一种快速检测弗氏柠檬酸杆菌免疫层析试纸条的研制

Development of a colloidal gold immunochromatographic test strip for detection of Citrobacter freundii

为了制备弗氏柠檬酸杆菌快速检测试纸条,联合卵黄抗体技术和胶体金免疫层析技术,采用弗氏柠檬酸杆菌抗原免疫母鸡,制备抗弗氏柠檬酸杆菌卵黄抗体(IgY);采用柠檬酸三钠还原法制备胶体金颗粒,用以标记IgY;在结合垫上包被用胶体金标记的IgY,硝酸纤维膜上包被弗氏柠檬酸杆菌抗原和羊抗鸡IgY作为检测线和质控线;将样品垫、金标结合垫、硝酸纤维膜和吸水垫组装成试纸条,测试其灵敏度、特异性、稳定性,并与ELISA结果进行比较.结果表明:该试纸条检测线为105 cfu?mL^(-1);试纸条与恶臭假单胞菌、铜绿假单胞菌、迟钝爱德华菌、哈维氏弧菌和溶藻弧菌均无交叉反应;该试纸条在4℃保存4个月后依然十分稳定;具有易操作,特异性、灵敏度高,不需要专业的操作人员和特殊的仪器设备,5～10 min即可获得结果等优势,适合弗氏柠檬酸杆菌的现场检测.

The current study was aimed to establish a rapid test strip for Citrobacter freundii by using egg yolk antibody and colloidal gold immunochromatography assay. Hens were immuned with C. freundii antigen. Antibody against C. freundii was purified from the yolks. The colloidal gold particles combining with yolk antibodies (IgY) were prepared by citric acid three sodium reduction method. The labeled colloidal gold-IgY complex was wrapped on the binding pad, and the nitric acid fiber membrane was coated with C. freundii antigen and Sheep anti-chicken IgY as the test line and the control line respectively. The sample pad, gold conjugate pad, nitrocellulose membrane and absorbent pad were assembled into test strips and sealed with desiccant. The sensitivity, specificity and stability of the test strip were tested, and test results were compared with those from ELISA. The detection results can be obtained within 5-10 min. The sensitivity of the test strip was 105 cfu·mL^-1, and there was no cross reaction with Pseudomonas putida, P. aeruginosa, Edwardsiella tarda, Vibrio harveyi and V. alginolyticus. The strip was stable and efficient after being stored at 4 ℃ for 4 months. As detection with the strip is easy to operate and does not require professional operator and special instrument, it is very suitable for the on-site detection of C. freundii.