Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

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. IMAGES:

本研究开发了一种将力显微镜（force microscopy）与特异性配体（specific ligands）介导的分子识别技术相结合的方法，可用于单事件水平的分子识别研究以及生物表面（biosurfaces）位点定位。为达成该研究目标，研究人员通过柔性间隔基（flexible spacer）将多克隆抗人血清白蛋白（anti-human serum albumin）抗体共价连接至力显微镜探针（force microscope tip），制备得到一款传感探针。该探针可通过形态独特的力信号检测单条抗体-抗原识别事件，其解离力（unbinding force）为244±22皮牛（pN）。分析结果显示，观测到的解离力源自单条抗原结合片段（Fab fragments）与人血清白蛋白分子的解离。研究发现，该抗体的两条Fab片段可独立结合且结合概率均等。柔性连接结构赋予抗体6纳米的动态结合范围（dynamical reach），使其在60毫秒的相遇时长下结合概率可达0.5。这使得该探针可在横向扫描过程中快速且可靠地检测抗原位点，定位精度达1.5纳米。本研究表明，该方法在表征分子识别复合物的速率常数与动力学特性，以及开展膜等生物表面的分子测绘方面具有应用前景。图像：