Abstract
Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. In this paper, we describe a sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The polyclonal antibody (pAb) and monoclonal antibodies (mAbs) specific to B. cereus were generated from rabbit antiserum and mouse ascites, respectively, using the octanoic acid/saturated ammonium sulfate precipitation method and protein A-sepharose columns. IgG-isotype mAbs were specially developed to undergo a novel peripheral multiple sites immunization for rapid gain of hybridomas and a subtractive screen was used to eliminate cross reactivity with closely related species such as Bacillus thuringiensis, B. subtilis, B. licheniformis and B. perfringens. The linear detection range of the method was approximately 1 × 104–2.8 × 106 cells/mL with a detection limit (LOD) of 0.9 × 103 cells/mL. The assay was able to detect B. cereus when the samples were prepared in meat with various pathogens. The newly developed analytical method provides a rapid method to sensitively detect B. cereus in food specimens.
Highlights
Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world
The subtractive screen largely enhanced the specificities of the generated monoclonal antibodies (mAbs), which ensured the specificity of the sandwich enzyme-linked immunosorbent assay (ELISA)
The preparation of soluble antigen plays a key role in the enhancement of the coating capacity for indirect ELISA, which facilitates successful subtractive selection
Summary
Bacillus cereus is increasingly recognized as one of the major causes of food poisoning in the industrialized world. We describe a sensitive double-antibody sandwich enzymelinked immunosorbent assay (ELISA) that was developed for rapid detection of B. cereus in food to minimize the risk of contamination. The bacterium causes two distinct forms of gastroenteritis connected to food poisoning, the emetic and the diarrheal types. Due to the increasing incidence of B. cereus food-borne illness and the wide spread distribution of B. cereus in food, rapid detection methods are required for diagnostic purposes and for the prevention of food contamination and food-borne outbreaks. Studies describing the identification techniques of B. cereus are mainly based on the use of selective media and biochemical tests These methods involve enrichment, plating and specific identification and are laborious, time-consuming and require trained personnel to detect traces of B. cereus cells in food[11]. Some immunological assays have been made commercially available, such as the B. cereus Enterotoxin Test Kit, commercial kits are not yet available for whole cells of B. cereus strains[1]
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