高致病性猪繁殖与呼吸综合征病毒基因缺失标记疫苗ELISA鉴别诊断方法的建立

DEVELOPMENT OF ELISA DIFFERENTIATION DIAGNOSIS FOR A DUALMARKER VACCINE OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

为了配合(Porcine reproductive and respiratory syndrome virus,PRRSV)基因标记疫苗株(rHN4-Δ25+NP49株)的临床抗体鉴别诊断应用,本研究以标记疫苗中缺失的25个氨基酸多肽作为包被抗原,通过对ELISA反应条件的优化,确定抗原最适包被浓度为500 ng/孔,血清最佳稀释度为1:40,同时确定其阴阳性临界值S/P判定标准为0.15,批内和批间重复实验结果显示其变异系数均低于10%,表明该方法具有良好的重复性。对临床血清检测结果显示与IDEXX试剂盒检测结果的符合率为94.84%,采用25 aa负标记ELISA方法检测HuN4-F112免疫猪血清,结果显示从免疫后21 d可检测25 aa特异性抗体,该抗体至少可持续存在126 d。本研究建立的ELISA检测方法为今后PRRSV基因工程标记弱毒疫苗株在临床鉴别诊断的应用提供了有利保障。

To develop a ELISA method for detection of antibody against 25aa in Nsp2 region which was deleted from a dual-marker PRRSV vaccine strain rHN4-Δ25＋NP49. The 25aa peptide was synthesized artificially and used as antigen to coat the microplate for ELISA. The optimal coating concentration of antigen was 500 ng/well. optimal serum dilution was 1：40, and the cut off S/P value was 0.15. The reproducibility test showed that the coefficients of variation for intra-and inter-assay were lower than 10%. 25aa-ELISA method was used to test clinical samples and compared with commercial PRRSV-detection-kit （IDEXX）. The results showed that coincidence of two methods was 94.84%. Then 25aa-ELISA was used to test HuN4-F112 immunized serum samples from 1 to 126 days post immunization （dpi）. Specific antibody against 25aa was detected during 21 to 126 dpi. The vaccine immunized pigs could be differentiated from naturally infected pigs with wild type PRRSVs. Therefore, this study set up a foundation for further investigation of the differentiation diagnosis for a dual-marker vaccine rHN4-Δ25＋NP49.