Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)

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.

Anti-capsule human monoclonal antibodies protect against hypervirulent and pandrug-resistantKlebsiella pneumoniae

“…Further, mAbs were screened for their ability to induce complement-dependent killing of ST147 NDM-1 Kp 41 . For this purpose, we developed a high-throughput luminescence-based serum bactericidal assay (L-SBA) that measures ATP content as a proxy of Kp viability 42,43 . Four dilutions per mAb were tested in the presence of an exogenous source of complement and antibodies causing a reduction greater than 30% in bacterial viability were considered as positive hits.…”

Setup and Characterization of a High-Throughput Luminescence-Based Serum Bactericidal Assay (L-SBA) to Determine Functionality of Human Sera against Shigella flexneri

“…Since up to 12 plates can be run in a day by a single operator, the assay has a high-throughput; moreover, our assay uses standard reagents and requires only a luminometer to detect ATP, making it simple enough to be adopted by any laboratory worldwide. We have also demonstrated the possibility to further increase the throughput of our L-SBA method by performing the assay in 384-well format [ 38 ], and thus the possibility to further increase the already high-throughput if necessary, especially for large clinical or sero-epidemiological studies.…”

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella

“…The traditional SBA method (CFU-based) had limitations in terms of time and operator dependency. To overcome these weaknesses, we developed a high-throughput SBA method based on a luminescent readout (L-SBA), both in 96-wells [29,30] and 384-wells [31] plate formats. Similar methodology has additionally been optimized and characterized to evaluate the functionality of antibodies elicited by a multivalent GMMA-based vaccine against Shigella on human sera, reported elsewhere [32].…”

“…Despite that, SBA has been rarely adopted for large epidemiological studies and/or clinical samples analysis because it is time-consuming and labor-intensive [28]. To overcome these bottlenecks, we developed a luminescence-based high-throughput SBA (L-SBA) in 96-well [29,30] and 384-well plate format [31]. The assay had been already characterized intra-laboratory in terms of specificity, linearity, and precision by using human-serum L-SBA [32].…”