摘要
[目的]为建立一种新型、高灵敏度,可直观与定量分析的快速检测方法,本研究创新使用具有氧化还原酶活性的合金纳米材料,将其作为标记物制备免疫层析试纸。待反应结束,使用TMB显色剂对免疫反应进行级联放大,可以显著提高检测的灵敏度,不仅可直观判断,也可精确定量分析,这为免疫层析技术标志物筛选与敏感性提高开拓了新的思路与方法。[方法]采用超声水浴法,通过抗坏血酸(AA)还原K2Ptcl4和Na2Pdcl4成功出制备Pt-Pd合金纳米颗粒,将其用于标记口蹄疫A型纯化抗原FMDV-146S,作为捕获抗原纳米标记物,再将纯化的A型表达抗原及其抗体(FMDV-146S-Ab)包被在硝酸纤维素膜(NC)上,分别作为检测线(T线)和质控线(C线),经条件优化,建立口蹄疫A型病毒抗体Pt-Pd合金纳米颗粒试纸条检测方法。对该免疫层析检测技术进行方法学考核,检测结果定量分析并建立标准曲线,同时与LPB-ELISA进行结果比对。[结果]该试纸条可在10 min内完成定性和定量检测,定量检测灵敏度为1:28/test,线性范围1:26/test—1:29/test;检测A型FMDV阳性血清,敏感性为98%,检测FMDV阴性血清、O型和Asia 1型的FMDV阳性血清以及其他动物常见病毒病阳性血清,特异性为94.8%;该试纸条重复检测效果良好;与OIE推荐LPB-ELISA法比较,相关性好,相关系数为0.9112。[结论]本研究开拓采用Pt-Pd合金纳米颗粒为标记物建立免疫层析方法,并首次应用于口蹄疫病毒抗体检测,结果证实其具备较高灵敏度,相比胶体金灵敏度可提高24倍,同时具备可操作性与重现性。该研究是快速免疫层析技术的新尝试,为合金类纳米材料应用提供新的思路,未来具有实践应用前景。
【Objective】A novel immunochromatographic test strip with peroxidase-like activity of Pt-Pd bimetal nanoparticles(NPs)as a marker(shortened as NPs test strip)was developed.The signal amplification was based on Pt-Pd bimetal NPs possessing high peroxidase-like activity toward 3,3,5,5’-tetramethylbenzidine,which could produce characteristic colored bands.It could not only improve the sensitivity of detection,but also completed a quantitative analysis of results with reader.This method provided a rapid and effective screening means for monitoring.【Method】Pt-Pd bimetal NPs were prepared by K2 Ptcl4 and Na2 Pdcl4,which were reduced by ascorbic acid(AA).The mixture was continuously sonicated in water bath.The purified antigen of FMDV serotype A was coupled with Pt-Pd bimetal NPs.The purified 146 S particles of FMDV serotype A(FMDV-146 S)and the antibodies against FMDV-146 S(FMDV-146 S-Ab)were blotted on nitrocellulose membrane as test line and control line,respectively.The sensitivity,specificity and stability of this NPs test strip were assessed by testing known positive and negative FMDV serum and positive serum against other animals’disease.Parallel tests of the positive FMDV serum by LPB-ELISA were performed.【Result】This established method could be accomplished qualitatively as well as semi-quantitative detection in 10 minutes,the sensitivity was up to 1:28/test,and the linear range was from 1:26/test-1:29/test.The detection sensitivity for the serum positive for FMDV type A was 98%,and the detection sensitivity for FMDV-negative serum,serum positive for FMDV type O and type Asia 1 as well as sera positive for other common viral diseases in animals was 94.8%.The differences between batches were small.Test serum samples showed that the strip results were coincident with LPB-ELISA,R2=0.9112.【Conclusion】An immunochromatographic assay based on Pt-Pd bimetal nanoparticles was established innovatively and applied to the detection of FMDV antibodies.The results demonstrated its high sensitivity,which was a 24-fold increase as compared with the colloidal gold.In addition,this novel assay also displayed excellent maneuverability and reproducibility.The established method represented a new attempt in fast immunochromatographic assay and probably predicted an alternative and highly promising application of the alloy nanoparticles.
作者
孙燕燕
李昕
林密
李峰松
包艳芳
陈夏辉
杨光
曾巧英
蒋韬
SUN YanYan;LI Xin;LIN Mi;LI FengSong;BAO YanFang;CHEN XiaHui;YANG Guang;ZENG QiaoYing;JIANG Tao(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences/Key Laboratory of Veterinary Etiological Biology/National Foot-and-Mouth Disease Reference Laboratory,Lanzhou 730046)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第3期653-661,共9页
Scientia Agricultura Sinica
基金
国家重点研发计划(2016YFD0500702-2)。
