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.

A microengraving method for rapid selection of single cells producing antigen-specific antibodies

Abstract

Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research1. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Schematic diagram depicting method for preparation of engraved arrays of secreted products from a mixture of cells.
Figure 2: Two methods for detection of antibodies on the surface of a glass slide after microengraving.
Figure 3: Selection of a hybridoma from a polyclonal mixture and validation of the specificity of its antibody.

References

  1. 1

    Brekke, O.H. & Sandlie, I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat. Rev. Drug Discov. 2, 52–62 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Fuller, S.A., Takahashi, M. & Hurrell, J.G.R. in Current Protocols in Molecular Biology (eds. Ausubel, F.M. et al.) 11.8.1–11.8.2, (John Wiley & Sons, Inc., New York, 2003).

    Google Scholar 

  3. 3

    Yokoyama, W.M. in Current Protocols in Immunology (eds. Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M. & Strober, W.) 2.5.1–2.5.17, (John Wiley & Sons, Inc., New York, 1995).

    Google Scholar 

  4. 4

    Davis, J.M., Pennington, J.E., Kubler, A.M. & Conscience, J.F. A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods 50, 161–171 (1982).

    CAS  Article  Google Scholar 

  5. 5

    Rueda, A.Z. & Coll, J.M. Cloning of myelomas and hybridomas in fibrin clots. J. Immunol. Methods 114, 213–217 (1988).

    CAS  Article  Google Scholar 

  6. 6

    Herzenberg, L.A. et al. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin. Chem. 48, 1819–1827 (2002).

    CAS  Google Scholar 

  7. 7

    Carroll, S. & Al-Rubeai, M. The selection of high-producing cell lines using flow cytometry and cell sorting. Expert Opin. Biol. Ther. 4, 1821–1829 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Harlow, E. & Lane, D . Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

    Google Scholar 

  9. 9

    He, M. et al. Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets. Anal. Chem. 77, 1539–1544 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Khademhosseini, A. et al. Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays. Lab Chip 5, 1380–1386 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Yamamura, S. et al. Single-cell microarray for analyzing cellular response. Anal. Chem. 77, 8050–8056 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Ostuni, E., Chen, C.S., Ingber, D.E. & Whitesides, G.M. Selective deposition of proteins and cells in arrays of microwells. Langmuir 17, 2828–2834 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Kim, H., Doh, J., Irvine, D.J., Cohen, R.E. & Hammond, P.T. Large area two-dimensional B cell arrays for sensing and cell-sorting applications. Biomacromolecules 5, 822–827 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Barry, R. & Soloviev, M. Quantitative protein profiling using antibody arrays. Proteomics 4, 3717–3726 (2004).

    CAS  Article  Google Scholar 

  15. 15

    Kang, I.K., Ito, Y., Sisido, M. & Imanishi, Y. Gas permeability of the film of block and graft copolymers of polydimethylsiloxane and poly(gamma-benzyl L-glutamate). Biomaterials 9, 349–355 (1988).

    CAS  Article  Google Scholar 

  16. 16

    Lee, J.N., Jiang, X., Ryan, D. & Whitesides, G.M. Compatibility of mammalian cells on surfaces of poly(dimethylsiloxane). Langmuir 20, 11684–11691 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Hendershot, L.M. & Sitia, R. in Molecular Biology of B Cells (eds. Honjo, T., Alt, F. & Neuberger, M.) (Elsevier Science Ltd., London, 2003).

    Google Scholar 

  18. 18

    Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    CAS  Article  Google Scholar 

  19. 19

    Ljunggren, H.G., Oudshoorn-Snoek, M., Masucci, M.G. & Ploegh, H.L. High-resolution one-dimensional isoelectric focusing of mouse MHC class I antigens. Identification of natural and experimentally induced class I antigens. Immunogenetics 32, 440–450 (1990).

    CAS  Article  Google Scholar 

  20. 20

    De Masi, F. et al. High throughput production of mouse monoclonal antibodies using antigen microarrays. Proteomics 5, 4070–4081 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Traggiai, E. et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Pasqualini, R. & Arap, W. Hybridoma-free generation of monoclonal antibodies. Proc. Natl. Acad. Sci. USA 101, 257–259 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Schatz, P.J. Use of peptide libraries to map the substrate specificity of a peptide modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Bio/Technology 11, 1138–1143 (1993).

    CAS  PubMed  Google Scholar 

  24. 24

    Garboczi, D.N., Hung, D.T. & Wiley, D.C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89, 3429–3433 (1992).

    CAS  Article  Google Scholar 

  25. 25

    Lee, J.N., Park, C. & Whitesides, G.M. Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices. Anal. Chem. 75, 6544–6554 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank Jennifer Love (Whitehead Institute Center for Microarray Technology) and Jessie Dausman for technical assistance. This research was supported by grants from the National Institutes of Health and National Academies Keck Futures Initiative, and used facilities at the Center for Nanoscale Systems at Harvard University supported by the NSF under the National Nanotechnology Infrastructure Network. J.C.L. is a Gilead Sciences Fellow of the Life Sciences Research Foundation. G.M.G. is supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research (NWO).

Author information

Affiliations

Authors

Contributions

J.C.L. and J.L.R. developed and implemented the methodology for microengraving, and prepared hybridomas for screening. G.M.G. synthesized the peptide-loaded MHC I tetramers used for immunization and subsequent screening. A.G.V. characterized the antibodies for specificity and isotype. J.C.L. and H.L.P. designed and supervised the project. All authors contributed to the preparation and writing of the manuscript.

Corresponding authors

Correspondence to J Christopher Love or Hidde L Ploegh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Analysis of efficiency for loading microwells with cells. (DOC 169 kb)

Supplementary Fig. 2

Measurement of the viability of individual cells in a closed microenvironment over time. (DOC 26 kb)

Supplementary Fig. 3

Fluorescence micrographs of two microarrays of anti-H-2Kb (red) and anti-ovalbumin (green) prepared sequentially using the same array of microwells loaded with Y3 and Hyb 099-01 cells. (DOC 408 kb)

Supplementary Fig. 4

Fluorescence micrographs of the regions from which the images shown in Figure 3 of the manuscript are taken. (DOC 388 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Love, J., Ronan, J., Grotenbreg, G. et al. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24, 703–707 (2006). https://doi.org/10.1038/nbt1210

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing