![]() ![]() The method presented here uses 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as a soluble redox mediator in the presence of glucose and horseradish peroxidase (HRP) for the indirect measurement of glucose oxidase activity on IMS-captured anthrax spores detected with monoclonal antibody-glucose oxidase (GOX) conjugate. In this study, IMS is combined with amperometry, which offers the advantages of being very sensitive, robust, and economical and can be made compact and easy to use in the field. The large surface area available on magnetic beads for the immobilization of antibodies allows for an efficient and sensitive capture of spores, although the binding specificity and limit of detection generally depend on the antibodies and binding conditions used. IMS has been used previously in combination with other techniques for the detection of Bacillus anthracis spores in various matrices. Immunomagnetic separation (IMS) is a technique in which antibodies specific to a target organism are immobilized on magnetic beads to enable fast and efficient concentration and purification of the target from crude samples, thus reducing or eliminating any matrix effects from contaminants in subsequent testing. More sensitive detection methods such as ELISA or PCR typically use time-consuming protocols and instruments not suitable for field use or require sample enrichment or clean-up prior to testing to avoid inhibition by environmental contaminants. Current field-deployable methods of anthrax spore detection such as lateral-flow devices (LFDs) lack sensitivity and quantitative ability, and are subjectively interpreted. The potential threat of anthrax spores being used as a biological weapon calls for a quick and reliable means of field detection that is portable, cost-effective, and easy to operate. 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. 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). ![]() Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. 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). Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. 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. ![]() Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. ![]()
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