L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. 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.
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. Bactericidal activity is subsequently calculated. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. 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. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. 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. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement.
Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies.
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