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Biomedical subjects

A M Egorov

Publications and source records attributed to A M Egorov.

At least 55 records · Page 3Linked to original sources
PubMed1996

Chemiluminescent immunoenzyme biosensor with a thin-layer flow-through cell. Application for study of a real-time bimolecular antigen-antibody interaction.

A simple flow enzyme system for real-time continuous monitoring of interaction of biological molecules has been developed. It relies upon a thin-layer flow-through cell placed directly into the measuring compartment of the luminometer. One ligand (antibody) is immobilized on the inner surfaces of the flow cuvette, and a second ligand (antigen) labeled with a peroxidase molecule moves through the flow cell. The quantity of the complex on the surface of the cell may be monitored by measurement intensity of chemiluminescence in the reaction of peroxidase label with substrates (p-iodophenol, luminol and hydrogen peroxide). In such a way one can detect in a real-time regime the kinetics of association (or dissociation) of the complex labeled ligand-receptor on the surface of the cuvette. Due to the small thickness of the flow cell the diffusion limitations of interaction for two kinds of biomolecules (soluble and immobilized) are negligible, so the resulting intensity of chemiluminescent signal reflects the kinetics of interaction between soluble and immobilized components. The system may be successfully used for molecular recognition studies, analyzing the kinetics of bimolecular interaction and for concentration determination.

Antigen-Antibody Reactions↗
PubMed1994

Horseradish peroxidase isozyme C. A comparative study of native and recombinant enzyme produced by E. coli transformants.

The purification and refolding of the recombinant horseradish peroxidase produce by E. coli transformants are described. The recombinant enzyme is of 34 kDa and has an isozyme spectrum similar to Sigma type VI horseradish peroxidase. The specific activity of the refolded peroxidase is of about 2000 U/mg with ABTS as a substrate. The recombinant and native enzyme are similar with respect to their catalytic properties in the reaction of enhanced chemiluminescence. Operational and thermal stability of the refolded peroxidase is two to three times lower than for the native one.

Enzyme Stability↗
PubMed1993

Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements.

Two mutants of bacterial formate dehydrogenase from Pseudomonas sp.101 (EC 1.2.1.2, FDH)-C255S (FDH-S) and C255M (FDH-M), were obtained and its properties were studied. Both mutations provided the high resistance to inactivation by Hg2+. Slow inactivation of mutants by DTNB reveals the presence in FDH molecule of another essential cysteine residue. Specific activities of FDH, FDH-S and FDH-M were 16, 16 and 9.5 U/mg of protein, respectively. Km on formate was 7.5, 7.5 and 20 mM and Km on NAD(+)-0.1, 0.3 and 0.6 mM for FDH, FDH-S and FDH-M, respectively. Mutations of Cys255 on Ser or Met resulted in increasing of enzyme stability at 25 degrees C and decreasing of thermostability (above 45 degrees C). Data obtained show that Cys255 is unique residue for providing both enzyme thermostability and catalytically optimal binding of coenzyme.

Amino Acid Sequence↗
PubMed1993

New immunoassay technique using antibody immobilized on a membrane and a flow cuvette as reaction vessel.

Immunoenzymatic detection systems have been developed using human IgG as a model antigen. A membrane with covalently immobilized specific antibodies was placed into a specially constructed ultranarrow flow cuvette and solutions containing the antigen and antibody-peroxidase conjugate were then successively passed through the flow capillary cell. After washing, the membrane was placed into the substrate solution and the intensity of developed colour on the membrane was recorded visually or by a reflection spectrophotometer. The lower detection limit was about 5 x 10(-11) M and the overall analysis time was 10 min. Photoimmobilization was used to immobilize the antibody and thereby permitting control of the protein surface concentration on the membrane as well as the dimensions and shape of the activated region.

Flow Injection Analysis↗
PubMed1993

Detection system for membrane immunoassay based on the trapping of a highly colored intermediate of the peroxidase reaction.

The oxidation of ortho-dianisidine by membrane bound horseradish peroxidase in the presence of sodium dextran sulfate affords a dark-green insoluble product, identified as an unstable meriquinone intermediate previously reported in literature. Cationic and unsubstituted dextrans do not stabilize the intermediate. The highest yield of the intermediate is observed at pH 4.0-5.0 and concentration of dextran sulfate ca. 0.5%. New highly sensitive detection system for peroxidase has been developed on the basis of ortho-dianisidine oxidation to the meriquinone intermediate in the presence of sodium dextran sulfate. Under certain conditions, with lowered sodium dextran sulfate concentrations, a progressive further oxidation of the green intermediate to the yellow-brown final product is observed on passing to higher enzyme concentrations. This finding opens a possibility to develop a detection system in which the color of the mixture of reaction products serves as a measure of the enzyme concentration.

Chemistry Techniques, Analytical↗
PubMed1993

Preparation of conjugates of progesterone with bovine serum albumin in the reversed micellar medium.

Progesterone-BSA (bovine serum albumin) conjugates which contain up to 47 steroid molecules linked to a BSA molecule have been prepared by the activated ester method, the conjugation step being carried out in reversed micellar solutions of sodium di(2-ethylhexyl) sulphosuccinate (AOT) in octane. The number of incorporated steroid molecules increases on passing to increased water/AOT ratios at the given activated steroid/BSA ratio. The results show that the reversed micellar medium would be useful for preparation of conjugates of hydrophobic steroids with proteins in respect to simplicity and ease in obtaining conjugates with high steroid/protein ratios.

Dioctyl Sulfosuccinic Acid↗
PubMed1993

Formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101: gene cloning and expression in Escherichia coli.

Using two degenerate 20- and 23-mer oligonucleotide probes, the gene encoding NAD(+)-dependent formate dehydrogenase (FDH) (EC 1.2.1.2) was shown to lie within a 3.5 kb PstI fragment of the chromosomal DNA of methylotrophic bacterium Pseudomonas sp. 101. A phasmid library was prepared in the lambda pSL5 vector with partial EcoRI-digested DNA from Pseudomonas sp. 101. The 12 clones selected contained three types of phasmid: lambda pFDH1, lambda pFDH2 and lambda pFDH3 (with inserts of 15, 17.6 and 18.5 kb respectively). The inserts contained the same 15 kb EcoRI and 3.5 kb PstI fragments and included the complete FDH gene. Further subcloning of the insert resulted in plasmid pFDH2 and a 2.32 kb HindIII-BgIII fragment. Active enzyme was expressed in Escherichia coli TG1 (pFDH2) strain under control of a lac promoter.

Amino Acid Sequence↗
PubMed1993

[Determination of phenobarbital by polarized fluoroimmunoanalysis. The effect of immunogen structure and tracer on specificity and detection limit].

The influence of the immunogen's tracer's structure on properties of antibodies to phenobarbital was studied by the polarisation fluoroimmunoassay method. It was shown that in homogeneous method of polarisation fluoroimmunoassay the titer of antibodies to phenobarbital (1/1000-1/10,000) is affected by the position of linker binding the antigen with carrier-protein and by the structure of the linker. The affinity constant ((1-4) x 10(9) M-1), cross-reactivity, limit of the detection (0.6-2.6 micrograms/ml) phenobarbital with different combinations of antibodies and tracers were calculated. The antisera grew the more heterogeneous in affinity and specificity, the longer became the linker. The more heterogeneous antibodies are preferable in the class specific assay of barbiturates.

Animals↗
PubMed1992

[A comparative analysis of the immunochemical determination of gentamycin by fluorescence polarization and quenching].

A polarization fluorescence immunoassay (PFIA) for gentamicin with using a set of reagents made in this country was developed. One ml of fluorescein-labeled gentamicin (50 nM) and 100 microliters of antiserum are added to 50 microliters of the sample and the fluorescence polarization is measured. The time of the assay is 10 to 15 minutes, the range of the measurable concentrations is 0 to 800 ng/ml, the sensitivity of the method is 5 ng/ml and the accuracy is 5.8-10.3 per cent. A fluorescence quenching immunoassay (FQIA) for gentamicin was also developed. Determination of gentamicin by the FQIA does not require the use of a specific polarization fluorimeter. Its linear calibrating dependence is more convenient. However, its accuracy and sensitivity are 3 times lower than those of the PFIA.

Drug Monitoring↗
PubMed1991

A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays.

The properties of a peroxidase from Arthromyces ramosus (ARP) in the chemiluminescent reaction of luminol oxidation have been studied. These were compared with the properties of horse radish peroxidase (HRP) in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence (ECL) reaction. By means of the stop-flow technique, ARP was shown to have an enzymatic activity toward luminol higher than that toward HRP. ARP can efficiently catalyze luminol oxidation in the absence of substrate enhancer. pH and substrate concentrations were optimized to determine ARP with the highest sensitivity. The detection limit of ARP was 5 x 10(-13) M, the same as that for HRP in the ECL reaction. The data on the use of ARP as a label in enzyme immunoassay of human IgG are presented. ARP was shown to have all the advantages of HRP as a label in chemiluminescent enzyme immunoassays: (i) high signal intensity, (ii) slow decay of luminescence, (iii) high signal/noise ratio, and (iv) as a consequence of (i)-(iii), high detection sensitivity. However, the low thermostability of ARP can limit the potential fields of its application.

Enzyme Stability↗
PubMed1991

[Determination of the primary structure of Eco-R1-fragment No. 5 of the rat tyrosine aminotransferase gene in the "Applied Biosystems" 370 automatic sequencer].

EcoRI-fragment No. 5 of the rat tyrosine aminotransferase gene containing exons K, L, intron 11, and a part of the 3'-nontranslatable region was digested with several restriction endonucleases (BspRI, Sau3A, BamHI, Ecl136II, AluI), the subfragments obtained were cloned into M13mp19 and sequenced using the Sanger technique with dye-labelled primers on the automated sequencer "Applied Biosystems", model 370A. The sequences were combined by means of a PC-GENE package and original programs to yield the primary structure (1064 b. p.) of the above fragment No. 5, adjacent to the previously sequenced EcoRI-fragment No. 4 (3677 b.p.).

Amino Acid Sequence↗