Chesla S E, Selvaraj P, Zhu C
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta 30332-0405, USA.
Biophys J. 1998 Sep;75(3):1553-72. doi: 10.1016/S0006-3495(98)74074-3.
We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.
我们报告了一种测量正向和反向动力学速率常数kf0和kr0的新方法，用于细胞黏附中锚定在相对表面上的单个受体和配体的结合。该方法不仅检测一对细胞之间的黏附；它还主要探测单个受体-配体键。其理念是量化黏附概率对接触持续时间以及受体和配体密度的依赖性。该实验是对现有微量移液器实验方案的扩展。分析基于小系统动力学概率公式的解析解。此方法被用于检测中国仓鼠卵巢细胞转染体上表达的Fcγ受体IIIA（CD16A）与包被于人红细胞上的人源或兔源免疫球蛋白G（IgG）之间的相互作用，发现其遵循单价双分子结合机制。对于CD16A - hIgG相互作用，测得的速率常数为Ackf0 = (2.6 +/- 0.32) x 10(-7) 微米4 s-1和kr0 = (0.37 +/- 0.055) s-1，对于CD16A - rIgG相互作用，测得的速率常数分别为Ackf0 = (5.7 +/- 0.31) X 10(-7) 微米4 s-1和kr0 = (0.20 +/- 0.042) s-1，其中Ac为接触面积，估计为3微米2的百分之几。
