牛D型产气荚膜梭菌肠毒血症dot-ELISA诊断方法的建立  

Development of a dot-enzyme linked immunosorbent assay for detection of bovine antibody against Clostridium perfringens type D

作　　者：李云霄[1] 金鑫[1] 单雪梅[1] 张营[1] 任春宇[1] 

机构地区：[1]延边大学农学院,吉林龙井133400

出　　处：《中国兽医科学》2009年第3期246-250,共5页Chinese Veterinary Science

基　　金：吉林延边朝鲜族自治州科技攻关重点项目(2004ybkj013)

摘　　要：以纯化的菌体蛋白为诊断抗原,建立了牛D型产气荚膜梭菌特异性抗体的斑点酶联免疫吸附试验(dot-ELISA)检测方法,确定了各组分的最适反应条件:抗原包被浓度为1.95μg/mL,血清稀释度为1∶320,二抗稀释度为1∶2 000,封闭液为30 mL/L脱脂乳,抗原、血清、二抗和封闭液的作用温度及时间均为37℃30 min。用该dot-ELISA检测30份牛血清,纯化抗原比粗提抗原的阳性检出率高16.7%;dot-ELISA的灵敏度是琼脂免疫扩散试验(AGID)法的43倍;用dot-ELISA法对100份牛血清样本进行检测,阳性率为59.0%;经特异性试验、重复性试验和与AGID法比较,表明用纯化抗原建立的方法具有良好的特异性、敏感性和重复性。A dot enzyme linked immunosorbent assay（dot-ELISA） for detection of cattle＇s antibody against Clostridium perfringens type D using purified antigen was developed. The optimal conditions for the dot-ELISA assay were that antigen concentration, serum dilution and dilution of the HRP-labeled rabbit anti-bovine IgG were 1.95μg/mL, 1：320, 1：2000, respectively and blocking agent was 30 mL/L skimmed milk at 37℃ for 30 min. Positive rate of 30 bovine serum samples in dot-ELISA using the purified antigen was 16.7% higher than that using the crude antigen. Sensitivity of the dot-ELISA was 43 times that of AGID. Positive rate of 100 bovine serum samples collected from Yanbian Prefecture was 59.0% by dot-ELISA. Specificity,reproducibility and comparison tests showed that dot-ELISA was specific, sensitive and reproducible.

关 键 词：牛 D型产气荚膜梭菌 纯化抗原 斑点酶联免疫吸附试验 

分 类 号：S852.616[农业科学—基础兽医学] R446.61[农业科学—兽医学]