Isolation of Antigen-Specific, Antibody-Secreting Cells Using a Chip-Based Immunospot Array

  • Hiroyuki KishiEmail author
  • Tatsuhiko Ozawa
  • Hiroshi Hamana
  • Eiji Kobayashi
  • Atsushi Muraguchi
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)


Antigen-specific monoclonal antibodies are useful tools to detect very small amounts of antigenic materials and are applicable for antibody therapeutics. To produce mouse monoclonal antibodies, a hybridoma between B lymphocytes and myeloma cells is used to produce antigen-specific monoclonal antibodies. However, a good hybridoma system is not available to obtain human monoclonal antibodies. To produce antigen-specific human monoclonal antibodies, transformation of B lymphocytes with Epstein-Barr viruses or a phage-display system is used. Here, we describe the screening of antigen-specific, antibody-secreting cells using microwell array chips to obtain antigen-specific human monoclonal antibodies. The system can be applied to screen antigen-specific, antibody-secreting cells from any animal species.

Key words

Antigen-specific antibody Antibody-secreting cell Microwell-array chip Immunospot array assay on a chip 



This work was supported by grants from the Toyama Medical Bio-Cluster Project and Hokuriku Innovation Cluster for Health Science of the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also supported by JSPS KAKENHI Grant Number JP16H06499 (H.K.), the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science (MEXT) and the Japan Agency for Medical Research and Development (AMED) (A.M.), and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101077 (T.O.).


  1. 1.
    Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497CrossRefGoogle Scholar
  2. 2.
    Kozbor D, Roder JC (1981) Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique. J Immunol 127:1275–1280PubMedGoogle Scholar
  3. 3.
    Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR et al (2004) An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med 10:871–875CrossRefGoogle Scholar
  4. 4.
    McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552–554CrossRefGoogle Scholar
  5. 5.
    Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12:433–455CrossRefGoogle Scholar
  6. 6.
    Ozawa T, Kinoshita K, Kadowaki S, Tajiri K, Kondo S, Honda R et al (2009) MAC-CCD system: a novel lymphocyte microwell-array chip system equipped with CCD scanner to generate human monoclonal antibodies against influenza virus. Lab Chip 9:158–163CrossRefGoogle Scholar
  7. 7.
    Tajiri K, Kishi H, Tokimitsu Y, Kondo S, Ozawa T, Kinoshita K et al (2007) Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity. Cytometry A 71:961–967CrossRefGoogle Scholar
  8. 8.
    Tokimitsu Y, Kishi H, Kondo S, Honda R, Tajiri K, Motoki K et al (2007) Single lymphocyte analysis with a microwell array chip. Cytometry A 71:1003–1010CrossRefGoogle Scholar
  9. 9.
    Yamamura S, Kishi H, Tokimitsu Y, Kondo S, Honda R, Rao SR et al (2005) Single-cell microarray for analyzing cellular response. Anal Chem 77:8050–8056CrossRefGoogle Scholar
  10. 10.
    Jin A, Ozawa T, Tajiri K, Obata T, Kondo S, Kinoshita K et al (2009) A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 15:1088–1092CrossRefGoogle Scholar
  11. 11.
    Love JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24:703–707CrossRefGoogle Scholar
  12. 12.
    Deutsch M, Deutsch A, Shirihai O, Hurevich I, Afrimzon E, Shafran Y et al (2006) A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells. Lab Chip 6:995–1000CrossRefGoogle Scholar
  13. 13.
    Biran I, Walt DR (2002) Optical imaging fiber-based single live cell arrays: a high-density cell assay platform. Anal Chem 74:3046–3054CrossRefGoogle Scholar
  14. 14.
    Zaimoku Y, Takamatsu H, Hosomichi K, Ozawa T, Nakagawa N, Imi T et al (2017) Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia. Blood 129:2908–2916CrossRefGoogle Scholar
  15. 15.
    Kurosawa N, Yoshioka M, Fujimoto R, Yamagishi F, Isobe M (2012) Rapid production of antigen-specific monoclonal antibodies from a variety of animals. BMC Biol 10:80CrossRefGoogle Scholar
  16. 16.
    Giudicelli V, Chaume D, Lefranc MP (2004) IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res 32:W435–W440CrossRefGoogle Scholar
  17. 17.
    Bernasconi NL, Traggiai E, Lanzavecchia A (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298:2199–2202CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hiroyuki Kishi
    • 1
    Email author
  • Tatsuhiko Ozawa
    • 1
  • Hiroshi Hamana
    • 1
  • Eiji Kobayashi
    • 1
  • Atsushi Muraguchi
    • 1
  1. 1.Department of Immunology, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan

Personalised recommendations