Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Thanaporn Liangsupree; Evgen Multia; Patrik Forssén; Torgny Fornstedt; Marja-Liisa Riekkola

doi:10.1016/j.bios.2022.114151

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Biosens Bioelectron. 2022 Jun 15:206:114151. doi: 10.1016/j.bios.2022.114151. Epub 2022 Mar 2.

Authors

Thanaporn Liangsupree¹, Evgen Multia¹, Patrik Forssén², Torgny Fornstedt², Marja-Liisa Riekkola³

Affiliations

¹ Department of Chemistry, P.O. Box 55, FI-00014, University of Helsinki, Finland.
² Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88, Karlstad, Sweden.
³ Department of Chemistry, P.O. Box 55, FI-00014, University of Helsinki, Finland. Electronic address: marja-liisa.riekkola@helsinki.fi.

PMID: 35259607
DOI: 10.1016/j.bios.2022.114151

Abstract

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_d 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.

Keywords: Adaptive interaction distribution algorithm; Exomere; Exosome; Extracellular vesicle; Kinetics; Quartz crystal microbalance.

MeSH terms

Biosensing Techniques*
Extracellular Vesicles* / chemistry
Intercellular Adhesion Molecule-1 / analysis
Intercellular Adhesion Molecule-1 / metabolism
Kinetics
Quartz Crystal Microbalance Techniques
Tetraspanins / analysis
Tetraspanins / metabolism

Substances

Tetraspanins
Intercellular Adhesion Molecule-1