Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen

Abstract

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Figure

Graphic representation of minimum detectable concentration (MDC), reliable detection limit (RDL), and reactivity threshold. The MDC is the concentration of anti-protective antigen antibody (anti-PA) corresponding to the interpolated intersection of the lower asymptote of the upper 95% confidence limit of the 4-parameter logistic log fit of the standard curve data. The RDL is the concentration of anti-PA antibody corresponding to the interpolated intersection of the lower asymptote of the upper 95% confidence limit with the lower 95% confidence limit of the standard’s data. The reactivity threshold was determined as the upper 95% confidence limit of the frequency distribution from log₁₀-transformed optical density (OD) values of control human sera tested at 1/50 dilution. This OD value was converted to an anti-PA immunoglobulin (Ig) G concentration by using the standard curve calibration factor. Where this calculated value is below the MDC of the assay, the MDC was selected as the default reactivity threshold.