Feasibility of Using a Luminescence-Based Method to Determine Serum Bactericidal Activity against Neisseria gonorrhoeae

Abstract

Development of a vaccine to limit the impact of antibiotic resistant Neisseria gonorrhoeae is now a global priority. Serum bactericidal antibody (SBA) is a possible indicator of protective immunity to N. gonorrhoeae, but conventional assays measure colony forming units (CFU), which is time-consuming. A luminescent assay that quantifies ATP as a surrogate measure of bacterial viability was tested on N. gonorrhoeae strains FA1090, MS11 and P9-17 and compared to CFU-based readouts. There was a linear relationship between CFU and ATP levels for all three strains (r > 0.9). Normal human serum (NHS) is a common source of complement for SBA assays, but needs to be screened for non-specific bactericidal activity. NHS from 10 individuals were used for serum sensitivity assays-sensitivity values were significantly reduced with the ATP method for FA1090 (5/10, p < 0.05) and MS11 (10/10, p < 0.05), whereas P9-17 data were comparable for all donors. Our results suggest that measuring ATP underestimates serum sensitivity of N. gonorrhoeae and that the CFU method is a better approach. However, mouse anti-P9-17 outer membrane vesicles (OMV) SBA titres to P9-17 were comparable with both methods (r = 0.97), suggesting this assay can be used to rapidly screen sera for bactericidal antibodies to gonococci.

Evaluating vaccine-elicited antibody activities againstNeisseria gonorrhoeae:cross-protective responses elicited by the 4CMenB meningococcal vaccine

“…The latter could be accelerated using automated colony counting. Our attempts to use the metabolic dye alamarBlue as a surrogate for colony counting for SBA did not yield substantial differences between sensitive and resistant conditions with the low numbers of Gc used for SBA, which agrees with Clow et al's finding that a luminescence-based alternative for SBA was not as quantitative as CFU enumeration (74). However, these alternative approaches could be optimized in the future to enable higher-throughput analyses of vaccine candidates, as for N. meningitidis (75).…”

Further Evaluation of Enterohemorrhagic Escherichia coli Gold Nanoparticle Vaccines Utilizing Citrobacter rodentium as the Model Organism

“…Therefore, we wanted to confirm that the antibodies produced following s.c. immunization exhibited similar functionality. To elucidate the bactericidal effects of sera antibodies, we employed a high-throughput, luminescence-based assay that has been used as a convenient and consistent approach to evaluate serum bactericidal activity against several pathogens [29,30]. For this method, C. rodentium was mixed with either 10% active sera, heat-inactivated sera, or heat-inactivated sera supplemented with naïve mouse sera obtained from mice immunized with AuNP-protein or adjuvant-only controls.…”

“…For this method, C. rodentium was mixed with either 10% active sera, heat-inactivated sera, or heat-inactivated sera supplemented with naïve mouse sera obtained from mice immunized with AuNP-protein or adjuvant-only controls. To determine bacterial viability, and therefore, the bactericidal properties of the sera, PBSwashed bacteria-sera suspensions were transferred to a microtiter plate after exposure to the sera, along with an equivalent amount of BacTiter-Glo reagent, which generates a luminescent signal proportional to amount of ATP, and therefore, to live bacteria, present in the sample [29,30]. The luminescence reading of each sample was measured and bacterial survival was calculated as a % of the luminescence given by C. rodentium in PBS alone, and bacterial killing in the presence of immune sera was standardized to killing by sera from adjuvant-only-treated mice.…”

A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values

“…Serum bactericidal assays measuring extracellular adenosine triphosphate (ATP) as a proxy for bacterial lysis have been developed for several pathogens. [18][19][20][21] In addition to ATP, adenylate kinase activity, which catalyzes the reaction 2 adenosine diphosphate (ADP) ! ATP 1 adenosine monophosphate (AMP), can also be used as a proxy for cell lysis.…”