I. P. Andreeva

High-sensitivity express immunochromatographic method for detection of plant infection by tobacco mosaic virus

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object — tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2–4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1–0.2 ml tested solution (extract from 10–20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.

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.

Quantitative Lateral Flow Immunoassay for Total Prostate Specific Antigen in Serum

ABSTRACT A simple method for the rapid determination of prostate-specific antigen (PSA) in serum is reported using a lateral flow immunoassay with gold nanoparticles as the label. The method uses the intensity of colored test lines to determine PSA from 0.3 to 30 ng/mL. The limit of detection was 0.3 ng/mL and the coefficient of variation was less than 10%. The analysis time was approximately 20 min. The novel method showed good correlation with enzyme linked immunosorbent assay (ELISA) measurements of prostate-specific antigen concentration in human serum with a linear regression coefficient of 0.985. The developed system was stable for at least 12 months when stored from +4 to 30°C and has potential application for clinical practice.

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.

Synthesis, SAR and molecular docking study of novel non-β-lactam inhibitors of TEM type β-lactamase

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type β-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.

Study of catalytic properties of recombinant β-lactamases TEM-1 and TEM-171 of the molecular class A

Homogeneous preparations of recombinant β-lactamases TEM-1 and TEM-171 of molecular class A, differing by an amino acid substitution of valine at position 84 to isoleucine (Val84Ile), was obtained. The kinetic parameters of the β-lactamase TEM-171 were determined using a chromogenic substrate CENTA (KM eff = 23 μM, Kcat = 102 s–1). The competitive inhibition of recombinant β-lactamases TEM-1 and TEM-171 by tazobactam was ascertained. The values of the inhibition constants in the hydrolysis of the CENTA substrate amount to 0.057 and 0.047 μM for TEM-1 and TEM-171, respectively. It was shown that the Val84Ile mutation leads to a decrease of TEM-171 enzyme thermal stability by 1.5 times.

Lateral flow immunoassay of human troponin-I

Abstractan immunochromatographic assay for a rapid determination of troponin-I, in which gold nanoparticles were used as visual tags, was developed. The optimum analysis conditions were determined. The detection limit of the assay was 1 ng/mL, and the coefficient of variation did not exceed 10%.

Immunochromatographic rapid analysis of human heart-type fatty acid-binding protein for acute myocardial infarction diagnosis

A method of immunochromatographic assay for rapid detection of human heart-type fatty acid-binding protein (h-FABP) as an early marker of acute myocardial infarction was developed. Gold nanoparticles were used as a visual label. The optimum conditions for assay were determined. The limit of detection in the assay was 1.5 ng/mL, and the variation coefficient did not exceed 8%. Using the developed test system, the clinical diagnosis of acute myocardial infarction (N = 10) was confirmed, and the results of testing the serum of healthy individuals (N = 25) were negative.

Bacterial TEM-Type Serine Beta-Lactamases: Structure and Analysis of Mutations

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2000 members: it includes genetically and functionally different bacterial enzymes capable to degrade the beta-lactam antibiotics. Beta-lactamases of molecular class A with serine residue in the active center are the most common ones. In the context of studies of the mechanisms underlying of evolution of the resistance, TEM type beta-lactamases are of particular interest due to their broad polymorphism. To date, more than 200 sequences of TEM type beta-lactamases have been described and more than 60 structures of different mutant forms of these enzymes have been presented in the Protein Data Bank. We have considered here the main structural features of the enzymes of this type with particular attention to the analysis of key mutations determining drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed a BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the TEM type beta-lactamases.

Cloning and expression of NDM-1 metallo-β-lactamase gene and study of the catalytic properties of the recombinant enzyme

A gene-expression system has been designed to express the NDM-1 metallo-β-lactamase gene in E. coli cells. This system enables the synthesis of the recombinant protein in a soluble and active form. A method for the isolation and purification of the recombinant enzyme has been developed. The yield of the homogeneous protein preparation was 10–15 mg per liter of E. coli culture medium. The catalytic parameters of the recombinant NDM-1 β-lactamase were measured for ampicillin (Km = 185 μM and kcat = 585 s–1) and meropenem (Km = 85 μM and kcat = 160 s–1). These values correlate well with the literature data. The catalytic parameters for the chromogenic CENTA substrate (Km = 14 μM and kcat = 290 s–1) were obtained for the first time.