摘要
用差速离心法纯化的猪轮状病毒(RV)分别免疫仔猪和兔,制备了猪抗RV和兔抗RV超免疫血清,并用层析方法进行了纯化,建立了检测RV的双抗体夹心ELISA方法。结果表明,该双抗体夹心ELISA的最佳反应条件为:猪抗RV IgG包被浓度为4μg/mL,兔抗RV IgG最佳工作浓度为3.5μg/mL,样品反应时间为90 min,酶标抗体工作浓度为1∶8 000,以D450 nm≥0.161作为阳性判定标准。该ELISA的重复性变异系数小于10%,最低检测限为1.25μg/mL,并与猪瘟病毒、猪伪狂犬病病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪大肠杆菌和沙门菌等病原无交叉反应,该ELISA试剂在室温和4℃条件下至少可保存4个月。用该ELISA方法和RV金标检测卡检测70份临床粪便样品,结果显示,ELISA的阳性检出率为22.9%,而金标检测卡的阳性检出率为20.0%。表明,建立的双抗体夹心ELISA方法具有特异性好、灵敏性高和重复性好等优点,可用于RV的快速检测。
Two kinds of hyperimmune sera,porcine-anti-rotavirus IgG and rabbit-anti-rotavirus IgG,were prepared by inoculating respectively piglets and rabbits with porcine rotavirus（RV） purified by diffe-rential centrifugation,and purified by affinity chromatography.A double antibody sandwich ELISA for detection of RV was developed based on the two kinds of IgG.The optimal coating concentration of porcine-anti-rotavirus IgG was 4 μg/mL,the optimal working concentration of rabbit-anti-rotavirus IgG was 3.5 μg/mL,the reaction time of sample was 90 min,and the optimal working dilution of HRP-labelled goat-anti-rabbit IgG was 1∶8 000.The positive standard value was 0.161（D 450 nm）.The coefficient of variation of reproducibility was less than 10%,and at least 1.25 μg/mL antigen could be detectable.The ELISA had no cross-reaction with classical swine fever virus,porcine pseudorabies virus,porcine transmissible gastroenteritis virus,porcine epidemic diarrhea virus,Escherichia coli and Salmonella.The prepared plates and reagents could preserved at least four months at room temperature and 4 ℃.Seventy clinical fecal samples were detected by the ELISA and the colloidal gold card,and the positive ratio was 22.9% and 20.0% respectively.The results revealed that the ELISA possessed good specificity and reproducibility,and higher sensitivity,indicating a suitable method for rapid detection of RV.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第8期723-727,共5页
Chinese Veterinary Science
基金
教育部"长江学者和创新团队发展计划"创新团队项目(IRTO848)
四川省教育厅资助项目(08zb075)
