Cheung Jane W C, Walker Gilbert C
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
Langmuir. 2008 Dec 16;24(24):13842-9. doi: 10.1021/la802452v.
The fibronectin (Fn) binding conformation on mica and ultraflat poly(D,L-lactide-co-glycolide) (UPLGA) was characterized using atomic force microscopy (AFM). AFM topographic images showed that Fn was in an extended form on mica and in a compact structure on UPLGA. With immuno-AFM, an antibody (Ab(hep)) was used to characterize the Fn binding conformation. When Fn opens its binding site for an antibody upon adsorption to a surface, the resulting Fn-antibody complex creates an additional peak in the sample's height distribution. Immuno-AFM uses this change to detect antigen-antibody binding. In this letter, height histograms (distributions) were generated using the mean true height of molecules, which was measured by examining the histogram for each individual molecule and subtracting the mica background. Mean true height values were obtained from the histograms and showed that Fn and Ab(hep) formed complexes on mica, signifying that one of the heparin binding sites on Fn was open when Fn was adsorbed to mica. The mean true height of the Fn-antibody complex from the histogram is greater than expected, suggesting that the antibody had pulled the extended "arms" of Fn together and caused an Fn conformation change upon binding. The height histograms can illustrate the Fn binding conformation and other antigen-antibody binding.
使用原子力显微镜（AFM）对纤连蛋白（Fn）在云母和超平聚（D，L-丙交酯-共-乙交酯）（UPLGA）上的结合构象进行了表征。AFM形貌图像显示，Fn在云母上呈伸展形式，而在UPLGA上呈紧密结构。利用免疫AFM，使用一种抗体（Ab(hep)）来表征Fn的结合构象。当Fn吸附到表面时打开其抗体结合位点，所形成的Fn-抗体复合物会在样品的高度分布中产生一个额外的峰。免疫AFM利用这种变化来检测抗原-抗体结合。在本信函中，通过检查每个单独分子的直方图并减去云母背景来测量分子的平均真实高度，从而生成高度直方图（分布）。从直方图中获得的平均真实高度值表明，Fn和Ab(hep)在云母上形成了复合物，这意味着当Fn吸附到云母上时，Fn上的一个肝素结合位点是开放的。直方图中Fn-抗体复合物的平均真实高度大于预期，表明抗体将Fn的伸展“臂”拉到了一起，并在结合时导致了Fn构象的变化。高度直方图可以说明Fn的结合构象以及其他抗原-抗体结合情况。
