摘要
目的建立以金磁微粒为载体的免疫PCR HIV-1 p24检测体系。方法以金磁微粒作为免疫PCR的载体并通过载体的特异性和非特异性反应验证其可行性;以小鼠抗HIV-1 p24单克隆抗体作为捕获抗体包被金磁微粒,生物素化的羊抗p24多克隆抗体作为检测抗体;报告DNA用生物素标记的引物通过PCR扩增预先制备,并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;并对检测的灵敏度和线性检测范围进行分析。结果金磁微粒上偶联抗体的效率可〉95%,偶联抗体的一致性较好,几乎不产生非特异性吸附,分离与洗涤步骤更简便更彻底;免疫PCR检测p24的灵敏度为0.1ng/L,比ELISA法检测的灵敏度高1.5×10^4倍,线性检测范围为p24浓度在0.1～100ng/L。结论金磁微粒是较理想的免疫PCR反应载体,金磁微粒为载体的免疫PCR检测HIV-1 p24是一种可兹应用的高灵敏度的检测方法。
OBJECTIVE To establishan immuno-PCR assay with the carriers of gold-magnetic particles for detection of HIV-1 p24. METHODS The feasibility of using gold-magnetic particles as the carriers was verified. The gold- magnetic particles were coated with mouse anti-p24 monoclonal antibody as the capture antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound through streptavidin to biotinylated polyclonal antibody as the detection antibody. HIV-1 p24 sandwiched by two antibodies was detected by amplifying the reporter DNA using PCR. RESULTS The efficiency of gold-magnetic particles coated with mouse anti-p24 monoclonal antibody could reach up to 95%. Furthermore, the amount of antibodies immobilization was consistent among different batches of gold-magnetic particles and there was nearly without nonspecific adsorption. The detection limit of immuno-PCR assay was 0.1 ng/L, an approximately 1..5 ×10^4-fold higher compared with an enzyme-linked immunosorbent assay. The linear range of p24 concentration was 0. 1-100 ng/L. CONCLUSIONS Gold-magnetic particle is one of the ideal immuno-PCR reaction carriers. The immuno-PCR for detection of HIV-1 p24 reported in this article is indicated to be a promising detection method.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2008年第3期301-304,355,共5页
Chinese Journal of Nosocomiology
基金
第三军医大学科研基金(XG20040125)
