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Nat Protoc. 2016 Aug;11(8):1531-53. doi: 10.1038/nprot.2016.091. Epub 2016 Jul 28.

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

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Institute for Microbiology, ETH Zurich, Zurich, Switzerland.
Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Centre d'Immunologie et des Maladies Infectieuses (CIMIParis UMRS 1135), F75013, Paris, France.
Maurice Müller Laboratories, Departement Klinische Forschung, Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Bern, Switzerland.
Department of Biomedicine, University Hospital Basel, Basel, Switzerland.
AP-HP, Groupement Hospitalier Pitié-Salpêtriàre, Département d'Immunologie, F75013, Paris, France.


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 10(5) 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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