hum. Antibod. 9, 111–124.
Skerra, A. and Plückthun, A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038–1041.
Schmiedl, A., Breitling, F., Winter, C., Queitsch, I., and Dübel, S. (2000) Effects of unpaired cysteines on yield, solubility and activity of different recombinant antibody constructs expressed in E. coli. J. Immunol. Methods 242, 101–114.
Kontermann, R. and Dübel, S. (eds.) (2001) Antibody Engineering. Springer-Verlag, New York (ISBN 3-540-41354-5).
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Zhou, H., Fisher, R. J., and Papas, T. S. (1994) Optimization of primer sequences for mouse scFv repertoire display library construction. Nucleic Acids Res. 22, 888–889.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Toleikis, L., Broders, O., Dübel, S. (2004). Cloning Single-Chain Antibody Fragments (scFv) from Hybridoma Cells. In: Decler, J., Reischl, U. (eds) Molecular Diagnosis of Infectious Diseases. Methods in Molecular Medicine™, vol 94. Humana Press. https://doi.org/10.1385/1-59259-679-7:447
Download citation
DOI: https://doi.org/10.1385/1-59259-679-7:447
Publisher Name: Humana Press
Print ISBN: 978-1-58829-221-6
Online ISBN: 978-1-59259-679-9
eBook Packages: Springer Protocols