The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of Campylobacter jejuni in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.Campylobacter enteritis is a significant cause of morbidity throughout the world, with an estimated annual incidence of 400 million cases (15). In the United States, it has been estimated that 2.4 million cases occur annually, resulting in 120 to 360 deaths (17). Campylobacter jejuni is the species most commonly associated with the disease, although the prevalence of Campylobacter coli infections rivals that of C. jejuni in some countries (7). Despite the low mortality rate associated with Campylobacter enteritis, the disease has a significant economic impact, with an estimated annual cost of nearly $1 billion in the United States alone (17).Campylobacters inhabit the intestinal tracts of a variety of mammals and birds (14), and they are transmitted from host to host via the fecal-oral route. In humans, infection results from the ingestion of contaminated foods, with as few as 500 CFU having been reported to cause disease (20). Campylobacters have been isolated from foods of both animal and nonanimal origin (13). Contaminated raw milk and untreated water are commonly associated with outbreaks of the disease (7); however, poultry, in particular chicken, is the most commonly implicated food source (18). Widespread contamination of poultry carcasses occurs during processing due to the release of intestinal contents during evisceration. Contamination rates of chicken carcasses as high as 93 to 98% (2, 18) have been reported, with C. jejuni counts on retail chicken often exceeding 10 3 cells per 100 g (1). Both the ubiquitousness and the potentially low infectious dose of these pathogens make their presence in the food supply a significant health hazard.Due to the prevalence of Campylobacter species in the food supply, routine and reliable monitoring for these pathogens is necessary in order to reduce their impact upon human health. Cultivation methods involving enrichment, isolation, and biochemical characterization require 4 to 5 days to complete (4, 5). Due to the perishable nature of many food items, a more rapid detection method is necessary to feasibly monitor the potential sources of these pathogens. For this reason, we have developed an immunocapture PCR method for the detection of Campylobacter in foods. The assay utilizes an immunomagnetic capture technique to isolate the bacteria from food samples, followed by PCR amplification of the genomic DNA encoding rRNA. The PCR products are detected in a chromogenic assay, thus per...

Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts.

Portable detection and quantitation methods for Bacillus 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 for Bacillus anthracis spores. 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.

Antibody-based detection assays are generally robust, a desirable characteristic for in-the-field use. However, to quantify the colorimetric or fluorescent signal, these assays require expensive and fragile instruments which are ill-suited to in-the-field use. Lateral flow devices (LFDs) circumvent these barriers to portability but suffer from poor sensitivity and subjective interpretation. Here, an antibody-based method for detectingBacillus anthracisspores via amperometric signal generation is compared to ELISA and LFDs. This amperometric immunoassay uses antibody conjugated to magnetic beads and glucose oxidase (GOX) along with the electron mediator 2, 6-dichlorophenolindophenol (DCPIP) for production of a measurable current from a 0.4 V bias voltage. With similar sensitivity to ELISA, the assay can be completed in about 75 minutes while being completely powered and operated from a laptop computer. Immunoassay amperometry holds promise for bringing low-cost, quantitative detection of hazardous agents to the field.

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