Interlaboratory Comparison of the Anthrax Lethal Toxin Neutralization Assay used to Assess Functional Antibodies in Multiple Species

Precision Bioassay, Burlington, VT; Battelle Eastern Science and Technology Center, Aberdeen MD; Battelle Biomedical Research Center, West Jefferson, OH; Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD; Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD; Centers for Disease Control and Prevention, Atlanta, GA; Emergent BioSolutions, Lansing MI; VaxGen Inc., Brisbane, CA; USAMRIID, Fort Detrick, MD

^* To whom correspondence should be addressed. Email: lynnf{at}niaid.nih.gov.

	Abstract

The anthrax lethal toxin neutralization assay (TNA) will likely be used as a correlate of protection to bridge efficacy of new anthrax vaccines in animal models to immunogenicity 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 behavior 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 a human reference material. Dilutional linearity of samples from each species was also similar, with spike to ED₅₀ slopes of less than 1.2 for all species. Agreement among the laboratories to consensus values was within 10% of the ED₅₀ values for all samples, and within 7.5% of the NF₅₀ values for all samples. The relative standard deviation combining all laboratories and species was 45% for the ED₅₀ values and 35% for the NF₅₀ values. These precision data suggest that the NF₅₀ readout may normalize 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.