Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Journal name:
Nature Protocols
Volume:
6,
Pages:
668–676
Year published:
DOI:
doi:10.1038/nprot.2011.322
Published online

Abstract

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.

At a glance

Figures

  1. A flow chart summarizing the protocol.
    Figure 1: A flow chart summarizing the protocol.

    Lymphocytes are prepared from immunized mice or vaccinated volunteers (Steps 1–21) and then applied to the chip (Steps 22–25). Cells are cultured in wells of a chip whose surface is coated with anti-IgG (Steps 26,27). Antigen-specific IgG-secreting cells are detected on the chip and retrieved using a microcapillary (Steps 28–39). Antigen-specific antibody-secreting cells can be detected and retrieved within 7 h. The protocol requires a microwell array chip and a relatively experienced researcher. Parts of the flow chart were derived from our previously published paper6.

  2. Cell retrieval using a micromanipulator.
    Figure 2: Cell retrieval using a micromanipulator.

    (a) Position of capillary for cell retrieval. (b) Position of capillary and micromanipulator for cell retrieval (retrieval position). (c) Position of capillary and micromanipulator for cell ejection (ejection position). (d) Transfer of a cell into a PCR tube.

  3. Identification of antigen-specific ASCs using ISAAC.
    Figure 3: Identification of antigen-specific ASCs using ISAAC.

    TransChromo Mice that have an artificial chromosome containing human Igγ and human Igκ genes were immunized with a recombinant human TRAIL-R1/Fc fusion protein, and TRAIL-R1 (TR1)-specific ASCs in CD138+ splenocytes were detected using ISAAC. Antibody cDNAs were recovered from individual cells and used for antibody production (Table 3). The antigen specificity of the produced antibodies was analyzed using ELISA. x axis, antibodies analyzed; y axis, optical density (OD). PC, serum from a TRAIL-R1-immunized mouse; closed columns, optical densities from TRAIL-R1-coated wells; open columns, optical densities from uncoated wells. Error bars indicate s.e.m. (n=3). The Committee on Animal Experiments at the University of Toyama approved the protocols for the animal experiments. The results were derived, with permission, from our published paper16.

References

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Affiliations

  1. Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

    • Aishun Jin,
    • Tatsuhiko Ozawa,
    • Hiroyuki Kishi &
    • Atsushi Muraguchi
  2. Department of Immunology, College of Basic Medical Science, Harbin Medical University, Harbin, China.

    • Aishun Jin
  3. The Third Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

    • Kazuto Tajiri
  4. Central Research Institute, Toyama Industrial Technology Center, Toyama, Japan.

    • Tsutomu Obata

Contributions

H.K. conceived of the ISAAC method. H.K. and A.M. supervised the project. A.J. designed and performed experiments. T. Obata developed the chip and A.J. tested it. A.J., T. Ozawa and K.T. tested and showed the utility of the protocol.

Competing financial interests

H.K. and A.M. are unsalaried directors of SC World Inc.

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