title: Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.
abstract: Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)μM (0.551ng/ml) for PA83 and 2.51×10(-5)μM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex.
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA. Electronic address: RAStoddard@cdc.gov.
authors.forename: Robyn A
authors.initials: RA
authors.lastname: Stoddard
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Conrad P
authors.initials: CP
authors.lastname: Quinn
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Jarad M
authors.initials: JM
authors.lastname: Schiffer
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Boyer
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Goldstein
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Bagarozzi
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Soroka
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Dauphin
authors.affiliation: Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd., Atlanta, GA 30333, USA.
authors.forename: Alex R
authors.initials: AR
authors.lastname: Hoffmaster
mesh_terms: D000881:Anthrax / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D022122:Anthrax Vaccines / Q000276:immunology; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000097:blood*; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D042241:Early Diagnosis; D005063:Europium; D015200:Fluoroimmunoassay; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D011237:Predictive Value of Tests; D015203:Reproducibility of Results
chemical_list: D022122:Anthrax Vaccines; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D015415:Biomarkers; C030325:anthrax toxin; D005063:Europium
keywords: Anthrax toxin; Bacillus anthracis; Europium; Immunoassays; Protective antigen; Time resolved fluorescence