Hybridoma cDNA can be difficult to clone using V region PCR. This is due to mutations in primer regions as well as the presence of other expressed V genes in the hybridoma. In this chapter, the use of RACE to clone V genes is described. This avoids some of the problems associated with the use of V gene PCR.
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
Carroll WL, Mendel E, Levy S (1988) Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol Immunol 25:991–995
Coloma MJ, Larrick JW, Ayala M, Gavilondo-Cowley JV (1991) Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR. Biotechniques 11(152–4):56
Doenecke A, Winnacker EL, Hallek M (1997) Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines. Leukemia 11:1787–1792
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002
Gavilondo-Cowley JV, Coloma MJ, Vazquez J, Ayala M, Macias A, Fry KE, Larrick JW (1990) Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells. Hybridoma 9:407–417
Honegger A, Pluckthun A (2001) The influence of the buried glutamine or glutamate residue in position 6 on the structure of immunoglobulin variable domains. J Mol Biol 309:687–699
Jung S, Spinelli S, Schimmele B, Honegger A, Pugliese L, Cambillau C, Pluckthun A (2001) The importance of framework residues H6, H7, and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody V(H) domains. J Mol Biol 309:701–716
Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Pluckthun A (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201:35–55
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554
Persic L, Roberts A, Wilton J, Cattaneo A, Bradbury A, Hoogenboom H (1997a) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187:9–18
Persic L, Righi M, Roberts A, Hoogenboom HR, Cattaneo A, Bradbury A (1997b) Targeting vectors for intracellular immunisation. Gene 187:1–8
Pescatori M, Bradbury A, Bouet F, Gargano N, Mastrogiacomo A, Grasso A (1995) The cloning of a cDNA encoding a protein (latrodectin), which co-purifies with the alpha-latrotoxin from the black widow spider, Latrodectus tredecimguttatus (Theridiidae). Eur J Biochem 230:322–328
Rohatgi S, Ganju P, Sehgal D (2008) Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from a small number of B cells. J Immunol Methods 339:205–219
Ruberti F, Bradbury A, Cattaneo A (1993) Cloning and expression of an anti-nerve growth factor (NGF) antibody for studies using the neuroantibody approach. Cell Mol Neurobiol 13:559–568
Ruberti F, Cattaneo A, Bradbury A (1994) The use of the RACE method to clone hybridoma cDNA when V region primers fail. J Immunol Methods 173:33–39
Sblattero D, Bradbury A (2000) Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 18:75–80
Storb U, Arp B, Wilson R (1980) Myeloma with multiple rearranged immunoglobulin kappa genes: only one kappa gene codes for kappa chains. Nucleic Acids Res 8:4681–4687
Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D (2000) Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity. J Immunol Methods 233:167–177
Zack DJ, Wong AL, Stempniak M, Weisbart RH (1995) Two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma: implications for isotypic exclusion. Mol Immunol 32:1345–1353
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
Bradbury, A. (2010). Coning Hybridoma cDNA by RACE. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_2
Download citation
DOI: https://doi.org/10.1007/978-3-642-01144-3_2
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01143-6
Online ISBN: 978-3-642-01144-3
eBook Packages: Springer Protocols