Abstract
Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Processed sequencing files (quality trimmed, merged and barcode assigned) for all data generated in this study were deposited at Sequence Read Archive (SRA) under accession PRJNA529803.
Change history
22 May 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41587-020-0563-7
References
Georgiou, G. et al. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat. Biotechnol. 32, 158–168 (2014).
Eyer, K. et al. Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring. Nat. Biotechnol. 35, 977–982 (2017).
Bannard, O. & Cyster, J. G. Germinal centers: programmed for affinity maturation and antibody diversification. Curr. Opin. Immunol. 45, 21–30 (2017).
Gunn, B. M. & Alter, G. Modulating antibody functionality in infectious disease and vaccination. Trends Mol. Med. 22, 969–982 (2016).
Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M. & Corcoran, L. M. The generation of antibody-secreting plasma cells. Nat. Rev. Immunol. 15, 160–171 (2015).
Meijer, P. J. et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006).
Di Niro, R. et al. High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat. Med. 18, 441–445 (2012).
Robinson, W. H. Sequencing the functional antibody repertoire–diagnostic and therapeutic discovery. Nat. Rev. Rheumatol. 11, 171–182 (2015).
DeKosky, B. J. et al. High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nat. Biotechnol. 31, 166–169 (2013).
Busse, C. E., Czogiel, I., Braun, P., Arndt, P. F. & Wardemann, H. Single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes. Eur. J. Immunol. 44, 597–603 (2014).
McDaniel, J. R., DeKosky, B. J., Tanno, H., Ellington, A. D. & Georgiou, G. Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes. Nat. Protoc. 11, 429–442 (2016).
Wang, B. et al. Functional interrogation and mining of natively paired human VH:VL antibody repertoires. Nat. Biotechnol. 36, 152–155 (2018).
Adler, A. S. et al. A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library. MAbs 10, 431–443 (2018).
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).
Story, C. M. et al. Profiling antibody responses by multiparametric analysis of primary B cells. Proc. Natl Acad. Sci. USA 105, 17902–17907 (2008).
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).
Shembekar, N., Hu, H., Eustace, D. & Merten, C. A. Single-cell droplet microfluidic screening for antibodies specifically binding to target cells. Cell Rep. 22, 2206–2215 (2018).
Bradbury, A. R., Sidhu, S., Dubel, S. & McCafferty, J. Beyond natural antibodies: the power of in vitro display technologies. Nat. Biotechnol. 29, 245–254 (2011).
Fitzgerald, V. & Leonard, P. Single cell screening approaches for antibody discovery. Methods 116, 34–42 (2017).
Klein, A. M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015).
Gurney, M. E., Heinrich, S. P., Lee, M. R. & Yin, H. S. Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons. Science 234, 566–574 (1986).
Liotta, L. A. et al. Tumor cell autocrine motility factor. Proc. Natl Acad. Sci. USA 83, 3302–3306 (1986).
Park, C. S. et al. Therapeutic targeting of tetraspanin8 in epithelial ovarian cancer invasion and metastasis. Oncogene 35, 4540–4548 (2016).
Anna, S. L., Bontoux, N. & Stone, H. A. Formation of dispersions using “flow focusing” in microchannels. Appl. Phys. Lett. 82, 364–366 (2003).
Mazutis, L. et al. Single-cell analysis and sorting using droplet-based microfluidics. Nat. Protoc. 8, 870–891 (2013).
Abate, A. R., Chen, C. H., Agresti, J. J. & Weitz, D. A. Beating Poisson encapsulation statistics using close-packed ordering. Lab Chip 9, 2628–2631 (2009).
Odegard, V. H. & Schatz, D. G. Targeting of somatic hypermutation. Nat. Rev. Immunol. 6, 573–583 (2006).
Roost, H. P. et al. Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity. Proc. Natl Acad. Sci. USA 92, 1257–1261 (1995).
Murugan, R et al. Clonal selection drives protective memory B cell responses in controlled human malaria infection.Sci. Immunol. 3, eaap8029 (2018).
Sorouri, M., Fitzsimmons, S. P., Aydanian, A. G., Bennett, S. & Shapiro, M. A. Diversity of the antibody response to tetanus toxoid: comparison of hybridoma library to phage display library. PLoS One 9, e106699–106614 (2014).
Baret, J. C. et al. Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab-on-a-Chip 9, 1850–1858 (2009).
Duffy, D. C., McDonald, J. C., Schueller, O. J. & Whitesides, G. M. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal. Chem. 70, 4974–4984 (1998).
Siegel, A., Bruzewicz, D., Weibel, D. & Whitesides, G. Microsolidics: fabrication of three-dimensional metallic microstructures in poly(dimethylsiloxane). Adv. Mater. 19, 727–733 (2007).
Zilionis, R. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat. Protoc. 12, 44–73 (2017).
Rohatgi, S., Ganju, P. & Sehgal, D. Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells. J. Immunol. Methods 339, 205–219 (2008).
R Core Team (R Foundation for Statistical Computing, Vienna, Austria, 2017).
Beaton, D., Chin Fatt, C. R. & Abdi, H. An ExPosition of multivariate analysis with the singular value decomposition in R. Computational Stat. Data Analysis 72, 176–189 (2014).
Acknowledgements
We would like to thank M. Holsti and W. Somers (Pfizer, BioMedicine Design) for advice on the technology development and critical reading of the manuscript; R. Nicol (Whitehead Institute, MIT Center for Genome Research) for advice on barcoded sequencing; at the Institut Pasteur, H. Mouquet for advice on antibody sequence selection, and F. Jönsson for advice on flow cytometry analysis; T. Kirk and S. Foulon (ESPCI Paris) for their help developing the barcoded hydrogel beads; S. N. Stewart (HiFiBio Therapeutics Inc.) for her help producing and analysing recombinant antibodies; the Institut Pierre-Gilles de Gennes (IPGG) for use of clean room facilities and the laser engraver (CII08, Axyslaser). This work was supported by the French Agence Nationale de la Recherche (ANR-14-CE16-0011 project DROPmAbs), by the Centre d’Innovation et Recherche Technologique (Citech) through the Institut Carnot Pasteur Microbes & Santé (ANR 16 CARN 0023-01), by BPI France (OSIRIS and CELLIGO projects) and by the French ‘Investissements d’Avenir’ program via the CELLIGO project and grant agreements ANR-10-NANO-02, ANR-10-IDEX-0001-02 PSL, ANR-10-LABX-31 and ANR-10-EQPX-34. NGS was performed by the ICGex NGS platform of the Institut Curie and the Institut de Biologie Intégrative de la Cellule (I2BC) platform (Gif-sur-Yvette) supported by the grants ANR-10-EQPX-03 and ANR-10-INBS-09-08 (France Génomique Consortium), by the Canceropole Ile-de-France and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS- 4654). C.C. acknowledges financial support from CONCYTEC, Peru. P.C.H. is a scholar in the Pasteur - Paris University (PPU) International PhD program and was also supported by the Fondation pour la Recherche Médicale (FRM; FDT201904008240). K.E. acknowledges financial support from the Swiss National Science Foundation and The Branco Weiss Fellowship - Society in Science.
Author information
Authors and Affiliations
Contributions
C.B., P.B., A. Gérard, A.J. and A.D.G. designed and supervised the study; C.B., P.B. and A.D.G. secured funding; L.B.-R., P.C.-H., C.C., M.D., R.D., K.E., S. Elllouze., S. Essono., A. Godina, K.G., B.I., B.J., R.K., V.M., P.M., S.M., G.M., C.O., Y.P., A.P., M.R., O.R.-L., G.R., A.S.-C., B. Saudemont., B. Shen., S.N.S. and A.W. performed the experiments; J.B., C.B., P.B., P.E., A. Gérard, A.D.G., A.J., G.A.M., G.M., C.N. and A.W. analyzed the data, and P.B., A. Gérard, A.D.G. and A.W. wrote the paper.
Corresponding authors
Ethics declarations
Competing interests
A.D.G. and C.B. are co-founders of Seven Pines Holding BV, and HiFiBiO Therapeutics SAS is a subsidiary of Seven Pines Holding BV. Patents have been filed on some aspects of this work and future development of the platform, and the inventors may receive payments related to exploitation of these under their employer’s rewards to inventors scheme.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary information
Supplementary Figures 1–26, Supplementary Tables 1 and 2, and Supplementary Note 1.
Supplementary Video 1
Cell and hydrogel bead co-compartmentalization into ~1 nl droplets. The aqueous phase containing cells and hydrogel beads, as well as lysis and reverse transcription reagents (right) were co-compartmentalized at the T-junction (middle) to form ~1 nl droplets (left) containing reagents necessary to lyse cells and initiate reverse transcription of VH–VL mRNA to form barcoded cDNA.
Supplementary Table 3
Expressed recombinant IgGs tested for binding to TT, GPI or TSPAN8-overexpressing cells
Supplementary Table 4
List of unique VH–VL pairs identified across sorts of splenocytes from TT, GPI and TSPAN8-overexpressing cell immunized mice.
Supplementary Table 5
Sequences of single-cell barcode indexes.
Rights and permissions
About this article
Cite this article
Gérard, A., Woolfe, A., Mottet, G. et al. High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics. Nat Biotechnol 38, 715–721 (2020). https://doi.org/10.1038/s41587-020-0466-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41587-020-0466-7
This article is cited by
-
Light-field flow cytometry for high-resolution, volumetric and multiparametric 3D single-cell analysis
Nature Communications (2024)
-
Design automation of microfluidic single and double emulsion droplets with machine learning
Nature Communications (2024)
-
Adaptive immune receptor repertoire analysis
Nature Reviews Methods Primers (2024)
-
A mini review on recent progress of microfluidic systems for antibody development
Journal of Diabetes & Metabolic Disorders (2024)
-
Droplet-based microfluidics
Nature Reviews Methods Primers (2023)
