Fluorescent multivalent opsonophagocytic assay for measurement of functional antibodies to Streptococcus pneumoniae

Abstract

We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.

FIG. 1.

Signal-to-noise ratios for five alamar blue reaction mixtures with various bacterial inocula. Signal was defined as mean fluorescence in the presence of bacteria. Noise was defined as the mean fluorescence of the reagent blanks in the absence of bacteria for each corresponding buffer condition. The total incubation period was 4 h. Reaction mixtures contained various concentrations of THYE and a constant concentration (10%) of alamar blue. The mixtures were tested with several concentrations of S. pneumoniae serotype 6B, reference strain DS2212-94 (63 to 4,000 CFU in a 20-μl well volume). At 4,000 CFU, the signal ranged from 82,358 FU with 25% THYE to 93,714 FU for 100% THYE. The blanks ranged from 5,966 FU with 25% THYE to 15,660 FU with 100% THYE. Thus, the optimized formulation (greatest signal-to-noise ratio) was obtained with 25% THYE.

FIG. 2.

Identity graphs comparing fmOPA titers to the reference multilaboratory OPA titers (19) for seven serotypes as shown in panels A to G. The solid line is the linear regression; the broken line is the line of identity. The size of the data point represents the number of serum samples that are at that coordinate (n = 24).

FIG. 2.

Identity graphs comparing fmOPA titers to the reference multilaboratory OPA titers (19) for seven serotypes as shown in panels A to G. The solid line is the linear regression; the broken line is the line of identity. The size of the data point represents the number of serum samples that are at that coordinate (n = 24).