Method for obtaining liposomal-immunoperoxidase conjugate
SUBSTANCE: liposomes are used as a matrix for activated ferment - horseradish peroxidase. To 5 mg of horseradish peroxidase oxygenated with a periodate method there added is 1 ml of liposome suspension in 0.01 M solution of carbonate-bicarbonate buffer at pH 9.5, and subjected to ultrasonic treatment during 1 min. Then it is incubated for 1 hour; immobilised with immunoglobulins in concentration of 5 mg during 2 hours at the temperature of 22±4°C; stabilised with 5 mg of sodium borane with further gel-chromatographic cleaning.
EFFECT: invention allows obtaining liposomal-immunoperoxidase conjugate for indication of infectious agents in an immunoenzymometric analysis and increasing service life of a preparation up to 6 years.
1 tbl, 3 ex
The invention relates to biotechnology and can be used in the production of immune-peroxidase conjugates for indication of infectious diseases in the enzyme-linked immunosorbent assay (ELISA).
Enzymes - specific protein substances with the ability to accelerate chemical reactions with subjectyou structure and exposed to the negative influence of temperature, buffer changes, the concentration of hydrogen ions and so the shelf Life is limited and varies from 6 months (for liquid medications, at a storage temperature of minus 4°C) up to 1 year (for lyophilised, at a storage temperature of 4-6°C).
A method of obtaining enzyme conjugate, comprising the activation of the enzyme and immobilization with immunoglobulins .
A method of obtaining enzyme conjugate, comprising lyophilization immunoperoxidase conjugate in solution of bovine serum albumin (BSA) and store at 4°C .
The disadvantage of the methods is the limited shelf life - up to 1 year.
The purpose of the invention to increase the shelf life immunoperoxidase conjugate with the preservation of its physico-chemical and immunological parameters by fitting the parameters of the immobilization of the enzyme to the matrix.
The technical result of the invention is achieved by the fact that the quality of the matrix are used liposomes (with adjustable composition, size, surface properties, non-toxicity, does not inactivate the enzyme, allowing to operate with high activity and so on), immobilized activated horseradish peroxidase associated with immunoglobulins are purified from unbound components.
You need to design and select the method of immobilization and to determine the influence of the latter on the enzyme. It is known that the enzyme is able to perform the role of a specific catalyst in compliance with mainly two conditions. First, the enzyme must be in the right conformation. Secondly, he should be able to change its conformation in the process of catalysis.
As a matrix, you need to choose one that would not hinder the penetration of the substrate and not "chained" enzyme, depriving him of mobility necessary to perform its inherent function.
Taking into account the above factors and taking into account the properties of liposomes, it should be noted that the most appropriate matrix for the enzyme is horseradish peroxidase and the task at hand - getting immobilized immunoperoxidase conjugate are liposomes.
The nature of the preparation of immobilized enzyme depends on the nature containing the enzyme phase. Is there a way to attach the enzyme using electrostatically who or other non-covalent binding mechanisms. On the other hand, the enzyme does not need to be attached to the carrier, and can be kept inside it. While the media forms around the enzyme setuptool matrix, the cells of which are so small that a molecule of the enzyme can be released from the network, but at the same time large enough for the penetration of low molecular weight substrates . One of the variants of this method lies in using a double phospholipid layer as the enzyme phase. In this case, the enzyme can be in aqueous solution, surrounded by a phospholipid barrier, or be dissolved in the hydrophobic part of the double layer.
Technology of production of liposomal-immunoperoxidase conjugate consisted of several stages: obtaining liposomes; the inclusion of the enzyme, immobilization with immunoglobulins; purification from unbound components, lyophilization and control. Liposomes obtained using chloroform-ethanol mixture, and the rate of diffusion of cations through the membrane increases. The raw material used gomogenizirovannykh the brain of a pig. After extraction was performed separation of lipids by adding 20% of the volume of the extract of 0.9% solution of sodium chloride. After incubation for 2-3 h lipids were separated. The value of the quantity of lipids ranged from 30 to 50 mg/ml of the lipid Composition was determined by means of thin layer of HRO is ecografia on the plates "Silufol" .
Lipids obtained as described above, consisted of phosphatidylcholine, phosphatidylserine, phosphatidylinositol and cholesterol. Then spent the evaporation of the extract lipids on a rotary evaporator to form a film of lipids on the walls of the flask. This step is very important due to the fact that the film lipids must be thin enough, otherwise undesired inclusion of a significant amount of dry phospholipid in a system of concentric bilayers, as a closed membrane will effectively prevent further access of water and salt. Then he washed the dried lipids with chloroform (based on 50 mg of lipids 3.5 ml of chloroform), was stirred flask to dissolve the precipitate, contributed 1.5 ml of 0.9% solution of sodium chloride, and chloroform was removed in a rotary evaporator. Determination of the size of liposomes was performed at the electron microscope and was calculated by the formula. The size of the resulting liposomes was 100-200 nm.
When receiving liposomal-immunoperoxidase conjugate initially conducted research in terms of the inclusion of the enzyme in liposomes. The most appropriate method was the inclusion of pre-activated horseradish peroxidase: (5±0,1) mg of horseradish peroxidase was dissolved in 1 ml of a 0.3 M solution of sodium carbonate acid was added to 0.025 ml of 0.32% solution of formalin. The mixture was mixed on a mixer in the course is e (30±5) minutes at a temperature of (22±4)°C and added 1 ml of a 0.04 M solution of periodate sodium, continuing to stir for (30±5) minutes at a temperature of (22±4)°C. Further contributed to 1.0 ml of a 0.16 M solution of ethylene glycol (to reduce surface tension), gently stirring in a period of (60±5) minutes at a temperature of (22±4)°C. At the end of the procedure, the drug is activated peroxidase should be transparent, odorless, greenish-brown color. The activated peroxidase was transferred into a dialysis bag and were dialyzed against 1 l of 0.01 M carbonate-bicarbonate buffer (CBS), pH of 9.5, for (19±1) h on the stirrer at a temperature of 2-8°C in the refrigeration chamber. Thus, the activation of the enzyme periodate oxidation, to form aldehyde groups.
Further, the step of receiving liposomal complex-immunoperoxidase conjugate was as follows. To 5 mg of oxidized periodate method peroxidase was added 1 ml of a suspension of liposomes in 0.01 M CBS pH of 9.5. The suspension was subjected to ultrasonic treatment (44 kHz) for 1 min, then incubated 1 h To liposomes immobilized horseradish peroxidase, was added tularemia immunoglobulins (Ig G) in an amount of from 2.5 to 10 mg in 3 ml of 0.01 M CBS pH of 9.5. Immobilization of immunoglobulins with activated horseradish peroxidase was carried out at a temperature of (22±4)°C, stirring at Shuttle for 2 h, stabilized 5 mg of borohydride in the refrigerator at a temperature of 4-6°C. the Probability is about, this peroxidase was included in the membrane of liposomes, and the aldehyde groups of the carbohydrate part of the enzyme interacted with the amino groups of immunoglobulins.
For purification of the conjugate from unbound enzyme used gel chromatography. The conjugate was applied on the column, 100×12 mm with Sephadex G-100, equilibrated with 0.1 M solution of the FSB pH of 7.2. This resulted in the fractionation of the mixture of molecules into zones depending on their size. Therefore, the first peak came liposomal-immunoperoxidase conjugate, then the unbound antibodies and the enzyme. The elution was performed in the same buffer, and fractions were collected in 2.0 ml and explored in the enzyme-linked immunosorbent assay (ELISA).
In the result set, which is essential for obtaining a quality product is the activation of the enzyme at the optimum time of immobilization 2 h and the protein concentration of immunoglobulins 5 mg/ml
To increase the shelf life of the medication was dispensed in ampoules, pre-adding 1% BSA, froze and liofilizirovanny for (18±2) h to a final temperature of 25°C. In a final product was controlled by the physico-chemical (solubility, color, transparency, loss in weight on drying) and immunological (activity, specificity, sensitivity) properties after drying and during storage is ment for 6 years (time of observation). The sensitivity and specificity of the conjugate was determined by ELISA with homologous and heterologous strains by the method of Dark M.F., Adams A.N. .
The table presents the results of physico-chemical properties and sensitivity of the drugs. Developed preparations meet the requirements of the regulations applicable to diagnostic drugs, and their stability is superior to traditional enzyme conjugates 6 times (observation period).
Thus, as a result of research developed the manufacturing technology of immobilized enzyme preparation that greatly increases its stability. This is due to the fact that the immobilization effectively prevents the harmful effects of environment on enzyme preparation, he becomes a prisoner in the matrix (liposomes), i.e. protected, in the absence of physical and catalytic restrictions, the matrix does not prevent the penetration of the substrate due to efficient method of obtaining liposomes, activate the enzyme and immobilization of antibodies, temperature selection, time parameters, holding gentle cleaning from unbound components.
The opportunity for practical application of the proposed method is illustrated by examples of its specific implementation using the receiving entirety of the claimed features.
Example 1. Getting the plague liposomal-immunoperoxidase conjugate and its control in ELISA
Pre-spent activated horseradish peroxidase: (5±0,1) mg of horseradish peroxidase was dissolved in 1 ml of a 0.3 M solution of sodium carbonic acid, was added to 0.025 ml of 0.32% solution of formalin. The mixture was stirred in a stirrer for (30±5) minutes at a temperature of (22±4)°C and added 1 ml of a 0.04 M solution of periodate sodium, continuing to stir for (30±5) minutes at a temperature of (22±4)°C. Further contributed to 1.0 ml of a 0.16 M solution of ethylene glycol, gently stirring in a period of (60±5) minutes at a temperature of (22±4)°C. the Activated peroxidase was transferred into a dialysis bag and were dialyzed against 1 l of 0.01 M solution of CBS pH of 9.5 for (19±1) h on the stirrer at a temperature of 2-8°C in the refrigeration chamber.
Obtaining complex liposomally-immunoperoxidase conjugate was carried out as follows: 5 mg of oxidized periodate method peroxidase was added 1 ml of a suspension of liposomes in a 0.01 M solution CBS pH of 9.5. The suspension was subjected to ultrasonic treatment for 1 min, then incubated 1 h To liposomes immobilized horseradish peroxidase, was added plague immunoglobulins (Ig G) in an amount of 5 mg per 3 ml of 0.01 M CBS pH of 9.5. Immobilization of immunoglobulins with activated horseradish peroxidase was carried out at a temperature of (22±4)°C, stirring at Shuttle for 2 h,stabilized 5 mg sodium borohydride in the refrigerator at a temperature of 4-6°C for 2 hours For purification of the conjugate from unbound enzyme used gel chromatography. The conjugate was applied on a column with Sephadex G-100, equilibrated with 0.1 M solution of the FSB pH of 7.2. This resulted in the fractionation of the mixture of molecules into zones depending on their size. In the first peak came conjugate, then the unbound antibodies and the enzyme. The elution was performed with the same buffer solution. The activity was tested in ELISA. The drug was dispensed in ampoules, pre-adding to the finished product 1% BSA, froze and liofilizirovanny for (18±2) h to a final temperature of 25°C. In a final product was controlled by the physico-chemical and immunological properties after drying and during storage. The sensitivity of the conjugate in ELISA with homologous strains was 1×104M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (observation period).
Example 2. Getting tularemia liposomal-immunoperoxidase conjugate and its control in ELISA
Was obtained analogously to example 1, except that instead of plague used tularensis antibodies. The sensitivity of the conjugate in ELISA with homologous strains was 5×104M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (the period of observation is possible).
Example 3. Getting brucellosis liposomal-immunoperoxidase conjugate and its control in ELISA
Was obtained analogously to example 1, except that instead of plague used Brucella antibodies. The sensitivity of the conjugate in ELISA with homologous strains was 1×105M.K./ml, in the absence of cross-reactions with heterologous strains. The shelf life is 6 years (observation period).
Sources of information
1. Nakane, P.K. Buffer-labelled antibody - a new method of conjugation / P.K.Nakane, A.Kawaoi // J. Histochem. Cytochem. - 1974. - Vol.22. - P.506-508.
2. RF patent №2276362, publ. 10.05.2006, "Method of detecting the virus Crimean-Congo hemorrhagic fever (KGL) in biological and field", the authors Vasilenko NF, Efremenko VI, Tyumentseva I.S. and other bull. No. 13.
3. Trevin M. Immobilized enzymes. - M.: Mir, 1983. - P.23.
4. Cats M. Technique of lipid. M.: Mir, 1975. - C-82.
5. Clark M.F., Adams A.N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant virus // J. Gen. Virol. - 1977. - V.36, No. 3. - P.475-480.
The method of obtaining liposomal-immunoperoxidase conjugate for indication of infectious diseases in immune-enzyme analysis, characterized by the fact that as a matrix for the activated enzyme is horseradish peroxidase - use liposomes to 5 mg of oxidized periodate method of horseradish peroxidase was added 1 ml of suspension lipo is ω 0.01 M solution of carbonate-bicarbonate buffer at pH of 9.5, subjected to ultrasonic treatment for 1 min, incubated for 1 h, immobilized with immunoglobulin at a concentration of 5 mg for 2 hours at a temperature of 22±4°C, stabilized 5 mg of sodium borohydride and subsequent gel chromatographic purification.
SUBSTANCE: invention refers to biotechnology, namely to a method for Mycobacterium leprae antibody test. The method involves the immunoenzyme blood serum test for Mycobacterium leprae antibodies. A test antigen in the immunoenzyme test is an aqueous suspension of Mycobacterium lufu cultured in a thermostate on the Lowenstein-Jensen medium for 7 days at temperature 37°C, heated at 100°C for 1.5 hours on a water bath.
EFFECT: presented invention enables simplifying the method for Mycobacterium leprae antibody test.
SUBSTANCE: versions of an antibody or its fragment, which are specific in relation to β-amyloid protein, are proposed. Each version is characterised by the fact that it includes H- and L-chains, or areas VH and VL, each of which contains three corresponding CDR. The following is described: polypeptide VL, polypeptide VH, as well as coding nucleic acid, expression vector containing it and a cell carrying the vector, which are used for obtaining an antibody or its functional fragment. The following is proposed; a test kit, versions of pharmaceutical composition, a mixture to be used as a medicine based on the antibody or its functional fragment. Versions of the method used for production of an antibody are described: using a cell, nucleic acid or a vector. A composite preparation method, as well as an in vitro amyloid disease diagnostics method, a method for determination of a degree of loading with in vitro amyloidogenic patches, a method for curing or relief of actions of amyloid disease, which use an antibody or its functional fragment, are described. Inventions can be used in therapy and diagnostics of Alzheimer disease and other enlisted amyloid diseases.
EFFECT: proposed inventions provide new antibodies that bind the epitope contained in the area of 12-23 protein αβ1-42; with that, residues 15-20 have a fundamental importance.
44 cl, 18 dwg, 9 tbl, 16 ex
SUBSTANCE: relative CD4+FOXP3+ T-cell count in peripheral blood of the patients suffering with haemoblastoses is determined after autologous stem haemopoietic cell transplantation in a period of the lymphocyte recovery to >500 cell/mcl. If observing an increase of the CD4+FOXP3+T-raeTOK count more than 9.1%, the development of an early relapse in the post-transplantation period.
EFFECT: using the given method enables grouping the patients suffering haemoblastoses as suffering a high-risk of the development of an early relapse of the primary disease following autologous stem haemopoietic cell transplantation and in need of the additional courses of the radiation therapy and chemotherapy for the purpose of improving the efficacy of autologous stem haemopoietic cell transplantation.
SUBSTANCE: specific cell-mediated immunity is assessed by modified lymphocyte blast transformation reaction (mLBTR) with PPD-L tuberculine; the level of interferon-gamma (IFN-γ) with PPD-L is tested with using a small volume (200 mcl) of whole blood and 4 dilutions of the PPD-L antigens. If the cell immunity stimulation index is more than 3.2 and IFN-γ with PPD-L more than 7.0, an active tuberculosis process, while the mLBTR with PPD-L more than 3.2 and IFN-γ with PPD-L less than 7.0 show an inactive tuberculosis infection.
EFFECT: using the given method enables more specific diagnosis of the tuberculosis infection, and assessed effectiveness of the conducted treatment.
SUBSTANCE: analytical tip (100) designed to conduct a visually detectable agglutination reaction after sucking reactants and a sample includes: a first opening (110) for applying negative or positive pressure to the inner volume of the analytical tip; a sample chamber (120); at least one side chamber (130) situated on the periphery of the sample chamber; a detection chamber (150) which is linked by a liquid to the sample chamber; a transition zone (140) between the detection chamber and the sample chamber for rotary mixing of the sample; a second opening (160) for sucking reactants and the sample into the inner volume of the analytical tip or for removal thereof from the inner volume of the analytical tip. The group of inventions also relates to a method of conducting an agglutination reaction in said analytical tip and a set for conducting visually detectable agglutination reactions having said analytical tip and reactants for agglutination.
EFFECT: analysis automation according to the type of agglutination reaction, thereby increasing safety and reliability thereof.
18 cl, 10 dwg, 1 tbl, 1 ex
SUBSTANCE: patients are examined for the intracellular levels of cardiac protein troponin T (cTnT) or cardiac protein troponin I (cTnl) in the cardiac tissue. The values appearing to be reduced by min. 50% in relation to the control and normal values is a sign of the cardiac involvement in the patient. The method of treating involves detecting the patients with the cardiac involvement by the declared method and introducing the therapeutically effective amount of GGF2 and a proteasome inhibitor. The increasing intracellular levels of the protein cTnT or cTnl in the patient's cardiac tissue after GGF2 has been introduced, are a positive sign of the effective introduction of GGF2 for treating the patient with the cardiac involvement.
EFFECT: using the declared group of inventions enables fast and accurate diagnosis of the cardiac involvement in the patient, as well as fast and accurate estimation of the effectiveness of the conducted therapy.
19 cl, 26 dwg, 1 ex
SUBSTANCE: invention described an early diagnostic technique for the developing pelvic prolapse in the females of reproductive age with no clinical signs, consisting in sampling the vaginal wall tissue, conducting the histochemical analysis, determining the quantity and state of elastin fibrils, the content of matrix proteins LOXL1 and FBLN5, wherein the developing pelvic prolapse is diagnosed if observing the reduction in elastin fibrils and a fragmentation thereof in a combination with the reduction in matrix protein LOXL1 and FBLN5 in the epithelium, vascular endothelium, fibroblast, extracellular matrix as related to the same in the healthy females.
EFFECT: method enables assessing the pathophysiological processes observed at the initial preclinical signs of the developing pelvic prolapse that will enable the more accurate interpretation of the causes of the developing pelvic floor dysfunction, and also the working-out of the basic principles to prevent the development of the given pathology in the females of reproductive age.
1 dwg, 1 tbl
SUBSTANCE: blood serum is tested by ELISA to find the content of oxidised low-density lipoproteins (oxLDL). If the oxLDL concentration exceeds 400 ng/ml, a high risk of the clinical complications of atherosclerosis is stated.
EFFECT: availability, low price and ease of the clinical trial using the ELISA widely used in laboratory diagnosis.
SUBSTANCE: histological examination of injured skin biopsy materials is performed by optical microscopy, immunophenotyping of infiltrate cells. The similar histologic pattern described as corneal layer detachment, nonuniform acanthosis, severe hyperkeratosis, sudden vascular distention, dermal oedema, diffuse infiltrates with lymphocyte penetration into the epidermis, the same immunophenotype of the infiltrate cells presented by CD3+, CD4+, CD43+, CD45RO+, CD2- CD5- lymphocytes; the above is added with the morphometric studies of antigen-representing cells in histologic specimens of the injured skin biopsy materials; that is followed by the antigen-representing cell count per 1 mm of the epidermis length, measurement of the area and nucleocytoplasmic relation. If observing an increase of the antigen-representing cell count per 1 mm of the epidermis length in 2.8 times and more as compared to the normal value in addition to an increase of the antigen-representing cell area in 1.36 times and more as compared to the normal value and an increase of the nucleocytoplasmic relation of the antigen-representing cells in 1.65 times and more as compared to the normal value, erythroderma is diagnosed. If observing a decrease of the antigen-representing cell count in 1.3 times and more as compared to the normal value in addition to a decrease of the antigen-representing cell area in 1.9 times and more as compared to the normal value and a decrease of the nucleocytoplasmic relation of the antigen-representing cells in 2 times and more as compared to the normal value, Sezary's syndrome is diagnosed.
EFFECT: higher diagnostic accuracy of the initial clinical presentations of Sezary's syndrome accompanying the benign inflammatory dermatoses.
1 cl, 2 tbl, 3 ex
SUBSTANCE: method for simultaneous determination of two analytes via bioluminescent molecular microanalysis involves treating the solid phase with a biospecific reagent, separating the unreacted liquid phase, treating the solid phase with an enzyme-containing conjugate, separating the liquid phase and the solid phase and analysing the solid phase, wherein the biospecific reagent for the first treatment of the solid phase is two immunoglobulins or one immunoglobulin with dual specificity or two oligonucleotides, and final treatment of the solid phase is carried out simultaneously with two enzyme-containing conjugates consisting of, respectively, recombinant Ca2+-dependent photoprotein obelins with different bioluminescence characteristics and molecules of different biospecificity, where the recombinant obelins used are obelin W92F; H22E and obelin Y138F, with subsequent analysis on a tablet bioluminometer with fast moving wideband colour filters with spectral and time separation of signals.
EFFECT: improved method.
3 ex, 4 dwg
SUBSTANCE: what is presented is a kit of reagents for immune-enzyme assay of the endogenous bioregulator antibodies in blood serum for detecting addiction disorders by two-site enzyme immunoassay in human blood serum in vitro. The kit comprises a 96-well demountable stripped plate with its surface sequentially coated by all conjugates: βendorphins, serotonin, dopamine, histamine, orphanin and angiotensin, as well as containers containing positive and negative reference samples, horseradish peroxidase-labeled anti-species anti-human antibodies, phosphate-buffered saline containing Tween 20, a substrate buffer solution, a tetramethyl benzidine substrate solution, and a stop solution.
EFFECT: more accurate detection of the addiction diseases, including alcoholism, drug addiction, chemical abuse, food addiction and gambling by using the kit of six labels.
3 tbl, 9 ex
SUBSTANCE: invention relates to biotechnology. The indicator strip has an electrode layer and a substrate having a channel on it. The channel has a first zone, a second zone and a third zone lying in series. A first antibody is localised in the first zone. Saccharide and peroxidase are localised in the first or second zone. A second antibody, meant for identifying an antigen determinant different from the antigen determinant of the same antigen identified by the first antibody, is immobilised in the second zone. A substrate reagent which contains saccharide oxidase is localised in the third zone.
EFFECT: indicator strip for quantitative determination of analysed substance in a liquid and a method for quantitative determination using said indicator strip are disclosed.
21 cl, 10 dwg
SUBSTANCE: substratum o-dianisidine is introduced in blood plasma diluted in phosphate-citrate buffer (pH 5.5). After hydrogen peroxide added in the concentration of 2 mM, the substratum oxidation rate is evaluated by sepectrophotometry in the presence of the myeloperoxidase inhibitor - 4-aminobenzoic acid hydrazide. Peroxidase activity of blood plasma haemoglobin is described as the substratum o-dianisidine oxidation rate.
EFFECT: method enables fast and reliable evaluation of haemoglobin contribution to total peroxidase activity of blood plasma that is applicable in prediction of the developing diseases and choosing a therapeutic approach.
2 dwg, 1 tbl, 3 ex
SUBSTANCE: test strap or electrochemical sensor for measurement of analysed substance amount in biological fluid, for instance, content of glucose in whole blood, includes self-size-limiting composition of reagents, in which system of enzymes is used for reaction with analysed substance, reacting system is mixed with water-soluble swelling polymer matrix, containing small water-insoluble particles with nominal size approximately from 0.05 to 20 mcm, preferably approximately from 1 to 10 mcm. Weight ratio of water-insoluble particles to water-soluble swelling polymer matrix constitutes from 1/2 to 2/1.
EFFECT: composition of reagents is applied on non-porous base with formation of thin layer approximately 6-16 mcm thick and ensures fast and stable response to sample introduction, remaining insensitive to sample volume.
52 cl, 10 dwg, 4 ex
SUBSTANCE: during immunoenzymometric analysis, a solid-phase antigen is immobilised on the surface of a solid phase. Further, a sample and a preparation which contains specific antigens with which free mycotoxin and the solid phase compete to bind are added. An anti-specific immunoenzymometric conjugate is then added and finally results are evaluated using a substrate-indicator mixture. The solid-phase antigen used is a conjugate of T-2 mycotoxin with an antigen of fraction 1 of a pestilential microbe. Rabbit hyperimmune serum is used at the competitive binding step.
EFFECT: invention increases sensitivity and specificity during detection of T-2 mycotoxin in liquid samples.
SUBSTANCE: invention relates to ecology field and can be used for definition of water integral pollution, and also of aqueous extracts by organic and inorganic compounds. It is possible usage of invention for definition of presence of these substances in air quality after its barbotage through the water. Method includes incubation of water-soluted pollutants with biological test-system and its quantitative assessment by caused by them 50% value reduction of maximal luminal - dependent chemiluminescence in comparison with control. In the capacity of test-system it is usedc complex multienzyme preparation made of horseradish root, allowing oxidase - peroxidase activity, in combination with sulfhydric reactant, which is taken in a finite concentration not less than 0.1 mcg/ml and not more than 100 mcg/ml.
EFFECT: invention provides increasing of sensitiveness of detection method of environmental pollution and also ability of integral and quantitative assessment of surrounding objects pollution.
1 dwg,1 tbl, 5 ex
FIELD: analytical methods.
SUBSTANCE: invention relates to dye composition suitable for analyte detection tests in glucose determination tests. For that, invention proposes 8-anilino-10-naphthalenesulfonate capable of reacting with 3-methyl-2-benzothiazolinone hydrazone hydrochloride or its analogue to give colored product characterized by reduced shift as compared to colored product obtained in reaction of 8-anilino-10-naphthalenesulfonate analogue substituted by alkyl radical in phenyl group with 3-methyl-2-benzothiazolinone hydrazone hydrochloride or its analogue on condition that, when indicated alkyl radical is methyl it can be present in 3', 4', or 5' positions. Invention also discloses above-mentioned reaction product, method of preparation thereof, composition based on this product, and method of detecting presence of analyte in sample utilizing title compound.
EFFECT: increased analyte detection accuracy.
15 cl, 3 tbl, 5 ex
SUBSTANCE: crushed biomass of horseradish root is soaked in 0.1 M sodium phosphate buffer solution pH 7.0, with preliminary blown nitrogen in presence of 5 mcM hemin solution and 5 mcM calcium chloride. The extract is separated by decantation followed by filtration and concentrating by ultrafiltration through ultrafilters with a pore size of less than 30 kilodalton. The enzyme extract is saturated with ammonium sulfate to 35% saturation and applied to a column with phenyl-sepharose after extensive washing of the buffer with sulfate, the active fractions are removed with a gradient of ammonium sulfate (35%-0%) and increasing the pH to 8.0. The enzyme is purified by gel filtration on Toyopearl HW55F, dialysed and dried lyophilisation.
EFFECT: use of hemin-containing buffers at a stage of extraction and gel filtration enables to obtain highly active enzyme with high acceleration of enzyme production process and improving its catalytic characteristics and stability.
7 cl, 1 tbl