Hinterdorfer P, Baumgartner W, Gruber H J, Schilcher K, Schindler H
Institute for Biophysics, University of Linz, Austria.
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3477-81. doi: 10.1073/pnas.93.8.3477.
A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.
通过将力显微镜与特定配体的分子识别相结合，已开发出一种用于在单事件水平上研究分子识别以及生物表面位点定位的方法。为实现这一目标，通过柔性间隔物将抗体（抗人血清白蛋白，多克隆）共价连接到力显微镜的尖端，设计了一种传感器。该传感器能够通过独特形状的力信号检测单个抗体 - 抗原识别事件，解离力为244±22 pN。分析表明，观察到的解离力源于人血清白蛋白分子中单个Fab片段的解离。发现抗体的两个Fab片段独立结合且概率相等。柔性连接为抗体提供了6纳米的动态结合范围，使得结合概率很高，在相遇时间为60毫秒时为0.5。这使得在横向扫描期间能够快速可靠地检测抗原位点，位置精度为1.5纳米。结果表明，该方法有望用于表征分子识别复合物的速率常数和动力学，以及用于生物表面（如膜）的分子图谱绘制。
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