溶藻弧菌免疫胶体金快速检测试纸条的制备

Development of Immunochromatographic Colloidal Gold Test Strip for Rapid Detection of Vibrio alginolyticus

采用溶藻弧菌ATCC17749菌体作为抗原免疫新西兰大白兔,制备兔抗溶藻弧菌多克隆抗体;利用柠檬酸钠还原氯金酸法制备胶体金及胶体金-抗体结合物,并将其喷涂在玻璃纤维膜上冷冻干燥;在硝酸纤维素膜上包被兔抗溶藻弧菌多克隆抗体和羊抗兔IgG分别作为检测线和控制线;将处理后的玻璃纤维素膜、硝酸纤维膜、样品垫及吸水纸组装成双抗夹心试纸条,并对试纸条灵敏度、特异性、稳定性进行评价。结果表明,制备的兔抗溶藻弧菌多克隆抗体的效价可达1∶512 000;胶体金溶胶的最佳制备条件为:加入1.8 mL 10 mg/mL的柠檬酸三钠,500 W微波炉中火加热10 min;试纸条检测线和质控线的抗体最佳包被量分别为8.0 mg/mL和1.0 mg/mL;试纸条检测灵敏度为1×106cfu/mL,特异性试验显示,试纸条与哈氏弧菌、副溶血弧菌、霍乱弧菌、拟态弧菌、美人鱼弧菌、弗氏弧菌、河流弧菌、创伤弧菌等8株菌无交叉反应;稳定性试验表明,试纸在4℃干燥条件下能稳定保存5个月以上。制备的溶藻弧菌多克隆检测试纸条可灵敏、较特异地检测源自病鱼的溶藻弧菌,整个检测过程可在10 min内完成,适用于溶藻弧菌的快速检测。

To develop a sensitive, rapid and specific gold immunchromatography assay for detection of Vibrio alginolyticus, Anti-V. alginolyticus polyclonal antibodies were obtained from the New Zealand white rabbits immunized with V. alginolyticus （ATCC17749）. Colloidal gold was prepared through reducing HAuCl4？4H2O by sodium citrate and conjugated with the polyclonal antibodies as the detecting reagent. The immunogold was sprayed on the gold conjugate pad of a chosen glass fiber membrane and was dried by lyophilization. The polyclonal antibodies and sheep anti-rabbit immunoglobulins were To develop a sensitive, rapid and specific gold immunchromatography assay for detection of Vibrio alginolyticus, Anti-V. alginolyticus polyclonal antibodies were obtained from the New Zealand white rabbits immunized with V. alginolyticus （ATCC17749）. Colloidal gold was prepared through reducing HAuCl4？4H2O by sodium citrate and conjugated with the polyclonal antibodies as the detecting reagent. The immunogold was sprayed on the gold conjugate pad of a chosen glass fiber membrane and was dried by lyophilization. The polyclonal antibodies and sheep anti-rabbit immunoglobulins were separately coated onto the same nitrocellulose membrane for sample detection and quality-control, respectively. The nitrocellulose membrane, gold conjugate pad, sample pad, filter paper and absorbent pad were assembled to prepare the strips. The detection specificity and sensitivity of this strip were evaluated. The titer of the rabbit anti-V. alginolyticus polyclonal antibody antiserum determined by ELISA was up to 1 to 512 000. The optimal conditions for preparing colloidal gold are as follows：added 1.8 mL 1% sodium citrate, heated in 500 W microwave oven for 10 min. The test line and the control line are sprinkled respectively with the concentration of 8.0 and 1.0 mg/mL. The detection sensitivity of the test was 1×10^6 cfu/mL. The strip showed no cross-reaction with V. harveyi, V. parahaemolyticus, V. cholera, V. mimicus, V. damsela, V. fischeri, V. fluvialis, V. fluvialis, V. vulnficus. The strips remained stable after preservation at 4 ℃ for 5 months. The test strip generated in this study has high specificity, sensitivity and can yield results in 10 min. It implies that this test strip has great potential use in the detection of V. alginolyticus.