Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

Abstract

Murine monoclonal antibodies (mAbs) are widely used but have limitations if administered in humans. The use of chimeric or humanized mAbs can reduce immunogenicity. The first step in producing such mAbs is to clone murine variable genes from a hybridoma, but it is possible to amplify both functional and aberrant variable genes, as they coexist in the hybridoma. During the development of a murine–human chimeric antibody, we have cloned from a hybridoma the functional heavy chain variable region (VH) and light chain variable region (VL) genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen. In this study, we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1, the development of a method to distinguish between the functional and abundant aberrant VL transcripts, and the origins of these aberrant genes. The aberrant VL gene is derived from OUR-1 cells, while the aberrant VH gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells. The aberrant VH and VL genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Kuenen B, Witteveen PO, Ruijter R, Giaccone G, Dontabhaktuni A, Fox F et al. A phase I pharmacologic study of necitumumab (IMC-11F8), a fully human IgG1 monoclonal antibody directed against EGFR in patients with advanced solid malignancies. Clin Cancer Res 2010; 16: 1915–1923.

    Article  CAS  Google Scholar 

  2. Azinovic I, DeNardo GL, Lamborn KR, Mirick G, Goldstein D, Bradt BM et al. Survival benefit associated with human anti-mouse antibody (HAMA) in patients with B-cell malignancies. Cancer Immunol Immunother 2006; 55: 1451–1458.

    Article  CAS  Google Scholar 

  3. Chen Z, Moayeri M, Crown D, Emerson S, Gorshkova I, Schuck P et al. Novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody. Infect Immun 2009; 77: 3902–3908.

    Article  CAS  Google Scholar 

  4. Riley CJ, Engelhardt KP, Saldanha JW, Qi W, Cooke LS, Zhu Y et al. Design and activity of a murine and humanized anti-CEACAM6 single-chain variable fragment in the treatment of pancreatic cancer. Cancer Res 2009; 69: 1933–1940.

    Article  CAS  Google Scholar 

  5. Huang YJ, Chen IC, Yu CM, Lee YC, Hsu HJ, Ching AT et al. Engineering anti-vascular endothelial growth factor single chain disulfide-stabilized antibody variable fragments (sc-dsFv) with phage-displayed sc-dsFv libraries. J Biol Chem 2010; 285: 7880–7891.

    Article  CAS  Google Scholar 

  6. Stadlmann J, Pabst M, Kolarich D, Kunert R, Altmann F . Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides. Proteomics 2008; 8: 2858–2871.

    Article  CAS  Google Scholar 

  7. Carroll WL, Mendel E, Levy S . Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol Immunol 1988; 25: 991–995.

    Article  CAS  Google Scholar 

  8. Laurent TC, Mertens P, Dierick JF, Letesson JJ, Lambert C, de Bolle X . Functional, molecular and structural characterisation of five anti-Brucella LPS mAb. Mol Immunol 2004; 40: 1237–1247.

    Article  CAS  Google Scholar 

  9. Nicholls PJ, Johnson VG, Blanford MD, Andrew SM . An improved method for generating single-chain antibodies from hybridomas. J Immunol Methods 1993; 165: 81–91.

    Article  CAS  Google Scholar 

  10. Cochet O, Martin E, Fridman WH, Teillaud JL . Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived V kappa by PNA-mediated PCR clamping. Biotechniques 1999; 26: 818–820.

    Article  CAS  Google Scholar 

  11. Ostermeier C, Michel H . Improved cloning of antibody variable regions from hybridomas by an antisense-directed RNase H digestion of the P3-X63-Ag8.653 derived pseudogene mRNA. Nucleic Acids Res 1996; 24: 1979–1980.

    Article  CAS  Google Scholar 

  12. Duan L, Pomerantz RJ . Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections. Nucleic Acids Res 1994; 22: 5433–5438.

    Article  CAS  Google Scholar 

  13. Juste M, Muzard J, Billiald P . Cloning of the antibody kappa light chain V-gene from murine hybridomas by bypassing the aberrant MOPC21-derived transcript. Anal Biochem 2006; 349: 159–161.

    Article  CAS  Google Scholar 

  14. Irani Y, Tea M, Tilton RG, Coster DJ, Williams KA, Brereton HM . PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts. J Immunol Methods 2008; 336: 246–250.

    Article  CAS  Google Scholar 

  15. KĂ¼temeier G, Harloff C, Mocikat R . Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction. Hybridoma 1992; 11: 23–32.

    Article  Google Scholar 

  16. Lawler AM, Lin PS, Gearhart PJ . Adult B-cell repertoire is biased toward two heavy-chain variable-region genes that rearrange frequently in fetal pre-B cells. Proc Natl Acad Sci USA 1987; 84: 2454–2458.

    Article  CAS  Google Scholar 

  17. Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 1997; 201: 35–55.

    Article  CAS  Google Scholar 

  18. Vidarsson G, van de Winkel JG, van Dijk MA . Multiplex screening for functionally rearranged immunoglobulin variable regions reveals expression of hybridoma-specific aberrant V-genes. J Immunol Methods 2001; 249: 245–252.

    Article  CAS  Google Scholar 

  19. Zhao P, Liang X, Kalbfleisch J, Koo HM, Cao B . Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum Antibodies 2003; 12: 129–135.

    Article  CAS  Google Scholar 

  20. Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF . Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. In: Barbas CF, Burton DR, Scott JK, Silverman GJ (eds.) Phage Display: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 2001: 9.37–9.52.

    Google Scholar 

  21. Coloma MJ, Hastings A, Wims LA, Morrison SL . Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. J Immunol Methods 1992; 152: 89–104.

    Article  CAS  Google Scholar 

  22. Roque-Navarro L, Mateo C, Lombardero J, Mustelier G, FernĂ¡ndez A, Sosa K et al. Humanization of predicted T-cell epitopes reduces the immunogenicity of chimeric antibodies: new evidence supporting a simple method. Hybrid Hybridomics 2003; 22: 245–257.

    Article  CAS  Google Scholar 

  23. Sugahara T, Nishimoto S, Miyazaki Y . Effects of polyamines on proliferation and IgM productivity of human–human hybridoma, HB4C5 cells. Cytotechnology 2008; 57: 115–122.

    Article  CAS  Google Scholar 

  24. Chen Z, Moayeri M, Zhao H, Crown D, Leppla SH, Purcell RH . Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding. Proc Natl Acad Sci USA 2009; 106: 13487–13492.

    Article  CAS  Google Scholar 

  25. Rohatgi S, Ganju P, Sehgal D . Systematic design and testing of nested (RT-) PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells. J Immunol Methods 2008; 339: 205–219.

    Article  CAS  Google Scholar 

  26. Lee KW, Hur BU, Song SY, Choi HJ, Shin SH, Cha SH . Methods for rapid identification of a functional single-chain variable fragment using alkaline phosphatase fusion. BMB Rep 2009; 42: 731–736.

    Article  CAS  Google Scholar 

  27. Wang D, Lu TY, Liu HB . Characterization and genetic diversity of the sturgeon Acipenser schrenskii Ig heavy chain. Immunobiology 2009; 214: 359–366.

    Article  CAS  Google Scholar 

  28. Miranda LR, Duval M, Doherty H, Seaman MS, Posner MR, Cavacini LA . The neutralization properties of a HIV-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain. J Immunol 2007; 178: 7132–7138.

    Article  CAS  Google Scholar 

  29. Goodyear CS, O'Hanlon GM, Plomp JJ, Wagner ER, Morrison I, Veitch J et al. Monoclonal antibodies raised against Guillain–Barré syndrome-associated Campylobacter jejuni lipopolysaccharides react with neuronal gangliosides and paralyze muscle–nerve preparations. J Clin Invest 1999; 104: 697–708.

    Article  CAS  Google Scholar 

  30. Fang W, Mueller DL, Pennell CA, Rivard JJ, Li YS, Hardy RR et al. Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. Immunity 1996; 4: 291–299.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr David Petillo for DNA sequencing, Ms Tessa Grabinski for technical and editing assistance and Mr David Nadziejka for scientific review and editing.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Brian Cao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ding, G., Chen, X., Zhu, J. et al. Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes. Cell Mol Immunol 7, 349–354 (2010). https://doi.org/10.1038/cmi.2010.33

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cmi.2010.33

Keywords

This article is cited by

Search

Quick links