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Biomedical subjects

I G Kharitonenkov

Publications and source records attributed to I G Kharitonenkov.

At least 91 records · Page 5Linked to original sources
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[Stat-diagnosis of influenza using lanthanide immunofluorescence analysis].

A test system for influenza diagnosis has been developed on the basis of monoclonal antibodies to influenza A and B virus proteins using the principles of lanthanide immunofluorescence analysis. The diagnostic test system was shown to be highly specific in detecting influenza A and B virions in model systems. For the one-step variant of a double sandwich used in the study, the total time of diagnostic experiment using clinical materials was shown to be reduced to 15-20 min.

Antibodies, Monoclonal↗
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[Indication of the influenza virus in clinical samples using immunoenzyme and lanthanide immunofluorescence analyses].

A comparative study of the potentials of enzyme-immunoassay (EIA) and lanthanide immunofluorescent assay (LIFA) for influenza virus detection in nasopharyngeal aspirates and nasopharyngeal washings was carried out. Monospecific and polyclonal antisera to hemagglutinin polyclonal antisera to matrix protein as well as polyclonal and monoclonal antisera to nucleoprotein were used. The nasopharyngeal aspirates were shown to contain virus-specific antigens in the amounts sufficient for influenza virus detection with polyclonal and monoclonal antibodies. There was a good correlation between chromophore results by EIA and fluorescence level in LIFA. The sensitivity of the test systems used (EIA and LIFA) was shown to be insufficient for the detection of virus-specific material in the nasopharyngeal washings. Besides, there was no correlation between the results obtained by different test systems. It was concluded that nasopharyngeal aspirates should be collected for rapid influenza diagnosis by EIA and LIFA.

Antibodies, Monoclonal↗
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[Use of an immunoperoxidase method in situ for detecting recombinant clones of vaccinia virus containing an expressed gene of influenza viral hemagglutinins].

The vaccine L-IVP strain of vaccinia virus (VV) was used to construct the recombinant viral clones containing the influenza A hemagglutinin gene. The recombinant T plasmid was obtained with HA gene inserted in the vector pGS-20 (B. Moss) under the 7.5 K promoter of VV. A homologous recombination technique was used to insert the gene with the flanking TK sequences into vaccinia virus genome. The recombinant clones were selected by dot-hybridization with [32P]-labeled HA-probe. These recombinants were analysed for HA gene expression by the indirect immunoperoxidase method in situ using the peroxidase conjugate of the staphylococcal A-protein. This technique allows to obtain stable stained preparation and analyse the protein behavior at the ultrastructural level.

Clone Cells↗
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[Isolation and characteristics of monoclonal antibodies to influenza virus types A and B].

Monoclonal antibodies (MCA) to influenza type A (10F) and B (5H and 6H) viruses have been prepared. By immunoblotting method, MCA 10F were found to be specific for NP-protein of influenza A virus, and MCA 5H and 6H to be specific for hemagglutinin of influenza B virus. It was established that the 10F clone interacted with all the investigated influenza A virus strains with different antigenic formulae (H1N1, H2N2, H3N2) and could be used for typing of this virus type. Clones 5H and 6H react specifically with hemagglutinins of influenza B viruses isolated in 1940, 1979, and 1983 which makes them useful for diagnosis of influenza B.

Animals↗
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[Effect of a new antiviral compound 1-morpholinomethyltetrahydro-2(1H)-pyrimidinone on the interaction of influenza virus proteins with flat lipid membranes].

The antiviral preparation 1-morpholinomethyltetrahydro-2(1H)-pyrimidinone (abbreviation DD-13) inhibiting reproduction of certain enveloped viruses belonging to groups of orthomyxoviruses and alphaviruses, influenza type A viruses among them, inhibited adsorption and insertion of influenza virus M protein into model bilayer lipid membranes. The preparation did not interact directly with lipid bilayers but, after pretreatment of M protein with it, inhibited M protein interaction with the membranes: adsorption and insertion into the bilayer DD-13 did not affect the interaction of influenza virus surface glycoproteins with the model lipid membranes. It is concluded that the DD-13 preparation, not interacting with the membranes directly, in native systems may modify the protein-lipid interactions at the stages of virus penetration into the cell, penetration of M protein-coated nucleocapsid into the cell nucleus through the nuclear membrane, as well as at the stage of virus particle assembly on the plasma membrane of the infected cell.

Adsorption↗
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[The molecular mechanism of the antiviral action of 2-(1'-aminoethyl)-bicyclo (2.2.1) heptane hydrochloride].

The interaction of 2-(1'-aminoethyl)-bicyclo (2.2.1) heptane hydrochloride possessing anti-influenza activity with flat bilayer lipid membranes was studied. The compound was shown to adsorb and build-in into the lipid bilayer changing the charge, modulus of transverse elasticity, and ion permeability of the membranes. Pre-addition of the preparation into the medium washing the membrane prevents interaction of influenza virus matrix protein with the membrane. The compound also destroys M-protein aggregates in the aqueous phase. The possible role of the observed effect of the compound in its antiviral action is discussed.

Adsorption↗
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[Immunoenzyme test system based on antibody F(ab)2-fragments for detecting the NP protein of the influenza virus].

A test system has been proposed for the detection of influenza A virus NP protein by solid-phase enzyme immunoassay in a modified system of "double" antibodies: the primary antibodies were F(ab)2-fragments of immunoglobulin class G isolated from rabbit antiserum specific for NP protein which was also used as the source of secondary antibodies. The antigen-bound antibodies were detected by protein A (from St. aureus) conjugate with horseradish peroxidase. The high specificity and sensitivity of the test system (0.3 ng/ml NP protein) and a minimal level of nonspecific reactions were demonstrated. The advantages of this method are discussed.

Animals↗
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[Effect of remantadine and amantadine on the interaction of influenza virus proteins with model lipid membranes].

The influence of adamantane derivatives (remantadine and amantadine) on the surface charge of lipid bilayer when they are adsorbed on an artificially formed bilayer lipid membrane (BLM) was studied. The presence of the final number of binding centres on the BLM surface for remantadine and amantadine molecules and the cooperative nature of interaction of these compounds with the lipid bilayer were demonstrated. The features of interaction of influenza virus proteins isolated from the virion--M protein and a mixture of surface glycoproteins--were studied. Both antiviral compounds were shown to reduce markedly M protein adsorption on the lipid bilayer and to affect negligibly the sorption properties of the surface glycoproteins. Remantadine molecules interact directly with M protein molecules reducing their hydrophobic properties and, thereby, reducing the affinity of this viral polypeptide to lipid bilayer.

Adamantane↗
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[Effect of the matrix protein of the influenza virus on oxidative phosphorylation in preparations of isolated liver mitochondria from white mice].

The effect of matrix proteins (M proteins) of influenza A/MRC-11 and B/Lee viruses on oxidative phosphorylation in preparations of isolated mitochondria of white mice liver was studied. M proteins were shown to cause disorders in the system of oxidative phosphorylation leading to reduction of the coefficient of respiratory control and amplification coefficient recorded by the polarographic method. It was also shown that an anti-influenza compound 2-(1'-aminoethyl)-bicyclo(2.2.1)heptane hydrochloride reduced the damaging effect of influenza virus A/MRC-11 M protein but had no influence on the effect on mitochondrial respiration of influenza B/Lee virus M protein.

Animals↗
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[Enhanced immunogenicity of influenza virus surface proteins when incorporated into liposomes].

A method for incorporation of influenza virus surface proteins into monolayer liposomes has been developed on the basis of removal of dialysed non-ionic detergent (beta-octyl glucoside) from a mixture of viral proteins and lipids. The resulting lipid-protein complexes (virosomes) are similar to intact virus particles by their morphological parameters. The immunogenicity of hemagglutinin incorporated into virosomes was significantly higher than that in the preparation of surface proteins isolated from virions which was inoculated into mice with or without complete Freund's adjuvant. The high immunogenicity of hemagglutinin incorporated into liposomes correlated with their high hemagglutinating activity. The possibility of using virosomes as subunit influenza vaccines is discussed.

Animals↗
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[Interaction of isolated influenza virus hemagglutinin with flat lipid membranes].

The interaction of isolated influenza virus hemagglutinin with cell membranes was studied on the model of flat lipid membranes from lecithin and azolectin. Protein molecules were shown to adsorb on the membrane, and permeability of the latter increases when a certain concentration is reached. At the stages of adsorption and penetration into the lipid bilayer the hemagglutinin (HA) showed positive cooperation in interaction with the membranes. At the stage of HA adsorption on the membrane in the presence of M protein, transmembrane interaction occurs between M-protein and HA. It is concluded that molecular mechanisms participate in the HA interaction with cell membrane of the infected host cell.

Adsorption↗
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[Isolation of the internal proteins of the influenza virus by preparative polyacrylamide gel electrophoresis for obtaining monospecific antisera].

The principal possibility of isolation of internal proteins (M and NP) of influenza type A (H1N1 and H3N2) and B viruses by SDS-PAG preparative electrophoresis and preparation of monospecific antisera to these proteins was demonstrated. The resulting preparations may be used for testing the biological objects by enzymeimmunoassay.

Animals↗
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[Development of an immunoenzyme test system for the quantitative demonstration of influenza virus hemagglutinin in biological specimens].

A test system for the detection and quantitation of influenza virus hemagglutinin (HA) in biological specimens by solid-phase enzyme immunoassay (SPEIA) is described. The test system implies sequential adsorption on polystyrene base of anti-HA guinea pig gammaglobulins, detectable HA-containing antigen, monospecific anti-HA rabbit serum, and peroxidase antirabbit conjugate. Adsorption of the HA-containing antigen is run in the presence of a high concentration of nonionic detergent. The developed method is highly sensitive (0.3-0.5 ng in a specimen) and permits the detection and quantitation of HA in whole influenza virions, preparations of surface glycoproteins and in preparations of hemagglutinin recovered from virus particles. The analysis (after pre-sensitization of the polystyrene adsorbent) takes from 2 to 21/2 hours. The possibility of using the developed test system for control and standardization of inactivated influenza vaccines (whole-virion, split, and subunit) is discussed.

Animals↗
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[Role of carbohydrates in the manifestation of the hemagglutinating and neuraminidase activities of the influenza virus].

Suspensions of influenza virions (MRC-11, X-7, and A/PR8/34 strains) were treated with a mixture of glycosidases including alpha-D-mannosidase, alpha-L-fucosidase, beta-D-galactosidase, and beta-D-N--acetylglucosaminidase for the study of the influence of carbohydrate part of influenza virus surface glycoproteins on the hemagglutinating and neuraminidase activity. The availability of oligosaccharide chains for the effect of glycosidases was found to change in relation to the functional status of the glycoprotein molecule. When carbohydrate residues were cleaved from neuraminidase in virus particle, its enzymatic activity did not change, whereas the hemagglutinating activity of virions after treatment with glycosidases decreased more than 50-fold.

Carbohydrates↗