Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization.
Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50=36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO(2) concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesus macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept=0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics.
Li
Han
H
Microbial Pathogenesis & Immune Response Laboratory, Meningitis and Vaccine Preventable Diseases Branch, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, United States.
Soroka
Stephen D
SD
Taylor
Thomas H
TH
Stamey
Karen L
KL
Stinson
Kelly Wallace
KW
Freeman
Alison E
AE
Abramson
Darbi R
DR
Desai
Rita
R
Cronin
Li X
LX
Oxford
J Wade
JW
Caba
Joseph
J
Pleatman
Cynthia
C
Pathak
Sonal
S
Schmidt
Daniel S
DS
Semenova
Vera A
VA
Martin
Sandra K
SK
Wilkins
Patricia P
PP
Quinn
Conrad P
CP
D000818:Animals; D000881:Anthrax / Q000276:immunology* / Q000517:prevention & control; D022122:Anthrax Vaccines / Q000276:immunology; D000907:Antibodies, Bacterial / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology*; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002460:Cell Line; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D018448:Models, Immunological; D009500:Neutralization Tests / Q000379:methods*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin