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An ex vivo method for rapid generation of monoclonal antibodies (ADLib system)

Nature Protocols volume 1, pages 1502–1506 (2006)Cite this article

Abstract

Here, we describe a protocol for using the ADLib (Autonomously Diversifying Library) system to rapidly generate specific monoclonal antibodies using DT40, a chicken B-cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci. We previously developed the ADLib system on the basis of our finding that gene conversion in DT40 cells was enhanced by treatment of the cells with a histone deacetylase inhibitor, trichostatin A (TSA). TSA treatment evolves a diversified library of DT40 cells (ADLib), in which each cell has different surface IgM specificity. Antigen-specific DT40 cells are selected from ADLib using antigen-conjugated magnetic beads, and their specificity can be examined by various immunological assays, using culture supernatant containing secreted IgM. The whole process from selection to screening can be completed in about 1 week. Thus, the ADLib system will accelerate biological studies, including drug discovery and design.

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Figure 1: Typical result of screening ELISA after the selection to raise anti-rabbit IgG mAbs.
Figure 2: Specificity of anti-chicken lysozyme chicken antibodies.

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References

  1. Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  Google Scholar 

  2. Griffiths, A.D. & Duncan, A.R. Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 9, 102–108 (1998).

    Article  CAS  Google Scholar 

  3. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

    Article  CAS  Google Scholar 

  4. Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455 (1994).

    Article  CAS  Google Scholar 

  5. Ohta, K., Shibata, T. & Nicolas, A. Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J. 13, 5754–5763 (1994).

    Article  CAS  Google Scholar 

  6. Yamada, T. et al. Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot. EMBO J. 23, 1792–1803 (2004).

    Article  CAS  Google Scholar 

  7. Reynaud, C.A., Anquez, V., Dahan, A. & Weill, J.C. A single rearrangement event generates most of the chicken immunoglobulin light chain diversity. Cell 40, 283–291 (1985).

    Article  CAS  Google Scholar 

  8. Reynaud, C.A., Anquez, V., Grimal, H. & Weill, J.C. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48, 379–388 (1987).

    Article  CAS  Google Scholar 

  9. Reynaud, C.A., Dahan, A., Anquez, V. & Weill, J.C. Somatic hyperconversion diversifies the single Vh gene of the chicken with a high incidence in the D region. Cell 59, 171–183 (1989).

    Article  CAS  Google Scholar 

  10. McCormack, W.T. & Thompson, C.B. Somatic diversification of the chicken immunoglobulin light-chain gene. Adv. Immunol. 48, 41–67 (1990).

    Article  CAS  Google Scholar 

  11. McCormack, W.T. & Thompson, C.B. Chicken IgL variable region gene conversions display pseudogene donor preference and 5′ to 3′ polarity. Genes Dev. 4, 548–558 (1990).

    Article  CAS  Google Scholar 

  12. Seo, H. et al. Rapid generation of specific antibodies by enhanced homologous recombination. Nat. Biotechnol. 23, 731–735 (2005).

    Article  CAS  Google Scholar 

  13. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).

    Article  CAS  Google Scholar 

  14. Arakawa, H., Hauschild, J. & Buerstedde, J.M. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295, 1301–1306 (2002).

    Article  CAS  Google Scholar 

  15. Harris, R.S., Sale, J.E., Petersen-Mahrt, S.K. & Neuberger, M.S. AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr. Biol. 12, 435–438 (2002).

    Article  CAS  Google Scholar 

  16. Buerstedde, J.M. et al. Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 9, 921–927 (1990).

    Article  CAS  Google Scholar 

  17. Takata, M. et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508 (1998).

    Article  CAS  Google Scholar 

  18. Sonoda, E. et al. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17, 598–608 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Drs. S. Takeda, M. Takata, T. Yamada, K. Hirota, T. Fukuda and K. Okuhara for helpful advice and discussion; Mr. H. Sasanuma for helpful advice on antibody selection; Ms. K. Arai, A. Nakamura, M. Masuoka, M. Asakawa and Mr. N. Harigai for their experimental support; and all members of Chiome Bioscience Inc. for their help. This work was supported by grants from the REDS (Rational Evolutionary Design of Advanced Biomolecule, Saitama) project, basic research from the Bio-oriented Technology Research Advancement Institution (to T. Shibata and K. Ohta), the New Energy and Industrial Technology Development Organization (to M. Fujiwara) and grants-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K. Ohta).

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Authors and Affiliations

  1. Chiome Bioscience Inc., RIKEN, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama-ken, Japan

    Hidetaka Seo, Shu-ichi Hashimoto & Kyoko Tsuchiya

  2. Genetic System Regulation Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama-ken, Japan

    Hidetaka Seo, Shu-ichi Hashimoto, Kyoko Tsuchiya, Waka Lin & Kunihiro Ohta

  3. REDS Group (Saitama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST), Saitama Small Enterprise Promotion Corporation, 3-12-18 Kamiaoki, Kawaguchi, 333-0844, Saitama, Japan

    Waka Lin

  4. Shibata Distinguished Scientist Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama-ken, Japan

    Takehiko Shibata

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  1. Hidetaka Seo
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  2. Shu-ichi Hashimoto
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  5. Takehiko Shibata
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  6. Kunihiro Ohta
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Correspondence to Hidetaka Seo or Kunihiro Ohta.

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Competing interests

1. We are applying patents that are based on the research. 2. Some authors are founders, employees, and stock-holders of Chiome Bioscience, Inc., a private venture company in Tokyo, Japan.

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Seo, H., Hashimoto, Si., Tsuchiya, K. et al. An ex vivo method for rapid generation of monoclonal antibodies (ADLib system). Nat Protoc 1, 1502–1506 (2006). https://doi.org/10.1038/nprot.2006.248

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