[Development of an Immunochromatographic Test System for the Detection of Helicobacter pylori Antigens].

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.

[Development of an immunochromatographic test system for the detection of human enidermal growth factor].

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.

[Development of immunochromatographic test system for the rapid detection of lipopolysaccharide antigen and cells of causative agent of bovine brucellosis].

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.

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products.

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).

[Immunochromatographic technique for express determination of ampicillin in milk and dairy products].

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.

Rapid pretreatment-free immunochromatographic assay of chloramphenicol in milk.

A pretreatment-free immunochromatographic assay for detection of chloramphenicol (CAP) in milk was developed. The assay is based on competition between CAP molecules in the sample and immobilized CAP-protein conjugate for binding to monoclonal anti-CAP antibodies conjugated with colloidal gold particles (average diameter 30nm). The assay is carried out in the course of sample flowing along test strip with immobilized reactants, and its results can be detected by the naked eye or by a photometric device. Effect of the concentration of immunoreactants on assay characteristics was studied. The assay protocol with maximal sensitivity and reliability was optimized using measured values of brightness of lines. Detection limit for CAP is 10ngmL(-1). Assay duration is 10min, and it can be carried out at room temperature without any additional devices and reactants. The developed test strip has been applied to CAP detection in dairy products.

Interaction of plum pox virus with specific colloidal gold-labeled antibodies and development of immunochromatographic assay of the virus.

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in samples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by "sandwich"-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.

[Immunochromatographic and latex-agglutination systems for diphtheria toxin detection].

The use of two monoclonal antibody types specific to different epitopes of diphtheria toxin systems have been developed to reveal diphtheria corynebacteria toxigenicity rapidly based on immunochromatographic and latex-agglutination detection of the diphtheria toxin. The methods have been tested on a sample of 36 clinical isolates. The possibility of significant detection of the toxigenic properties of the Corynebacterium strain, grown for 1 day, has been demonstrated. The developed methods allow for the detection of diphtheria toxin in concentrations of 3-4 ng/ml. The developed test systems are a perspective tool for diphtheria diagnostics because of significant time shortening as compared to traditional microbiological methods.

[Development of immunochromatographic test systems for express detection of plant viruses].

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rod-shaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 microg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.

Rapid polyelectrolyte-based membrane immunoassay for the herbicide butachlor.

Oppositely charged water-soluble polyelectrolytes were used in the developed membrane immunoenzyme assay for the herbicide butachlor. High-affinity and rapid binding between polyanion polymethacrylate and polycation poly(N-ethyl-4-vinylpyridinium) was applied to separate reacted and free immunoreactants. Competitive immunoassay format with peroxidase-labeled antigen was realized. The insoluble colored product of the peroxidase reaction was formed by bound labeled immune complexes and was reflectometrically detected. The assay combines short duration (15 min), high sensitivity (0.03 g/mL) and availability for out-of-laboratory testing. Different image processing algorithms were used to determine the herbicide content. Low variation coefficients of the measurements in the proposed quantitative assay, namely 4.8-9.0% for the range of antigen concentrations from 0.1 to 3.0 ng/mL, are evidence of the assay effectiveness. Possibility to control the butachlor content in mineral, artesian, and drinking water was demonstrated.

[Oxygenase activity of cytochrome P-450 electrochemically reduced in the presence of methylviologen]. / Oksigenaznaia aktivnost' tsitokhroma P-450, vosstanovlennogo élektrokhimicheski v prisutstvii metilviologena.

An electrochemical system of cytochrome P-450 reduction in the presence of the water-soluble redox carrier methylviologen has been developed. In this system cytochrome P-450 effectuates a steady-state demethylation of dimethylaniline and hydroxylation of aniline. The results of control experiments suggest that the above reactions are mediated by cytochrome P-450. The effect of the peroxidase reaction is excluded by an addition of high concentrations of catalase to the incubation mixture. At the same time the hydroxylation of these substrates is accompanied by methylviologen demethylation.

[Some structural properties of 80S acid protein from pea ribosomes]. / Nekotorye kharakteristiki struktury kislogo belka 80S-ribosom gorokha.

The 80S acid protein from pea ribosomes similar to the L7/L12 protein from E. coli was studied. This protein was found to be rich in alanine (18 mol.%) and to contain an acid amino acids excess over basic ones, the ratio of basic amino acids to acid ones was 0.42. As in the case of other eukaryotic L7/L12 homologs studied, the N-terminal amino acid of the protein is methionine. Using the double immunodiffusion technique, no crossreaction of E. coli anti-L7/L12 with 80S acid protein from pea ribosomes was observed. It was assumed that the protein molecule contains conservative sites responsible for the specific functioning of eukaryotic L7/L12 homologs.

Ribosomal acidic proteins of eucaryotic cells. Isolation of a protein from pea seedlings equivalent to E. coli L7/L12.

By the method of ethanol-salt extraction with ion-exchange chromatography on CM-cellulose an acidic protein of pea 80S ribosomes was isolated. This protein located in the large subunit, had a molecular weight of 14 000 and an IEP of 4.7. The protein is partially phosphorylated, alanine-rich and has methionine at the N-terminal position. Based on these characteristics and on the comparative study of tryptic hydrolyzates of the plant protein and E. coli L7/L12, the protein so obtained is found to be homologous to the L7/L12 of the procaryotic ribosomes.

[Acid proteins of eukaryotic cell ribosomes. Isolation of the protein similar to E. coli L7/L12 from 80S pea ribosomes]. / Kislye belki ribosom éukarioticheskikh kletok. Vydelenie belka, skhodnogo s L7/L12 E. coli, iz 80S-ribosom gorokha.

Using ethanol-salt extraction and ion-exchange chromatography, an acid protein was isolated from 80S pea ribosomes. The protein is localized in the large subunit, is phosphorylated and has a molecular weight of 14000 and pI of 4.7. These features and the results of a comparative study of tryptic hydrolysates of the plant protein and of the protein under study suggest that the latter is homologous to the ribosomal protein L7/L12 of a prokaryotic type.