基于量子点技术的猪伪狂犬病病毒gB抗体免疫层析试纸研究

Study on Quantum Dots-based Fluorescent Immunochromatographic Strip for gB Antibody of Porcine Pseudorabies Virus

【目的】建立一种基于量子点(quantum dots,QDs)技术的猪伪狂犬病病毒(Pseudorabies virus,PRV)gB抗体免疫层析试纸,为PRV疫苗免疫效果评估提供一种快速、简便的检测技术。【方法】选用昆虫细胞表达系统表达的gB蛋白,采用羧基修饰的水溶性QDs,在偶联剂1-乙基-3[3-二甲基氨基丙基]碳二亚胺盐酸盐(EDC)的作用下制备gB-QDs荧光标记物。将金黄色葡萄球菌蛋白A(Staphylococcus aureus protein A,SPA)和抗gB蛋白的单克隆抗体分别固定在硝酸纤维素膜上作为检测线和质控线,将样品垫、标记垫、吸水垫、支撑底板按生产工艺组装成基于QDs的荧光免疫层析试纸。检测该试纸的特异性、敏感性及与商品化ELISA试剂盒的符合率。【结果】敏感性试验结果显示,该试纸的敏感性为1∶6400。特异性试验结果显示,该试纸与猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)阳性血清均无交叉反应,特异性为100%。通过检测田间猪血清样品,对比商品化ELISA检测试剂盒,结果显示,113份田间猪血清样品中,两种方法检测结果不一致的血清有11份,结果一致的血清有102份,该试纸与商品化ELISA试剂盒的符合率为90.3%。【结论】本研究制备的试纸检测线紫外灯下显色清晰可见,辨识度高,具有较高的特异性和敏感性,可用于PRV gB抗体的检测。

【Objective】The objective of this study was to develop a Pseudorabies virus(PRV)gB antibody detection strip based on quantum dots(quantum dots,QDs)technique and provide a rapid and simple detection technique for evaluating the immune efficacy of PRV vaccines.【Method】In this study,PRV gB protein expressed by insect cell expression system and carboxyl-modified water-soluble QDs were used,gB-QDs fluorescent coupling compounds were prepared under the action of the coupling agent EDC.Staphylococcus aureus protein A(SPA)and the monoclonal antibodies of anti-gB protein were fixed on the nitrocellulose membrane as detection line and control line,respectively.Sample pads,quantum dots labeled pads,absorbent pads,and support plates were assembled into quantum dots-based fluorescent immunochromatographic strip(QDs-FICS)according to the production process.Then,the sensitivity,specificity of the QDs-FICS and the coincidence rate of it with commercial ELISA kit were detected.【Result】The sensitivity test results showed that the sensitivity of QDs-FICS was 1∶6400.The specificity of QDs-FICS showed that the QDs-FICS was highly specific to anti-PRV serum and had no cross-reaction with Classical swine fever virus(CSFV),Foot-and-mouth disease virus(FMDV),Porcine circovirus type 2(PCV2)and Porcine reproductive and respiratory syndrome virus(PRRSV)positive serum,the specificity of it was 100%.Through the detection of field pig serum samples and comparing with commercial ELISA kit,the results showed that among the 113 field pig serum samples,11 were inconsistent between the two methods,and 102 were consistent with the results,the coincidence rate of the QDs-FICS with the commercial ELISA kit was 90.3%.【Conclusion】The color of the test line of the developed strip was clearly visible with high recognition under ultraviolet lamp.The QDs-FICS had a high specificity and sensitivity,it could be used to detect the antibody of PRV gB protein.