猪瘟病毒中和性单克隆抗体制备及阻断ELISA方法建立

Selection of neutralizing mAbs and establishment of a blocking ELISA for detecting antibodies against CSFV

采用毕赤酵母表达的猪瘟病毒(CSFV)重组E2蛋白免疫BALB/c小鼠,经间接免疫荧光(IFA)筛选,获得了17株特异性单抗。经病毒中和试验(VNT)验证,上述单抗均具有CSFV中和活性,其中单克隆抗体3H9的病毒中和效价最高,大于1∶10240。采用HRP标记的CSFV单抗3H9为阻断抗体,配合酵母表达的CSFV E2蛋白作为包被抗原,建立了一种用于检测猪血清中CSFV中和抗体的阻断ELISA方法。该方法优化后的抗原包被浓度为0.5μg/mL,阻断抗体稀释度为1∶8000。通过对100份阴性猪血清的检测,确定该方法的阻断率cut-off值为30%。该方法敏感性高,对中和抗体效价低至1∶16的血清检测仍呈阳性;特异性强,仅与CSFV抗体阳性猪血清呈阳性反应,与其他猪病病毒及牛病毒性腹泻病毒(BVDV)抗体阳性猪血清均呈阴性反应;良好的可重复性,批内、批间变异系数分别为0.90%~3.21%、1.89%~6.57%,均小于10%。对188份现有猪血清检测结果显示,该方法与病毒中和试验和IDEXX猪瘟病毒抗体检测试剂盒的符合率分别为96.3%、94.7%。用该方法对南京市浦口区某养殖场猪瘟疫苗免疫猪血清进行检测,抗体阳性率为80.8%(189/234),证明了该疫苗免疫效果良好。此外,该方法为一步法,与现有方法相比,检测过程缩短至40 min。试验结果表明,利用酵母表达E2蛋白并制备中和抗体,建立了一种更加高效检测猪瘟中和抗体的阻断ELISA方法。

To obtain a highly effective neutralizing monoclonal antibodies and establish an ELISA method for detecting swine serum neutralizing antibodies against CSFV,the recombinant E2 protein of CSFV expressed by Pichia pastoris was used,in this study,to immunize mice and to screen the monoclonal antibodies.17 strains of CSFV specific monoclonal antibodies were obtained,all of which had IFA and virus neutralization activity.The antibody named 3H9 had the highest neutralization activity,of which the neutralizing valence was above than 1∶10240.The recombinant CSFV E2 protein was used as a coating antigen and HRP-3H9 as a blocking antibody to establish a blocking ELISA for detecting swine serum against CSFV.The results were that this method had an antigen coating concentration of 2μg/mL,and a blocking antibody dilution of 1∶8000.The cut-off value of the present method was 30%by the detection of 100 samples of negative swine serum against CSFV.The minimum neutralizing antibody titer detectable by this method was 1∶16,indicating that this method had a high sensitivity.The specificity test showed that only the positive serum against CSFV was detected as positive,while the sera against the other swine disease viruses and BVDV were negative,indicating a high specificity of this method.The repeatability test showed that the intra-and interbatch coefficient of variation of this method was below 10%,implying a high repeatability.Comparisons of this method with the virus neutralization test and the IDEXX CSFV Ab test ELISA kit presented coincidence rates of 96.3%and 94.7%respectively.The present method was used to detect the swine serum samples collected from pigs immunized with the attenuated CSFV vaccine in a pig farm and the resulting positive rate of antibodies against CSFV was 80.8%(189/234).Compared with the conventional blocking method,the present assay was a onestep easy and rapid method taking only 40 minutes for a whole detection process.This blocking ELISA for detection of CSFV neutralizing antibodies in swine serum was accurate,easy and fast,which would serve as a more effective means for the efficient evaluation of CSF vaccines.