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Effective high-throughput isolation of fully human antibodies targeting infectious pathogens

Nature Protocols volume 16, pages 3639–3671 (2021)Cite this article

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Abstract

As exemplified by the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there is a strong demand for rapid high-throughput isolation pipelines to identify potent neutralizing antibodies for prevention and therapy of infectious diseases. However, despite substantial progress and extensive efforts, the identification and production of antigen-specific antibodies remains labor- and cost-intensive. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of potent antigen-specific antibodies against human immunodeficiency virus 1, hepatitis C virus, human cytomegalovirus, Middle East respiratory syndrome coronavirus, SARS-CoV-2 and Ebola virus. It is based on computationally optimized multiplex primer sets (openPrimeR), which guarantee high coverage of even highly mutated immunoglobulin gene segments as well as on optimized antibody cloning and production strategies. Here, we provide the detailed protocol, which covers all critical steps from sample collection to antibody production within 12–14 d.

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Fig. 1: Critical steps in single-cell B-cell cloning workflows.
Fig. 2: Detailed experimental outline.
Fig. 3: Anticipated results for the different stages of the antibody isolation pipeline.

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Acknowledgements

We thank M. Nussenzweig and members of the Nussenzweig lab for valuable support as well as intital protocols on single B-cell cloning and antibody production. We thank all members of the Klein lab for helpful discussion. This work was funded by grants from the European Research Council (ERC-StG639961, to F.K.), the German Centre for Infection Research (DZIF, to F.K., H.G. and L.G.), the Bill and Melinda Gates Foundation (BMGF, INV-002143 to F.K.), the German Research Foundation (DFG) (CRC 1279 and CRC 1310 to F.K.) and the COVIM: NaFoUniMedCovid19 (FKZ: 01KX2021 to F.K.).

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  1. These authors contributed equally: Lutz Gieselmann, Christoph Kreer.

Authors and Affiliations

  1. Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany

    Lutz Gieselmann, Christoph Kreer, Meryem Seda Ercanoglu, Nathalie Lehnen, Matthias Zehner, Philipp Schommers, Julian Potthoff, Henning Gruell & Florian Klein

  2. German Center for Infection Research, Partner Site Bonn-Cologne, Cologne, Germany

    Lutz Gieselmann, Philipp Schommers, Henning Gruell & Florian Klein

  3. Department I of Internal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany

    Philipp Schommers

  4. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Florian Klein

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Contributions

L.G., C.K. and F.K. conceptualized the workflow. L.G, C.K, M.S.E., M.Z., N.L., H.G., P.S. and J.P. established protocols and performed experiments. F.K. and C.K. supervised the development of the protocol. L.G., C.K. and F.K. wrote the manuscript. All authors were involved in editing of the final manuscript.

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Correspondence to Florian Klein.

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Key references using this protocol

Ehrhardt, S. A. et al. Nat. Med. 25, 1589–1600 (2019): https://doi.org/10.1038/s41591-019-0602-4

Schommers, P. et al. Cell 180, 471–489.e22 (2020): https://doi.org/10.1016/j.cell.2020.01.010

Kreer, C. et al. Cell 182, 843–854.e12 (2020): https://doi.org/10.1016/j.cell.2020.06.044

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Gieselmann, L., Kreer, C., Ercanoglu, M.S. et al. Effective high-throughput isolation of fully human antibodies targeting infectious pathogens. Nat Protoc 16, 3639–3671 (2021). https://doi.org/10.1038/s41596-021-00554-w

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