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 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. 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.

Patterns of antibody response in humans to the anthrax vaccine adsorbed (AVA) primary (six-dose) series☆

“…A serum concentration of IgG to PA ≥ 3 g/mL was observed in all subjects after vaccination. This level of antibody, which represents the lower limit of quantification for undiluted serum using an ELISA analogous to that applied in this study [9], was reached by 39.5% of subjects after the first injection, by a total of 96.5% after the second injection, and by 100% after the third injection ( Table 4). The mean number of days after the first vaccine dose needed to reach 3 g/mL was 24.2 (range = 13-63 days).…”

“…Optical density (OD) values were read within 30 min by using a Bio-Tek ELx808 plate with 405 and 490 nm filters, and KC4 software (Bio-Tek Instruments, Inc., Winooski, VT). OD values were converted to immunoglobulin concentration ( g/mL) by using a standard curve calibration factor [9]. Titers were calculated as the reciprocal of the highest dilution of the test serum yielding a mean OD value ≥ the cut-off value for the assay.…”

The Critical Role of Pathology in the Investigation of Bioterrorism-Related Cutaneous Anthrax

“…Serology and reverse transcription (RT)-PCR were performed by other laboratories at CDC. [42][43][44] § Contained a small, superficial, shave biopsy that was inadequate for pathologic evaluation.…”

Zoonotic Infections Among Employees from Great Smoky Mountains and Rocky Mountain National Parks, 2008–2009

“…for standard reference serum AVR801 was used to determine the concentration of anti-PA IgG in lg/mL of test serum; the lower limit of quantification of the anti-PA ELISA was 3.7 mg/mL anti-PA IgG in undiluted serum samples; diagnostic sensitivity was 98.6% and diagnostic specificity was 98.4% (Quinn et al 2002(Quinn et al , 2004Semenova et al 2004) Anti-PA IgG concentration ‡ to the assay lower limit of quantification (3.7 lg/mL)…”

“…Pathogens tested and specific methods of serologic testing for each of the agents evaluated have been described elsewhere (Table 1; Lindsey et al 1976;Dikken and Kmety 1978;Brown et al 1981;Dalton et al 1995;Plikaytis et al 1996;Nicholson et al 1997;Johnson et al 2000Johnson et al , 2005Martin et al 2000;Quinn et al 2002Quinn et al , 2004Dumler 2004;Semenova et al 2004;Jones et al 2007). Baseline seropositivity was interpreted as prior infection.…”