牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。

Preparation of monoclonal antibodies against bovine infectious rhinotracheitis virus and development of a blocking ELISA

To establish a blocking ELISA assay for detecting bovine serum antibody against the infectious bovine rhinotracheitis virus(IBRV),the sucrose density gradient untracentrifugation purified IBRV immunogen was used for immunization.A monoclonal antibody(MAb)was obtained and designated as cp-1-1,which showed good reactivity with IBRV in indirect ELISA,indirect immunofluorescence assay(IFA)and western blot.IFA showed that the MAb cp-1-1 only reacted positively with IBRV,and did not react with bovine viral diarrhea virus(BVDV)or bovine parainfluenza virus type 3(BPIV3),indicating its good specificity.Mass spectrometry revealed that the MAb cp-1-1 recognized epitope locating on the VP8 protein of IBRV.Subsequently,the purified IBRV was used as a coating antigen,and the MAb cp-1-1 was used as a detection antibody to establish a blocking ELISA for detecting serum antibodies against IBRV.The diagnostic method has an antigen coating amount of 0.89μg/well,a sample dilution of1:2,a detection MAb amount of 1.3μg/well,and a secondary antibody dilution of 1:5000.Using 50 bovine serum samples with weak virus neutralization antibody titers to IBRV(virus neutralizing antibody titer 1:4-1:16 to IBRV)as the standard reference sera,the percent inhibition 52.06%was determined as the cut-off value of the method.When the percent inhibition was higher than 52.06%,the sample was considered positive.Otherwise,the sample was considered as negative.Specificity test showed that only the serum against IBRV was positive,while the sera against BVDV,BPIV3,bovine adenovirus type 3(BADV-3)and type O FMDV showed negative results,indicating that the method had good specificity.The minimum neutralizing antibody titer detectable by this method is 1:4,which is consistent with the sensitivity of the virus neutralization test,indicating that the method had good sensitivity.Repeatability tests showed that the intra-and inter-assay coefficients of variation of the method were less than 10%,showing good repeatability.The coincidence rate of this method with the virus neutralization was 98.46%.This method was used to monitor the sera collected from cattle immunized with IBRV inactivated vaccine in a cattle farm and the positive rate of antibody against IBRV was 99.51%(205/206).Monitoring of 801 bovine sera collected from 8 provinces(including municipalities and autonomous regions),IBRV-positive rate was 41.6%(333/801).The blocking ELISA developed in this study can be used for IBRV vaccine immunological surveillance and sero-epidemiological investigation provides technical support for prevention and control of infectious bovine rhinotracheitis(IBR)in China..