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Biosensors Jul 2019Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology... (Review)
Review
Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology for lateral flow (immunochromatographic) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qualitative conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quantitative information about the content of a target compound in a sample mixture. This review describes the main options for detecting, processing, and interpreting immunochromatographic analysis results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examined.
Topics: Biosensing Techniques; Chromatography, Affinity; Data Analysis; Humans; Image Processing, Computer-Assisted; Light; Magnetics; Photometry; Point-of-Care Systems; Point-of-Care Testing; Reagent Strips; Software
PubMed: 31319629
DOI: 10.3390/bios9030089 -
Journal of Pharmaceutical and... Sep 2015The rates at which biological interactions occur can provide important information concerning the mechanism and behavior of these processes in living systems. This... (Review)
Review
The rates at which biological interactions occur can provide important information concerning the mechanism and behavior of these processes in living systems. This review discusses several analytical methods that can be used to examine the kinetics of biological interactions. These techniques include common or traditional methods such as stopped-flow analysis and surface plasmon resonance spectroscopy, as well as alternative methods based on affinity chromatography and capillary electrophoresis. The general principles and theory behind these approaches are examined, and it is shown how each technique can be utilized to provide information on the kinetics of biological interactions. Examples of applications are also given for each method. In addition, a discussion is provided on the relative advantages or potential limitations of each technique regarding its use in kinetic studies.
Topics: Animals; Biological Products; Chromatography, Affinity; Electrophoresis, Capillary; Humans; Kinetics; Protein Binding; Surface Plasmon Resonance
PubMed: 25700721
DOI: 10.1016/j.jpba.2015.01.042 -
Molecules (Basel, Switzerland) Oct 2023The identification of weak-affinity ligands targeting membrane proteins is of great interest in Fragment-Based Drug Design (FBDD). Recently, miniaturized weak affinity...
The identification of weak-affinity ligands targeting membrane proteins is of great interest in Fragment-Based Drug Design (FBDD). Recently, miniaturized weak affinity chromatography (WAC) has been proposed as a valuable tool to study interactions between small ligands and wild-type membrane proteins embedded in so-called nanodisc biomimetic membranes immobilized on GMA-co-EDMA monoliths in situ-synthesized in capillary columns (less than one microliter in volume). In this proof-of-concept study, the achievable affinity range was limited to medium affinity (low micromolar range). The present work investigates different strategies to extend the affinity range towards low affinities, either by increasing the density of membrane proteins on the chromatographic support or by reducing non-specific interactions with the monolith. The combination of the use of a new and more hydrophilic monolithic support (poly(DHPMA-co-MBA)) and a multilayer nanodisc grafting process (up to three layers) allows a significant increase in the membrane protein density by a more than three-fold factor (up to 5.4 pmol cm). Such an increase in protein density associated with reduced non-specific interactions makes it possible to extend the range of detectable affinity, as demonstrated by the identification and characterization of affinities of very low-affinity ligands (Kd values of several hundred micromolar) for the adenosine receptor AAR used as a model protein, which was not possible before. The affinity was confirmed by competition experiments.
Topics: Membrane Proteins; Chromatography, Affinity; Methylmethacrylates; Drug Design; Ligands
PubMed: 37894592
DOI: 10.3390/molecules28207113 -
Analytical Chemistry Nov 2023Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process....
Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography-mass spectrometry (LC-MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.
Topics: Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, Liquid; Mass Spectrometry; Recombinant Proteins
PubMed: 37883730
DOI: 10.1021/acs.analchem.3c02180 -
Methods in Enzymology 2021Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium...
Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is routinely used to screen a variety of overexpression conditions (e.g., host, medium, inducer concentration, post-induction temperature and/or incubation time) and to assess the purity of the final product, its limitations, including aberrant protein migration due to compositional eccentricities or incomplete denaturation, often preclude firm conclusions regarding the extent of overexpression and/or purification. Therefore, we recently reported an automated liquid chromatography-mass spectrometry-based strategy that couples immobilized metal affinity chromatography (IMAC) with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS) to directly analyze cell lysates for the presence of target proteins. IMAC-OBE-nMS can be used to assess whether target proteins (1) are overexpressed in soluble form, (2) bind and elute from an IMAC resin, (3) oligomerize, and (4) have the expected mass. Here, we use four poly-His-tagged proteins to demonstrate the potential of IMAC-OBE-nMS for expedient optimization of overexpression and purification conditions for recombinant protein production.
Topics: Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Histidine; Mass Spectrometry; Recombinant Proteins
PubMed: 34752295
DOI: 10.1016/bs.mie.2021.07.007 -
Biomolecules Oct 2022Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of...
Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.
Topics: Chromatography, Affinity; Intrinsically Disordered Proteins; Protein Conformation
PubMed: 36358915
DOI: 10.3390/biom12111566 -
Metallomics : Integrated Biometal... Sep 2022Copper is essential in a host of biological processes, and disruption of its homeostasis is associated with diseases including neurodegeneration and metabolic disorders....
Copper is essential in a host of biological processes, and disruption of its homeostasis is associated with diseases including neurodegeneration and metabolic disorders. Extracellular copper shifts in its speciation between healthy and disease states, and identifying molecular components involved in these perturbations could widen the panel of biomarkers for copper status. While there have been exciting advances in approaches for studying the extracellular proteome with mass spectrometry-based methods, the typical workflows disrupt metal-protein interactions due to the lability of these bonds either during sample preparation or in gas-phase environments. We sought to develop and apply a workflow to enrich for and identify protein populations with copper-binding propensities in extracellular fluids using an immobilized metal affinity chromatography (IMAC) resin. The strategy was optimized using human serum to allow for maximum quantity and diversity of protein enrichment. Protein populations could be differentiated based on protein load on the resin, likely on account of differences in abundance and affinity. The enrichment workflow was applied to plasma samples from patients with Wilson's disease and protein IDs and differential abundancies relative to healthy subjects were compared to those yielded from a traditional proteomic workflow. While the IMAC workflow preserved differential abundance and protein ID information from the traditional workflow, it identified several additional proteins being differentially abundant including those involved in lipid metabolism, immune system, and antioxidant pathways. Our results suggest the potential for this IMAC workflow to identify new proteins as potential biomarkers in copper-associated disease states.
Topics: Chromatography, Affinity; Copper; Humans; Mass Spectrometry; Proteome; Proteomics
PubMed: 35929804
DOI: 10.1093/mtomcs/mfac059 -
ChemistryOpen May 2022DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent...
DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4-targeting synthetic or natural compounds, we developed an affinity chromatography-based assay, named G-quadruplex on Oligo Affinity Support (G4-OAS), by synthesizing G4-forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on-support elongation and deprotection of the G4-forming oligonucleotides can be performed, along with the successive affinity chromatography-based assay, renamed as G-quadruplex on Controlled Pore Glass (G4-CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4-CPG assay are reported.
Topics: Antineoplastic Agents; Chromatography, Affinity; G-Quadruplexes; Ligands
PubMed: 35608081
DOI: 10.1002/open.202200090 -
Journal of Clinical Microbiology Jul 2017Malaria is a potentially life-threatening disease requiring rapid diagnosis and treatment. Although microscopic examination of thick and thin blood films remains the... (Review)
Review
Malaria is a potentially life-threatening disease requiring rapid diagnosis and treatment. Although microscopic examination of thick and thin blood films remains the gold standard for laboratory diagnosis, rapid antigen tests and nucleic acid amplification methods may also play a useful role in detection of acute infection. This review discusses the advantages and disadvantages of the commonly used diagnostic methods and provides important practice points for optimal malaria test utilization.
Topics: Chromatography, Affinity; Diagnostic Tests, Routine; Humans; Malaria; Microscopy; Nucleic Acid Amplification Techniques
PubMed: 28404673
DOI: 10.1128/JCM.02562-16 -
Journal of Chromatography. B,... Oct 2014Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding... (Review)
Review
Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.
Topics: Chromatography, Affinity; Humans; Kinetics; Protein Binding; Proteins
PubMed: 24572459
DOI: 10.1016/j.jchromb.2014.01.026