title: Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.
abstract: Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU) at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.
authors.affiliation: GSK Vaccines Institute for Global Health (GVGH) S.r.l., Siena, Italy.
authors.forename: Francesca
authors.identifier: http://orcid.org/0000-0003-1239-8696
authors.initials: F
authors.lastname: Necchi
authors.affiliation: GSK Vaccines Institute for Global Health (GVGH) S.r.l., Siena, Italy.
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
authors.affiliation: GSK Vaccines Institute for Global Health (GVGH) S.r.l., Siena, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Rondini
mesh_terms: D000255:Adenosine Triphosphate / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D001419:Bacteria / Q000276:immunology* / Q000378:metabolism; D001770:Blood Bactericidal Activity / Q000276:immunology*; D016971:Citrobacter freundii / Q000276:immunology / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007106:Immune Sera / Q000097:blood / Q000276:immunology*; D016014:Linear Models; D051379:Mice; D050296:Microbial Viability / Q000276:immunology; D009345:Neisseria meningitidis / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000276:immunology / Q000378:metabolism; D012763:Shigella flexneri / Q000276:immunology / Q000378:metabolism; D012764:Shigella sonnei / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity
chemical_list: D007106:Immune Sera; D000255:Adenosine Triphosphate