TY - JOUR TI - Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor AU - Liangsupree, Thanaporn AU - Multia, Evgen AU - Forssén, Patrik AU - Fornstedt, Torgny AU - Riekkola, Marja-Liisa T2 - Biosensors and Bioelectronics AB - 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 Kd 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. DA - 2022/06/15/ PY - 2022 DO - 10.1016/j.bios.2022.114151 DP - ScienceDirect VL - 206 SP - 114151 J2 - Biosensors and Bioelectronics LA - en SN - 0956-5663 UR - https://www.sciencedirect.com/science/article/pii/S0956566322001919 Y2 - 2022/03/07/20:47:56 KW - Adaptive interaction distribution algorithm KW - Exomere KW - Exosome KW - Extracellular vesicle KW - Kinetics KW - Quartz crystal microbalance ER -