M. Yu. Rubtsova

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).

Synthesis of protein A conjugates with silver nanoparticles

The single-stage synthesis of the conjugates of protein A with silver nanoparticles is performed. The dependence of the dispersity of the prepared sols on the composition of reaction mixture is investigated by optical spectroscopy and transmission electron microscopy. The relative stability of the obtained preparations with respect to the coagulation by NaCl is estimated.

Multiparametric determination of genes and their point mutations for identification of beta-lactamases.

More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by antibiotic resistance of microorganisms that are the causative agents of infectious diseases. Several mechanisms for the resistance of Enterobacteriaceae have been established, but the main one is the enzymatic hydrolysis of the antibiotic by specific enzymes called beta-lactamases. Beta-lactamases represent a large group of genetically and function-ally different enzymes of which extended-spectrum beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid localization of the encoded genes, the distribution of these enzymes among the pathogens increases every year. Among ESBLs the most widespread and clinically relevant are class A ESBLs of TEM, SHV, and CTX-M types. TEM and SHV type ESBLs are derived from penicillinases TEM-1, TEM-2, and SHV-1 and are characterized by several single amino acid substitutions. The extended spectrum of substrate specificity for CTX-M beta-lactamases is also associated with the emergence of single mutations in the coding genes. The present review describes various molecular-biological methods used to identify determinants of antibiotic resistance. Particular attention is given to the method of hybridization analysis on microarrays, which allows simultaneous multiparametric determination of many genes and point mutations in them. A separate chapter deals with the use of hybridization analysis on microarrays for genotyping of the major clinically significant ESBLs. Specificity of mutation detection by means of hybridization analysis with different detection techniques is compared.

Chemiluminescent immunoassay: Application of a portable scanning luminometer for the determination of 2,4‐dichlorophenoxyacetic acid in microtiter and membrane strip format

A sensitive competitive immunoassay with chemiluminescent detection for the determination of 2,4‐dichlorophenoxyacetic acid (2,4‐D) was carried out in a microtiter and membrane strip format. The application of a portable scanning luminometer for the detection of light emission in polystyrene strips and from the surface of a porous membrane resulted in an increase in the sensitivity of the assay and a wider detection range when compared to conventional colorimetric detection. With the assay in microtiter strip format, 2,4‐D can be determined within the range 0.005–1 μg l‐1 with an IC50 at 0.04 p.g l‐1. Different immobilization techniques involving adsorption and covalent immobilization on different kinds of modified nylon were examined to optimize the attachment of specific antibodies to the surface of porous membranes. Photoimmobilization on positively charged nylon modified with p‐azidobenzoic acid was found to result in higher sensitivity. The application of a membrane strip format allows the quick quan...

Enzyme-linked immunosorbent assay of chlorampenicol in foodstuff

A test-system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chloramphenicol (CAP) in foodstuff has been developed. The detection limit of the method was 0.05 μg/l. The procedures for milk samples preparation of various fat content and chicken muscles were optimized. Before the analysis milk was diluted 5-fold with a buffer. The detection limit for milk was 0.3 μg/l; recoveries varied from 74 to 118%. Two protocols for chicken muscles preparation were elaborated; extraction with buffer (the express method) and extraction with acetonitrile. The detection limits of CAP in chicken muscles were 0.5 and 0.3 μg/kg, respectively; recovery values were 71–107% and 95–115%, respectively. The results of residual amounts of CAP detection in foodstuff by ELISA and HPLC-MS were in good correlation.

Recombinant horseradish peroxidase: production and analytical applications.

Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

Bioluminescent analysis in a biomembrane-like medium consisting of surfactant, hydrocarbon solvent, and water: ATP determination using firefly luciferase

ATP is the most important coupling agent between exergonic and endergonic processes in living organisms. It is either consumed or regenerated in every metabolic sequence. Hence, accurate, sensitive, and facile methods are required for ATP determination. The most promising (specific) assay involves the firefly luciferase-catalyzed reaction in which a single enzyme uses ATP as a substrate and produces light as a At present,6 aqueous solution is the most widely used medium for carrying out the bioluminescent assay. Colloidal solutions of water in organic solvents have been recently demonstrated to be more promising media for some enzymatic reactions.’-’* In the colloidal case, enzyme molecules are entrapped in the inner cavities of hydrated surfactant reversed micelles. Such a microheterogeneous solution has proven13 to be quite a favorable environment for firefly luciferase as well. We now report a striking enhancement of the sensitivity of the bioluminescent response to ATP concentration in the Brij 96-cyclohexane-water system, as compared to that in aqueous solution. Under the conditions of our experiments, this ternary system exists in the form of a cubic lattice composed of reversed micelles of surfactant.14 A similarity between some of the structural and environmental aspects of such systems and those of biomembranes has been recently discussed.” Firefly luciferase was isolated from Luciola mingrelica and was purified using ion-exchange chromatography on DEAE-Sephadex.I6 The protein concentration in aqueous solution was determined spectrophotometrically a t 280 nm. Stock solutions of the purified enzyme and substrates (luciferin and ATP) were prepared in 0.05 M Tris-acetate buffer, pH 7.8, containing 2 m M EDTA and 10 mM MgS04. A typical

Study of the monoclonal antibody - insulin interaction

We generated four stable hybridoma lines producing monoclonal antibodies. All antibodies were as reactive with insulin from other species as with swine insulin. The apparent affinity of the monoclonal antibodies for insulin varied in the range from 2 X 10(8) M-1 to 2 X 10(10) M-1. According to their affinity constants some antibodies could be used for the preparation of immunosorbent, and others for the development of immunoassay methods. Bioluminescent cofactor immunoassay is a method of the choice for the detection of insulin concentration in physiological fluids due to its extreme sensitivity and reproducibility. High affinity antibodies of clones I and II may serve as immunospecific constituents for immunoassay method. Epitope specificity of all monoclonal antibodies has not been studied yet in detail and will be the subject of further investigation.

The study of the role of mutations M182T and Q39K in the TEM-72 β-lactamase structure by the molecular dynamics method

Synthesis of β-lactamases is one of the common mechanisms of bacterial resistance to β-lactam antibiotics such as penicillins and cephalosporins. The widespread use of antibiotics resulted in appearance of numerous extended-spectrum β-lactamase variants or inhibitor-resistant β-lactamases. In TEM type β-lactamases mutations of 92 residues have been described. Several mutations are functionally important and they determine the extended substrate specificity. However, roles of the most so-called associated mutations, located far from the active site, remain unknown. We have investigated the role of associated mutations in structure of β-lactamase TEM-72, which contains two key mutations (G238S, E240K) and two associated mutations (Q39K, M182T) by means of molecular dynamics simulation. Appearance of the key mutations (in 238 and 240 positions) caused destabilization of the protein globule, characterized by increased mobility of amino acid residues. Associated mutations (Q39K, M182T) exhibited opposite effect on the protein structure. The mutation M182T stabilized, while the mutation Q39K destabilized the protein. It appears that the latter mutation promoted optimization of the conformational mobility of β-lactamase and may influence the enzyme activity.