Zhang Mei-Yun, Dimitrov Dimiter S
NCI-Frederick, National Institutes of Health, Frederick, MD, USA.
Methods Mol Biol. 2009;525:175-86, xv. doi: 10.1007/978-1-59745-554-1_9.
HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against gp140 (covalently linked gp120 and the extracellular portion of gp41) has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a panel of gp41-specific human monoclonal antibodies from an HIV-1 immune library derived from long-term nonprogressors. These antibodies recognize conformational epitopes in gp41 and exhibited, to various extents, neutralization activity in assays based on spreading infection in peripheral blood mononuclear cells. The CAP methodology is generally applicable for selection of antibodies specific for any epitope that is not a dominant epitope in the antigen. It is superior to a traditional pre-depletion method in avoiding potential loss of target-specific antibodies.
HIV包膜糖蛋白（Env）由两个非共价结合的亚基组成：gp120和gp41。针对gp140（共价连接的gp120和gp41的胞外部分）进行噬菌体展示抗体文库淘选，大多筛选出了抗gp120抗体。在没有gp120的情况下，天然gp41不稳定。使用gp41片段作为抗原，只能筛选出中和活性相对较弱的抗体。为了在完整Env的背景下增强对gp41特异性抗体的筛选，我们开发了一种称为竞争性抗原淘选（CAP）的方法。利用CAP，我们从来自长期不进展者的HIV-1免疫文库中鉴定出一组gp41特异性人单克隆抗体。这些抗体识别gp41中的构象表位，并在基于外周血单个核细胞中扩散感染的试验中不同程度地表现出中和活性。CAP方法通常适用于筛选针对抗原中任何非显性表位的特异性抗体。在避免靶标特异性抗体的潜在损失方面，它优于传统的预清除方法。
