检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用

doi:10.11843/j.issn.0366-6964.2014.10.016

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (10): 1693-1698.doi: 10.11843/j.issn.0366-6964.2014.10.016

检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用

张改梅^1#，贾红^1#，侯绍华¹，鑫婷¹，郭晓宇¹，袁维峰¹，刘淑清¹，吴竞¹，高新桃²，李明¹，董瑞凯¹，朱鸿飞^1*

（1.中国农业科学院北京畜牧兽医研究所，北京 100193；2.中国农业科学院研究生院，北京 100081）

收稿日期:2014-03-13 出版日期:2014-10-23 发布日期:2014-10-23
通讯作者: 朱鸿飞(1965-)，研究员，博士生导师，E-mail:bioclub@vip.sina.com
作者简介:张改梅(1990-)，女，山西阳泉人，硕士，主要从事动物疫苗与分子疫苗的研究，E-mail：gaimei0809@126.com，#共同第一作者，在本研究中承担同等量的研究工作，共享第一作者
基金资助:
国家863计划项目 (2012AA101302)；中央级公益性科研院所基本科研业务费专项资金项目（2014ywf-zd-1）；中国农业科学院科技创新工程（ASTIP-IAS-11）

Establishment of a Double Antibody Sandwich ELISA against Bovine IFN-γ and Its Preliminary Application in Clinical Diagnosis of Bovine Tuberculosis

ZHANG Gai-mei^1#，JIA Hong^1#，HOU Shao-hua¹，XIN Ting¹，GUO Xiao-yu¹，YUAN Wei-feng¹，LIU Shu-qing¹，WU Jing¹，GAO Xin-tao²，LI Ming¹，DONG Rui-kai¹，ZHU Hong-fei^1*

（1.Institute of Animal Sciences of CAAS，Beijing 100193，China；2.Graduate School of Chinese Academy of Agricultural Sciences，Beijing 100081，China）

Received:2014-03-13 Online:2014-10-23 Published:2014-10-23

摘要/Abstract

摘要：

本研究旨在建立牛IFN-γ体外释放法，为牛结核病的免疫学诊断提供一种快速、有效的新检测技术。分别以获得的牛IFN-γ单克隆抗体Bo-mAb₁作为捕获抗体，以Bo-mAb₂^H作为检测抗体，建立了基于双抗体夹心ELISA（DAS-ELISA）的牛IFN-γ体外释放法。该方法的最低检出限为3.12 ng•mL^－1，可特异性检测出重组牛IFN-γ（Bac-BoIFN-γ）及天然BoIFN-γ，而与猪、犬、鸡的天然IFN-γ无交叉反应。批内、批间变异系数小于5.11%。临床检测结果显示，与结核菌素皮内变态试验（TST）、进口Bovigam^TM试剂盒检测的符合率分别为88.7%和95.3%。结果表明，DAS-ELISA法具有良好的敏感性、特异性及精确度，可用于临床样品的检测，为监测牛结核病特别是其早期感染提供了强有力的检测技术手段。

Abstract:

This study was aimed to establish a Bovine IFN-γ Release Method in vitro，which could provide a new，rapid，effective detection technology for the immunological diagnosis of bovine tuberculosis.The bovine IFN-γ release method based on the double antibody sandwich ELISA (DAS-ELISA) was established，in which，Bo-mAb₁against bovine IFN-γ was used as the capture antibody and HRP-labeled Bo-mAb₂^H as the detection antibody．Our results showed that the detection limit of this ELISA was 3.12 ng•mL^－1.This method could specifically detect bovine IFN-γ both from blood plasma and recombinant protein，showing no cross-reaction with porcine，canine as well as chicken IFN-γ.The intra-assay and inter-assay coefficient of variability were both within 5.11%.When compared with the tuberculin skin test，and the Bovigam^TM kit，our preliminary clinical results showed that the coincidence rate was 88.7% and 95.3%，respectively.As a result，a conclusion can be made that this DAS-ELISA method shows good sensitivity，specificity and accuracy，and could apply to processing large quantities of clinical samples.Our research contributes to the early detection of bovine tuberculosis.

中图分类号:

张改梅，贾红，侯绍华，鑫婷，郭晓宇，袁维峰，刘淑清，吴竞，高新桃，李明，董瑞凯，朱鸿飞. 检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用[J]. 畜牧兽医学报, 2014, 45(10): 1693-1698.

ZHANG Gai-mei，JIA Hong，HOU Shao-hua，XIN Ting，GUO Xiao-yu，YUAN Wei-feng，LIU Shu-qing，WU Jing，GAO Xin-tao，LI Ming，DONG Rui-kai，ZHU Hong-fei. Establishment of a Double Antibody Sandwich ELISA against Bovine IFN-γ and Its Preliminary Application in Clinical Diagnosis of Bovine Tuberculosis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(10): 1693-1698.

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检测牛IFN-γ双抗体夹心ELISA的建立及在牛结核病诊断上的初步应用

Establishment of a Double Antibody Sandwich ELISA against Bovine IFN-γ and Its Preliminary Application in Clinical Diagnosis of Bovine Tuberculosis

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