...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Mapping epitopes and antigenicity by site-directed masking
【24h】

Mapping epitopes and antigenicity by site-directed masking

机译：通过定点掩蔽定位表位和抗原性

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (beta-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to beta-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with beta-lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops.
机译：在这里，我们描述了一种将抗体结合定位到折叠抗原表面的方法。我们首先创建了一组突变抗原（β-内酰胺酶），其中单暴露于表面的残基突变为半胱氨酸。然后，我们将半胱氨酸残基化学束缚在固相上，从而掩盖了以每个半胱氨酸残基为中心的表面斑，并阻止了抗体与该表面区域的结合。通过这些手段，我们绘制了一些针对β-内酰胺酶的单克隆抗体的表位。此外，通过将多克隆抗血清样品去除到被掩盖的抗原上，并测量每个被去除的抗血清样品到未掩盖的抗原上的结合，我们绘制了23个不同表位的抗原性。在弗氏佐剂中用β-内酰胺酶免疫小鼠和兔子后，我们发现抗血清与天然和变性抗原都发生了反应，并且抗体反应主要针对天然抗原的裸露灵活环区域。相比之下，在PBS中免疫后，我们发现抗血清与变性抗原的反应很弱，并且抗体反应在抗原表面的分布更均匀。我们建议变性的抗原（在弗氏佐剂的乳化过程中产生）引发的抗体主要结合天然蛋白的柔性区域，这解释了抗原性和骨架柔性之间的相关性。因此，在疫苗接种或自然感染期间抗原的变性将有望使抗体反应集中于柔性环。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号