Abstract
Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens.
Highlights
Serum bactericidal assay (SBA) represents a method to determine in vitro the ability of antibodies present in serum to kill bacteria through complement activation
We demonstrated the performance of this method and the equivalence of results compared to the traditional colony-forming units (CFU)-based method against several pathogens, including Citrobacter freundii, Salmonella serovars Typhimurium and Enteritidis, Shigella flexneri serotypes 2a and 3a, Shigella sonnei, Neisseria meningitidis [8] and S
384-well forage, sera dilution volume and method for dispensing buffers or removing supernatants) mat using half of the reaction volume used for the 96-well format (50 μL rather than 100 established with 96-well-plates luminescence-based high-throughput SBA (L-SBA), we optimized the 384-well format μL, maintaining same proportion of reagents)
Summary
Serum bactericidal assay (SBA) represents a method to determine in vitro the ability of antibodies present in serum to kill bacteria through complement activation. In the SBA, bacteria are mixed with dilutions of heat-inactivated serum in the presence of exogenous complement. The number of live bacteria is determined at each serum dilution after a certain amount of time. The dilution of serum resulting in killing 50% of bacteria in the reaction represents the bactericidal antibody titer [4,5]. The traditional SBA methods had some bottlenecks, mainly represented by the need for manually plating onto agar plates and counting the colony-forming units (CFU) both at the beginning and at the end of incubation at each serial dilution. The assay is considered time consuming and labor intensive for screening large datasets, even when automated colony-counting systems are in place
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