Validation of an Anti-Protective Antigen ELISA for Quantitative IgG Evaluation in B. anthracis Immunized Horses

The potency test for anthrax vaccines has historically involved the challenge of actively or passively immunized laboratory animals with a fully virulent strain of Bacillus anthracis. Lethal challenge studies with the archetypal virulent strains such as B. anthracis Ames strain present considerable difficulties in laboratory management and handling and are too inefficient for the early evaluation of alternative preventative and therapeutic interventions. An ELISA for the evaluation of antibody response to protective antigen (PA) in horses immunized with the Sterne 34F2 strain spore vaccine was developed. The objective of this work was to study the performance of this assay in terms of the guidelines set forth by the International Conference on Harmonics (ICH) and the Center for Biologics Evaluation and Research (CBER) for analytical procedures. We have demonstrated a working range for this assay (73-1581 EU/ml) on the bases of the following parameters: linearity (25 and 1,662 EU/ml, r2 = 0.9988, p < 0.001), accuracy (94.8 105.4 %, recovery within the range of 25 and 1,662 EU/ml), precision (≤ 17.6 % CV, repeatability; ≤ 15.7 and ≤ 13.1 % CV, intermediate precision per day and per analyst, respectively), limit of detection (2.25 EU/ml) and limit of quantitation (25 EU/ml). The assay was also demonstrated to be specific for the evaluation of anti-PA IgG antibodies. Based on the assay performance characteristics it was determined that the assay was adequate for use in B. anthracis immunogenicity testing in horses.