Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules
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Multia, Evgen. "Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules." Doctoral School in Natural Sciences Dissertation Series (2021).
This doctoral thesis describes the development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules. The focus of the study was on subpopulations of lipoproteins and extracellular vesicles (EVs) that are important in the detection of different diseases, such as atherosclerotic cardiovascular diseases and cancer, and may even possess therapeutic potential. In the thesis, immunoaffinity chromatography (IAC) with selective antibodies immobilized on the monolithic disk columns were utilized for the selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) was able to fractionate relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Continuous flow quartz crystal microbalance (QCM) and partial filling affinity capillary electrophoresis (PF-ACE) were employed to study the affinity of the interactions between the antibody and lipoproteins.
The first step was to develop a method to study interactions between antibody and lipoproteins to select a high affinity antibody useful for the isolation of lipoprotein subpopulations by IAC. The interaction data obtained with PF-ACE was analyzed to determine the heterogeneity of the interactions with adsorption energy distribution calculations, while the QCM data was processed with interaction maps. The affinity constants obtained with QCM and PF-ACE agreed well with each other.
Next, the IAC methods were developed to capture EVs of different cellular origins from human plasma using anti-CD9 monoclonal antibody (mAb), while anti-CD61 mAb was exploited to capture platelet-derived EVs. The anti-apolipoprotein B-100 (anti-apoB-100) mAb was exploited to immunocapture apoB-100 containing lipoproteins. The anti-apoB-100 mAb was also characterized by the PF-ACE and QCM studies. Appropriate elution conditions were found for the IAC methods, which has often been an issue with magnetic beads-based immunoaffinity methods.
Since IAC allowed selective isolation of EVs and lipoproteins, a size-based separation to their subpopulations with AsFlFFF was introduced as a successive step. This enabled additional characterization of subpopulations by nanoparticle tracking analysis, western blotting, electron microscopy, capillary electrophoresis coupled with laser-induced fluorescent detection, zeta potential measurements, as well as free amino acids and glucose analysis with hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Finally, IAC was successfully on-line coupled to AsFlFFF, resulting in quick and automated isolation and fractionation of the subpopulations of EVs and lipoproteins. The constructed IAC-AsFlFFF system was able to process reliably 18–38 samples in 24 h with only minor operator involvement, resulting in highly reproducible and gentle fractionation of EV subpopulations in the size range of exomeres and exosomes.
Polymeric monolithic disk columns were utilized for the first time for the IAC-based isolation of EVs and their subpopulations from human plasma, and for the detection of exomeres in CD9+ EVs and CD61+ platelet-derived EVs from human plasma samples. The results demonstrated that CD61+ EVs are potentially taking part in gluconeogenesis based on free amino acids and glucose present as cargo.View full article