猪伪狂犬病病毒gB蛋白抗体竞争化学发光酶联免疫检测方法的建立

Establishment of Competitive Chemiluminescent Enzyme Immunoassay for Detecting Antibodies against gB Protein of Pseudorabies Virus

为了建立一种快速定量检测猪伪狂犬病病毒(PRV)gB蛋白抗体的竞争化学发光酶联免疫检测方法,以大肠杆菌表达并纯化的猪伪狂犬病病毒gB重组蛋白作为包被抗原,以辣根过氧化物酶标记的抗gB蛋白单克隆抗体作为酶标抗体,以国家参考品稀释制备的校准品绘制标准曲线实现定量检测,成功建立的PRV-gB-CLEIA在45 min内即可完成检测,可检测到最大稀释倍数为1∶2 048稀释的国家参考品;与猪瘟等其他5种病毒抗原的标准阳性血清无交叉反应;批内变异系数为1.13%~9.47%,批间变异系数为2.43%~14.07%,重复性和稳定性较好。通过对采集的180份临床血清检测结果进行比较,该方法与中和试验阳性符合率为94.00%,阴性符合率为96.92%,总符合率为96.11%,明显优于商品化ELISA试剂盒。本研究建立的PRV-gB-CLEIA检测方法可以用于PRV gB抗体的快速定量检测。

A competitive and quantitative chemiluminescent enzyme immunoassay(CLEIA) for rapidly detecting antibody against gB protein of Pseudorabies Virus(PRV) was established by using the purified recombinant gB protein expressed in E. coli as coating antigen and horseradish peroxidase labelled monoclonal antibody(MAb) against gB protein as enzyme labelled antibody, and calibrator diluted from national reference to draw standard curves for quantitative detection. The successfully established PRV-gB-CLEIA which could complete the test within 45 min had no cross-reaction with the standard positive serum of other five viral antigens such as swine fever and could detect the national reference with the maximum dilution ratio of 1:2 048. The coefficient variation of intra-assay was between 1.13% and 9.47%, and inter-assay between 2.43% and 14.07%. By comparing the detection results of 180 clinical serum samples collected, the positive coincidence rate between the method and neutralization test was 94.00%, and the negative coincidence rate was 96.92%, and the total coincidence rate was 96.11%, which was obviously superior to commercial ELISA kit. The PRV-gB-CLEIA established in this study could be used for the rapid and quantitative detection of PRV gB antibody.