猪瘟野毒感染抗体阻断ELISA检测方法的建立与初步评价

以猪瘟野毒E2蛋白为包被抗原、辣根过氧化物酶标记的猪瘟野毒单抗作为酶标抗体,建立检测猪瘟野毒抗体的阻断ELISA方法。猪瘟野毒E2最适包被浓度为0.03μg/mL,待检血清最适稀释度为1∶4,酶标猪瘟野毒单抗稀释度为1∶1 000。用建立的阻断ELISA方法检测369份临床阴性血清,计算阻断率,确定临界值,阻断率>40%为猪瘟野毒抗体阳性,阻断率≤40%为猪瘟野毒抗体阴性。用建立的ELISA方法检测84份血清,其中78份为免疫猪瘟疫苗的血清,6份为猪瘟病毒感染血清。结果显示,78份免疫血清均检测为猪瘟野毒抗体阴性,6份猪瘟感染血清均检测为猪瘟野毒抗体阳性。因此可初步判定该方法可用于鉴别诊断猪瘟病毒自然感染动物和C株疫苗免疫动物的血清抗体,并为临床检测猪瘟野毒抗体提供便捷、快速,精准的检测工具,对猪瘟的临床诊断、预防以及猪瘟净化工作具有非常重要的参考意义。

Establishment and preliminary evaluation of the blocking ELISA for detection of antibody against wild-type CSFV

In this study, a blocking ELISA method for detecting antibodies against wild classical swine fever virus(CSFV) was established using E2 as a coating protein and horseradish peroxidase-labeled swine fever virus monoclonal antibody as an enzyme-labeled antibody. The results showed that the optimal concentration of E2 was 0.03 μg/mL, the optimal dilution of the serum to be tested was 1:4, and the dilution of the enzyme-labeled CSFV Monoclonal Antibody was 1:1 000. The established blocking ELISA method was used to detect 369 clinically negative serum samples, and the blocking rate was calculated to determine the cut-off value. The blocking rate >40% indicated the wild CSFV antibody to be positive, and the blocking rate ≤40% proved the wild CSFV antibody to be negative. 84 serum samples were tested by the established ELISA method, of which 78 were immunized with the swine fever vaccine and 6 were infected with the swine fever virus. The results showed that all the 78 immunized serum samples were negative for wild CSFV antibody, and 6 infected samples were positive for wild CSFV antibody. Therefore, it might be preliminarily concluded that the present method might be used to identify the serum antibodies of animals naturally infected with swine fever virus and of those immunized with the C strain vaccine. The method was a convenient, rapid and accurate tool for the clinical detection of swine fever virus wild-type antibodies, and provided important reference for the clinical diagnosis, prevention and eradication of swine fever.