Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41
Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41
复制标题
DOI：
10.1177/1087057103259155
复制
发表时间：
2003-12-01
影响因子：
--
通讯作者：
Jiang, SB
中科院分区：
文献类型：
--
作者：
Liu, SW;Boyer-Chatenet, L;Jiang, SB
人类免疫缺陷病毒I型(HIV-1)囊膜糖蛋白gp41在病毒侵入过程中起着重要作用。在病毒与靶细胞膜的融合过程中，gp41胞外区的N-端和C-端七肽重复序列(HR)结合形成一个6-螺旋束，与融合活性gp41核心相对应。任何阻止分别来自N-末端和C-末端HR区的N-肽和C-肽之间的gp41 6-螺旋束形成的化合物，都可能抑制HIV-1介导的膜融合。基于这一原理，我们以前建立了用N-肽N36和C-肽C34和特异性识别gp41 6-螺旋束的单抗(NC-1)进行药物筛选的夹心酶联免疫吸附试验(ELISA)。用异硫氰酸荧光素(FITC)标记的C34代替C34，直接检测荧光强度，从而建立了一种直接检测荧光强度的荧光免疫吸附分析方法。与夹心酶联免疫吸附试验相比，该方法具有相似的敏感性和特异性，但更为快速、经济和方便。使用集成的机器人样本处理系统，该方法已被应用于针对gp41的HIV-1融合抑制剂的大规模高通量有机化合物筛选。
The human immunodeficiency virus type I (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.