title: Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry.
abstract: Portable detection and quantitation methods forBacillus anthracis(anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity forBacillus anthracisspores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field.
authors.affiliation: Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. dfw808@yahoo.com.
authors.forename: David F
authors.initials: DF
authors.lastname: Waller
authors.affiliation: Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. hewbrian@gmail.com.
authors.forename: Brian E
authors.initials: BE
authors.lastname: Hew
authors.affiliation: Holatron Systems, LLC., 833 Ilaniwai Street, Suite 2, Honolulu, HI 96813, USA. charlie@holatron.com.
authors.forename: Charlie
authors.initials: C
authors.lastname: Holdaway
authors.affiliation: Mike Jen Software Services, 1035 Aster Ave #2103, Sunnyvale, CA 94086, USA. mike.jen@gmail.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Jen
authors.affiliation: Black Ivory Biotech, Inc., P.O. Box 893128, Mililani, HI 96789, USA. biogabe@gmail.com.
authors.forename: Gabriel D
authors.initials: GD
authors.lastname: Peckham
mesh_terms: D001408:Bacillus anthracis; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D023482:Bioterrorism; D004566:Electrodes; D007118:Immunoassay; D018189:Immunomagnetic Separation; D013171:Spores, Bacterial
keywords: amperometry; anthrax spores; biodetection; bioterrorism; immunoassay; portable assay