免疫PCR检测HIV-1 p24的实验研究

Laboratory study of HIV-1 p24 by immuno-PCR

目的以HIV-1 p24为检测对象,建立以金磁微粒为固相载体的免疫PCR检测技术。方法以鼠抗HIV-1 p24单克隆抗体作为捕获抗体,包被金磁微粒,以生物素化的羊抗p24多克隆抗体作为检测抗体。报告DNA为甘蓝型油菜肉桂酰辅酶A还原酶基因的一个片段,用生物素标记的引物通过PCR扩增预先制备,通过链亲和素的桥联标记生物素化的多抗,被两抗体夹心捕获的人重组HIV-1 p24抗原通过PCR扩增报告DNA的方式予以检测。并且优化了免疫PCR的检测条件,对检测灵敏度进行了分析。结果为降低非特异性扩增,链亲和素和用于标记的DNA的浓度分别控制在0.1mg/L和10ng/L。免疫PCR检测的灵敏度为0.1ng/L,比ELISA法检测的灵敏度高1.5×104倍。结论金磁微粒为载体的免疫PCR检测HIV-1 p24抗原是一种灵敏度较高的检测方法。

Objective To establish an immuno-PCR detection method with the carrier of golden-magnetic particles for laboratory detection of HIV-1 p24 antigen. Methods Using mouse anti-p24 monoclonal antibody as the capture antibody coating golden-magnetic particles,using biotinylated goat anti-p24 polyclonal antibody as the detection antibody. The reporter DNA is a fragment of cinnamoyl coenzyme A reucing enzyme gene of Brassica napus L, and it was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated polyclonal antibody. Human recombinant p24 antigen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. And then the detection conditions were optimized and the sensitivity was anlyzed. Results To reduce the effect of nonspecific amplification, the optimal concentration of streptavidin and DNA label were determined to be 0. lmg/ L and 10ng/ L,respectively. The detection limit of the immuno-PCR assay was 0. 1ng/ L, an approximately 1.59× 10^4-fold higher compared with an enzyme-linked immunosorbent assay. Conclusion The immuno-PCR for detection HIV-1 p24 antigen is indicated to be a high sensitive detection method.