Some hybridoma cell lines are very low producers, difficult to cultivate, or antibody production is lost upon prolonged culture. In these cases, a recombinant "hybridoma immortalization" can rescue a valuable antibody for further unlimited propagation in E. coli or other recombinant production systems. Further, monoclonal antibodies with promising specificity, but low affinity, can be improved by phage display, or mouse antibodies can be humanised for therapy. This chapter describes sets of thoroughly validated oligonucleotide primers for a PCR procedure to isolate and clone the DNA of antibody Fv fragments (antigen binding region) from mouse and rat hybridoma cell lines. The protocol includes a colony staining assay to identify E. coli clones producing scFv antibody fragments.
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
Breitling F, Dübel S (1999) Recombinant antibodies. Wiley, New York, ISBN:0-471-17847-0
Brocks B, Garin-Chesa P, Behrle E, Park JE, Rettig WJ, Pfizenmaier K, Moosmayer D (2001) Species-crossreactive scFv against the tumor stroma marker “fibroblast activation protein” selected by phage display from an immunized FAP-/- knock-out mouse. Mol Med 7:461–469
Dübel S, Breitling F, Klewinghaus I, Little M (1992) Regulated secretion and purification of recombinant antibodies in E.coli. Cell Biophys 21:69–79
Dübel S, Breitling F, Fuchs P, Zewe M, Gotter S, Moldenhauer G, Little M (1994) Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers. J Immunol Methods 175:89–95
Evan GI, Lewis GK, Ramsay G, Bishop M (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616
Fuchs P, Breitling F, Little M, Dübel S (1997) Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc. Hybridoma 16:227–233
Liu Z, Song D, Kramer A, Martin AC, Dandekar T, Schneider-Mergener J, Bautz EK, Dübel S (1999) Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster. J Mol Recognit 12:103–111
Moosmayer D, Dübel S, Brocke B, Watzka H, Hampp C, Scheurich P, Little M, Pfizenmaier K (1995) A single chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity. Ther Immunol 2:31–40
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schiweck W, Buxbaum B, Schatzlein C, Neiss HG, Skerra A (1997) Sequence analysis and bacterial production of the anti-c-myc antibody 9E10: the V(H) domain has an extended CDR-H3 and exhibits unusual solubility. FEBS Lett 414:33–38
Strebe N, Guse A, Schüngel M, Schirrmann T, Hafner M, Jostock T, Hust M, Müller W, Dübel S (2009) Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies. J Immunol Methods 341:30–40
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer-Verlag
About this protocol
Cite this protocol
Strebe, N., Breitling, F., Moosmayer, D., Brocks, B., Dübel, S. (2010). Cloning of Variable Domains from Mouse Hybridoma by PCR. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_1
Download citation
DOI: https://doi.org/10.1007/978-3-642-01144-3_1
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01143-6
Online ISBN: 978-3-642-01144-3
eBook Packages: Springer Protocols