Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples

Abstract

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10(3) spores was achieved by incubating spores simultaneously with capture and detection antibodies ("liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.

Development of Antibodies against Anthrose Tetrasaccharide for Specific Detection of Bacillus anthracis Spores

“…With our capture ELISA for the immunological detection of B. anthracis spores, we have a rapid and sensitive tool with a detection limit lower than the detection limits of most commercially available assays (11). The specificity and the sensitivity of the pc115 antibody in the capture ELISA in combination with polyclonal spore antibodies pc114 and H64-BA IgY make it a conclusive and reliable tool for the application and the development of new detection assays.…”

“…In addition to PCR techniques, which could be hampered by inhibitory factors, and inefficient means for the preparation of DNA from spores, the rapid immunological detection of B. anthracis from complex specimens is currently possible only when the spore concentrations are relatively high and when handheld test kits are used (12). In cases of lower concentrations, time-consuming cultivation and later identification would take too long in an emergency situation (11). Thus, there is a crucial need for a sensitive and highly specific means of identification of B. anthracis spores in environmental samples that urgently need to be tested.…”

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification ofBacillus anthracisSpores in Suspicious White Powders and Environmental Samples

“…However, PCR requires clean samples in a small volume and is not generally suitable for field use. 42,43 Other methods of detection have included fluorescence-based sandwich immunoassays on glass slides, 44,45 peptide functionalized surface-enhanced Raman spectroscopy (SERS), piezo-electric based detection, 43,46 PCR combined with fluorescence resonance energy transfer (FRET), 47 and aptamers and bacteriophage. 43 While each of these methods holds some promise for laboratory-based detection, none is currently appropriate for field use to rapidly screen unknown environmental samples (ie, white powders) for the presence of B. anthracis spores.…”

Ultra-sensitive fluorescence detection using integrated waveguide technology

“…We have previously reported applications using solid-phase IWT, where analytes are captured on the inner surface of an open ended capillary tube and analyzed using an instrument called Signalyte TM (Li, 2005a). This format has been used to detect and quantify Escherichia coli O157 (Zhu et al, 2005) and Bacillus anthracis spores (Hang et al, 2008), where dection limits were 10 cells and 1000 spores, respectively. Based on these previous studies, a new generation intrument was developed to optimize assays based on liquid phase IWT, called Signalyte TM -II; the instrument is shown in Figure 2.…”