title: Validation of the anthrax lethal toxin neutralization assay.
abstract: A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED(50)) to the reference standard ED(50). Accuracy was maintained, even in samples with minimal neutralizing capacity, and linearity was noted when sample dilutions resulted in accurate prediction of the Y(max)and Y(min). Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58).
authors.affiliation: Product Development and Regulatory Affairs, US Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick, Frederick, MD 21702-5011, USA. Donna.Hering@det.amedd.army.mil
authors.forename: Donna
authors.initials: D
authors.lastname: Hering
authors.forename: William
authors.initials: W
authors.lastname: Thompson
authors.forename: John
authors.initials: J
authors.lastname: Hewetson
authors.forename: Stephen
authors.initials: S
authors.lastname: Little
authors.forename: Sarah
authors.initials: S
authors.lastname: Norris
authors.forename: Judith
authors.initials: J
authors.lastname: Pace-Templeton
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry*; D001408:Bacillus anthracis; D001427:Bacterial Toxins / Q000737:chemistry*; D002455:Cell Division; D002460:Cell Line; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D066298:In Vitro Techniques; D051379:Mice; D009500:Neutralization Tests / Q000379:methods*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
chemical_list: D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D013778:Tetrazolium Salts; D013844:Thiazoles; C030325:anthrax toxin; C022616:thiazolyl blue