Interlaboratory comparison of results of an anthrax lethal toxin neutralization assay for assessment of functional antibodies in multiple species

Abstract

The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED(50)) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED(50)s for all samples and within 7.5% of the quotients of the test sample ED(50) and the reference standard ED(50) (NF(50)s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED(50)s and 35% for the NF(50)s. These precision data suggest that the NF(50) readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision.

FIG. 1.

Box-and-whisker plots of 4PL model curve shape parameters for sera from reference material and three species from four laboratories (laboratories A, C, E, and G); sample sizes are indicated at the top. Only values associated with curves identified as “full” by the data reduction software are shown. Filled dots, medians; boxes, first to third quartiles; whiskers, extensions of 1.5 interquartile ranges past the boxes; open dots, data that were beyond the whiskers. Note the comparable ranges of the y-axis scales for the lower and upper asymptotes. Four estimated upper asymptotes were greater than the upper limit of the graph (all for NHP sera from laboratory G, with values of 3.6, 3.8, 5.5, and 7.0). Note the log scale of the y axis for slope.

FIG. 2.

Estimated differences (dots) with 90% CIs (triangles) for parameter estimates for each species relative to the results for the reference material, expressed as a percentage of the reference curve depth for asymptotes and a percentage of the reference 4PL model slope for that parameter. In equivalence testing, only the farther of the two confidence limits (thus, a one-sided 95% confidence limit) is considered.

FIG. 3.

Dilutional linearity plots for the 23 specimens that were assayed at more than one spiked concentration. Each row of panels depicts the specimens from a particular species. Solid lines have the specimen-specific intercept and species-specific slope estimated in the dilutional linearity model; dashed lines have the same intercept but a slope equal to 1.

FIG. 4.

Plots of residuals relative to consensus estimates of neutralizing activity. Patterns for both ED₅₀ (A) and NF₅₀ (B) are shown. Points show the residuals (on the log scale; the y-axis scale is exponentiated for clarity, i.e., 2^residual). Solid line, mean ratio; dashed lines, 2 standard deviations above and below the mean.

FIG. 5.

Bland-Altman plots of ratio (y axis) between two laboratories' reported NF₅₀s relative to the geometric mean of the two laboratories' reported NF₅₀ (x axis). The y-axis scale is exponentiated for clarity, i.e., 2^residual. Solid line, mean ratio; dashed lines, 2 standard deviations above and below the mean.