鸡柔嫩艾美耳球虫特异性抗体EtMIC4-N蛋白BAS-ELISA检测方法的建立

doi:10.11924/j.issn.1000-6850.casb15010225

中国农学通报 ›› 2015, Vol. 31 ›› Issue (14): 44-50.doi: 10.11924/j.issn.1000-6850.casb15010225

所属专题：生物技术；畜牧兽医

鸡柔嫩艾美耳球虫特异性抗体EtMIC4-N蛋白BAS-ELISA检测方法的建立

刘新月¹,王黎霞²,宋丽聪¹,赵晨璐¹,张建军¹,安健¹

(¹北京农学院动物科学技术学院,北京 102206；²北京农业职业学院畜牧兽医系,北京 102442)

收稿日期:2015-01-29 修回日期:2015-03-18 接受日期:2015-03-25 出版日期:2015-06-02 发布日期:2015-06-02
通讯作者: 安健

Establishment of BAS-ELISA Method with Recombinant EtMIC4-N Protein for the Detection of Antibodies Against Chicken Eimeria tenella

Liu Xinyue¹, Wang Lixia², Song Licong¹, Zhao Chenlu¹, Zhang Jianjun¹, An Jian¹

(¹College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206;²Beijing Vocational College of Agriculture, Beijing 102442)

Received:2015-01-29 Revised:2015-03-18 Accepted:2015-03-25 Online:2015-06-02 Published:2015-06-02

摘要/Abstract

摘要： 为了检测柔嫩艾美耳球虫感染鸡血清中特异性抗体水平，原核表达纯化EtMIC4-N蛋白，采用免疫印迹法确定其反应原性，再以纯化后的蛋白为包被抗原，以生物素标记的羊抗鸡IgG为二抗，以HRP结合链霉亲和素为酶标抗体，建立了检测柔嫩艾美耳球虫抗体的BAS-ELISA方法。经检测，EtMIC4-N蛋白最佳包被浓度为7 μg/mL，最适封闭液为1% BSA，最佳血清稀释度为1:80，二抗稀释度为1:100000，酶标抗体稀释度为1:100000，最适显色时间为15 min，组内和组间变异系数均小于10%。通过动物感染试验验证，结果表明建立的BAS-ELISA可以用于检测柔嫩艾美耳球虫特异性抗体的检测，并且具有简便，快速，灵敏，成本低等特点。

关键词: 土壤酸化, 土壤酸化, pH值, 宣恩县

Abstract: To quickly detect the specific antibody levels in serum of chickens which infected by Eimeria tenlla, EtMIC4-N protein was expressed and purified by Escherichia coli. The reactogenicity of EtMIC4-N protein was identified by Western Bloting. A BAS-ELISA method was established using EtMIC4-N protein as the coating antigen and biotin conjugated goat anti-chicken IgG antibody as the second antibody, streptavidin peroxidase conjugated as the enzyme-labeled antibody. The results indicated that the best concentration of coating antigen was 7 μg/mL, the best blocking buffer was 1% BSA, the best dilution of sera samples, the second antibody, the enzyme-labeled antibody was 1:80, 1:100000, 1:100000 respectively, the best chromogenic reaction time was 15 min, and coefficients of variation in intra-batch and inter-batch were less than 10%. The animal experiment results suggested that BAS-ELISA, which was simple, rapid, sensitive and low-cost, could be used to detect the antibodies against chicken Eimeria tenlla.

刘新月,王黎霞,宋丽聪,赵晨璐,张建军,安健. 鸡柔嫩艾美耳球虫特异性抗体EtMIC4-N蛋白BAS-ELISA检测方法的建立[J]. 中国农学通报, 2015, 31(14): 44-50.

Liu Xinyue,Wang Lixia,Song Licong,Zhao Chenlu,Zhang Jianjun and An Jian. Establishment of BAS-ELISA Method with Recombinant EtMIC4-N Protein for the Detection of Antibodies Against Chicken Eimeria tenella[J]. Chinese Agricultural Science Bulletin, 2015, 31(14): 44-50.

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鸡柔嫩艾美耳球虫特异性抗体EtMIC4-N蛋白BAS-ELISA检测方法的建立

Establishment of BAS-ELISA Method with Recombinant EtMIC4-N Protein for the Detection of Antibodies Against Chicken Eimeria tenella

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