猪圆环病毒2型血清中和抗体阻断ELISA检测方法的建立及应用

Development and application of a blocking ELISA for detection of serum neutralizing antibody against porcine circovirus type 2

利用PCV2-Cap蛋白中和性单克隆抗体建立了一种检测猪血清中PCV2中和抗体的阻断ELISA方法。用该方法与免疫过氧化物酶单层细胞试验对353份试验猪血清样品进行平行检测,结果两种方法的符合率为96.0%;该方法的敏感性为97.9%,特异性为92.4%,且与其他几种猪病毒参考阳性血清无交叉反应。该阻断ELISA与血清中和试验的检测结果呈显著正相关(r=0.997 0),其敏感性还优于血清中和试验。用阻断ELISA对PCV2灭活疫苗免疫与攻毒试验猪血清样品进行检测,结果显示,疫苗接种后第2周就能检测到PCV2中和抗体,4周后阳转率达100%。另外,用该方法对东北三省不同地区送检的703份血清样品进行检测,结果阳性检出率为73.0%。表明,东北三省猪场中的PCV2感染率较高,危害严重。

A blocking ELISA was developed for detecting porcine serum neutralizing antibody against porcine circovirus type 2（PCV2） based on a neutralizing monoclonal antibody（McAb） to PCV2-Cap protein. There was 96.0 % coincidence rate between the blocking ELISA and the immunoperoxidase monolayer assay based on detection of 353 swine serum samples. The sensitivity and specificity of the blocking ELISA was 97.9% and 92.4%, respectively, and there was no cross reaction with the positive sera infected with porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, transmissible gastroenteritis virus of swine,porcine epidemic diarrhea virus,classical fever virus, or porcine reproductive and respiratory syndrome virus, respectively. The detection results of neutralizing antibody against PCV2 by the blocking ELISA was significant positive correlation（r=0. 997 0） with that by the serum neutralization test（SNT）, and the sensitivity of the blocking ELISA was higher than that of SNT. Neutralizing antibodies could be detected from the sera of the swine vaccinated with PCV2 inactivated vaccine after two weeks,all sera was positive after four weeks. 703 serum samples collected from Heilongjiang, Jilin and Liaoning provinces showed 73.0% positive rates,indicating that PCV2 infection rate in the swine herds in the three provinces was high and the damage caused by PCV2 was serious.