摘要
为建立一种快速、简便、适应现场应用的水牛大片形吸虫检测方法,本试验利用抗大片形吸虫ES抗原单克隆抗体杂交瘤细胞株7D1和7D4分别制备和纯化得到单克隆抗体7D1和7D4;采用柠檬酸三钠还原法制备颗粒大小为30nm的胶体金,再用胶体金标记纯化单克隆抗体7D1,在硝酸纤维素膜的质控带和检测带处分别包被羊抗鼠IgG二抗和单克隆抗体7D4,经反应条件优化,组装成了大片形吸虫免疫检测试纸条。经测试该检测试纸条对ES抗原的检测下限为0.94μg/mL,与胰扩盘吸虫、前后盘吸虫、支原体、弓形虫、伊氏锥虫均无交叉反应,特异性好。采集广西地区334份水牛新鲜粪样并用试纸条检测,阳性率为38.62%。结果表明,试纸条方法操作简单、快速、易于判定,特异性好,敏感性高,适合基层大面积普查和现场检测大片形吸虫。
To establish a rapid,simple,sensitive and adapting field application assay for water buffalo Fasciola gigantica,the monoclonal antibodies of hybridoma cell strains 7D1 and 7D4against excretory-secretary antigen（ES Ag）of Fasciola gigantica were prepared and purified.Meanwhile,the colloidal gold particle was prepared by the sodium cirrate reduction method,then the affinity purified 7D1 monoclone antibodies was labeled with the 30 nm colloidal gold particles.The 7D4 monoclonal antibodies and the goat anti-mouse IgG antibody using as secondary antibodies were coated on the surface of nitrocellulose filter（NC）membrane as the test line（T line）and control line（C line）,respectively.The immune colloidal gold test strip was developed by optimizing the experiment conditions.The test results showed the prepared strip had a detecting prescribed minimum of 0.94μg/mL of Fasciola gigantica excretory-secretary antigen,and it had no any cross reaction with the antigens of Eurytrema pancreaticum,Paramphistome,Chlamydia,Toxoplasma gondii and Trypanosoma evansi.Using the prepared strip to investigate the 334 fresh feces from buffaloes in Guangxi and the positive rate was 38.62%.These results indicated that this strip method was simple,rapid and easy determination,good specificity,high sensitivity,and adapted field application assay for Fasciola gigantica.
作者
王华俊
刘宇
蒋薇
赵雪丽
曹伟伟
陈汉忠
闫若潜
WANG Hua-jun LIU Yu JIANG Wei ZHAO Xue-li CAO Wei-wei CHEN Han-zhong YAN Ruo-qian(College of Animal Science and Technology , Guangxi University, Nanning 530005, China Henan Centre for Animal Diseases Control ＆ Prevention, Zhengzhou 450008, China)
出处
《中国畜牧兽医》
CAS
北大核心
2017年第1期254-261,共8页
China Animal Husbandry & Veterinary Medicine
基金
广西教育厅科研项目
关键词
大片形吸虫
单克隆抗体
胶体金
试纸条
Fasciola gigantica
monoclonal antibody
colloidal gold
test strip
