中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
5期
448-452
,共5页
董华凰%刘建礼%朱红%张桂云%孟令章%邢文革%邱茂锋%肖瑶%姚均%潘品良%蒋岩
董華凰%劉建禮%硃紅%張桂雲%孟令章%邢文革%邱茂鋒%肖瑤%姚均%潘品良%蔣巖
동화황%류건례%주홍%장계운%맹령장%형문혁%구무봉%초요%요균%반품량%장암
HIV-1核心蛋白质p24%金纳米粒子%聚合酶链反应%电泳,琼脂凝胶
HIV-1覈心蛋白質p24%金納米粒子%聚閤酶鏈反應%電泳,瓊脂凝膠
HIV-1핵심단백질p24%금납미입자%취합매련반응%전영,경지응효
HIV-1 core protein p24%Gold nanoparticles%Polymerase chain reaction%Electrophoresis,agar gel
目的 建立以金纳米探针结合终端PCR技术超灵敏检测HIV-1 p24抗原的方法.方法 采用单克隆抗体1G12和1D4建立的夹心ELISA法检测合成的HIV-1 p24抗原.挑选拟南芥基因组中一段47 bp的DNA作为条形码DNA,与5′端修饰巯基的捕获DNA(条形码DNA的互补序列)杂交形成双链DNA.在金纳米粒子表面连接1D4,并与双链DNA通过巯基连接,制成金纳米探针.微孔板上包被1G12,与待检的重组HIV-1 p24抗原及金纳米探针夹心结合.将结合的条形码DNA通过加热解离下来作为检测信号,设计并合成引物进行PCR检测及4％凝胶电泳分析,进而鉴定p24抗原的含量,然后与ELISA法灵敏度进行比较.结果 采用单克隆抗体1G12和1D4建立夹心ELISA法,检测HIV-1 p24抗原最低检测限为1000 pg/ml,采用相同抗体对建立金纳米探针法,通过PCR凝胶电泳法间接检测HIV-1 p24抗原,显示PCR产物电泳条带的强弱与所检测p24抗原含量具有良好的对应关系,最低检测限可达到1 pg/ml.结论 金纳米探针结合PCR凝胶电泳法可以对HIV-1 p24抗原进行检测,与ELISA法比较,其灵敏度可提高3个数量级.
目的 建立以金納米探針結閤終耑PCR技術超靈敏檢測HIV-1 p24抗原的方法.方法 採用單剋隆抗體1G12和1D4建立的夾心ELISA法檢測閤成的HIV-1 p24抗原.挑選擬南芥基因組中一段47 bp的DNA作為條形碼DNA,與5′耑脩飾巰基的捕穫DNA(條形碼DNA的互補序列)雜交形成雙鏈DNA.在金納米粒子錶麵連接1D4,併與雙鏈DNA通過巰基連接,製成金納米探針.微孔闆上包被1G12,與待檢的重組HIV-1 p24抗原及金納米探針夾心結閤.將結閤的條形碼DNA通過加熱解離下來作為檢測信號,設計併閤成引物進行PCR檢測及4％凝膠電泳分析,進而鑒定p24抗原的含量,然後與ELISA法靈敏度進行比較.結果 採用單剋隆抗體1G12和1D4建立夾心ELISA法,檢測HIV-1 p24抗原最低檢測限為1000 pg/ml,採用相同抗體對建立金納米探針法,通過PCR凝膠電泳法間接檢測HIV-1 p24抗原,顯示PCR產物電泳條帶的彊弱與所檢測p24抗原含量具有良好的對應關繫,最低檢測限可達到1 pg/ml.結論 金納米探針結閤PCR凝膠電泳法可以對HIV-1 p24抗原進行檢測,與ELISA法比較,其靈敏度可提高3箇數量級.
목적 건립이금납미탐침결합종단PCR기술초령민검측HIV-1 p24항원적방법.방법 채용단극륭항체1G12화1D4건립적협심ELISA법검측합성적HIV-1 p24항원.도선의남개기인조중일단47 bp적DNA작위조형마DNA,여5′단수식구기적포획DNA(조형마DNA적호보서렬)잡교형성쌍련DNA.재금납미입자표면련접1D4,병여쌍련DNA통과구기련접,제성금납미탐침.미공판상포피1G12,여대검적중조HIV-1 p24항원급금납미탐침협심결합.장결합적조형마DNA통과가열해리하래작위검측신호,설계병합성인물진행PCR검측급4％응효전영분석,진이감정p24항원적함량,연후여ELISA법령민도진행비교.결과 채용단극륭항체1G12화1D4건립협심ELISA법,검측HIV-1 p24항원최저검측한위1000 pg/ml,채용상동항체대건립금납미탐침법,통과PCR응효전영법간접검측HIV-1 p24항원,현시PCR산물전영조대적강약여소검측p24항원함량구유량호적대응관계,최저검측한가체도1 pg/ml.결론 금납미탐침결합PCR응효전영법가이대HIV-1 p24항원진행검측,여ELISA법비교,기령민도가제고3개수량급.
Objective To establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.Methods Sandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.Results Sandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.Conclusion The new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.
