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

Moor, Kathrin; Fadlallah, Jehane; Toska, Albulena; Sterlin, Delphine; Balmer, Maria Luisa; Macpherson, Andrew; Gorochov, Guy; Larsen, Martin; Slack, Emma (2016). Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry. Nature protocols, 11(8), pp. 1531-1553. Nature Publishing Group 10.1038/nprot.2016.091

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Gastroenterologie / Mukosale Immunologie
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > DBMR Forschung Mu35 > Forschungsgruppe Gastroenterologie / Mukosale Immunologie

04 Faculty of Medicine > Department of Gastro-intestinal, Liver and Lung Disorders (DMLL) > Clinic of Visceral Surgery and Medicine > Gastroenterology

UniBE Contributor:

Balmer, Maria Luisa and Macpherson, Andrew

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1754-2189

Publisher:

Nature Publishing Group

Language:

English

Submitter:

Lilian Karin Smith-Wirth

Date Deposited:

26 Apr 2017 16:38

Last Modified:

26 Apr 2017 16:38

Publisher DOI:

10.1038/nprot.2016.091

PubMed ID:

27466712

BORIS DOI:

10.7892/boris.94394

URI:

https://boris.unibe.ch/id/eprint/94394

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