Winters-Hilt Stephen, Morales Eric, Amin Iftekhar, Stoyanov Alexander
The Research Institute for Children, 200 Henry Clay Ave, New Orleans, LA 70118, USA.
BMC Bioinformatics. 2007 Nov 1;8 Suppl 7(Suppl 7):S20. doi: 10.1186/1471-2105-8-S7-S20.
The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.
We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed.
Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.
联合国/国际理论物理中心纳米孔探测器为研究单个抗体的结合及构象变化提供了一种新方法。许多关于抗体功能的关键问题仍未解决，而纳米孔探测器能够以全新方式解决这些问题。
我们提供的证据表明，不同形式的通道阻断可能与同一种抗体相关，我们将这些不同的阻断与抗体在探测器纳米级通道中“捕获”的不同方向联系起来。我们通过通道电流的降低直接检测抗体的存在。添加抗原后阻断模式的变化表明可间接检测抗体/抗原结合。同样，对DNA发夹锚定抗体进行了研究，其中DNA连接在抗体Fc区域底部的羧基末端，与游离（未结合）IgG抗体相比，（冗长的）捕获阻断类型明显更少。还观察到了离液剂的引入及其对蛋白质 - 蛋白质相互作用的影响。
基于纳米孔的方法最终可能对蛋白质之间结合时发生的复杂构象“协商”进行直接分析。
