Liangsupree Thanaporn, Multia Evgen, Forssén Patrik, Fornstedt Torgny, Riekkola Marja-Liisa
Department of Chemistry, P.O. Box 55, FI-00014, University of Helsinki, Finland.
Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88, Karlstad, Sweden.
Biosens Bioelectron. 2022 Jun 15;206:114151. doi: 10.1016/j.bios.2022.114151. Epub 2022 Mar 2.
Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9, CD63, and CD81 EV subpopulations of <50 nm exomeres and 50-80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants K ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63 EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV - ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.
连续流动石英晶体微天平（QCM）被用于研究细胞外囊泡亚群（外泌体样小泡和外泌体大小的细胞外囊泡）与四种亲和配体之间的结合动力学：抗四跨膜蛋白单克隆抗体（抗CD9、抗CD63和抗CD81）以及重组细胞间黏附分子-1（ICAM-1）或CD54蛋白。使用我们最近开发的在线耦合免疫亲和色谱-不对称流场流分级系统，分离并分级得到了高纯度的小于50nm外泌体样小泡和50-80nm外泌体的CD9、CD63和CD81细胞外囊泡亚群。专门为分析复杂生物相互作用设计的自适应相互作用分布算法（AIDA），采用四步程序可靠估计速率常数分布的异质性程度。外泌体样小泡大小的细胞外囊泡与抗四跨膜蛋白抗体之间的相互作用显示出两个具有相当结合动力学的相互作用位点，估计解离常数K范围从纳摩尔到飞摩尔。与外泌体相比，外泌体样小泡表现出略高的亲和力。CD63细胞外囊泡与抗四跨膜蛋白抗体的亲和力最高。还研究了参与细胞外囊泡细胞内化的细胞外囊泡亚群与ICAM-1的相互作用。细胞外囊泡-ICAM-1相互作用也具有高亲和力（纳摩尔到皮摩尔范围），与抗四跨膜蛋白抗体和细胞外囊泡的相互作用相比，总体亲和力较低。我们的研究结果证明，QCM是一种在样品浓度有限的情况下进行动力学研究的有价值的无标记工具，并且先进的算法，如AIDA，对于正确确定动力学异质性至关重要。据我们所知，这是首次关于血浆来源的细胞外囊泡亚群与抗四跨膜蛋白抗体和ICAM-1之间相互作用的动力学研究。
