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1.
Biomed Khim ; 57(5): 554-61, 2011.
Article in Russian | MEDLINE | ID: mdl-22629606

ABSTRACT

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range 60-1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radio-immunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.983, respectively. The developed test system is stable for at least 9 months when stored at 4 degrees C and can be used in clinical practice.


Subject(s)
Cyclosporine/blood , Enzyme-Linked Immunosorbent Assay , Immunosuppressive Agents/blood , Antibodies, Monoclonal , Humans , Limit of Detection , Reagent Kits, Diagnostic
2.
Prikl Biokhim Mikrobiol ; 43(4): 471-8, 2007.
Article in Russian | MEDLINE | ID: mdl-17929576

ABSTRACT

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.


Subject(s)
Anti-Bacterial Agents/analysis , Escherichia coli/metabolism , Serum , Anti-Bacterial Agents/pharmacology , Biosensing Techniques , Escherichia coli/drug effects , Gentamicins/analysis , Gentamicins/pharmacology , Immune Sera , Luminescence , Streptomycin/analysis , Streptomycin/pharmacology , Tetracycline/analysis , Tetracycline/pharmacology
3.
Vopr Med Khim ; 46(4): 410-8, 2000.
Article in Russian | MEDLINE | ID: mdl-11075424

ABSTRACT

The conjugation of the drugs with vector molecules enables to obtain therapeutic preparation, which may be transported to the selected target organ. In the present work the methods of conjugation of antineoplastic enzyme L-lysine alpha-oxidase with antibodies were elaborated. Conjugates were worked out through the attachment of amino groups on the antibody surface either with the aldehyde groups which were created in L-lysine alpha-oxidase molecule (0.2% of initial enzymatic activity) or with the aldehyde groups of cross-linking molecules. Maximal (78%) L-lysine alpha-oxidase activity in conjugates was observed when oxidized peroxidase which contained the aldehyde groups was used as crosslinking agent. The glutaraldehyde method yielded 70% of initial enzyme activity.


Subject(s)
Antibodies/chemistry , Lysine/chemistry , Animals , Cross-Linking Reagents , Drug Carriers , Equidae , Glutaral , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/immunology , Immunoglobulin G , Mice , Oxidation-Reduction , Rabbits , Spectrophotometry
4.
Probl Endokrinol (Mosk) ; 40(4): 48-51, 1994.
Article in Russian | MEDLINE | ID: mdl-7971912

ABSTRACT

A quick reliable homogeneous polarization fluoroimmunoassay (PFIA) of progesterone was developed. The assay is carried out with Abbott TDx (USA) polarization fluorometer and it takes 5-7 min to analyze 10 samples by this method. The range of progesterone concentrations determined is 1 to 1000 ng/ml. Fluorescein labeled progesterone-3-carboxymethyloxime which was used as tracer (labeled antigen) during analysis has been synthesized and purified. Two types of PFIA were developed: one making use of rabbit antiserum to progesterone-3-carboxymethyloxime conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), in the other antiserum to progesterone-11-hemisuccinate-BSA (or KLH) is used. Different combinations of the tracer and antibodies were used. The sensitivity of heterologous PFIA (with antibodies to immunogen heterologous to tracer by structure) was higher than that of homologous PFIA. The test is sufficiently sensitive and specific. The method is particularly valuable for determination of progesterone in model solutions (using buffer standards).


Subject(s)
Fluorescence Polarization Immunoassay , Progesterone/analysis , Animals , Rabbits , Reproducibility of Results , Sensitivity and Specificity
5.
Steroids ; 58(11): 547-50, 1993 Nov.
Article in English | MEDLINE | ID: mdl-7505959

ABSTRACT

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.


Subject(s)
Micelles , Progesterone/chemistry , Serum Albumin, Bovine/chemistry , Dioctyl Sulfosuccinic Acid , Octanes , Spectrophotometry
6.
Anal Biochem ; 211(2): 274-8, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8317703

ABSTRACT

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.


Subject(s)
Chemistry Techniques, Analytical/methods , Dianisidine/analysis , Horseradish Peroxidase/metabolism , Dextran Sulfate , Hydrogen-Ion Concentration , Immunoenzyme Techniques , Membranes, Artificial , Nylons , Oxidation-Reduction , Spectrophotometry/methods
7.
Biull Eksp Biol Med ; 114(12): 629-31, 1992 Dec.
Article in Russian | MEDLINE | ID: mdl-1292695

ABSTRACT

The influence of met-enkephalin on specific antibody production by lymphocytes from mouse lymph nodes was studied in vitro in productive phase of immune response. It was shown that the peptide did not influence secretion of IgM-antibody to T-independent antigen-trinitrobenzensulfoacidic group, but suppressed secretion of IgG-antibody to T-dependent antigens both during primary and secondary response. The efficiency of superlow concentrations of the peptide (10(-15)-10(-14) M) for the response to ovalbumin, but not for the response to bovine gamma-globulin was shown. All effects of met-enkephalin were naloxone-reversible. The existence of individual distribution in dose-dependences of peptide action on antibody secretion in response to ovalbumin was demonstrated.


Subject(s)
Antibodies/drug effects , Enkephalin, Methionine/pharmacology , Lymphocytes/drug effects , Animals , Antibodies/analysis , Antibody Specificity/drug effects , Dose-Response Relationship, Drug , Female , Immunization/methods , Lymph Nodes/drug effects , Lymph Nodes/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Time Factors
8.
Biokhimiia ; 57(11): 1664-70, 1992 Nov.
Article in Russian | MEDLINE | ID: mdl-1336982

ABSTRACT

The binding of the opioid antagonist [3H]-naloxone to immunocompetent cells of the mouse, F1(CBA x C57B1/6), in medium 199 has been studied. The binding was reversible and reached a maximum during 15-20 min at 37 degrees C. The stereospecificity profile was proven to correspond to mu-type receptors. The binding curve was characterized by high positive cooperativity (nH = 2.3, IC50 = 5 nM). Mitogenic stimulation by Con A, SEA, and ML caused an increase in the number of receptors. Besides, stimulation by an antigen (ovalbumin) changed the binding parameters. The distribution of binding sites for naloxone on various immunocompetent cells was investigated. The maximal number of sites was found on lymphocytes of lymph nodes and bone marrow cells. A conclusion is drawn that both T- and non-T-cells play a role in naloxone binding.


Subject(s)
Antigens/immunology , B-Lymphocytes/metabolism , Mitogens , Naloxone/metabolism , T-Lymphocytes/metabolism , Animals , B-Lymphocytes/immunology , Female , Lymphoid Tissue/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Receptors, Opioid/metabolism , Substrate Specificity , T-Lymphocytes/immunology , Tritium
9.
Immunol Lett ; 32(1): 27-30, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1323526

ABSTRACT

The influence of Met-enkephalin on mitogenic stimulation of mouse splenocytes was investigated. Met-enkephalin (ME) was shown to suppress proliferation induced by Concanavalin A and activate proliferation induced by Staphylococcus enterotoxin A. Both effects were revealed at low (down to 10(-14) M) concentration of pentapeptide. Naloxone reversed ME influence on cell activation. The number of receptors for naloxone was shown to increase up to 2.5-fold during mitogenic activation. The difference in expression of various types of opioid receptors at mitogenic stimulation was demonstrated by ligand displacement experiments.


Subject(s)
Enkephalin, Methionine/pharmacology , Lymphocyte Activation/drug effects , Receptors, Opioid/physiology , Animals , Binding, Competitive , Concanavalin A/pharmacology , Diprenorphine/metabolism , Diprenorphine/pharmacology , Enterotoxins/pharmacology , Female , Mice , Mice, Inbred C57BL/immunology , Mice, Inbred CBA/immunology , Naloxone/pharmacology , Pain/physiopathology , Receptors, Opioid/drug effects , Receptors, Opioid, delta , Receptors, Opioid, mu , Sensory Thresholds , Spleen/cytology
10.
Bioorg Khim ; 17(1): 35-41, 1991 Jan.
Article in Russian | MEDLINE | ID: mdl-2064622

ABSTRACT

Properties of four types of monoclonal antibodies to horse-radish peroxidase were investigated. The dissociation constants and molecular-weight composition of the immune complexes were determined. The antibodies are shown to be directed to different epitopes on the polypeptide chain. Results of the theoretical prediction of the epitope localisation are presented. The interaction between the antibodies and peroxidase isozymes were studied.


Subject(s)
Antibodies, Monoclonal/immunology , Horseradish Peroxidase/immunology , Amino Acid Sequence , Blotting, Western , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Immunoglobulin G/immunology , Molecular Sequence Data , Sequence Homology, Nucleic Acid
11.
J Immunol Methods ; 131(2): 213-22, 1990 Aug 07.
Article in English | MEDLINE | ID: mdl-2202761

ABSTRACT

In this communication some of the advantages and constraints in the use of ELISA (enzyme-linked immunosorbent assay) procedures to evaluate antigen-antibody dissociation constants (Kd) are discussed and experimental conditions under which the effective Kd is close to the true value are proposed. Interactions between horseradish peroxidase (POD), human myoglobin and insulin with mono- and polyclonal antibodies (McAb and PcAb) were used to demonstrate that ELISA can be used to determine the average Kd, characterizing the interaction between antigens and PcAb. The Kd values obtained by ELISA were similar to those determined by luminescent immuno-cofactor analysis (LICA).


Subject(s)
Antigen-Antibody Complex/metabolism , Animals , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Horseradish Peroxidase/immunology , Insulin/immunology , Mice , Rabbits
12.
Bioorg Khim ; 16(4): 476-81, 1990 Apr.
Article in Russian | MEDLINE | ID: mdl-2165407

ABSTRACT

The influence of sodium metaperiodate concentration on kinetics and conversion degree of peroxidase carbohydrate moiety as well as the effect of the oxidation degree of the carbohydrate moiety on the composition, structure and properties of insulin-peroxidase conjugates were studied. The initial rate of peroxidase's oxidation is directly proportional to the periodate concentration; the oxidation rate constant of peroxidase carbohydrate moiety is 1.23 x 10(-3) M-1 min-1. At the molar ratio of metaperiodate to peroxidase 150:1 or higher, the maximal quantity of aldehyde groups (62 +/- 2) in the peroxidase molecule is formed and the oxidation of each carbohydrate chain leads to the formation of eight aldehyde groups. The molecular mass composition of the insulin-peroxidase conjugates was studied by HPLC. The conjugates proved to be multicomponent mixtures of oligomers (53, 83, 128, 174, 268, 440 kD and higher). The insulin-peroxidase molar ratio in the fractions of the conjugates with molecular masses higher than 83 kD is 8:1. It was shown that the affinity of insulin-peroxidase conjugates to antibodies depends on the oxidation degree of peroxidase used for production of conjugates.


Subject(s)
Carbohydrate Metabolism , Glycoproteins/metabolism , Insulin/metabolism , Peroxidase/metabolism , Immunoenzyme Techniques , Kinetics , Macromolecular Substances , Oxidation-Reduction
13.
Lab Delo ; (2): 25-8, 1990.
Article in Russian | MEDLINE | ID: mdl-1692361

ABSTRACT

Indirect solid-phase enzyme immunoassay (EIA) was used for the titration of antibodies to insulin in human blood sera. The modification suggested by the authors permits assessing the immunoglobulins G and M insulin-binding ability. The technique was optimized for all the stages of analysis and a method for estimating the insulin-binding ability of IgG in the sera diluted 1:80 and of IgM in the sera diluted 1:320 was developed. Fifty blood sera of patients with insulin-dependent diabetes mellitus were analyzed. EIA results were in good correlation with the findings of radioimmunoassay.


Subject(s)
Insulin Antibodies/analysis , Diabetes Mellitus, Type 1/blood , Humans , Immunoenzyme Techniques , Radioimmunoassay
14.
J Biolumin Chemilumin ; 4(1): 164-76, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2678913

ABSTRACT

The mechanism of peroxidase-catalysed oxidation of luminol by H2O2 was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction was also studied. The close approach of an effector molecule to the active site of the enzyme was found to inhibit the enhanced chemiluminescent reaction. Novel homogeneous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfully tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10(-9) M for IgG and 10(-11) M for T4, the overall time of the assay being 5-15 min.


Subject(s)
Immunochemistry , Immunoenzyme Techniques , Luminescent Measurements , Haptens , Horseradish Peroxidase , Humans , Immunoglobulin G/analysis , Insulin/analysis , Insulin Antibodies/analysis , Kinetics , Luminol , Oxidation-Reduction , Substrate Specificity , Thyroxine/analysis
15.
Biokhimiia ; 53(11): 1858-63, 1988 Nov.
Article in Russian | MEDLINE | ID: mdl-3251550

ABSTRACT

Monoclonal antibodies to horseradish peroxidase were obtained. The interaction of two antibody clones with the enzyme was studied. Antibodies of one clone were found to inhibit the enzyme activity during the oxidation of 2.2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) diammonium salt and the cooxidation of luminol and luciferin. The latter was concomitant with a complete inhibition of the peroxidase activity. The values of binding constants as determined by the solid phase immunoenzymatic and homogeneous methods are equal to (1.2 +/- 0.5).10(8) M-1 and (1.8 +/- 0.2).10(11) M-1, respectively.


Subject(s)
Antibodies, Monoclonal , Horseradish Peroxidase/immunology , Peroxidases/immunology , Antibodies, Monoclonal/isolation & purification , Firefly Luciferin/metabolism , Luminescent Measurements , Luminol/metabolism
16.
Article in Russian | MEDLINE | ID: mdl-3318243

ABSTRACT

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%.


Subject(s)
Antigens/analysis , Immunoenzyme Techniques , Insulin Antibodies/analysis , Adsorption , Animals , Antibody Specificity , Binding Sites, Antibody , Evaluation Studies as Topic , Guinea Pigs , Immunoenzyme Techniques/instrumentation , Indicators and Reagents , Insulin , Time Factors
17.
Biokhimiia ; 52(9): 1531-8, 1987 Sep.
Article in Russian | MEDLINE | ID: mdl-3315010

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal , Insulin Antibodies/immunology , Insulin/immunology , Animals , Antigen-Antibody Reactions , Binding Sites, Antibody , Immunoglobulin Fab Fragments/immunology , Insulin/metabolism , Kinetics , Models, Biological , Swine
18.
Antibiot Med Biotekhnol ; 32(8): 579-83, 1987 Aug.
Article in Russian | MEDLINE | ID: mdl-3674839

ABSTRACT

The effect of potassium orthophosphate on growth of the mycelium, its ATP contents and biosynthesis of the macrotetrolide antibiotic nonactin by Str. chrysomallus var. macrotetrolidi was studied. Direct dependence of the ATP contents in the mycelium on the amount of the phosphate added to the medium and consumed by the developing actinomycete was shown. Changes in the intracellular content of ATP depended also on the mycelium age. It was characterized by two peaks. Hemin was detected in the actinomycete mycelium. Its levels were sufficiently high and depended on the mycelium age and cultivation conditions, in particular on the phosphate content in the medium. Higher levels of nonactin biosynthesis were characteristic of the mycelium with lower contents of ATP, proteins and hemin. Intensive production of the antibiotic proceeded at the background of decreasing levels of ATP in the mycelium.


Subject(s)
Adenosine Triphosphate/metabolism , Anti-Bacterial Agents/biosynthesis , Culture Media/metabolism , Dose-Response Relationship, Drug , Hemin/metabolism , Macrolides , Phosphates/metabolism , Streptomyces/growth & development , Streptomyces/metabolism , Time Factors
19.
Article in Russian | MEDLINE | ID: mdl-3303764

ABSTRACT

The possibility of using two variants of the enzyme-linked fluorescent cofactor immunoassay for the determination of antibody binding constants has been demonstrated. The determination of binding constants for antibodies isolated by affinity chromatography techniques has been carried out. These techniques permit the isolation of fractions, differing in their affinity by 5-10 times, from the whole population of antibodies.


Subject(s)
Antibody Affinity , Immune Sera/immunology , Immunoglobulin G/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites, Antibody , Binding, Competitive , Chromatography, Affinity/methods , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Rabbits
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