EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

An in vivo passive protection assay for the evaluation of immunity in AVA-vaccinated individuals.

Samples of human plasma from anthrax vaccine adsorbed (AVA, BioThrax)-vaccinated individuals were used to demonstrate passive protection of A/J mice from a lethal challenge with the Sterne strain of anthrax bacteria. The maximum concentration of human anti-protective antigen IgG in mouse sera 24 h after injection of 260 microg of anti-PA IgG was 134 microg/ml, declining to 91 microg/ml at 72 h (half-life=101.7 h). Mice showed significant survival (p

Intralaboratory validation of cell-free translation assay for detecting ricin toxin biological activity.

A cell-free translation (CFT) assay for determining ricin biological activity was validated. The statistical data from the validation study showed a high level of precision within and between runs of the assay. The assay was specific for determining ricin biological activity in food-based matrixes and discriminated ricin from other ribosome-inactivating proteins. The mean bias (relative error) between measured ricin concentrations of 3 validation samples and their nominal concentrations was 1.1, 6.6, and 20.3%, while the coefficient of variation (CV) was 14.1, 7.7, and 13.5%, respectively, demonstrating good precision, accuracy, and linearity. The CVs of ricin concentrations in 2 ricin-containing samples calculated from a dilution series were <5 and <12%, respectively, demonstrating very good parallelism. The analyte stability of ricin-containing samples stored for 1 month either at 4 or -20 degrees C, the stability of ricin stock solutions, and the results of assays executed by different analysts and using different luminometers were evaluated. The statistical validation data confirmed that the 4-parameter logistic equation, y = (a - d)/[1 + (x/c)b] + d, provided an accurate representation of a sigmoidal relationship between the measured response and the observed ricin concentration for the CFT assay.

Evaluation of a botulinum fragment C-based ELISA for measuring the humoral immune response in primates.

An enzyme-linked immunosorbent assay (ELISA) using botulinum neurotoxin serotype B recombinant fragment C (rBoNTB(HC)) was developed to measure specific humoral immune responses of monkeys vaccinated with a vaccine consisting of rBoNTB(HC). Several fundamental parameters for a bioassay were evaluated. The evaluation results demonstrated that using BoNTB(HC) as the capture antigen led to a specific and sensitive ELISA for botulinum type B antibody with excellent precision, accuracy, and linearity. There was a good correlation (r=0.91) between ELISA titers and neutralization bioassay titers. Experimental results suggested that the ELISA could be useful for detecting botulinum type B antibody levels and may supplement mouse neutralization bioassays during planned clinical manufacturing and clinical trials of rBoNTB(HC) vaccine.

On-road facility to measure and characterize emissions from heavy-duty diesel vehicles.

In response to lingering concerns about the utility of dynamometer data for mobile source emissions modeling, the U.S. Environmental Protection Agency (EPA) has constructed an on-road test facility to characterize the real-world emissions of heavy-duty trucks. The facility was designed to effectively demonstrate the full range of vehicle operation and to measure the emissions produced. Since it began operation, the facility has been continuously upgraded to incorporate state-of-the-art technology. Its potential uses include collecting modal emissions data, validating dynamometer test parameters and results, and demonstrating new emission control technologies.