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. 1999 Sep;31(1-2):133–141. doi: 10.1023/A:1008063903946

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

Akira Ichikawa, Yoshinori Katakura, Kiichiro Teruya, Shuichi Hashizume, Sanetaka Shirahata
PMCID: PMC3449779  PMID: 19003133

Abstract

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method.

Keywords: cholera toxin B subunit, human monoclonal antibody, interleukin-2, interleukin-4, in vitro immunization

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References

  1. Alexander H, Harpprecht J, Podzuweit HG, Rautenberg P, Müller-Ruchholtz W. Human monoclonal antibodies recognize early and late viral proteins of human cytomegalovirus. Hum Antibod Hybridomas. 1994;5:81–90. [PubMed] [Google Scholar]
  2. Alfonso M, Lanne B, Ifversen P, Vázquez AM, Pérez R, Portoukalian J, Zeuthen J. Antigen-specific primary immune response of human B-lymphocytes after in vitro immunization with GM3 ganglioside. Hum Antibod Hybridomas. 1995;6:102–112. [PubMed] [Google Scholar]
  3. Borrebaeck CAK. Strategy for the production of monoclonal antibodies using in vitro activated B-cells. J Immunol Methods. 1989;123:157–165. doi: 10.1016/0022-1759(89)90219-6. [DOI] [PubMed] [Google Scholar]
  4. Boyd AW, Fisher DC, Fox DA, Schlossman SF, Nadler LM. Structural and functional characterization of IL-2 receptors on activated human B cells. J Immunol. 1985;134:2387–2392. [PubMed] [Google Scholar]
  5. Defrance T, Vanbervliet B, Aubry JP, Takabe Y, Arai N, Miyajima A, Yokota T, Lee F, Arai KI. B cell growth-promoting activity of recombinant human interleukin 4. J Immunol. 1987;139:1135–1141. [PubMed] [Google Scholar]
  6. Defrance T, Vanbervliet B, Pene J, Banchereau J. Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM. J Immunol. 1988;141:2000–2005. [PubMed] [Google Scholar]
  7. Glassy MC. Production methods for generating human monoclonal antibodies. Hum Antibod Hybridomas. 1993;4:154–165. [PubMed] [Google Scholar]
  8. Gupta KC, Agha R, Santos E, Brodeur BR. Isolation of human monoclonal antibodies binding to B fragment of diphtheria toxin. Hum Antibod Hybridomas. 1992;3:25–31. [PubMed] [Google Scholar]
  9. Hoffmann P, Jimenez-Diaz M, Loleit M, Tröger W, Wiesmüller KH, Metzger J, Jung G, Kaiser I, Stöcklin S, Lenzner S, Peters JH, Grimm R, Schäfer E, Bessler WG. Preparation of human and murine monoclonal antibodies: antigens combined with or conjugated to lipopeptides constitute potent immunogens for in vitro and in vivo immunizations. Hum Antibod Hybridomas. 1990;1:137–144. [PubMed] [Google Scholar]
  10. James K, Bell TG. Human monoclonal antibody production. J Immunol Methods. 1987;100:5–40. doi: 10.1016/0022-1759(87)90170-0. [DOI] [PubMed] [Google Scholar]
  11. Jelinek DF, Lipsky PE. Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells. J Immunol. 1987;139:1005–1013. [PubMed] [Google Scholar]
  12. Kawahara H, Shirahata S, Tachibana H, Murakami H. In vitro immunization of human lymphocytes with human lung cancer cell line A549. Hum Antibod Hybridomas. 1992;3:8–13. [PubMed] [Google Scholar]
  13. Koda K, Glassy MC. In vitro immunization for the production of human monoclonal antibody. Hum Antibod Hybridomas. 1990;1:15–22. [PubMed] [Google Scholar]
  14. Laroche-Traineau J, Clofent-Sanchez, Venzon G, Nurden AT. The ex vivo production of human monoclonal antibodies to glycoprotein IIb-IIIa complexes of Blood platelets. Hum Antibod Hybridomas. 1994;5:165–177. [PubMed] [Google Scholar]
  15. Paul WE, Ohara J. B-cell stimulatory factor-1/interleukin 4. Annu Rev Immunol. 1987;5:429–459. doi: 10.1146/annurev.iy.05.040187.002241. [DOI] [PubMed] [Google Scholar]
  16. Power DA, Gerkis V, d'Apice AJF. Production of human monoclonal antibodies to B lymphocyte cell surface antigens by in vitro immunization and human-human hybridoma technology. Hum Antibod Hybridomas. 1990;1:34–41. [PubMed] [Google Scholar]
  17. Sahasrabuddhe GC, Martin BA, Davis FM. Immunological evidence for the relation between low Mr secreted form of human B cell growth factor and an intracellular 60K proteins. Lymphokine Res. 1988;7:141–155. [PubMed] [Google Scholar]
  18. Schroeder TJ, First MR, Mansour ME, Hurtubise PE, Hariharan S, Ryckman FC, Munda R, Melvin DB, Penn I, Ballistreri WF, Alexander JW. Antimurine antibody formation following OKT3 therapy. Transplantation. 1990;49:48–51. doi: 10.1097/00007890-199001000-00010. [DOI] [PubMed] [Google Scholar]
  19. Shoji M, Kawamoto S, Sato S, Kamei M, Kato M, Hashizume S, Seki K, Yasumoto K, Nagashima A, Nakanishi H, Suzuki T, Imai T., Nomoto K, Murakami H. Specific reactivity of human monoclonal antibody AE6F4 against cancer cells in tissues and sputa from lung cancer patients. Hum Antibod Hybridomas. 1994;5:116–122. [PubMed] [Google Scholar]
  20. Shoji M, Kawamoto S, Setoguchi Y, Mochizuki K, Honjoh T, Kato M, Hashizume S, Hanagiri T, Yoshimatsu T, Nakanishi K, Yasumoto K, Nagashima A, Nakahashi H, Suzuki T, Imai T, Nomoto K, Murakami H. The 14–3–3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4. Hum Antibod Hybridomas. 1994;5:123–130. [PubMed] [Google Scholar]
  21. Yokota T, Arai N, deVries J, Spits H, Banchereau J, Zlotnik A, Rennick D, Howard M, Takabe Y, Miyatake S, Lee F, Arai KI. Molecular biology of interleukin 4 and interleukin 5 gene and biology of their products that stimulate B cells, T cells and hematopoietic cells. Immunol Rev. 1988;102:137–187. doi: 10.1111/j.1600-065X.1988.tb00744.x. [DOI] [PubMed] [Google Scholar]

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