Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout

• Published in: PloS one Vol. 12; no. 2; p. e0172163
• Main Authors: Necchi, Francesca, Saul, Allan, Rondini, Simona
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 13.02.2017; Public Library of Science (PLoS)
• Subjects: Adenosine Triphosphate - immunology; Adenosine Triphosphate - metabolism; Agar; Analysis; Animals; Antibodies; Antigens; Assaying; ATP; ATP (Adenosine triphosphate); Bacteria; Bacteria - immunology; Bacteria - metabolism; Bactericidal activity; Biology and Life Sciences; Blood Bactericidal Activity - immunology; Blood serum; Citrobacter; Citrobacter freundii; Citrobacter freundii - immunology; Citrobacter freundii - metabolism; Colonies; Complement; Complement system; Data acquisition; Data processing; Dilution; Glycerol; Health aspects; High-throughput screening (Biochemical assaying); High-Throughput Screening Assays - methods; Immune Sera - blood; Immune Sera - immunology; Immunoglobulins; Laboratories; Linear Models; Luminescence; Measurement; Medicine and Health Sciences; Metabolism; Methods; Mice; Microbial Viability - immunology; Microorganisms; Neisseria meningitidis; Neisseria meningitidis - immunology; Neisseria meningitidis - metabolism; Physical Sciences; Reproducibility of Results; Research and Analysis Methods; Salmonella; Salmonella enterica; Salmonella enterica - immunology; Salmonella enterica - metabolism; Serum bactericidal activity; Shigella; Shigella flexneri - immunology; Shigella flexneri - metabolism; Shigella sonnei - immunology; Shigella sonnei - metabolism; Species Specificity; Survival; Vaccines; Detroit Michigan; United States > US; Italy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0172163

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.