猪弓形虫感染免疫胶体金试纸条的制备与初步应用  

Development and preliminary application of colloidal gold immunochromatographic strip for the detection of Toxoplasma infection in swine

作　　者：寇金华 杨正涛 刘维建 高珺珊 李建华 杨举 张西臣 宫鹏涛 

机构地区：[1]吉林大学动物医学学院

出　　处：《中国兽医学报》2018年第1期82-87,共6页Chinese Journal of Veterinary Science

基　　金：吉林省科技发展计划资助项目(20130206023NY);国家公益性行业(农业)科研专项资助项目(201303042)

摘　　要：制备1种检测猪弓形虫血清循环抗原的免疫胶体金试纸条。利用柠檬酸三钠还原法制备40nm的胶体金溶液，标记抗弓形虫表面抗原sAG3（surfaceantigen3，sAG3）单克隆抗体（Anti-A—SAG3—7），用另1株抗弓形虫表面抗原sAG3单克隆抗体（Anti—A—SAG3—23）与羊抗小鼠IgG抗体分别包被于硝酸纤维素膜（NC膜）上的检测线和质控线，制备并优化免疫胶体金试纸条检测条件。胶体金溶液的最适pH值为加入4uL 0．2mol／L K2CO3时的pH值。Anti—A-SAG3—7的最适标记量为12ug。最佳NC膜为Miltipore 135。T、C线抗体最佳包被质量浓度均为0．25g／L，T、C线抗体最佳包被体积均为8uL。试纸条的灵敏度达1：160，与猪囊虫阳性血清无交叉反应。稳定性试验表明，该试纸条置于室温至少可保存1个月有效，置于4℃可保存3个月有效。对广东某地区、吉林某地区猪场的各94份猪待检血清进行检测，分别有17，10份为阳性血清，样品阳性率为18．08％，10．63％。本研究制备的胶体金试纸条具有操作简单，敏感特异等优点，适合临床基层单位对猪弓形虫病的早期诊断及流行病学调查。To develope one kind of colloidal gold immunochromatographic strips for the detection of Toxoplasma circulating antigen in swine,colloidal gold particle with a diameter of 40 nm was prepared by sodium citrat reduction method and then conjugated with SAG3 monoclonal antibody （Anti-A-SAG3-7） against Toxoplasma. Another SAG3 monoclonal antibody （Anti-A-SAG3-23） against Toxoplasma and goat-anti-mouse IgG were respectively coated on nitrocellulose membrane as the test line（T line）and control iine（C line）. And then reaction system was optimized. The opti- mal pH was reached when 4 uL 0.2 mol/L K2 CO3 was added to 1 mL colloid gold solution. The optimal spray amount of Anti-A-SAG3-7 was 12 ug. The best alternative nitrocellulose membrane was Millipore 135. The optimum spray concentration of antibodies at T line and C line were both 0.25 g/L and the best spray volume were both 0.8 uL. The sensitivity of the method was 1 ： 160. No cross reaction was detected with swine Cysticercosis serum. The stability test demonstrate that the strips could be stored for 1 month at room temperature,and at least 3 months at 4℃. 94 samples from Guangdong and 94 samples from Jilin were detected by the colloidal gold strips. The positive rates were 18.08%（17/94）, 10.63% （10/94） respectivly. In conclusion,the colloidal gold strip developed in our research has the adventages of simple operation,high sensitivity and specificity, which is suitable for the early diagnosis and epidemiological investigation of swine toxoplasmosis in clinical application.

关 键 词：刚地弓形虫 SAG3 循环抗原 胶体金 

分 类 号：S852.7[农业科学—基础兽医学]