| dct.abstract |
The literature part of this thesis contains the review of affinity chromatography using monolithic stationary supports in the separation and isolation of biomacromolecules, a technique known as affinity monolith chromatography (AMC). Affinity chromatography is a liquid separation technique operating on the principle of reversible binding of affinity ligands and target analytes. Experimentally, affinity chromatography involves the attachment of affinity ligands to the stationary support. By selecting appropriate ligands having high affinity and specificity towards the target, selective captures of analytes of interest are made possible, allowing their isolation from complex sample matrices. Subsequently, bound analyte species are released from the ligands by employing suitable elution solutions. In addition to the specificity, monolithic stationary phases offer a number of other benefits over conventional particulate supports, i.e., improved mass transfer characteristics, allowing convective rather than diffusional transport of analytes; and high permeability, permitting operations at high flow rates without suffering from backpressure. These benefits result in substantially reduced time requirements for isolation and separation while maintaining satisfactory separation efficiency. Different types of monolithic materials, including organic polymer-based monoliths (e.g., cryogels), inorganic monoliths (e.g., silica monoliths), and hybrid monoliths have been prepared and employed in AMC. A large range of affinity ligands, e.g., proteins, antibodies, immobilized metal ions, dye ligands, have been used with monolithic supports in different formats, and in different applications. The mentioned material-related topics, as well as recent applications of AMC, are discussed in detail in this review.
The experimental part of this thesis deals with the isolation of lipoproteins, and low-density lipoprotein (LDL) in particular, from human blood plasma using a newly developed AMC technique. LDL, a globular and major lipid carrier in blood, is diagnostically a highly relevant subclass of lipoproteins due to its involvement in the genesis of atherosclerosis. The currently most frequently employed method for lipoprotein isolation from blood plasma is ultracentrifugation. However, this method suffers from drawbacks, such as being time-consuming, requiring expensive equipment, and the possible exchange of lipids and lipoprotein subclasses during sample processing. Therefore, the first goal was to develop a faster LDL isolation protocol, capable of yielding LDL with good functionality and purity. Thus, the first section reports on the isolation of low-density lipoprotein (LDL) from human blood plasma by employing affinity monolith chromatography method using Convective Interaction Media (CIM) monolithic disk columns as stationary supports. Specifically, anti-apoB100-monoclonal antibody (mAb) was immobilized onto a CIM monolithic disk, providing a suitable capture medium for LDL through its major apolipoprotein, apolipoprotein B100 (apoB100). Other lipoprotein classes, namely very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL), also carry apoB100 and thus may be captured. To discriminate against these lipoproteins, and to obtain LDL with satisfactory purity, an additional CIM monolithic column was immobilized with a glycosaminoglycan, namely chondroitin-6-sulfate (C6S), which also binds lipoproteins, albeit with different specificity and interactions. Both of these affinity media were evaluated for LDL binding either individually or in combination. The quality of the isolated LDL was confirmed with different characterization techniques, such as size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzymatic cholesterol and triglyceride assays, and enzymatic-linked immunoassays (ELISAs) specific to apolipoprotein B100 and apolipoprotein E. The results from these multi-method characterizations confirmed the successful LDL isolation with good activity.
The second section of the thesis was devoted to quartz crystal microbalance (QCM) biosensor studies of LDL samples isolated from different individuals by different methods (affinity chromatography and conventional ultracentrifugation). A QCM sensor chip immobilized with anti-apoB100 mAb was used and challenged series of different LDL concentrations. The resulting sensorgrams were analyzed with a new numerical algorithm, namely Adaptive Interaction Distribution Algorithm (AIDA), permitting the determination of the number of analyte-receptor binding sites and the underlying kinetics. It was found that the obtained rate constant distributions, and clustering of antibody-LDL complexes were almost identical for all LDL samples, irrespective of sources or isolation techniques. For all samples, a total of five major complex clusters were identified. The major contributions of the two dominating clusters may arise from specific, yet heterogeneous LDL interactions at the antibody binding sites, while the other three clusters observed reflect most likely nonspecific low-affinity interactions from various sources, such as mass transfer effects, and the use of a non-orienting ligand immobilization chemistry. |
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