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ETEC菌毛蛋白K99抗体PPA-ELISA检测 方法的建立及初步应用
谢金文,白耀国,李书光,王金良,曲光刚,苗立中,沈志强
0
(山东省滨州畜牧兽医研究院 山东省畜禽用蜂胶疫苗研究开发推广中心,滨州 256600; 山东绿都生物科技有限公司,滨州 256600)
摘要:
以纯化的大肠埃希菌K99菌毛蛋白为包被抗原,建立检测大肠埃希氏菌K99 IgG抗体的间接ELISA方法。确定间接ELISA的最适反应条件,即抗原包被的ELISA板4℃过夜,最适抗原包被浓度为4.94 μg·mL-1,兔抗体稀释倍数为1:50,猪抗体稀释倍数为1:200,确定最佳封闭液为100 g·L-1脱脂奶粉,最佳封闭时间为0.5 h,血清反应时间为1 h,二抗最适浓度梯度为1:2000,反应时间为37℃ 0.5 h,底物室温反应时间为37℃ 10 min,阴阳性临界值兔血清为0.25、猪血清为0.35。试验证实该间接PPA-ELISA方法的特异性强、敏感性高、重复性好,可以为疫苗效力检验和流行病学调查提供参考方法。
关键词:  ETEC  K99  PPA-ELISA  抗体  检测  建立
DOI:10.13610/j.cnki.1672-352x.20150302.015
基金项目:山东省自然科学科学基金青年基金项目(ZR2014CQ009), 滨州市2013年科技发展计划项目(2013GG0304), 山东省现代农业产业技术体系生猪产业创新团队(SDAIT-06-011-14)共同资助。
Development and preliminary application of PPA-ELISA in detection of antibodies against K99 fimbrial protein
XIE Jinwen,BAI Yaoguo,LI Shuguang,WANG Jinliang,QU Guanggang,MIAO Lizhong,SHEN Zhiqiang
(Shandong Binzhou Animal Science & Veterinary Medicine Institute, Shandong Research & Development Center of Veterinary Propolis Vaccines, Binzhou,256600; Shandong Lvdu Biotechnology Co., Ltd. ,Binzhou,256600)
Abstract:
A PPA-ELISA method for detecting anti-K99 IgG levels was developed by using the porcine enterotoxigenic Escherichia coli (ETEC)-purified K99 fimbrial protein as the coating antigen. The PPA-ELISA was tested with serum samples obtained from rabbits and pigs. The optimal parameters affecting the method were also determined. They were: antigen coated ELISA plates at 4℃ overnight; the optimal coating concentration of K99 was 4.94 μg/mL; the optimal dilution of corresponding serum samples for rabbits was 1:50 and for pigs was 1:200; the optimal blocking solution was 100 g/L skimmed milk powder. The best blocking time, reaction time for serum samples, PPA concentration gradient, PPA reaction time and substrate reaction time at room temperature were 0.5 h, 1 h, 1:2000, 0.5 h, and 10 min, respectively. The positive-negative threshold values of rabbit and pig sera were 0.25 and 0.35, respectively. The results proved that the PPA-ELISA method was sensitive, specific and repeatable and can be applied to the vaccine potency test and epidemiological investigation.
Key words:  ETEC  K99  PPA-ELISA  antibody  detection  establishment

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