Search

To improve surge testing capability for a response to a release of Bacillus anthracis, the CDC anti-Protective Antigen (PA) IgG Enzyme-Linked Immunosorbent Assay (ELISA) was re-designed into a high throughput screening format. The following assay performance parameters were evaluated: goodness of fit (measured as the mean reference standard r 2 ), accuracy (measured as percent error), precision (measured as coefficient of variance (CV)), lower limit of detection (LLOD), lower limit of quantification (LLOQ), dilutional linearity, diagnostic sensitivity (DSN) and diagnostic specificity (DSP). The paired sets of data for each sample were evaluated by Concordance Correlation Coefficient (CCC) analysis. The goodness of fit was 0.999; percent error between the expected and observed concentration for each sample ranged from -4.6% to 14.4%. The coefficient of variance ranged from 9.0% to 21.2%. The assay LLOQ was 2.6 μg/mL. The regression analysis results for dilutional linearity data were r 2  = 0.952, slope = 1.02 and intercept = -0.03. CCC between assays was 0.974 for the median concentration of serum samples. The accuracy and precision components of CCC were 0.997 and 0.977, respectively. This high throughput screening assay is precise, accurate, sensitive and specific. Anti-PA IgG concentrations determined using two different assays proved high levels of agreement. The method will improve surge testing capability 18-fold from 4 to 72 sera per assay plate., (Published by Elsevier Ltd.)

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 microgram/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 micro g of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 microgram/ml, while the dynamic range was 0.06 to 1.7 microgram/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 microgram of anti-PA IgG per ml, the RDL was 0.016 microgram/ml, and the whole-serum equivalent MDC was 1.5 micrograms/ml. The dynamic range was 0.006 to 6.8 microgram/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

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.