Skip to main content

Thank you for visiting 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.

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring


Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells1,2,3,4,5. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening6,7.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: DropMap technology principle and calibration.
Figure 2: Ex vivo measurement of spleen and bone marrow single-cell IgG secretion rates and affinities using DropMap.
Figure 3: Evolution of single-cell secretion rates and affinities of anti-TT IgG-secreting cells over the time course of immunization.


  1. Nossal, G.J.V. & Mäkelä, O. Elaboration of antibodies by single cells. Annu. Rev. Microbiol. 16, 53–74 (1962).

    Article  CAS  Google Scholar 

  2. Helmreich, E., Kern, M. & Eisen, H.N. The secretion of antibody by isolated lymph node cells. J. Biol. Chem. 236, 464–473 (1961).

    CAS  PubMed  Google Scholar 

  3. Hibi, T. & Dosch, H.M. Limiting dilution analysis of the B cell compartment in human bone marrow. Eur. J. Immunol. 16, 139–145 (1986).

    Article  CAS  Google Scholar 

  4. Bromage, E., Stephens, R. & Hassoun, L. The third dimension of ELISPOTs: quantifying antibody secretion from individual plasma cells. J. Immunol. Methods 346, 75–79 (2009).

    Article  CAS  Google Scholar 

  5. Salmon, S.E. & Smith, B.A. Immunoglobulin synthesis and total body tumor cell number in IgG multiple myeloma. J. Clin. Invest. 49, 1114–1121 (1970).

    Article  CAS  Google Scholar 

  6. El Debs, B., Utharala, R., Balyasnikova, I.V., Griffiths, A.D. & Merten, C.A. Functional single-cell hybridoma screening using droplet-based microfluidics. Proc. Natl. Acad. Sci. USA 109, 11570–11575 (2012).

    Article  CAS  Google Scholar 

  7. Mazutis, L. et al. Single-cell analysis and sorting using droplet-based microfluidics. Nat. Protoc. 8, 870–891 (2013).

    Article  CAS  Google Scholar 

  8. Radbruch, A. et al. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6, 741–750 (2006).

    Article  CAS  Google Scholar 

  9. Eisen, H.N. Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses. Cancer Immunol. Res. 2, 381–392 (2014).

    Article  CAS  Google Scholar 

  10. Wine, Y. et al. Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response. Proc. Natl. Acad. Sci. USA 110, 2993–2998 (2013).

    Article  CAS  Google Scholar 

  11. Lavinder, J.J. et al. Identification and characterization of the constituent human serum antibodies elicited by vaccination. Proc. Natl. Acad. Sci. USA 111, 2259–2264 (2014).

    Article  CAS  Google Scholar 

  12. Nossal, G.J.V. & Lederberg, J. Antibody production by single cells. Nature 181, 1419–1420 (1958).

    Article  CAS  Google Scholar 

  13. Georgiou, G. et al. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat. Biotechnol. 32, 158–168 (2014).

    Article  CAS  Google Scholar 

  14. Tas, J.M. et al. Visualizing antibody affinity maturation in germinal centers. Science 351, 1048–1054 (2016).

    Article  CAS  Google Scholar 

  15. Czerkinsky, C.C., Nilsson, L.A., Nygren, H., Ouchterlony, O. & Tarkowski, A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65, 109–121 (1983).

    Article  CAS  Google Scholar 

  16. Saletti, G., Çuburu, N., Yang, J.S., Dey, A. & Czerkinsky, C. Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood. Nat. Protoc. 8, 1073–1087 (2013).

    Article  Google Scholar 

  17. Clargo, A.M. et al. The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method. MAbs 6, 143–159 (2014).

    Article  Google Scholar 

  18. Love, J.C., Ronan, J.L., Grotenbreg, G.M., van der Veen, A.G. & Ploegh, H.L. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 24, 703–707 (2006).

    Article  CAS  Google Scholar 

  19. Jin, A. et al. Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array. Nat. Protoc. 6, 668–676 (2011).

    Article  CAS  Google Scholar 

  20. Köster, S. et al. Drop-based microfluidic devices for encapsulation of single cells. Lab Chip 8, 1110–1115 (2008).

    Article  Google Scholar 

  21. Boitard, L. et al. Monitoring single-cell bioenergetics via the coarsening of emulsion droplets. Proc. Natl. Acad. Sci. USA 109, 7181–7186 (2012).

    Article  CAS  Google Scholar 

  22. Anna, S.L., Bontoux, N. & Stone, H.A. Formation of dispersions using “flow focusing” in microchannels. Appl. Phys. Lett. 82, 364–366 (2003).

    Article  CAS  Google Scholar 

  23. Foote, J. & Eisen, H.N. Kinetic and affinity limits on antibodies produced during immune responses. Proc. Natl. Acad. Sci. USA 92, 1254–1256 (1995).

    Article  CAS  Google Scholar 

  24. Poulsen, T.R., Meijer, P.J., Jensen, A., Nielsen, L.S. & Andersen, P.S. Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid. J. Immunol. 179, 3841–3850 (2007).

    Article  CAS  Google Scholar 

  25. Pilbrough, W., Munro, T.P. & Gray, P. Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS One 4, e8432 (2009).

    Article  Google Scholar 

  26. Sigal, A. et al. Variability and memory of protein levels in human cells. Nature 444, 643–646 (2006).

    Article  CAS  Google Scholar 

  27. Vieira, P. & Rajewsky, K. The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18, 313–316 (1988).

    Article  CAS  Google Scholar 

  28. Yokoyama, W.M. et al. Production of monoclonal antibodies. Curr. Protoc. Immunol. 102, 205 (2013).

    Article  Google Scholar 

  29. Greenfield, E.A. Antibodies: A Laboratory Manual. 2nd edn. (CSH Press, 2014).

  30. Bernasconi, N.L., Traggiai, E. & Lanzavecchia, A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298, 2199–2202 (2002).

    Article  CAS  Google Scholar 

  31. Duffy, D.C., McDonald, J.C., Schueller, O.J.A. & Whitesides, G.M. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal. Chem. 70, 4974–4984 (1998).

    Article  CAS  Google Scholar 

  32. Duda, R.O. & Hart, P.E. Use of the Hough transformation to detect lines and curves in pictures. Commun. ACM 15, 11–15 (1972).

    Article  Google Scholar 

Download references


We would like to thank the Institut Pierre-Gilles de Gennes (IPGG) for use of clean room facilities and the laser engraver (CII08, Axyslaser), and Pfizer for the generous gift of TT-Alexa488, and CHO cell lines secreting TT4, TT7, and TT10. TT11 and TT27 antibodies are Pfizer proprietary antibodies isolated from their collaboration with HiFiBiO. We thank Pfizer (M. Holsti, G. Cheung, and W. Somers) as well as HiFiBiO Team (A. Gérard, A. Woolfe, M. Reichen, A. Poitou, S. Essonno, R. Kumar, S. Ellouze, K. Grosselin, B. Shen, and C. Brenan) for identification, rapid cloning, and validation of TT11 and TT27 antibodies. This work received support from the French Investissements d'Avenir program under the grant agreements ANR-10-NANO-02, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31 and ANR-10- EQPX-34, by the French Agence Nationale de la Recherche (ANR-14-CE16-0011 project DROPmAbs), from Région Ile-de-France (DIM NanoK) and by the Institut Carnot Pasteur Maladies Infectieuses. K.E. acknowledges financial support from the 'Fondation Pierre-Gilles de Gennes', and the 'Swiss National Science Foundation' and the 'Society in Science—The Branco Weiss Fellowship'. C.C. acknowledges financial support from CONCYTEC, Peru. We would like to further acknowledge R. Henson for making the dscatter function for Matlab publicly available, M. Spitzer, J. Wildenhain, J. Rappsilber and M. Tyers for BoxPlotR that has been used to create the box plots, and B. Iannascoli (Unit of Antibodies in Therapy and Pathology, Institut Pasteur) for help with antibody production and cell lines.

Author information

Authors and Affiliations



K.E., A.J., A.D.G., J. Bibette, P.B. and J. Baudry designed the study; K.E., R.C.L.D., A.G., E.B.-L., C.N., A.D.G., J. Bibette and J. Baudry developed the technology; K.E., R.C.L.D., C.E.C., L.B.-R., V.M., G.M., P.E. and A.G. performed the experiments; K.E., A.D.G., P.B. and J. Baudry analyzed the data and wrote the paper.

Corresponding authors

Correspondence to Andrew D Griffiths, Pierre Bruhns or Jean Baudry.

Ethics declarations

Competing interests

J. Bibette and A.D.G. are co-founders of Seven Pines Holding BV, and HiFiBiO SAS is a subsidiary of Seven Pines Holding BV. Patents have been filed on some aspects of this work and the inventors may receive payments related to exploitation of these under their employer's rewards-to-inventors scheme.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Eyer, K., Doineau, R., Castrillon, C. et al. Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring. Nat Biotechnol 35, 977–982 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research