福建农业学报2017，Vol.32Issue(8)：823-827,5.DOI:10.19303/j.issn.1008-0384.2017.08.003
猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立
Double Antibody Sandwich ELISA for Detection of Porcine Epidemic Diarrhea Virus
摘要
Abstract
A double antibody sandwich ELISA (DAS-ELISA) was developed using the high specificity,mousederived monoclonal antibody (Mab) as the capture antibody and the rabbit-derived polyclonal antibody against porcine epidemic diarrhea virus (PEDV) as the detecting antibody.The optimal reaction conditions for DAS-ELISA was determined to include a coating concentration of 4.40 g · mL-1 for PEDV MAb E1 with 1 h incubation at 37℃,the use of 5％ BSA solution for blocking for 1 h,an application of 5.91 μg · mL-1 in concentration of rabbit polyclonal antibodies against PEDV,a 2 000 × dilution of HRP,and the positive OD equal or greater than 0.381 at 450 nm wave length on the spectrophotometer measurement.The developed method showed no cross-reaction between porcine rotavirus and transmissible gastroenteritis virus.The detection sensitivity of the method was 30 g· mL-1 (5× 10312);and,the coefficient variation of repetition,less than 10％.Furthermore,a total of 42 clinical samples were positively detected by the method in conjunction with RT-PCR at a rate of 92.30％.Consequently,it was concluded that the newly developed DAS-ELISA methodology was highly specific,sensitive,rapid,and hence,applicable for PEDV detection.关键词
猪流行性腹泻病毒/单克隆抗体/双抗体夹心ELISAKey words
porcine epidemic diarrhea virus/monoclonal antibody/double antibody sandwich ELISA分类
农业科技引用本文复制引用
王晨燕,王隆柏,吴学敏,陈如敬,车勇良,周伦江..猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立[J].福建农业学报,2017,32(8):823-827,5.基金项目
福建省科技计划项目——省属公益类科研院所基本科研专项(2017R1023-8) （2017R1023-8）
福建省农业科学院青年创新基金项目(MYQJ2015-2) （MYQJ2015-2）
福建省科技创新平台建设项目——福建省畜禽疫病防控技术重大研发平台(2014N2003-4) （2014N2003-4）
