Measuring two-dimensional receptor-ligand binding kinetics by micropipette
Measuring two-dimensional receptor-ligand binding kinetics by micropipette
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DOI：
10.1016/s0006-3495(98)74074-3
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发表时间：
1998-09-01
影响因子：
3.4
通讯作者：
Zhu, C
中科院分区：
文献类型：
--
作者：
Chesla, SE;Selvaraj, P;Zhu, C
我们报道了一种新的方法，用于测量正向和反向动力学速率常数，k（f）（o）和k（r）（o），用于在细胞粘附中锚定到相对表面的单个受体和配体的结合。该方法不仅检查单对细胞之间的粘附，它还主要探测单个受体-配体键。我们的想法是量化的依赖性的粘附概率的接触时间和密度的受体和配体。该实验是现有微量移液管方案的扩展。分析是基于小系统动力学的概率公式的解析解。该方法被应用于检查Fc γ受体IIIA（CD 16 A）表达的中国仓鼠卵巢细胞转染和免疫球蛋白G（IgG）的人或兔源包被在人红细胞，发现遵循单价生物分子结合机制之间的相互作用。测得的速率常数为A（c）k（f）（o）=（2.6 +/-0.32）x10（-7）μ m（4）s（-1）及k（r）（o）= CD 16 A-hIgG相互作用的（0.37 +/- 0.055）s（-1）和A（c）k（f）（o）=对于CD 16 A-rIgG相互作用，分别为（5.7 +/- 0.31）x 10-7 μ m（4）s（-1）和k（r）（o）=（0.20 +/- 0.042）s（-1），其中A（c）为接触面积，估计为3 μ m（2）的百分之几。
We report a novel method for measuring forward and reverse kinetic rate constants, k(f)(o) and k(r)(o), 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 immunogobulin 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 A(c)k(f)(o) = (2.6 +/- 0.32) x 10(-7) mu m(4) s(-1) and k(r)(o) = (0.37 +/- 0.055) s(-1) for the CD16A-hIgG interaction and A(c)k(f)(o) = (5.7 +/- 0.31) x 10-7 mu m(4) s(-1) and k(r)(o) = (0.20 +/- 0.042) s(-1) for the CD16A-rIgG interaction, respectively, where A(c) is the contact area, estimated to be a few percent of 3 mu m(2).