摘要
目的建立以金纳米探针结合终端PCR技术超灵敏检测HIV-1p24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-1p24抗原。挑选拟南芥基因组中一段47bp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-1p24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1134建立夹心ELISA法，检测HIV-1p24抗原最低检测限为1000pg／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-1p24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1pg／ml。结论金纳米探针结合PCR凝胶电泳法可以对HIV-1p24抗原进行检测，与ELISA法比较，其灵敏度可提高3个数量级。
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 sulthydryl, and the Gold Nanoparticle Probe was produced. 1GI2 was precoated in the micropahes, 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 electmphoresis, 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 1194, 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.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2012年第5期448-452,共5页
Chinese Journal of Laboratory Medicine
