ABSTRACT
The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by π-π stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.
ABSTRACT
BACKGROUND: Ciprofloxacin (CIP) and chloramphenicol (CAP) are relevant antibiotics of the fluoroquinolone (FQ) and amphenicol (AP) groups, respectively, widely used in veterinary practice and they contaminate agricultural products. In this study, a rapid and sensitive immunochromatographic assay (ICA) was developed for simultaneous detection of CIP and CAP in dairy products. The ICA was carried out in a direct competitive format using gold nanoparticles as a label. RESULTS: The ICA developed here allowed for the detection of CIP and CAP in Triton X-100-containing buffered saline (PBST) within 15 min with instrumental detection limits of 20 pg mL-1 and 0.5 ng mL-1 , respectively, and with a visual detection limit of 5 ng mL-1 for both antibiotics. The ICA showed cross-reactivity (69-160%) to 19 antibiotics in the FQ group and no cross-reactivity (<0.1%) to 2 antibiotics of the AP group. The ICA allowed detection of CIP and CAP in a panel of dairy products by employing a simple procedure of preliminary sample preparation. The detection limits for the two antibiotics were the same as in PBST. The analytical recoveries of CIP and CAP in dairy products ranged from 83% to 120%. CONCLUSION: The analytical characteristics of the test system allow its use for the detection of antibiotics in milk and dairy products during all steps of production. © 2019 Society of Chemical Industry.
Subject(s)
Anti-Bacterial Agents/analysis , Chloramphenicol/analysis , Dairy Products/analysis , Fluoroquinolones/administration & dosage , Immunoassay/methods , Animals , Cattle , Ciprofloxacin/analysis , Food Contamination/analysis , Gold/chemistry , Immunoassay/instrumentation , Limit of Detection , Metal Nanoparticles/chemistry , Milk/chemistryABSTRACT
This review is devoted to the analytical application of carbohydrate-binding proteins called lectins. The nature of lectins and the regularities of their specificity with respect to simple sugars and complex carbohydrate-containing biomolecules are discussed. The main areas of the modern analytical application of lectins are described. Lectin-affinity chromatography, histo- and cytochemical approaches, lectin blotting, microarray, and biosensor technologies as well as microplate analysis are considered in detail. Data on the use of lectins for the detection of cells and microorganisms as well as the study of protein glycosylation are summarized. The large potential of lectins as components of analytical systems used for the identification of glycans and the characteristics of their structure are substantiated.
Subject(s)
Lectins/analysis , Biosensing Techniques , Blotting, Western , Humans , Protein Array AnalysisABSTRACT
The high affinity and specificity of biological receptors determine the demand for and the intensive development of analytical systems based on use of these receptors. Therefore, theoretical concepts of the mechanisms of these systems, quantitative parameters of their reactions, and relationships between their characteristics and ligand-receptor interactions have become extremely important. Many mathematical models describing different bioassay formats have been proposed. However, there is almost no information on the comparative characteristics of these models, their assumptions, and predictive insights. In this review we suggested a set of criteria to classify various bioassays and reviewed classical and contemporary publications on these bioassays with special emphasis on immunochemical analysis systems as the most common and in-demand techniques. The possibilities of analytical and numerical modeling are discussed, as well as estimations of the minimum concentrations that may be detected in bioassays and recommendations for the choice of assay conditions.
Subject(s)
Biological Assay/methods , Biological Assay/standards , Models, Theoretical , Research/trends , Research Design/standardsABSTRACT
The effects of water-dispersed gold nanoparticles (8.0±0.9 nm in diameter) on the rat small intestinal mucosa and Peyer plaques, liver, and spleen were studied by electron microscopy. Water-dispersed gold nanoparticles injected into isolated intestinal loop not only accumulated in the small intestinal mucosa and Peyer plaques, but also penetrated into other organs, e.g. liver and spleen. Ultrastructural changes in the cells (hyperplasia of endoplasmic reticulum) were detected in the studied organs.
Subject(s)
Gold/toxicity , Intestine, Small/metabolism , Liver/metabolism , Metal Nanoparticles/toxicity , Spleen/metabolism , Animals , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Gold/pharmacokinetics , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestine, Small/drug effects , Intestine, Small/pathology , Liver/drug effects , Liver/pathology , Male , Microscopy, Electron, Transmission , Rats, Wistar , Spleen/drug effects , Spleen/pathology , Tissue DistributionABSTRACT
A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopaminefluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.350 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.
Subject(s)
Food Analysis/methods , Phenethylamines/analysis , Animals , Fluorescence Polarization Immunoassay/methods , HumansABSTRACT
Characteristics of the fluorescence polarization immunoassay (FPIA) as a mean for express control of antibiotic levels in various specimens and its advantages vs. other analytical tests are described. The developmental stages of the analytical procedure and its parameters are considered for chlorampnenicol as an example. The analysis is based on competitive interaction of anti-chloramphenicol antibodies with the chloramphenicol-fluorophore conjugate and the potential free chloramphenicol in the specimen. The experimental results of the comparison of the chloramphenicol FPIA with the use of two conjugates differing in the length of the bridge length between the antibiotic functional groups and fluorophore (fluorescein) are presented. The requirements to the choice of the antibody and conjugate concentrations providing highly sensitive detection are characterized. The detection limit of chloramphenicol in the FPIA was 10 ng/ml and the determination of the concentrations ranged from 20 ng/mI to 10 mcg/ml. The time of the assay was 10 min.
ABSTRACT
A rapid immunochromatographic assay was developed for the control of tetracycline (TC). The assay is based on the competition between immobilized TC-protein conjugate and TC in a tested sample for binding with polyclonal anti-TC antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. Conjugation of colloidal gold and the total immunoglobulin (IgG) fraction of polyclonal antibodies was used to increase the assay sensitivity to ensure low content of specific antibodies in the conjugate. This allowed effective inhibition of free TC and conjugate binding in the strip test zone. Photometric marker registration allows control of the reduction of binding, thereby enhancing detection sensitivity. The proposed assay allows TC to be detected at concentrations up to 20 ng/mL, exceeding the limit of detection of the known analogues, in a wide working range (more than two orders) of 60 pg/mL to 10 ng/mL, ensured through the use of polyclonal antibodies. The assay time is 10 min. The efficiency of the designed assay is shown to identify TC in milk; the degree of recovery of TC ranges from 90 to 112%. The precision of the concentrations measurements was no more than 10%.
ABSTRACT
A test system based on immunochromatography in the sandwich format and intended for express detection of Helicobacter pylori antigens has been developed. Contact of a sample with a test strip coated with immunochemical reagents triggers the movement of the liquid along the membrane components of the test strip, immunochemical interactions, and the formation of detection zones stained by gold nanoparticles. The concentration and kinetic dependences of the immunochemical interactions have been characterized. The reagent and membrane composition of the test system has been selected to provide a minimal detection limit. The detection of H. pylori cell wall antigens at concentrations as low as 0.3 µg/mL in aqueous solution and a suspension of a clinical sample of feces has been demonstrated; the assay duration was 10 minutes. Staining enhancement by the addition of silver salts allowed for a further reduction of the detection limit to 0.03 µg/mL. The developed test system can be used for field diagnostics.
Subject(s)
Antigens, Bacterial/isolation & purification , Chromatography, Affinity/methods , Helicobacter Infections/diagnosis , Helicobacter pylori/isolation & purification , Antigens, Bacterial/immunology , Biosensing Techniques , Gold , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Humans , Limit of Detection , Metal Nanoparticles/chemistryABSTRACT
Nanodispersed gold is widely used as a marker in different analytical systems. For such purposes, it is usually obtained by the reduction of salts. This work studied the potential analytical applications of nanodispersed gold obtained by laser ablation because gold produced with this method has no chemical coating. The nanoparticles produced were characterized by transmission electron microscopy and spectrophotometry. The average size of the particles was 24.5 nm. Concentration dependences of antibody immobilization on ablative gold were obtained. With the use of antibody-conjugated nanoparticles, an immunochromatographic system was constructed for the detection of zearalenone mycotoxin. This immunoassay was characterized by a detection limit of 0.1 ng/ml antigen with an assay duration of only 15 min, which is on par with current test systems comprising nanodispersed gold obtained by chemical reduction. The simplicity of ablative dispersing makes this a prospective method for the labeling of various antibodies for analytical use.
Subject(s)
Chromatography, Affinity , Gold/chemistry , Lasers , Metal Nanoparticles/chemistry , Zearalenone/analysis , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Limit of Detection , Zearalenone/immunologyABSTRACT
An immunochromatographic test was developed for the simultaneous detection of several compounds in a complex sample matrix. The system was designed in a 'traffic light' format comprising three lines of different colors on a test strip, thereby providing an easy tool with which to identify an analyte of interest based on the visible color of the line formed (qualitative analysis), and to determine the amount of the analytes present based on the fluorescence intensity of the lines (quantitative analysis). For the development of the multicolor immunochromatographic test, we used antibodies against antibiotics of three different classes as selective binders. Each antibody was labeled with water-soluble quantum dots with emission maximum at either 525, 585, or 625 nm. The test system exhibited high sensitivity, with limits of detection for ofloxacin, chloramphenicol, and streptomycin of 0.3, 0.12, and 0.2 ng mL(-1), respectively. These values are 80-200 times lower than those achievable with ELISA using the same antibodies. Using the 'traffic light' assay, these antibiotics could be detected in milk samples within 10 min without any sample preparation. The 'traffic light' assay also demonstrated a high degree of analyte detection when testing spiked milk samples (92-101%) and accuracy (quantitation error <8% of the mean).
Subject(s)
Anti-Bacterial Agents/analysis , Chromatography, Affinity/instrumentation , Food Analysis/instrumentation , Food Contamination/analysis , Milk/chemistry , Quantum Dots , Animals , Complex Mixtures/analysis , Equipment Design , Equipment Failure Analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Methods for registration of intermolecular interactions based on the phenomenon of surface plasmon resonance (SPR) have become one of the most efficient tools to solve fundamental and applied problems of analytical biochemistry. Nevertheless, capabilities of these methods are often insufficient to detect low concentrations of analytes or to screen large numbers of objects. That is why considerable efforts are directed at enhancing the sensitivity and efficiency of SPR-based measurements. This review describes the basic principles of the detection of intermolecular interactions using this method, provides a comparison of various types of SPR detectors, and classifies modern approaches to enhance sensitivity and efficiency of measurements.
Subject(s)
Surface Plasmon Resonance/methods , Nanoparticles , Sensitivity and SpecificityABSTRACT
The effects of water-dispersed Ag nanoparticles on the small intestinal mucosa, liver, and spleen of rats were studied by transmission electron microscopy. Acute experiments demonstrated penetration of Ag nanoparticles injected into the isolated intestinal loop into the intestinal mucosa, liver, and splenic tissues. Ultrastructural changes (lobed nucleus, megamitochondria) were found in the studied organs. These data indicated that injection of water-dispersed Ag nanoparticles into the gastrointestinal tract was followed by their penetration through the epithelium of the small intestinal mucosa into other organs, e.g. into the liver and spleen. This fact is essential for evaluation of potential risks of the nanoparticle effects on human health and environment.
Subject(s)
Intestinal Mucosa/cytology , Liver/cytology , Metal Nanoparticles/chemistry , Spleen/cytology , Animals , Hepatocytes/cytology , Intestine, Small/cytology , Male , Microscopy, Electron, Transmission , Mitochondria , Peyer's Patches/cytology , Rats , Rats, Wistar , Silver/chemistry , Tissue DistributionABSTRACT
A method was developed for the rapid detection of human epidermal growth factor based on a sandwich-format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid flow movement across the membrane components of the test strip, immunochemical reactions, and the formation of colored lines. Requirements on the configuration of the test system in order to achieve the lowest limit of detection were defined in the course of the development of the assay. It was shown that this method enables the detection of human epidermal growth factor within 5 min at concentrations as low as 10 pg/mL in aqueous solutions, urine, and the blood serum and plasma. The developed test system can be used for point-of-care diagnostics.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Chromatography, Affinity/methods , Epidermal Growth Factor/blood , Epidermal Growth Factor/urine , Membranes, Artificial , Chromatography, Affinity/instrumentation , Humans , Sensitivity and SpecificityABSTRACT
There was studied migration of artificial silver nanoparticles from polyethylene films destined for package of such foods as bread and poultry into model media reproducing physico-chemical properties and composition of said products. Modification of films was performed by 5- or 10-fold spraying of silver nanoparticles on the surface of package material. Model media were composed from water, alcohol and plant oil according to US FDA and Russian Federal Service for Surveillance of Consumer Rights Protection and Human Well-Being official recommendation. Nanoparticles were detected in model media by means of transmission electron and atomic force microscopy. Quantification of silver in nanoparticles migrating from films was performed by mass-spectrometry with inductively coupled plasma. The results obtained showed that silver migrated from films into test media in form of nanoparticles with mean diameter close to 10-20 nm. Migrated particles were partially aggregated to complexes with dimension about 50 nm with degree of aggregation depending on media composition. Quantification showed that amounts of silver nanoparticles migrating in foods did not exceed save level of this nanomaterial consumption even in aggravated conditions when almost all volume of product was consumed in form packaged in films modified with nanosilver.
Subject(s)
Food Contamination/analysis , Food Packaging/standards , Metal Nanoparticles/analysis , Silver/analysis , Mass Spectrometry , Microscopy, Atomic Force , Microscopy, Electron , Models, Theoretical , Plant Oils/analysis , Polyethylene/analysis , Water/analysisABSTRACT
A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/analysis , Brucella abortus/immunology , Brucellosis/diagnosis , Chromatography, Affinity , Lipopolysaccharides/analysis , Animals , Antibodies, Bacterial/chemistry , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Brucella abortus/isolation & purification , Brucellosis/immunology , Brucellosis/microbiology , Cattle , Color , Mice , Reagent Kits, Diagnostic , Reagent Strips , Sensitivity and Specificity , Time FactorsABSTRACT
Atomic force microscopy (AFM) can be used to make measurements in vacuum, air, and water. The method is able to gather information about intermolecular interaction forces at the level of single molecules. This review encompasses experimental and theoretical data on the characterization of ligand-receptor interactions by AFM. The advantage of AFM in comparison with other methods developed for the characterization of single molecular interactions is its ability to estimate not only rupture forces, but also thermodynamic and kinetic parameters of the rupture of a complex. The specific features of force spectroscopy applied to ligand-receptor interactions are examined in this review from the stage of the modification of the substrate and the cantilever up to the processing and interpretation of the data. We show the specificities of the statistical analysis of the array of data based on the results of AFM measurements, and we discuss transformation of data into thermodynamic and kinetic parameters (kinetic dissociation constant, Gibbs free energy, enthalpy, and entropy). Particular attention is paid to the study of polyvalent interactions, where the definition of the constants is hampered due to the complex stoichiometry of the reactions.
Subject(s)
Microscopy, Atomic Force/methods , Receptors, Cell Surface/chemistry , Animals , Humans , Kinetics , Ligands , Protein Binding , Receptors, Cell Surface/metabolism , ThermodynamicsABSTRACT
A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR-protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL(-1)) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16-250 ng mL(-1) its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR-protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).
Subject(s)
Dairy Products/analysis , Immunoassay/methods , Milk/chemistry , Streptomycin/analysis , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Cattle , Gold/chemistry , Mice , Streptomycin/immunologyABSTRACT
An immunochromatographic method for determination of beta-lactam antibiotic ampicillin has been developed. The method is based on the competitive interaction between antibiotic molecules contained in the sample and protein conjugate of penicillin immobilized on a membrane for binding with specific antibodies labeled with colloidal gold, which occurs during movement of the sample to be tested and reagents along the membrane. The completion of the test system ensures control of exceeding the maximum permissible content of the antibiotic in milk and dairy products (10 ng/ml). The possibility of testing milk, raw milk, and dairy products for 10 minutes at room temperature without sample preparation has been demonstrated.
Subject(s)
Ampicillin/analysis , Chromatography, Affinity/methods , Food Inspection/methods , Immunoassay/methods , Milk/chemistry , Animals , Antibodies/immunology , Antibodies/metabolism , Binding, Competitive , Cattle , Chromatography, Affinity/instrumentation , Female , Food Inspection/instrumentation , Gold Colloid/chemistry , Immobilized Proteins/chemistry , Immobilized Proteins/immunology , Immobilized Proteins/metabolism , Immunoconjugates/chemistry , Immunoconjugates/immunology , Immunoconjugates/metabolism , Membranes, Artificial , Serum Albumin/chemistry , Serum Albumin/metabolismABSTRACT
The actual methods of detection and identification of manufactured nanoparticles in both simple and complex multi-component matrix for assessing biological effects and safety of nanotechnology products have been reviewed. The detection of priority species of biologically active nanoparticles, which include fullerenes, single- and multi-walled carbon nanotubes, nanoparticles of silver, gold, titanium oxide, aluminum, cerium, zinc and silicon, has been given a special attention. The requirements for sample preparation have been discussed. The results of the successful application for the detection of manufactured nanoparticles in bioassays with methods of scanning and transmission electron microscopy, confocal laser scanning microscopy, atomic force microscopy, scanning tunneling microscopy, size exclusion chromatography, field-flow fractionation, electrophoretic, light scattering, spectrophotometry, fluorescent spectroscopy, X-ray and other spectrometry, mass spectrometry, "particle counters", immunochemistry have been reviewed. The possibilities and limitations of different techniques, and their complementarity have been analyzed.