Nyambi P N, Burda S, Bastiani L, Williams C
Department of Pathology, New York University School of Medicine, 10016, New York, NY, USA.
J Immunol Methods. 2001 Jul 1;253(1-2):253-62. doi: 10.1016/s0022-1759(01)00384-2.
This protocol describes a simple assay that can be used to study the nature of exposure of antigenic epitopes and antigenic relatedness of different intact, native HIV-1 strains. The assay is based on the principle that mAbs coated on microtiter wells bind to epitopes on the surface of intact, native virions. The bound virion is then lysed to release p24, which is then quantitated (pg/ml) to give a measure of the amount of virion bound to the mAb. High p24 levels released after lysis correlate with high level capture of virions by mAbs, and as such, reflect good exposure of the epitope on the virion. Likewise, binding patterns of a specific mAb with different virus strains reveal information on their antigenic relatedness. In establishing this assay, the nature of exposure of antigenic epitopes and the antigenic relatedness of six intact, native HIV-1 virions of clades A, B, C, D, F and G were examined using anti-HIV-1 mAbs directed at epitopes in the V2, V3, CD4bd and C5 of gp120, and in clusters I and II of the gp41 region. Analysis of the binding data shows that mAbs directed at epitopes in the V3, C5 and gp41 Cluster I region bound best to the viruses examined, suggesting that these are the regions most exposed and conserved on intact, native HIV-1 virions of different clades. Epitopes in the V2 and CD4bd of gp120, and in gp41 cluster II, are not exposed on intact, native virions.
本方案描述了一种简单的检测方法，可用于研究抗原表位的暴露性质以及不同完整天然HIV-1毒株的抗原相关性。该检测方法基于以下原理：包被在微量滴定孔中的单克隆抗体（mAb）与完整天然病毒粒子表面的表位结合。然后裂解结合的病毒粒子以释放p24，接着对p24进行定量（pg/ml），以衡量与mAb结合的病毒粒子数量。裂解后释放的高p24水平与mAb对病毒粒子的高捕获水平相关，因此反映了病毒粒子上表位的良好暴露。同样，特定mAb与不同病毒毒株的结合模式揭示了它们的抗原相关性信息。在建立该检测方法时，使用针对gp120的V2、V3、CD4结合域（CD4bd）和C5以及gp41区域的簇I和簇II中表位的抗HIV-1 mAb，检测了A、B、C、D、F和G六个分支的完整天然HIV-1病毒粒子的抗原表位暴露性质和抗原相关性。结合数据的分析表明，针对V3、C5和gp41簇I区域中表位的mAb与所检测的病毒结合最佳，这表明这些是不同分支的完整天然HIV-1病毒粒子上最暴露和保守的区域。gp120的V2和CD4bd以及gp41簇II中的表位在完整天然病毒粒子上未暴露。
