猪伪狂犬病病毒gE抗原时间分辨荧光免疫层析检测方法的建立

Establishment of time-resolved fluorescence immunochromatographic assay for detection of pseudorabies virus gE antigen

为建立一种简便、快速、特异、可定量检测样品中猪伪狂犬病病毒(PRV)gE抗原的时间分辨荧光微球免疫层析检测方法,采用单克隆抗体技术和荧光微球免疫层析技术,以羧基荧光微球和NC膜为载体将单克隆抗体进行标记和包被,制备PRV gE抗原检测试纸卡。优化了标记抗体、包被抗体的工艺,并通过试纸卡线性范围、最低检出限、特异性等性能指标对其进行评价。最终确定20μL荧光微球的标记抗体量为20μg,检测线包被抗体质量浓度为2 g/L时,检测时间为15 min,线性范围为1∶6.25~1∶200.00倍稀释的TCID50为1×10^8.6/0.1 mL的PRV抗原,最低检出限为1∶800倍稀释的TCID50为1×10^8.6/0.1 mL的PRV抗原,精密性小于10%,与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪流行性腹泻病毒(PEDV)及PRV Batha-K61株、PRV-HB2000株等均无交叉反应,室温干燥条件下至少保存10个月。该试纸卡与PRV gE与gD二重实时荧光PCR平行检测134份临床样本,两者总符合率为89.55%。初步建立了定量检测样品中PRV gE抗原时间分辨荧光免疫层析检测方法,有良好的临床应用价值。

To establish a simple,rapid,specific time-resolved fluorescence immunochromatographic assay for quantitative determination of pseudorabies virus(PRV)gE antigen in samples.We prepared test card by integrating monoclonal antibody technology and fluorescence immunochromatography technique.Carboxy fluorescent microspheres and nitrocellulose membrane were used as carriers for labeling and coating monoclonal antibody.We optimized the process by adjusting the amount of labeling and coating antibody.According to the linear range,the lowest detection limit,specific and other performance indicators,we evaluated the immunochromatographic assay for pseudorabies virus gE antigen.When the amount of labeled antibody was 20μg/20μL fluorescent microspheres,and the concentration of coated antibody on the test line was 2 g/L,the optimal reaction time was 15 minutes.Assay linear range was a 1∶6.25 to 1∶200.00-fold dilution TCID50 of 1×10^8.6/0.1 mL of PRV antigen,and the minimum detection limit was a 1∶800-fold dilution TCID50 of 1×10^8.6/0.1 mL of PRV antigen.The coefficient of variation were less than 10%.The test card had a nice specificity,no cross-reactivity to CSFV,PRRSV,PCV2,PPV,PEDV,PRV Batha-K61 strain and PRV-HB2000 strain,and could stored for at least 10 months at room temperature under the sealed condition for drying.By detecting 134 clinical samples in parallel with simultaneous detection of porcine pseudorabies virus gE and gD by duplex real-time PCR kit,the total compliance rate of both was 89.55%.The test card,initially established a fluorescence immunochromatographic detection method for quantitative detection of pseudorabies virus gE antigen in samples,has a good clinical application value.