Detection of complement-mediated antibody-dependent bactericidal activity in a fluorescence-based serum bactericidal...

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group B Neisseria meningitidis in both human and mouse antisera.