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

作者：Moor Kathrin; Fadlallah Jehane; Toska Albulena; Sterlin Delphine; Balmer Maria L; Macpherson Andrew J; Gorochov Guy; Larsen Martin^*; Slack Emma^*

来源：Nature Protocols, 2016, 11(8): 1531-1553.

DOI：10.1038/nprot.2016.091

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 mu g of antibody are required per sample. The technique requires 3-4 h of handson experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications.

• 出版日期2016-8