Liu Shuwen, Boyer-Chatenet Louise, Lu Hong, Jiang Shibo
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10021, USA.
J Biomol Screen. 2003 Dec;8(6):685-93. doi: 10.1177/1087057103259155.
The human immunodeficiency virus type 1 (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.
人类免疫缺陷病毒1型（HIV-1）包膜糖蛋白gp41在病毒进入过程中起重要作用。在病毒与靶细胞膜融合过程中，gp41胞外域的N端和C端七肽重复（HR）区域相互作用形成一个6螺旋束，即融合活性gp41核心。任何阻断分别源自N端和C端HR区域的N肽和C肽之间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，我们之前利用N肽N36、C肽C34和一种特异性识别gp41 6螺旋束的单克隆抗体（NC-1）建立了一种用于药物筛选的夹心酶联免疫吸附测定（ELISA）。在本研究中，通过使用异硫氰酸荧光素（FITC）偶联的C34替代C34，并直接检测荧光强度而非更复杂的酶促反应，开发了一种荧光免疫吸附测定（FLISA）。与夹心ELISA相比，这种FLISA具有相似的灵敏度和特异性，但速度更快、更经济且更方便。利用集成机器人样品处理系统，该测定已被应用于大规模高通量筛选针对gp41的HIV-1融合抑制剂的有机化合物。
