检测猪鼻支原体抗原的ELISA方法的建立及其应用  

Development and application of ELISA for the detection of Mycoplasma hyorhinis antigen

作　　者：朱红振 危艳武[1] 黄立平[1] 刘丹[1] 夏德利[1] 吴洪丽[1] 边海桥 冯力[1] 刘长明[1] ZHU Hong-zhen;WEI Yan-wu;HUANG Li-ping;LIU Dan;XIA De-li;WU Hong-li;BIAN Hai-qiao;FENG Li;LIU Chang-ming(State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of AgriculturalSciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处：《中国兽医科学》2019年第7期828-833,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0500602)

摘　　要：为了建立检测猪鼻支原体(Mhr)抗原的ELISA方法,本研究采用杂交瘤技术制备了抗Mhr菌体的特异性单克隆抗体(MAb),经亲和层析纯化后标记辣根过氧化物酶(HRP),建立了一种能够检测Mhr抗原的ELISA方法。ELISA反应条件优化结果为:HRP标记的MAb最适稀释度为1∶1000,该方法的批内和批间变异系数均小于6%,经超声波处理Mhr抗原效果更佳,最低抗原检出量为25ng。用该方法对已知猪肺炎支原体(Mhp)、猪圆环病毒2型(PCV2)抗原检测无交叉反应。本研究建立的检测Mhr抗原的ELISA方法具有良好的特异性、敏感性和重复性,可为Mhr体外培养物抗原定量检测提供可靠的技术手段。In order to establish an ELISA method for the detection of Mycoplasma hyorhinis(Mhr)antigen,a specific monoclonal antibody(MAb)against Mhr was prepared by hybridoma technique.The MAb is purified by affinity chromatography and labeled with horseradish peroxidase(HRP).An ELISA method was established for Mhr antigen detection.The optimal reaction conditions were that the HRP-conjugated IgG was dilution at 1∶1 000.The results of reproducibility test showed that the coefficient of intra-and inter-variations were less than 6%.The much better result could be obtained by means of Mhr antigen treatment by ultrasonication.The minimum detection quantity for Mhr antigen in this ELISA was 25 ng.No cross reactions were detected with Mycoplasma hyopneumoniae(Mhp)and PCV2 antigen in this assay.This ELISA method for Mhr antigen detection has good reproducibility,specificity and sensitivity.It provides an effective mean for the quantitative detection of Mhr in vitro culture antigens.

关 键 词：猪鼻支原体 单克隆抗体 抗原检测 ELISA 

分 类 号：S852.62[农业科学—基础兽医学]