Over the last two decades, the group of techniques called affinity probe CE has been widely used for the detection and the determination of several types of biomolecules with high sensitivity. These techniques combine the low sample consumption and high separation power of CE with the selectivity of the probe to the target molecule. The assays can be defined according to the type of probe used: CE immunoassays, with an antibody as the probe, or aptamer-based CE, with an aptamer as the probe. Immunoassays are generally divided into homogeneous and heterogeneous groups, and homogeneous variant can be further performed in competitive or noncompetitive formats. Interacting partners are free in solution at homogeneous assay, as opposed to heterogeneous analyses, where one of them is immobilized onto a solid support. Highly sensitive fluorescence, chemiluminescence or electrochemical detections were typically used in this type of study. The use of the aptamers as probes has several advantages over antibodies such as shorter generation time, higher thermal stability, lower price, and lower variability. The aptamer-based CE technique was in practice utilized for the determination of proteins in biological fluids and environmentally or clinically important small molecules. Both techniques were also transferred to microchip. This review is focused on theoretical principles of these techniques and a summary of their applications in research.
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1st ed, repr. 255 s.
- MeSH
- Philosophy MeSH
- Metaphysics MeSH
- Conspectus
- Věda. Všeobecnosti. Základy vědy a kultury. Vědecká práce
- NML Fields
- humanitní vědy a umění
Label-free affinity biosensors offer a promising platform for the development of a new generation of medical diagnostic technologies. Nevertheless, when such sensors are used in complex biological media, adsorption of non-targeted medium components prevents the specific detection of the analyte. In this work, we introduce for the first time a biosensor assay based on surface plasmon resonance (SPR) capable of diagnosing different stages of Epstein-Barr virus (EBV) infections in clinical serum samples. This was achieved by simultaneous detection of the antibodies against three different antigens present in the virus. To prevent the interference of the fouling from serum during the measurement, the SPR chips were coated by an antifouling layer of a polymer brush of poly[oligo(ethylene glycol) methacrylate] grown by surface-initiated atom transfer radical polymerization. The bioreceptors were then attached via hybridization of complementary oligonucleotides. This allowed the sensor surface to be regenerated after measurement by disrupting the complementary pairs above the oligonucleotides' melting temperature and attaching new bioreceptors. In this way, the same sensing surface could be used repeatedly. The procedure used in this work will serve as a prototype strategy for the development of label-free affinity biosensors for diagnostics in blood serum or plasma samples. This is the first example of detection of marker of a disease in clinical serum samples by an optical affinity biosensor.
- MeSH
- Equipment Failure Analysis MeSH
- Biosensing Techniques instrumentation MeSH
- Equipment Design MeSH
- Immunoassay instrumentation MeSH
- Epstein-Barr Virus Infections blood diagnosis immunology MeSH
- Humans MeSH
- Surface Plasmon Resonance instrumentation MeSH
- Antibodies, Viral immunology MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Kinetic and thermodynamic studies of ligand-receptor interactions are essential for increasing the understanding of receptor activation mechanisms and drug behavior. The characterization of molecular interactions on living cells in real-time goes beyond most current binding assays, and provides valuable information about the dynamics and underlying mechanism of the molecules in a living system. The effect of temperature on interactions in cell-based assays is, however, rarely discussed. In the present study, the effect of temperature on binding of monoclonal antibodies, cetuximab and pertuzumab to specific receptors on living cancer cells was evaluated, and the affinity and kinetics of the interactions were estimated at selected key temperatures. Changes in the behavior of the interactions, particularly in the on- and off-rates were observed, leading to greatly extended time to reach the equilibrium at 21°C compared with at 37°C. However, the observed changes in kinetic characteristics were less than a factor of 10. It was concluded that it is possible to conduct real-time measurements with living cells at different temperatures, and demonstrated that influences of the ambient temperature on the interaction behavior are likely to be less than one order of magnitude.
- Publication type
- Journal Article MeSH
BACKGROUND/AIMS: The determination of neuron-specific enolase (NSE) is relatively frequently requested in the differential diagnosis of small-cell lung carcinoma and non-small-cell lung carcinoma. The individual results of different immunoassays are often not comparable, which has been confirmed by long-term external quality assessments. In this study, we assessed the possible sources of these differences. METHODS: More than 3,000 NSE analyses were performed using seven different immunoassays: DELFIA (PerkinElmer), Elecsys 2010 or Modular Analytics E 170 (Roche), Kryptor (B.R.A.H.M.S.), the enzyme-linked immunosorbent assay DRG and three assays based on immunoradiometric assays (DiaSorin, Immunotech and Schering-CIS). The following parameters were evaluated: precision profile of the individual methods, linearity on dilution and modified recovery, comparability and discrimination of immunoassays, sensitivity, and specificity. RESULTS: There were differences in the correlation of values of certain low-concentration specimens. Some assays correlate well while others do not (up to fivefold difference), especially in the case of controls prepared synthetically. Therefore, the current non-standardized preparation of controls is questionable in our opinion. In the cutoff range, the difference in the results of native samples did not exceed its double value. The variation in values >100 microg/l obtained with different assays is <40%. CONCLUSION: Our results confirmed expected matrix interferences especially in the range of normal and cutoff NSE concentrations. Another source of discrepancies can be attributed to different antibody affinity to alphagamma- and gammagamma-enolase isoenzymes. Finally, improper settings of cutoff values also contribute to the different discrimination of the methods. Copyright (c) 2007 S. Karger AG, Basel.
- MeSH
- Adenocarcinoma enzymology blood MeSH
- Biological Assay MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Phosphopyruvate Hydratase blood MeSH
- Humans MeSH
- Carcinoma, Small Cell enzymology blood MeSH
- Biomarkers, Tumor metabolism MeSH
- Lung Neoplasms enzymology blood MeSH
- Carcinoma, Non-Small-Cell Lung enzymology blood MeSH
- Sensitivity and Specificity MeSH
- Carcinoma, Squamous Cell enzymology blood MeSH
- Case-Control Studies MeSH
- Carcinoma, Large Cell enzymology blood MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
Carbonyl-reducing enzymes are important in both metabolism of endogenous substances and biotransformation of xenobiotics. Because sufficient amounts of native enzymes must be obtained to study their roles in metabolism, an efficient purification strategy is very important. Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline) is a prospective anticancer drug and one of the xenobiotic substrates for carbonyl-reducing enzymes. A new purification strategy based on molecular recognition of carbonyl-reducing enzymes with oracin as a ligand is reported here. The type of covalent bond, ligand molecules orientation, and their distance from the backbone of the solid matrix for good stearic accessibility were taken into account during the designing of the carrier. The carriers based on magnetically active microparticles were tested by recombinant enzymes AKR1C3 and CBR1. The SiMAG-COOH magnetic microparticles with N-alkylated oracin and BAPA as spacer arm provide required parameters: proper selectivity and specificity enabling to isolate the target enzyme in sufficient quantity, purity, and activity.
- MeSH
- Alcohol Oxidoreductases isolation & purification MeSH
- Biological Assay MeSH
- Chromatography, Affinity MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Enzyme Assays methods MeSH
- Enzymes isolation & purification MeSH
- Ethanolamines chemistry MeSH
- Isoquinolines chemistry MeSH
- Ligands MeSH
- Magnetics * MeSH
- Microspheres MeSH
- Molecular Structure MeSH
- Antineoplastic Agents chemistry MeSH
- Schiff Bases chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.
- MeSH
- Chromatography, Liquid MeSH
- Protein Interaction Domains and Motifs genetics MeSH
- Humans MeSH
- Microfilament Proteins genetics isolation & purification MeSH
- Neoplasms genetics pathology MeSH
- Surface Plasmon Resonance * MeSH
- LIM Domain Proteins genetics isolation & purification MeSH
- Streptavidin chemistry MeSH
- Tandem Mass Spectrometry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Developmental responses to auxin are regulated by facilitated uptake and efflux, but detailed molecular understanding of the carrier proteins is incomplete. We have used pharmacological tools to explore the chemical space that defines substrate preferences for the auxin uptake carrier AUX1. Total and partial loss-of-function aux1 mutants were assessed against wild-type for dose-dependent resistance to a range of auxins and analogues. We then developed an auxin accumulation assay with associated mathematical modelling to enumerate accurate IC50 values for a small library of auxin analogues. The structure activity relationship data were analysed using molecular field analyses to create a pharmacophoric atlas of AUX1 substrates. The uptake carrier exhibits a very high level of selectivity towards small substrates including the natural indole-3-acetic acid, and the synthetic auxin 2,4-dichlorophenoxyacetic acid. No AUX1 activity was observed for herbicides based on benzoic acid (dicamba), pyridinyloxyacetic acid (triclopyr) or the 6-arylpicolinates (halauxifen), and very low affinity was found for picolinic acid-based auxins (picloram) and quinolinecarboxylic acids (quinclorac). The atlas demonstrates why some widely used auxin herbicides are not, or are very poor substrates. We list molecular descriptors for AUX1 substrates and discuss our findings in terms of herbicide resistance management.
- MeSH
- Arabidopsis metabolism MeSH
- Models, Biological MeSH
- Biological Assay MeSH
- Herbicides metabolism MeSH
- Indoles metabolism MeSH
- Inhibitory Concentration 50 MeSH
- Plant Roots growth & development MeSH
- 2,4-Dichlorophenoxyacetic Acid metabolism MeSH
- Indoleacetic Acids metabolism MeSH
- Mutation genetics MeSH
- Arabidopsis Proteins metabolism MeSH
- Seedlings growth & development MeSH
- Substrate Specificity MeSH
- Nicotiana cytology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Bulged DNA structures are of general biological significance because of their important roles in a number of biochemical processes. Compounds capable of targeting bulged DNA sequences can be used as probes for studying their role in nucleic acid function, or could even have significant therapeutic potential. The interaction of [Fe(2)L(3)](4+) metallosupramolecular helicates (L = C(25)H(20)N(4)) with DNA duplexes containing bulges has been studied by measurement of the DNA melting temperature and gel electrophoresis. This study was aimed at exploring binding affinities of the helicates for DNA bulges of various sizes and nucleotide sequences. The studies reported herein reveal that both enantiomers of [Fe(2)L(3)](4+) bind to DNA bulges containing at least two unpaired nucleotides. In addition, these helicates show considerably enhanced affinity for duplexes containing unpaired pyrimidines in the bulge and/or pyrimidines flanking the bulge on both sides. We suggest that the bulge creates the structural motif, such as the triangular prismatic pocket formed by the unpaired bulge bases, to accommodate the [Fe(2)L(3)](4+) helicate molecule, and is probably responsible for the affinity for duplexes with a varying number of bulge bases. Our results reveal that DNA bulges represent another example of unusual DNA structures recognized by dinuclear iron(II) ([Fe(2)L(3)](4+)) supramolecular helicates.
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
- MeSH
- Biological Assay MeSH
- Cytokines metabolism MeSH
- HEK293 Cells MeSH
- Protein Conformation MeSH
- Leukocytes, Mononuclear drug effects MeSH
- Humans MeSH
- Membrane Proteins chemistry metabolism MeSH
- Nucleotides, Cyclic chemical synthesis pharmacology MeSH
- Computer Simulation MeSH
- Gene Expression Regulation drug effects MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
