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Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry

Nature Protocols volume 11, pages 1531–1553 (2016)Cite this article

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Abstract

Antibacterial antibody responses that target surfaces of live bacteria or secreted toxins are likely to be relevant in controlling bacterial pathogenesis. The ability to specifically quantify bacterial-surface-binding antibodies is therefore highly attractive as a quantitative correlate of immune protection. Here, binding of antibodies from various body fluids to pure-cultured live bacteria is made visible with fluorophore-conjugated secondary antibodies and measured by flow cytometry. We indicate the necessary controls for excluding nonspecific binding and also demonstrate a cross-adsorption technique for determining the extent of cross-reactivity. This technique has numerous advantages over standard ELISA and western blotting techniques because of its independence from scaffold binding, exclusion of cross-reactive elements from lysed bacteria and ability to visualize bacterial subpopulations. In addition, less than 105 bacteria and less than 10 μg of antibody are required per sample. The technique requires 3–4 h of hands-on experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications.

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Figure 1: Overview of the protocol.
Figure 2: Detailed analysis of surface epitopes.
Figure 3: Increasing the target cell number is equivalent to increasing the dilution factor.
Figure 4: The effects of different contamination sources on acquired data.
Figure 5: Timing of serum IgG and IgA induction after confirmed K. pneumoniae sepsis secondary to a lung infection.
Figure 6: Bacterial flow cytometry with purified IgA from human breast milk and fecal water.
Figure 7: Effects of bacterial cryo-preservation and fixation on IgG binding.
Figure 8: Nonspecific antibody binding.
Figure 9: Use of irrelevant bacterial species and germ-free mice as negative controls in the murine system.
Figure 10: Controls in the human system.
Figure 11: Data analysis methods.
Figure 12: Vaccination with oral peracetic-acid-inactivated S. typhimurium and infection with live-attenuated S. typhimurium induce a similar magnitude of intestinal IgA response.
Figure 13: Bacterial flow cytometry reveals increased microbiota-specific serum antibodies in metabolic liver disease.

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Acknowledgements

We acknowledge J. Doré and C. Juste (Institut National de la Recherche Agronomique (INRA), UMR1319 Micalis, Jouy en Josas, France) for access to well-characterized bacterial strains, and C. Parizot and D. Gouas for technical help during initial experimental setup. The authors also acknowledge the following funders: M.L. was supported by the Institut national de la santé et de la recherche médicale (Inserm), Agence Nationale de la Recherche (MetAntibody, ANR-14-CE14-0013) and Fondation pour l'Aide a la Recherche sur la Sclerose En Plaques (ARSEP). E.S. was supported by an SNF Ambizione fellowship (PZ00P3_136742) and an ETH research grant (ETH-33 12-2).

Author information

Author notes

  1. Kathrin Moor, Jehane Fadlallah and Albulena Toska: These authors contributed equally to this work.

  2. Martin Larsen and Emma Slack: These authors jointly directed this work.

Authors and Affiliations

  1. Institute for Microbiology,

    Kathrin Moor, Albulena Toska & Emma Slack

  2. , ETH Zurich, Zurich, Switzerland

    Kathrin Moor, Albulena Toska & Emma Slack

  3. Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses (CIMIParis UMRS 1135), F75013, Paris, France

    Jehane Fadlallah, Delphine Sterlin, Guy Gorochov & Martin Larsen

  4. Maurice Müller Laboratories, Departement Klinische Forschung,

    Maria L Balmer & Andrew J Macpherson

  5. , Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Bern, Switzerland

    Maria L Balmer & Andrew J Macpherson

  6. Department of Biomedicine, University Hospital Basel, Basel, Switzerland

    Maria L Balmer

  7. Département d'Immunologie, AP-HP, Groupement Hospitalier Pitié-Salpêtriàre, F75013, Paris, France,

    Guy Gorochov & Martin Larsen

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Contributions

K.M., J.F., A.T. and D.S. generated the data shown in Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. M.L.B. generated the data shown in Figure 13. A.J.M. provided support during the development of the method and reviewed the manuscript. G.G. provided support for adaption of the method to clinical scenarios and reviewed the manuscript. M.L. further developed the method for application to human research, generated data shown in Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and wrote the manuscript. E.S. established the methodology and analysis protocols, generated data shown in Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and wrote the manuscript.

Corresponding authors

Correspondence to Martin Larsen or Emma Slack.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Raw data and calculations for graphs shown in Figures 11 and 12 (XLSX 343 kb)

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Moor, K., Fadlallah, J., Toska, A. et al. Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry. Nat Protoc 11, 1531–1553 (2016). https://doi.org/10.1038/nprot.2016.091

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