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Biomedical subjects

A M Egorov

Publications and source records attributed to A M Egorov.

At least 91 records · Page 5Linked to original sources
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

[Cooperative effects during interaction of monoclonal antibodies with insulin dimers].

The interaction of monoclonal antibodies of three types with the ATP-labeled insulin dimer was studied by the luminescent immunocofactor method. It was shown that the effective equilibrium binding constant increases at equimolar antigen/antibody concentrations. This can be due to the formation of multimolecular complexes between the antigens and antibodies. The feasibility of the binding constants increase during the formation of cyclic tetramolecular complexes is considered. A theoretical model for the description of interaction between the bivalent antigen and antibodies based on the increase of the binding constant during the formation of cyclic complexes is proposed. The coefficients of binding constant increase for antigens belonging to three different clones were calculated.

Animals↗
PubMed1987

[Immunoenzyme analysis of human IgG in the kinetic mode].

A kinetic sandwich enzyme-linked immunosorbent assay for the detection of human IgG (used as a model antigen) has been developed. Rabbit antihuman IgG has been used both for coating polystyrene microtitration plates and for the preparation of the conjugate of anti-human IgG with horse-radish peroxidase. The kinetics of the reaction of the antigen and the antibody-peroxidase conjugate with the reagents immobilized on polystyrene plates has been studied. The assay is optimized with respect to its sensitivity and the duration of intervals for every stage of the assay. The optimal time of the assay is about 10-15 minutes. The correlation between sensitivity and the duration of every stage of the assay has been established.

Adsorption↗
PubMed1987

[Immunoenzyme method of sequential saturation for determining antigens using the model of insulin].

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.

Adsorption↗
PubMed1986

NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1. Stabilization against temperature and urea induced inactivation.

Chemical modification of the NAD+-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1 results in considerable enzyme stabilization towards urea and temperature induced inactivation. The stabilizing effect was shown to originate from the suppression of hydrogenase tetramer dissociation. The magnitudes of the stabilizing effects (5-fold and more) were in agreement with the values predicted on the basis of the enzyme thermoinactivation mechanism postulated earlier. Hydrophobic interactions are considered to be critical for the stability of the enzyme quaternary structure. Various methods of hydrogenase immobilization were tested. The enzyme was immobilized with a high retention of activity on aminated silochrom via its carboxylic groups.

Acrolein↗
PubMed1986

[Determination of binding constants of antibodies fractionated according to their affinity to insulin].

The number of populations of antibodies differing in their affinity for insulin was determined in guinea pig antiserum. The antibody fractions differing in their affinity were obtained by affinity chromatography on an antigenic sorbent. Elution with a stepwise pH gradient from 4 to 2 differing in the number and form of the steps resulted in 10 fractions of antibodies obtained from individual animal sera. The binding constants of fractionated antibodies were determined by a bioluminescent immunocofactor method. With a change in elution pH from 4 to 2, the equilibrious binding constant of antibodies increased from 10(6) to 5 X 10(9) M-1.

Animals↗
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↗
PubMed1986

[Properties of the human anti-IgG-IgG system in various modifications of immunoenzyme analysis].

The physico-chemical characteristics of EIA, and in particular sensitivity, accuracy, reproducibility, can undergo essential changes with variations occurring in the course of the experiment, which makes it necessary to optimize conditions at each stage of the assay. For this purpose the human anti-IgG-IgG system has been studied in two modifications of EIA: the sandwich and competitive methods.

Antibodies, Anti-Idiotypic↗
PubMed1985

Biosynthesis, isolation and properties of NAD-dependent formate dehydrogenase from the yeast Candida methylica.

NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Candida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a two-step procedure, involving a sequential application of DEAE-cellulose ion-exchange chromatography and Sephadex G-200 gel filtration, and a single-step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. It consisted of two identical subunits with a relative molecular mass of 46 000, each containing one -SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 X 10(-4) M for NAD and 1.3 X 10(-2) M for formate. Formate dehydrogenase was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoate, cyanide and azide.

Aldehyde Oxidoreductases↗
PubMed1985

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.

Animals↗