牛病毒性腹泻病毒E2蛋白阻断ELISA抗体检测方法的建立

Development of blocking ELISA for detecting antibodies to bovine viral diarrhea virus E2 protein

为了建立一种检测牛病毒性腹泻病毒(BVDV)E2蛋白抗体的阻断ELISA方法,以BVDV-1重组E2蛋白作为包被抗原,辣根过氧化物酶(HRP)标记的BVDV-1 E2蛋白的单克隆抗体作为检测抗体,经过一系列的条件筛选与优化,建立阻断ELISA方法,并对该检测方法的特异性、敏感性和与中和试验结果的符合率进行测定。经优化后的最佳反应条件:抗原包被质量浓度为0.25 mg/L;酶标单抗稀释度为1∶2000,37℃孵育1 h;待检血清稀释度为1∶8,37℃孵育1.5 h;37℃避光显色10 min。使用该方法对87份BVDV阴性牛血清进行检测,确定当待检血清的阻断率≤21.13%时,该血清为阴性;阻断率≥26.86%时,该血清为阳性。该方法检测BVDV-2、边界病病毒、猪瘟病毒、牛副流感病毒3型、口蹄疫病毒、布鲁菌、副结核及牛支原体阳性血清均无交叉反应;最低可检出中和效价为8的阳性血清。用该方法检测105份临床血清样品,与血清中和试验总符合率为93.33%,且阻断率与中和抗体效价呈正相关。上述结果表明,本研究建立的阻断ELISA方法特异性好、灵敏度高、操作简便,为BVDV流行病学调查及免疫效果评估提供了一种新的技术手段。

To establish a blocking ELISA method for detecting antibodies against bovine viral diarrhea virus(BVDV)E2 protein,the recombinant E2 protein of BVDV-1 was used as the coating antigen and the horseradish peroxidase labeled monoclonal antibody of BVDV-1 E2 protein(HRP-1E2B3)was used as the detection antibody.The blocking ELISA method was established by optimizing the reaction conditions,and the specificity and sensitivity of the method were examined.Clinical bovine serum samples were tested and compared with neutralization test results.The optimized reaction conditions were:0.25 mg/L of the coating antigen,1:2000 diluted HRP-1E2B3 and incubated at 37℃ for 1 h;1:8 diluted testing serum and incubated at 37℃ for 1.5 h;color development at 37℃ for 10 min away from light.The criteria were determined based on 87 BVDV negative bovine serum samples with the blocking rate greater than 26.86% as positive.No cross reaction was revealed with the positive serum of BVDV-2,BDV,CSFV,BPIV3,FMDV,Brucella,para-tuberculosis,and Mycoplasma bovis.The diluted serum with a neutralization titer of 8 could be detected as positive.A total of 105 clinical serum samples were detected by blocking ELISA and neutralization test with a coincidence rate of 93.33%,for these two methods.The blocking rate was positively correlated with the titer of neutralizing antibody.These results indicated that the blocking ELISA method established in this study had good specificity,high sensitivity,and ease of operation,which provided a new technical tool for the epidemiological investigation and immune evaluation of BVDV vaccination.