Zhu Longjiao, He Jing, Cao Xiaohan, Huang Kunlun, Luo Yunbo, Xu Wentao
Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing, 100083, China.
Sci Rep. 2016 Mar 15;6:16092. doi: 10.1038/srep16092.
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 × 10(4)-2.8 × 10(6) cells/mL with a detection limit (LOD) of 0.9 × 10(3) 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.
蜡样芽孢杆菌日益被认为是工业化国家食物中毒的主要原因之一。在本文中，我们描述了一种灵敏的双抗体夹心酶联免疫吸附测定法（ELISA），该方法用于快速检测食品中的蜡样芽孢杆菌，以尽量降低污染风险。分别使用辛酸/饱和硫酸铵沉淀法和蛋白A-琼脂糖柱，从兔抗血清和小鼠腹水中制备出对蜡样芽孢杆菌具有特异性的多克隆抗体（pAb）和单克隆抗体（mAb）。专门开发了IgG同种型单克隆抗体，通过一种新型外周多位点免疫法快速获得杂交瘤，并采用消减筛选法消除与苏云金芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌和产气荚膜梭菌等密切相关菌种的交叉反应性。该方法的线性检测范围约为1×10⁴-2.8×10⁶个细胞/毫升，检测限（LOD）为0.9×10³个细胞/毫升。当样品在含有各种病原体的肉类中制备时，该测定法能够检测出蜡样芽孢杆菌。新开发的分析方法为灵敏检测食品标本中的蜡样芽孢杆菌提供了一种快速方法。
