兔病毒性出血症病毒抗体间接ELISA检测方法的建立

Establishment of indirect ELISA for detection of antibody against rabbit hemorrhagic disease virus

建立一种检测兔病毒性出血症病毒(RHDV)抗体的间接ELISA方法。对RHDV陕西分离株VP60基因进行原核表达,Western blot分析表达产物的免疫反应性;以纯化的蛋白为包被抗原建立ELISA方法,并对反应条件进行优化。结果表明,VP60蛋白在大肠杆菌中成功表达,产物约为42.34 ku的融合蛋白,具有良好的反应原性;优化的ELISA最佳工作条件为:重组抗原包被浓度2.9μg/mL,37℃2 h后4℃过夜,1%BSA 37℃封闭2 h,待检血清37℃孵育1 h,酶标抗体1∶10 000稀释,37℃作用1 h,37℃显色5 min,临界值为0.340;建立的ELISA方法特异性强、重复性好、敏感性高;临床检测180份样品,与血凝抑制试验的符合率为74.1%,与商品化试剂盒检测结果符合率为94.8%。该方法可用于临床样品的大批量检测。

Abstract： The aim of this study is to develop an indirect ELISA for detecting the antibody against rabbit hemorrhagic disease virus （RHDV）. The VP60 gene of RHDV Shanxi strain was expressed. The immunoreactivity of recombinant protein was analyzed by Western blot. Then the purified recombinant protein was used for establishment of an indirect ELISA for detection of anti-RHDV antibody. The results showed that the recombinant protein with the molecular weight of 42. 34 ku was successfully expressed in Escherichia coli. The optimal reac- tion condition were determined as followings ： coating antigen with a concentration of 2. 9 μg/mL at 4 ℃ overnight after 37 ℃ 2 h, 1% BSA as blocking agent at 37 ℃ 2 h, serum sample （1 ： 100） at 37 ℃ for 1 h, HRP labeled goat anti-chicken （ 1 ： 10000） at 37 ℃ for 1 h, the substrate TMB for ELISA being incubated at 37 ℃ for 5 min. The threshold of positive and negative sera was 0. 340 by statistical analysis. The ELISA assay was confirmed to have a good repeatability, sensitivity and specificity. The coincidence rate was 74. 1% with HI and 94. 8% with the commercial reagent in detecting 180 clinical sera. It suggests that the ELISA method can be used for detection of clinical samples.