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B B Dzantiev

Publications and source records attributed to B B Dzantiev.

At least 19 recordsLinked to original sources
PubMed2002

Antiperoxidase antibodies enhance refolding of horseradish peroxidase.

The effect of monoclonal antibodies on protein folding was studied using horseradish peroxidase refolding from guanidine hydrochloride as a model process. Among the five antiperoxidase clones tested, one was found to increase the yield of catalytically active peroxidase after guanidine treatment. The same clone also increased the activity of the native peroxidase by a factor of 2-2.5. While peroxidase refolding under standard conditions resulted in the recovery of only 7-8% of the initial catalytic activity, antibody-assisted refolding increased the yield to 50-100% (or 20-40% from the activity of native enzyme with antibodies). Kinetics of autorefolding and antibody-assisted refolding differed significantly. In the course of autorefolding the catalytic activity was recovered within the first 2.5 min and did not change further within a 2.5- to 60-min interval, whereas in the course of antibody-assisted refolding maximal catalytic activity was attained only in 60 min. The yield of active peroxidase for the antibody-assisted refolding depended linearly on the antibody concentration. The observed effect was strongly specific. Other antiperoxidase clones tested as well as nonspecific antithyroglobulin antibody affected neither kinetics, no the yield of peroxidase refolding.

Animals↗
PubMed2000

[A noninstrumental Immunoassay based on colloidal dyes].

Detecting labels based on water dispersions of colloidal textile dyes were developed that are useful in various analytical and diagnostic test systems for a simple visual assessment of the assay. Colored water-insoluble particles of dyes were used for the sorptional immobilization of streptavidin on their surface. The resulting streptavidin-dye (STR-DYE) complexes possessed a high visualizing capacity and were used for the combined detection of pesticides (simazine and 2,4-dichlorophenoxyacetic acid) by noninstrumental immunoassay (DYE-comb-assay, competitive dot-immunoassay in the comb format). The detection limits and the duration of our DYE-comb-assay (4 ng/ml, 20-25 min), HRP-comb-assay (competitive dot-immunoassay in the comb format using the enzymic conjugate of STR with horseradish peroxidase) (16 ng/ml), and the traditional competitive ELISA (12-16 ng/ml, 1.5 h) were compared. This DYE-comb-assay is simple enough and can be used under field conditions.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1999

Immunoassay techniques for detection of the herbicide simazine based on use of oppositely charged water-soluble polyelectrolytes.

Linear water-soluble polyelectrolytes, i.e., poly(methacrylate) polyanion and poly(N-ethyl-4-vinylpyridinium) polycation, were used as carriers for the reactants in immunoassay. The strength of ionic forces through distance and the cooperative binding of oppositely charged chains, the carriers interact with each other at an extremely high rate and affinity. These properties of the polyelectrolytes made it possible to carry out the immunochemical steps of the assay in true solution and then to quickly separate the resulting products from the reaction mixtures. The above approach was applied to an assay for the herbicide simazine. Both enzyme-linked immunosorbent assay (ELISA) and dot blot formats of the immunoassay were evaluated. In the ELISA format, the polycation was adsorbed on the surface of a microtiter plate. A tracer antigen (simazine) was allowed to interact in solution with components of the reaction mixture containing simazine-peroxidase conjugate, specific antibodies, and staphylococcal protein A conjugated with the polyanion, and then the mixture was added to the immobilized polycation. Quick separation of the immunoreactants was achieved due to formation of interpolyelectrolyte complexes between polycation and polyanion molecules. After washing, the microplate wells were filled with a solution of substrate, and the optical density of the reaction products was measured. In the second format, a solution of the same reaction mixture (after incubation) was filtered through a porous membrane, with the polycation adsorbed. The subsequent addition of substrate led to the development of colored spots. Sensitivity of the dot blot format was close to that of the traditional ELISA format using the same reactants, i.e., 0.5 ng/mL. However, the assay was much faster (assay time decreased from 100-120 to 45 min). Sensitivities of the dot immunoassay were 1 ng/mL for densitometric detection and 10 ng/mL for visual detection with a duration of 20 min. The techniques developed here were used for simazine determination in water, milk, and juices.

Electrolytes↗
PubMed1999

Laccase from Coriolus hirsutus as alternate label for enzyme immunoassay. Determination of pesticide 2,4-dichlorophenoxyacetic acid.

A new label--laccase from the fungus Coriolus hirsutus--was applied for solid-phase enzyme-linked immunosorbent assays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems permit the detection of 2,4-D in concentrations down to 10-20 ng/mL; time of the assays is 1.5-2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1997

Interaction between antibodies and hapten-protein conjugates of different composition: theoretical predictions and experimental data.

Mathematical models of competitive ELISAs with labelled antibody and with labelled antigen taking into account bivalent interactions between antibodies and hapten-protein conjugates were developed and analyzed. It was shown that in the kinetic model of the immunochemical reaction the conjugate composition influenced the amplitude of detected signal but not ELISA sensitivity. In the equilibrium model decreased sensitivity correlated with bivalent complexes formation. The predictions were tested experimentally using 2,4-dichlorophenoxyacetic acid (2,4-D) and testosterone as haptens. It was confirmed that increasing of the hapten : protein ratio resulted in formation of bivalent complexes with antibodies. The equilibrium binding constants for these complexes were two orders of magnitude higher than for monovalent ones. Optimal conjugate compositions have been chosen for ELISA of these haptens.

Animals↗
PubMed1995

[Interaction of cytokines with cellular receptors].

The cytokinin family includes biologically active polypeptide molecules secreted by haemopoietic and immunocompetent cells which control cell proliferation and differentiation. Cytokinin interactions with specific receptors of the cell surface results in oligomerization of these receptors, i.e. in association of two or more membrane molecules. It is becoming obvious that oligomerization of receptors is an indispensable stage in the manifestation by cytokinins of their biological activity. In this context, studies of regularities of cytokinin receptor interactions resulting in receptor oligomerization is important for both elucidation of molecular mechanisms underlying kinin action and construction of compounds having the properties of agonists (or antagonists) of cytokinin-induced oligomerization of membrane receptors. A conclusion is draw about the important role of polyvalent cytokinin interactions with cell receptors in the initiation of oligomerization and subsequent formation of functionally active receptor complex.

Cytokines↗
PubMed1994

A new visual enzyme immunoassay of methamphetamine using linear water-soluble polyelectrolytes.

A new visual enzyme immunoassay (EIA) technique has been developed. Oppositely charged synthetic linear water-soluble polyelectrolytes (poly-N-ethyl-4-vinyl-pyridine as polycation and polymethacrylate as polyanion) were used as carriers for reagent immobilization. The ability of these molecules to form an insoluble complex was applied for the separation of bound and free components of the immunoassay reaction mixture. This approach was realized in methamphetamine visual EIA. In the first stage of the assay two specific reactions took place during incubation of the analytical reagents with the probe to be analyzed: (1) competition between methamphetamine and hapten conjugated with peroxidase for the interaction with specific antibodies and (2) interaction of these antibodies with the protein A-polymethacrylate conjugate. As a result of these reactions the (polyanion-protein A)-antibody-(hapten-peroxidase) complex was formed. Then the reaction mixture was filtered through an Ultrabind membrane (0.45 microns) with adsorbed poly-N-ethyl-4-vinylpyridine, and the immunological complexes were immobilized to the membrane by electrostatic interaction. The level of peroxidase binding on the membrane was measured by diaminobenzidine substrate. The system described was optimized to achieve both high rapidity (20 min) and an appropriate sensitivity (0.4 micrograms/ml) for methamphetamine assay.

Animals↗
PubMed1990

Polyvalent interaction of antibodies with bacterial cells.

We have studied the physical-chemical characteristics of the interaction of peroxidase-labelled rabbit antibodies with Bacillus sp. bacterial cells. The antibodies are able to bind bivalently with two antigen sites on the bacterial cells with the formation of intramolecular "cyclic" complexes. A kinetic model is proposed suggesting the existence of monovalent and bivalent cell surface antigens. The equilibrium constant of the bivalent IgG binding to the bacterial cell is by two orders of magnitude higher as compared to monovalent Fab fragments. The intramolecular reaction between the free active site of the monovalently bound antibody and a free antigen site on the cell surface is the rate limiting step of the polyvalent interaction. Formation of the cyclic complexes seems to be accompanied by essential tension of bonds and deformation of the IgG molecule. Agglutination of bacterial cells was also studied. The cell agglomerate size dependence on the antibody concn has a threshold. Agglutination proceeds under conditions where the antigen-antibody binding on the cell surface is far from equilibrium.

Agglutination↗
PubMed1988

[Method of determining proteolytic activity by using a conjugate of bovine serum albumin with peroxidase].

The authors propose a method for determination of proteolytic activity, based on the hydrolysis of peroxidase-labeled molecules of bovine serum albumin immobilized on the surface of polystyrene microassay plates with the subsequent determination of peroxidase activity on the carrier or in the solution. The optimum conditions for the sorption of the labeled substrate have been established. The method permits the determination of bacillary alkaline protease at a concentration of 01. microgram/ml within 45 minutes. The determination of four proteases has demonstrated that this method shows good correlation with the routine one (r = 0.98), but is more sensitive and less time- and labor-consuming.

Amylases↗
PubMed1987

[Determination of beta-galactosidase activity on nitrocellulose plates using 5-bromo-3-indolyl-beta-D-galactopyranoside and tetrazolium salts].

A simple and convenient technique has been developed for detection of beta-galactosidase from E. coli on nitrocellulose sheets using a mixture of 5-bromoindol-3-yl-beta-D-galactopyranoside and nitro blue tetrazolium, which enables rapid detection of fmole (10(-15) mole) quantities of the enzyme at pH 9.5. The technique has the following advantages: the substrates are stable for a long period; reaction products give non-fading intense blue colour, resolution is extremely good with essentially no diffusion.

Chemical Phenomena↗
PubMed1987

[Patterns in the immunoenzyme analysis of bacterial cells].

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.

Adsorption↗
PubMed1987

[Modification of the immunoenzyme analysis of haptens as exemplified by testosterone determination].

The solid-phase enzyme immunoassay for testosterone (TS), permitting the determination of this hormone at concentrations of up to 0.5 ng/ml, has been developed. The method comprises the adsorption of TS conjugated with soya trypsin inhibitor in the wells of a standard polystyrene assay plate, competition between adsorbed TS and TS under test for the binding sites of specific antibodies, and the detection of antibodies bound to the carrier by means of peroxidase-labeled antispecific antibodies. Antisera to TS have been obtained by the immunization of rabbits with TS conjugated with bovine serum albumin of a known composition. These antisera are specific to TS and do not interact with estrogens and progesterone. The study of their cross reactions with eleven TS derivatives has demonstrated that antibodies reveal the presence of structural changes in ring D of the molecule of TS and are insensitive to variations in ring A. The determinant comprising the 17-OH-group essentially contributes to the binding of antibodies.

Adsorption↗
PubMed1986

[Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay].

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.

Chemical Phenomena↗
PubMed1986

[Development of a solid-phase immunoenzyme method for determining the antibody level in the blood sera of persons vaccinated with influenza vaccine].

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.

Adsorption↗
PubMed1986

[Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis].

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.

Antibodies, Bacterial↗
PubMed1985

[Cell wall antigenic determinants in ribosomal preparations of Streptococcus group A].

As revealed by double immunodiffusion and the competitive immunoenzyme assay, the immunization of animals with ribosomes isolated from group A streptococci leads to the formation of antibodies to M-protein and does not lead to the formation of antibodies to A-polysaccharide and lipoteichoic acid, which is seemingly caused by the presence of M-protein in ribosomes. Antiribosomal serum has permitted the detection of more antigenic determinants in M-protein than commercial antiserum to homologous M-protein. The immunoenzyme assay for the quantitative determination of ribosomes has been developed.

Animals↗