摘要
为建立一种快速、特异性强的用于检测兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)抗原的双抗夹心ELISA方法,本研究通过抗体配对实验,确定以抗兔出血症病毒结构蛋白VP60单克隆抗体9H9为捕获抗体,辣根过氧化物酶标记的9H9为检测抗体建立双抗夹心ELISA方法。通过优化,确定最佳条件为:0.5μg/m L稀释后的9H9作为捕获抗体,底物显色液37℃包被2 h 4℃过夜,1%明胶溶液37℃封闭2 h,用含10%胎牛血清的PBST按5μg/m L稀释后的辣根过氧化物酶标记的9H9作为检测抗体,37℃作用15 min。建立的ELISA方法与杯状病毒科其他病毒无交叉反应,应用本方法和RT-PCR方法对85份兔肝脏样品进行检测,证明双抗夹心ELISA方法具有良好的特异性和敏感性,有望作为临床快速检测的一种简便方法。
In this study, a rapid, specific double antibody sandwich ELISA （DAS-ELISA） method for detection of Rabbit hemorrhagic disease virus （RHDV） was developed. By using the pairing of monoclonal antibodies, it was confirmed that the method was developed using anti-VP60 monoclonal antibody 9H9 as capturing antibody and HRP-9H9 as detection antibody. The results showed that the optimal concentration of 9H9 was 0.5μg/mL. The optimal concentration of HRP-9H9 was 5μg/mL. The optimum condition of coating was 37℃ and then 4℃ overnight. The optimal blocking condition was PBST containing 1% gelatin and incubation at 37℃ for 2 h. The best diluent of detecting antibodies was PBST containing 10%FBS. The ELISA method had no cross-reactivity with other caliciviruses. In clinical tests, 85 samples isolated from rabbits were subjected to DAS-ELISA and RT-PCR. The results show that the DAS-ELISA method had a high level of specificity and sensibility could be used for rapid detection of RHD.
出处
《中国动物传染病学报》
CAS
北大核心
2016年第2期20-24,共5页
Chinese Journal of Animal Infectious Diseases
基金
公益性农业科研专项(201303046)
上海市科委创新项目(13391901602)
