| 1. |
- Forssén, Patrik, 1966-, et al.
(författare)
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Reliable Strategy for Analysis of Complex Biosensor Data
- 2018
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:8, s. 5366-5374
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Tidskriftsartikel (refereegranskat)abstract
- When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.
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| 2. |
- Liangsupree, Thanaporn, et al.
(författare)
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Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor
- 2022
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 206
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Tidskriftsartikel (refereegranskat)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 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
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| 3. |
- Liangsupree, Thanaporn, et al.
(författare)
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Rapid affinity chromatographic isolation method for LDL in human plasma by immobilized chondroitin-6-sulfate and anti-apoB-100 antibody monolithic disks in tandem
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.
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