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1.
Biomed Khim ; 57(5): 554-61, 2011.
Artigo em Russo | MEDLINE | ID: mdl-22629606

RESUMO

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.


Assuntos
Ciclosporina/sangue , Ensaio de Imunoadsorção Enzimática , Imunossupressores/sangue , Anticorpos Monoclonais , Humanos , Limite de Detecção , Kit de Reagentes para Diagnóstico
2.
Prikl Biokhim Mikrobiol ; 43(4): 471-8, 2007.
Artigo em Russo | MEDLINE | ID: mdl-17929576

RESUMO

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.


Assuntos
Antibacterianos/análise , Escherichia coli/metabolismo , Soro , Antibacterianos/farmacologia , Técnicas Biossensoriais , Escherichia coli/efeitos dos fármacos , Gentamicinas/análise , Gentamicinas/farmacologia , Soros Imunes , Luminescência , Estreptomicina/análise , Estreptomicina/farmacologia , Tetraciclina/análise , Tetraciclina/farmacologia
3.
Vopr Med Khim ; 46(4): 410-8, 2000.
Artigo em Russo | MEDLINE | ID: mdl-11075424

RESUMO

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.


Assuntos
Anticorpos/química , Lisina/química , Animais , Reagentes de Ligações Cruzadas , Portadores de Fármacos , Equidae , Glutaral , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/imunologia , Imunoglobulina G , Camundongos , Oxirredução , Coelhos , Espectrofotometria
4.
Probl Endokrinol (Mosk) ; 40(4): 48-51, 1994.
Artigo em Russo | MEDLINE | ID: mdl-7971912

RESUMO

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).


Assuntos
Imunoensaio de Fluorescência por Polarização , Progesterona/análise , Animais , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
Steroids ; 58(11): 547-50, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7505959

RESUMO

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.


Assuntos
Micelas , Progesterona/química , Soroalbumina Bovina/química , Ácido Dioctil Sulfossuccínico , Octanos , Espectrofotometria
6.
Anal Biochem ; 211(2): 274-8, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8317703

RESUMO

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.


Assuntos
Técnicas de Química Analítica/métodos , Dianisidina/análise , Peroxidase do Rábano Silvestre/metabolismo , Sulfato de Dextrana , Concentração de Íons de Hidrogênio , Técnicas Imunoenzimáticas , Membranas Artificiais , Nylons , Oxirredução , Espectrofotometria/métodos
7.
Biull Eksp Biol Med ; 114(12): 629-31, 1992 Dec.
Artigo em Russo | MEDLINE | ID: mdl-1292695

RESUMO

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.


Assuntos
Anticorpos/efeitos dos fármacos , Encefalina Metionina/farmacologia , Linfócitos/efeitos dos fármacos , Animais , Anticorpos/análise , Especificidade de Anticorpos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Imunização/métodos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fatores de Tempo
8.
Biokhimiia ; 57(11): 1664-70, 1992 Nov.
Artigo em Russo | MEDLINE | ID: mdl-1336982

RESUMO

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.


Assuntos
Antígenos/imunologia , Linfócitos B/metabolismo , Mitógenos , Naloxona/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos B/imunologia , Feminino , Tecido Linfoide/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Receptores Opioides/metabolismo , Especificidade por Substrato , Linfócitos T/imunologia , Trítio
9.
Immunol Lett ; 32(1): 27-30, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1323526

RESUMO

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.


Assuntos
Encefalina Metionina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Receptores Opioides/fisiologia , Animais , Ligação Competitiva , Concanavalina A/farmacologia , Diprenorfina/metabolismo , Diprenorfina/farmacologia , Enterotoxinas/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Camundongos Endogâmicos CBA/imunologia , Naloxona/farmacologia , Dor/fisiopatologia , Receptores Opioides/efeitos dos fármacos , Receptores Opioides delta , Receptores Opioides mu , Limiar Sensorial , Baço/citologia
10.
Bioorg Khim ; 17(1): 35-41, 1991 Jan.
Artigo em Russo | MEDLINE | ID: mdl-2064622

RESUMO

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.


Assuntos
Anticorpos Monoclonais/imunologia , Peroxidase do Rábano Silvestre/imunologia , Sequência de Aminoácidos , Western Blotting , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Hidrólise , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico
11.
J Immunol Methods ; 131(2): 213-22, 1990 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-2202761

RESUMO

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).


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Animais , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática , Cobaias , Peroxidase do Rábano Silvestre/imunologia , Insulina/imunologia , Camundongos , Coelhos
12.
Bioorg Khim ; 16(4): 476-81, 1990 Apr.
Artigo em Russo | MEDLINE | ID: mdl-2165407

RESUMO

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.


Assuntos
Metabolismo dos Carboidratos , Glicoproteínas/metabolismo , Insulina/metabolismo , Peroxidase/metabolismo , Técnicas Imunoenzimáticas , Cinética , Substâncias Macromoleculares , Oxirredução
13.
Lab Delo ; (2): 25-8, 1990.
Artigo em Russo | MEDLINE | ID: mdl-1692361

RESUMO

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.


Assuntos
Anticorpos Anti-Insulina/análise , Diabetes Mellitus Tipo 1/sangue , Humanos , Técnicas Imunoenzimáticas , Radioimunoensaio
14.
J Biolumin Chemilumin ; 4(1): 164-76, 1989 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2678913

RESUMO

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.


Assuntos
Imunoquímica , Técnicas Imunoenzimáticas , Medições Luminescentes , Haptenos , Peroxidase do Rábano Silvestre , Humanos , Imunoglobulina G/análise , Insulina/análise , Anticorpos Anti-Insulina/análise , Cinética , Luminol , Oxirredução , Especificidade por Substrato , Tiroxina/análise
15.
Biokhimiia ; 53(11): 1858-63, 1988 Nov.
Artigo em Russo | MEDLINE | ID: mdl-3251550

RESUMO

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.


Assuntos
Anticorpos Monoclonais , Peroxidase do Rábano Silvestre/imunologia , Peroxidases/imunologia , Anticorpos Monoclonais/isolamento & purificação , Luciferina de Vaga-Lumes/metabolismo , Medições Luminescentes , Luminol/metabolismo
16.
Artigo em Russo | MEDLINE | ID: mdl-3318243

RESUMO

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%.


Assuntos
Antígenos/análise , Técnicas Imunoenzimáticas , Anticorpos Anti-Insulina/análise , Adsorção , Animais , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Estudos de Avaliação como Assunto , Cobaias , Técnicas Imunoenzimáticas/instrumentação , Indicadores e Reagentes , Insulina , Fatores de Tempo
17.
Biokhimiia ; 52(9): 1531-8, 1987 Sep.
Artigo em Russo | MEDLINE | ID: mdl-3315010

RESUMO

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.


Assuntos
Anticorpos Monoclonais , Anticorpos Anti-Insulina/imunologia , Insulina/imunologia , Animais , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Fragmentos Fab das Imunoglobulinas/imunologia , Insulina/metabolismo , Cinética , Modelos Biológicos , Suínos
18.
Antibiot Med Biotekhnol ; 32(8): 579-83, 1987 Aug.
Artigo em Russo | MEDLINE | ID: mdl-3674839

RESUMO

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.


Assuntos
Trifosfato de Adenosina/metabolismo , Antibacterianos/biossíntese , Meios de Cultura/metabolismo , Relação Dose-Resposta a Droga , Hemina/metabolismo , Macrolídeos , Fosfatos/metabolismo , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Fatores de Tempo
19.
Artigo em Russo | MEDLINE | ID: mdl-3303764

RESUMO

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.


Assuntos
Afinidade de Anticorpos , Soros Imunes/imunologia , Imunoglobulina G/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva , Cromatografia de Afinidade/métodos , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Coelhos
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