猪瘟病毒中和抗体竞争抑制ELISA检测方法的建立

Establishment of a competitive inhibition ELISA for detection of neutralizing antibodies against classical swine fever virus

利用杆状病毒表达的猪瘟病毒(CSFV)重组E2蛋白作为包被抗原,辣根过氧化物酶标记的E2蛋白中和性单克隆抗体作为竞争抗体,建立了一种用于检测CSFV中和抗体的重组E2蛋白竞争抑制ELISA(CI-ELISA)方法。经筛选确定,CI-ELISA的抗原最佳包被浓度为3μg/mL,酶标竞争抗体的最佳稀释度为1∶4 000,阴性血清临界值为33%,阳性血清临界值为40%。CI-ELISA的检出下限为1∶44(中和抗体效价),与进口阻断ELISA试剂盒的符合率为85.00%,高于间接ELISA与进口阻断ELISA试剂盒的符合率(81.25%),CI-ELISA与其他主要病毒的阳性血清均无交叉反应。对采自我国4个省份的290份现地血清进行CI-ELISA检测,结果273份(94.1%)为CSFV抗体阳性。

Using the baculovirus-expressed recombinant E2 protein of classical swine fever virus（CSFV） as coating antigen and a anti-E2 neutralizing monoclonal antibody（McAb） 6E10 labeled with horseradish peroxidase （HRP） as competitive antibody. A competitive inhibition ELISA（CI-ELISA） for detection of neutralizing antibody against the E2 protein was developed and the reaction condition was optimized in CI EI.ISA. The optimal concentration of coating antigen in CI ELISA was 3 μg/mL,and the optimal dilution of HRP 6E10 was 1 ：4 000. The cut off value of 30% was set for the 90 known negative sera,and the cut off value of 40 % was set for the positive sera. The sensitivity of CI-ELISA was 1 ： 44 in neutralization titer. The agreement （85. 00%）of CI-ELISA with IDEXX Blocking ELISA Kit was higher than that （81.25%） of the indirect EIASA with the IDEXX Blocking EI.ISA Kit. No cross reaction was observed in CI-ELISA with anti-sera to bovine viral diarrhea virus, porcine circovirus type 2,porcine parvovirus,transmissible gastroenteritis virus,Japanese encephalitis virus, porcine rotavirus, porcine reproductive and re spiratory syndrome virus,porcine epidemic diarrhea virus,and porcine pseudorabies virus. Out of 290 porcine sera on swine farms in four provinces of China,94.1% （273/290） were positive CSFV by CI-ELISA.