Stabilizing composition for obtaining lyophilised preparations based on conjugate of antiimmunoglobulin g and horseradish peroxidase
(57) Abstract:The invention relates to the field of biotechnology and immunology, and can be used to obtain stable forms of immunobiological preparations, sokhranyayuschikh its own specific characteristics in a long time. A stabilizing composition includes a protein hydrolysate, sucrose, phosphate-saline buffer, bacteriostatic, bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.% : bovine serum albumin 0,1 - 3,0; sucrose 1,0 - 10,0; hydrolyzed protein 1,0 - 5,0; potassium chloride 2,0 - 8,0; EDTA (Trilon B) 0,03 - 0,2; bacteriostatic 0,01 - 0,02; phosphate-saline buffer else. As hydrolyzed protein used peptone or gelatin, and as bacteriostatic - thimerosal. The stabilizer provides the conservation of the biochemical properties of the drug conjugate anti-IgG and horseradish peroxidase in the process of freeze drying, storage and rehydration at room temperature. 1 C. p. F.-ly, 3 tables. The invention relates to the field of biotechnology and immunology, and can be used to obtain stable forms of immunobiological preparations, preserving their shirouma compositions of conjugates on the basis of protein A and antivitamin immunoglobulins [1, 2, 3]. The compositions of the stabilizers include carbohydrates in the form of sucrose or lactose, amino acids and BSA.The disadvantage of these stabilizing compounds is that they increase the stability of drugs in no more than 60% and thus does not give the option to save the drugs for 1 year. Using glucose or lactose as the carbohydrate component of the stabilizer to obtain conjugates specific activity of the preparations is reduced already at the first stage of freezing - thawing on 35-40%.The closest technical solution (prototype) is a stabilizing composition to obtain a lyophilized preparations on the basis of protein A and horseradish peroxidase (KG BA:HRP), including sucrose (1,3 - 6%, peptone 4 - 12% and phosphate-saline buffer (FSB) else .The disadvantages of this regulator is that it retains well the drug KG BA:HRP, but not fully protects the conjugate of antiimmunoglobulin G and horseradish peroxidase (KG anti - IgG:HRP) freeze at the stage of preparation, and also does not ensure the preservation of specific recognition of immunoglobulins after prolonged storage of the dry drug at positive temperatures.The problem is solved in that the stabilizing composition to obtain lyophilised preparations based on conjugate of antiimmunoglobulin G and horseradish peroxidase, including hydrolyzed protein, sucrose, phosphate-saline buffer and bacteriostatic, according to the invention, further comprises bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.%:
Bovine serum albumin - 0,1-3,0
Sucrose - 1,0-10,0
Hydrolyzed protein - 1,0-5,0
Potassium chloride - 2,0-8,0
EDTA (Trilon B) - 0,03-0,2
The bacteriostatic - 0,01-0,02
Phosphate-saline buffer - Rest
And as hydrolyzed protein used peptone or gelatin, and as bacteriostatic - thimerosal.A comparison of the proposed stabilizing composition with the known analogues shows that the distinctive features of the prototype signs are new, previously unknown property, namely the complete preservation of the activity of the drug KG anti-IgG: HRP at the stage of lyophilization and the increase compared to the prototype and other analogues of the term of his wounds at positive temperature is an economic decision criterion of "inventive step".Such preservation activity of the conjugate, probably caused by the following reasons. First, the hydrolysate in contrast to the native protein contains a short peptides, which are much lighter and easier to enter into interaction with both hydrophilic and hydrophobic groups on the surface of the conjugate unlike bulky polymer source of protein molecules. Secondly, the introduction of EDTA in the form of its soluble salts of the trylon B is known for its chelating properties, protects gamenow group of horseradish peroxidase from the damaging interactions with ions of heavy metals. In these interactions active metal ions are replaced by passive to the exchange of potassium ions with the introduction of KCl salt.Example 1. Technology of preparation of a stabilizing composition to obtain a freeze-dried preparations.First prepare the hydrolyzed protein by high-temperature splitting of a 10% aqueous solution of peptone according to GOST 13805-76  or gelatin according to GOST 11293-78  (pH = 7.0) at 135oC for 3.5 hours Hydrolyzed peptone and gelatin characterized by amino acid composition, viscosity, and fractional composition characterized by gel filtration and cryoscopy.Com is the so called 40oC under stirring until complete disappearance of the precipitate. The entire weight of the solution add the required amount of the trylon B. as bacteriostatic use of thimerosal in an amount of 0.01%.The resulting mixture was filtered through a glass filter, and then through the filter Millipor" with a pore size of 0.45 µm.The filtrate is cooled in snow bath and add to it with stirring, a solution of the drug KG anti-IgG: HRP, reaching breeding in 500 to 1000 times depending on the titer of the conjugate is determined in direct response by ELISA with standard immunoglobulins.The product is poured in hoods 0.2 ml in vials of 5 ml, closed with rubber stoppers and placed in a metal cassette for freezing. Freezing of the drug in vials carried out at a temperature of counter - 60oC for 10-12 hFrozen drug in vials are transferred into the freeze chamber lyophilic install TG-50, shelves which chilled to minus 35oC. the Temperature of the preparation at the stage of freeze dehydration support below the glass transition temperature (-30oC). The average heating rate of the material at the final stage is 6oC/min, time vyderzhivala 1,0 - 2.0 wt.%. The determination carried out by the method of . The vials with the product is sealed under vacuum.The activity of the conjugate control at all stages of preparation of the dry form: when filling, freezing and after lyophilization. As the ELISA test systems use specially selected from one high quality party kits Recombinant anti - HIV 1+2". ELISA is performed in accordance with the Instructions of applying the specified test system.To substantiate the quantitative content of components in stabilizing the composition prepared as described above options proposed composition (1-6) and structure-prototype (7). The data are given in table. 1.Example 2. The study of the preservation activity of the conjugate anti-IgG:HRP at the stage of lyophilization.Comparative studies on the preservation of the activity of the preparation containing the inventive stabilizing composition with different quantitative content of the components (compounds 1-6) or use of the stabilizer composition-prototype (part 7). The data are given in table. 2.The results show that the most stable during freezing and dehydration are variants of the drug from formulations 1 and 2, one of which sodl. 2, all the compounds ensure the preservation of the activity of the conjugate is at the stage of freeze cooking and within the accuracy of staging ELISA differences statistically insignificant.Example 3. The study of the activity of the drug containing different options for stabilizing compositions during storage
To verify the effectiveness of the stabilizers when stored at positive temperatures lay in thermostats 30 vials conjugate anti-IgG:HRP, with different ways of compounds stabilizers, and stores these drugs at a temperature of 4 and 37oC. the Results are shown in table. 3. The activity of the conjugate anti-IgG:HRP in storage is determined in % of the dry form of this medication is stored at -20oC ( = 5%).The results of the analysis table. 3 shows that during storage of the drug in 4oC for 6 months offer a stabilizing composition (N 1 and N 2) has higher characteristics of the conservation of the drug than the prototype. Characteristics are statistically significant during storage of the drug 2 months at 37oC. With increasing storage time of the preparation of the advantages of the proposed stabilizer become even more obvious.Mark is ralista protein and sucrose, necessary for the formation of the drug in tablet form with uniformly distributed pores. The total protein concentration and sucrose pick up at least 5%. The upper limit concentrations of native proteins is limited by their solubility in the FSB.The minimum concentration of EDTA is not securely protects the iron ion from the impurity ions in the solution, and the maximum concentration provides an excess of chelating agent which binds to and partially active iron.Thus, the economic efficiency of application of the invention consists in the following. The stabilizing composition (1 and 2) based on hydrolyzed protein, sucrose and EDTA (table. 2) allows freeze-dried conjugate anti-IgG:HRP without loss of activity. When storing conjugate anti-IgG:HRP at 37oC during 10 months of the inventive stabilizer composition (table. 3) ensures the preservation of the original activity by 80%, and the prototype is at the level of 50% of the initial activity. Thus, compared with the prototype of the proposed stabilizing composition reduces loss of specific activity of the drug in half when storing it for a long time (10 months or more).Industrial primaryvoices.com drugs.Sources of information
1. U.S. patent N 4504579, CL C 12 Q 1/28, C 12 N 9/96, publ. 19/6, (analog).2. Vorobyev, M. S., Shalamberidze A. G., Kahn A. N. and other Issues of Virology. - 1992, No. 2.3. Naofumi Hanafusa. - Contributions from the Institute of Low Temperature Science. Ser. B, 1972, N 17, p 1-38 (analog).4. The patent of Russian Federation N 2046342, CL G 01 N 33/535, C 12 N 9/96, publ. 1995 (prototype).5. GOST 13805-076. Peptone is an enzymatic. Gosstandart of the USSR, 1976.6. THE 11293-78. Food gelatin. Gosstandart of the USSR, 1978.7. MI 123. 39. 002-88. The method of determining the residual moisture of small quantities of material. - 1988. 7 C. 1. Stabilizing composition for obtaining lyophilised preparations based on conjugate of antiimmunoglobulin G and horseradish peroxidase, including hydrolyzed protein, sucrose, phosphate-saline buffer and bacteriostatic, characterized in that it further comprises bovine serum albumin, EDTA (Trilon B), and potassium chloride in the following ratio, wt.%:
Bovine serum albumin - 0,1 - 3,0
Sucrose - 1,0 - 10,0
Hydrolyzed protein - 1,0 - 5,0
Potassium chloride - 2,0 - 8,0
EDTA (Trilon B) - 0,03 - 0,2
The bacteriostatic - 0,01 - 0,02
Phosphate-saline buffer - Rest
2. the e bacteriostatic - thimerosal.
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: 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
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: 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: 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: 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: 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: 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
SUBSTANCE: group of inventions relates to biotechnology and can be applied in creation of analytic methods with application of peroxidases. Method includes preparation of substrate mixture, introduction of peroxidases in substrate mixture with the following registration of intensity of formed luminescence. Substrate mixture includes the following components, given in final concentrations: buffer solution 10-125 mM, luminol 0.05-8 mM, hydrogen peroxide 0.05-8 mM, 4-aminopyridines 0.1-10 mM, N-carboxyphenothiazine, or N-(carboxymethyl)phenothiazine, or N-(2-carboxyethyl)phenothiazine 0.1-10 mM. And pH of buffer solution constitutes 7.9-9.0.
EFFECT: invention ensures obtaining of high intensity of chemiluminescence and, accordingly, high sensitivity of peroxidase activity determination.
4 cl, 8 ex
SUBSTANCE: invention relates to field of analytical chemistry, namely to express-detection of explosive substances (ES) based on organic peroxides. Method is based on fixation of hydroxen peroxide, released in the process of explosive substance decomposition by indicator method. For this purpose change of indicator colour is fixed within 1 minute after contact with solid-phase material, possessing function of surface acidity and providing decomposition of ES to hydrogen oxide. Application of claimed method simplifies analysis of cyclic peroxides due to reduction of the number of analysis stages, as well as to elimination of liquid reagents, including concentrated acids and organic solvents.
EFFECT: invention provides carrying out express-analysis of trace quantities of peroxide ES outside laboratory in wide range of climatic conditions.
6 cl, 2 tbl, 7 ex