Method of obtaining high-titre antimicrobial serum for estimation of antigen activity of anti-staphylococcal vaccines
SUBSTANCE: invention relates to the field of biotechnology. Claimed is a method of obtaining a high-titre antimicrobial serum. Method includes application of strain S.aureus No1991, characterised by immunogenicity ED50=(50-100)×106 microbial cells, weak virulence LD50=(0.5-2.0)×109 microbial cells; its inactivation and drying with dimethylketone by double processing with 3 volumes of dimethylketone. After that, immunisation of animals is carried out: two times subcutaneously and two times intravenously. In conclusion serum isolation is carried out.
EFFECT: invention can be used in medicine for estimation of antigen activity of anti-staphylococcal vaccines.
The invention relates to medicine, namely to protivogribkovym vaccines and methods of assessing their antigenic activity. The aim of the invention is to create the most immunogenic non-toxic protivogribkovogo of the drug to obtain vysokotirazhnyh immune sera suitable for evaluation of antigenic activity of staphylococcal vaccines of different antigenic composition at all stages of their preparation, control of the final product and stability during storage.
The advantage of the invention is to standardize methods of control protivotrombovyh products intended for active specific prophylaxis and therapy of staphylococcal infections through the use of serological methods based on the use vysokotirazhnyh antimicrobial protivotrombovyh sera, for which we propose a method of preparing immunogenic, non-toxic and labellling drug.
Known methods for producing antistaphylococcal plasma and serum. The disadvantage of these methods is the induction of antibodies to individual factors of pathogenicity of Staphylococcus aureus and low level of antibodies[2, 3, 4, 9, 10].
Worldwide is the development of staphylococcal vaccines of new generation. Significant efforts in research and to mirceski organizations were focused on obtaining and testing of monovalent vaccines, consisting of capsular polysaccharides (Staphvax), alpha-toxin, other pathogenicity factors  Staphylococcus. Study most of these drugs was stopped at the stage of pre-clinical trials. The most well studied vaccine Staphvax, the effectiveness of which in clinical trials has not been confirmed. These data led the authors to the conclusion about the necessity to develop polyvalent staphylococcal vaccines with an optimal set of antigens[6, 7, 8].
In Russia, carried out research on the development of cell-free polyvalent antistaphylococcal vaccines based on water-soluble protein and polysaccharide antigens of the cell wall S.aureus. Preclinical studies have established low toxicity and significant protective activity of this drug . However, even using mnogokubovyh complex schemes of immunization with this vaccine does not provide vysokotirazhnyh sera. At the same time revealed a correlation between antibody titers and survival of rabbits on the model of generalized staphylococcal infection in rabbits (table 1), which determines the possibility of the use of serological methods for the evaluation of immunogenic activity of staphylococcal drugs.
|Immunogenic activity of dry staphylococcal vaccine in experiments on rabbits (antibody titers and protection on the model of generalized staphylococcal infection)|
|no experience||No. series||The multiplicity of immunization||Immunizing dose mg||Grafted||Unvaccinated|
|Reverse cf. the title at±m||Survived from the number of rabbits taken in the experience||Reverse cf. the title at±m||Survived from the number of rabbits taken in the experience|
When developing protivotrombovyh vaccines and in preclinical studies evaluating drugs produced in experiments on animals (mice, rabbits, rare monkeys). These studies are time-consuming. When you research often get different results due to different sensitivity the spine of mice, they are difficult to use on the stages of production, and, as shown by many studies, the protective activity established in mice, has not been confirmed in clinical trials . The claimed method of obtaining immunogenic non-toxic protivogribkovogo preparation for immunization of animals with the aim of obtaining vysokotirazhnyh immune sera will be used to assess immune staph drugs in serological (competitive ELISA and others) and immunochemical (immunoblot, immunodiffusion) reactions.
To achieve this purpose: 1) strain S.aureus 1991, isolated from a patient with pneumonia, characterized by high intra-specific protective activity of the ED50=(20-100)×106microbial cells with weak virulence - LD50>1×109microbial cells; 2) the most gentle method of inactivation of microbial mass dimethylketone; 3) short circuit immunization, consisting of two subcutaneous and two intravenous institutions (table 2).
|Comparative data protective and sensitizing activity of preparations derived from different strains of Staphylococcus|
|Strain||Protective activity||Sensitizing activity|
|No.||LD50, MIC. CL ×109||strain to infect||mouse||rabbits||Guinea pigs|
|survived/total||%±m||survived/total||Ms. cont. life, days±m||preparation from strain||the ratio p/introduction||index of anaphylactic shock, %±m|
|2003||0,2||-"-||4/20||20,0±8,9||the concentration is||the concentration is||2003||3-Crat||75,0±5,8|
|-"-||-"-||1986||6/28||21±7,6||the concentration is||the concentration is||the concentration is||the concentration is||the concentration is|
|1986||0,18||-"-||18/45||40±7,2||2/4||27,0±7,6||the concentration is||the concentration is||the concentration is|
|1991||1,0||1986||23/45||51±7,0||4/4||60 (observation period)||-"-||3-Crat||28,5±4,0|
Selection of antigenic components produced by inactivation and drying microbial mass of cells dimethylketone by 2-fold processing in the amount of 3 volumes relative to the volume of the microbial mass.
An example of the method.
Strain S.aureus 1991 (deposited in gisk named after. L.A. Tarasevich) were cultured on solid agar medium on the basis of pancreatic hydrolysate of casein at 37°C for 16-18 hours. The culture is washed off with distilled water. Possible cultivation in reactors by submerged cultivation for 7-8 hours at a temperature of 37°C or through managed processes of cultivation (waste and topping-up, continuous) polysynthetic environment. For example, the environment of salts KN2RHO4To2NRA4, (NH4)2SO4, MgSO4, sodium citrate, with the addition of glucose. and the yeast EXT the act.
Under cultivation in reactors branch of the microbial biomass from the culture medium produced by centrifugation or separation, it is also possible by microfiltration in a tangential flow.
Inactivation of microbial mass produced by 2-fold processing dimethylketone within 18-24 hours of 3 volume relative to the volume of the microbial mass. After the 2nd treatment get dry microbial mass, standartizeta by weight.
The obtained dry inactivated microbial mass is used to immunize animals (mice, rabbits). High protective activity can significantly reduce the immunization scheme to obtain vysokotirazhnyh sera to 2-3 courses:
year 1 - 2 times subcutaneously at intervals of 3-4 days;
2 year 2 times intraperitoneally with an interval of 3-4 days;
3 the course is carried out additionally under the scheme of the 2nd course only in case of insufficiency of 2 courses (table 3).
|The titer of the rabbit sera in TPHA in the dynamics of the 3 courses immunization staphylococcal antigen|
|Antigenic staphylococcal preparation, prepared by the claimed method||No. of sera of rabbits||Reverse antibody titer|
|before immunization||after 1 year p/immunization||after 2 courses/immunization||after 3 courses/immunization|
|P1 and 2<0,05; R2 and 3<0,01; P3 and 4<0,001|
1. Egorova NB, Ephraim V.N., Kurbatova EA, Gruber IM Experimental and clinical and immunological evaluation of acellular staphyococcus vaccine "Stafilov". Journe. microbiol. 2008. No. 6. - P.101-108.
2. Ivanova SP. Method of obtaining immune antistaphylococcal serum. Lab. Case. 1975. No. 5. - 306-308.
3. Mikhailova N.A. and others Experience the vaccine "pipol" for immunization of donors-volunteers. Immunology. - 2006. - V.27. No. 2. - P80-83.
4. Fedorovskaya E.A. and other Immune response of the donor when immunization staphylococcal toxoid. Medical business. 1989. No. 2. - P.70-72.
5. Otto M. Novel targeted immunotherapy approaches for Infection gets. Expert Opin Biol Ther. 2010. - Vol.10. No. 7. P - 1049-1059.
6. Poole-Warren LA, Hallett MD, Hone PW, SH Burden, Farrell PC. Vaccination for prevention of CAPD gets associated infection: results of a prospective multicentre clinical trial. Clin. Nephrol.1991. - Vol.35. No. 5. P.198-206. [PubMed: 1855327]
7. Schaffer AC, Lee J.C. Gets vaccines and immunotherapies. Infect. Dis. Clin. North Am. 2009 - Vol.23. No. 1. 153-71. [PubMed: 19135920]
8. Stranger-Jones Y.K., Bae So, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc. Natl. Acad. Sci. 2006. - Vol.103. No. 45. - P16942-7. [PubMed: 17075065]
9. Ekstedt R.D. Ann N.Y. Immune response to surface antigens of Staphylococcus aureus and their role in resistance to disease gets. N.Y. Acad. Sci., 1974. - Vol.236. - P.203-220.
10. Richter U., Karsch W.Z. Production of diagnostic antisera for the detection of gets enterotoxins A-e Gesamte. Hyg. 1980. - Vol.26. - N1. - P.53-55.
The method of obtaining vysokotirazhnoj antimicrobial serum for about Enki antigenic activity protivotrombovyh vaccines including the use of S.aureus strain No. 1991, high immunogenicity ED50=(50-100)×106microbial cells, weak virulence LD50=(0.5 to 2.0)×109microbial cells, inactivation of him and drying dimethylketone by 2-fold processing 3 volumes dimethylketone with subsequent immunization twice subcutaneously and intravenously twice and isolation of serum.
SUBSTANCE: invention relates to biotechnology, in particular to obtaining nutrient media for cultivation of a listeriosis causative agent. A nutrient medium contains fermentative hydrolysate of soya beans, fermentative hydrolysate from an activated embryo-egg mass of quails, sodium chloride, potassium phosphate 2-substituted 3-aqueous, sodium phosphate 2-substituted 12-aqueous, microbiological agar and distilled water in a specified component ratio.
EFFECT: invention makes it possible to simplify the nutrient solution for cultivation of the listeriosis causative agent.
SUBSTANCE: immobilised biocatalyst for microbial biotransformation of steroid compounds comprises cells of a microorganism having 1,2-dehydrogenase activity, included in the polyvinyl alcohol cryogel matrix. As cell having 1,2-dehydrogenase activity the immobilised biocatalyst comprises biomass of actinobacteria Pimelobacter simplex. The ratio of components in the biocatalyst is (wt %): actinobacteria cells Pimelobacter simplex - 1-5 (in dry substance), polyvinyl alcohol - 7-20, the aqueous phase - up to 100.
EFFECT: implementation of stereo, regioselective biotransformation of respective steroid substrates at their elevated initial concentration, obtaining the desired product with high yield, the ability to perform multiple, at least 30 cycles, use of the biocatalyst without loss of activity.
1 dwg, 1 tbl, 1 ex
SUBSTANCE: method comprises preparing a culture medium, its sterilisation and cooling. In the cooled culture medium the combined inoculum is added, containing separately activated by β-galactosidase bifidobacteria of strain Bifidobacterium longum B 379 M, propionic acid bacteria of strain Propionibakterium Freudenreichii Shermanii subsp. AC-2503, and the activated bacterial inoculum Lactobacillus acidophilus of viscous race taken in a ratio of 9:0.7:0.3, respectively, in an amount of 5% by volume of the culture medium. It is cultured for 14-16 hours at given process parameters, and the cells are separated from the culture liquid to obtain a bacterial mass. The resulting bacterial mass is mixed with the protective medium at a ratio of 1:1. It is bottled, sealed and frozen.
EFFECT: invention enables to intensify the process of fermentation and to improve the structural and mechanical properties of the clot.
2 cl, 7 dwg, 5 tbl, 3 ex
SUBSTANCE: invention relates to biotechnology and a recombinant strain of Escherichia coli bacteria - a producer of biologically active flagellin. The described strain is obtained by transformation of an E. coli BL21[DE3] cell culture with recombinant plasmid DNA pET151FliC, which is obtained based on a pET151FliC vector in which was embedded a fliC gene which codes biologically active flagellin, having a nucleotide sequence represented in Seq ID No 3. The strain is deposited in the Russian National Collection of Industrial Microorganisms (RCIM) of the Research Institute for Genetics and Selection of Industrial Microorganisms under No B-11369.
EFFECT: present solution has higher production capacity with respect to recombinant flagellin, which is an effective adjuvant.
1 dwg, 2 tbl, 3 ex
SUBSTANCE: strain Lactobacillus acidophilus No. 9-PS has biochemical activity and high acidity. The strain is deposited in the Departmental collection of beneficial microorganisms for agricultural purposes of Russian Agricultural Academy (RCAM) under the registration number of RCAM01850. The strain may find application in prevention and correction of disorders of microbiocenosis of the gastrointestinal tract.
EFFECT: invention enables to increase growth of mucous cultures of lactic acid bacteria and accelerates the process of colonisation of intestinal with beneficial microflora.
2 tbl, 3 ex
SUBSTANCE: strain Gluconacetobacter sucrofermentans H - 110 is a producer of bacterial cellulose. The strain is deposited in the Russian National Collection of Industrial Microorganisms under the registration number of strain Gluconacetobacter sucrofermentans RNCIM B-11267 and can be used in medicine, food and pharmaceutical industries.
EFFECT: invention enables to increase the yield of bacterial cellulose.
2 dwg, 6 ex
FIELD: food industry.
SUBSTANCE: inventions group relates to the field of microbiology and may be used in food industry. One proposes Lactobacillus sanfranciscensis DSM 22063 strain and Lactobacillus plantarum DSM 22064 strain; the both strains are able to perform complete gluten decomposition in flour. The strains may be used for production of a mixture for complete gluten decomposition in flour. Additionally, one proposes a method for preparation of dough of flour with completely decomposed gluten. The produced gluten- detoxified flour dough may be used for production of a baking mixture with completely decomposed gluten. The said dough may be used for yeast bakery goods production. Additionally, the baking mixture and gluten- detoxified dough may be used for manufacture of food products suitable for recovery of nutritional imbalance being the consequence of gluten-free food ration.
EFFECT: inventions group allows to manufacture a product with residual gluten concentration lower than 20 ppm.
24 cl, 7 dwg, 2 tbl, 5 ex
SUBSTANCE: bacterial strain Exiguobacterium mexicanum RNCIM B-11011 is proposed, having the ability to dispose quickly of oil, diesel fuel, motor oil, gas condensate.
EFFECT: strain can be used to clean soil and water reservoirs contaminated by crude oil and petroleum products in a wide temperature range.
3 tbl, 5 ex
SUBSTANCE: strain of bacteria Exguobacterium mexicanum RNCIM V-11011 is grown, and the suspension is made from it, which is applied in the cryomorphic soil and water environment. It is exposed under the specified parameters from 7 to 60 days and the quantitative content of oil and petroleum products in the test soil and water environment is determined.
EFFECT: invention enables to reduce the time of denaturation of oil and petroleum products and to reduce the concentration of oil and petroleum products in the soil and water environment.
3 tbl, 4 ex
SUBSTANCE: propanediol-oxydoreductase coding gene is introduced into an E.coli cell, which makes it possible to produce high levels of 1,2-propanediol, in fact, without 1,3-propanediol, with growing on glycerol as the only carbon source. Genes of glycerol dehydrogenase (gldA), dihydroxyacetone kinase (dhaK) and/or methylglyoxal synthase (mgsA) can be additionally inserted to express the said enzymes together with propanediol-oxydoreductase (fucO), as well as a gene of glycerol dehydratase or aldo-keto reductase in order to express the said glycerol dehydratase or aldo-keto reductase together with propanediol-oxydoreductase. The E.coli cell, transformed by the propanediol-oxydoreductase coding gene, can be defective in arabinose, methylglyoxal and/or dihydroxyacetonephosphate metabolism.
EFFECT: invention relates to the field of genetic engineering and can be used for recombinant production of 1,2-propanediol (1,2-PD).
9 cl, 4 dwg, 9 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed group of inventions relates to medicine, namely to cardiology, and deals with treatment and prevention of arterial hypertension. For this purpose pharmaceutical composition, containing activated potentiated antibodies to angiotensin II receptor and to endothelial NO-synthase, is introduced.
EFFECT: method ensures effective treatment of arterial hypertension due to synergic antihypertensive action of composition components.
11 cl, 2 ex, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed group relates to medicine, namely to cardiology, and deals with treatment of chronic heart failure. For this purpose pharmaceutical composition, containing activated potentiated forms of antibodies to angiotensin II receptor and to endothelial NO-synthase is introduced.
EFFECT: synergic action of pharmacological composition components ensures improvement of systolic function of left ventricle and increase of tolerance to physical load in said group of patients.
11 cl, 2 ex, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.
EFFECT: invention can be used in medicine.
74 cl, 14 dwg, 14 ex, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed invention relates to the field of immunology. Disclosed is a method of treating rheumatoid arthritis, chronic arthritis in children or Castelman's disease, application of an antibody in the said method, as well as application of an antibody in production of a medication for treatment of rheumatoid arthritis, chronic arthritis in children or Castelman's disease. The antibody by the claimed invention possesses an improved antigen-neutralising ability, pharmacokinetics, immunogenicity, safety and physicochemical properties and can be further applied in therapy of diseases, associated with the activation of the receptor IL-6.
EFFECT: claimed is the medication for treatment of rheumatoid arthritis, chronic arthritis in children or Castelman's disease, representing the antibody against the receptor IL-6, obtained on the basis of the antibody TOCILIZUMAB.
12 cl, 5 dwg, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biotechnology and immunology. Described are versions of antibodies, binding the GRM molecule, as well as their antigen-binding fragments, amino acid sequences of variable parts of which are presented in the claim materials. Nucleic acid, coding the said antibodies, is presented. Claimed is a method of obtaining the RGM-binding protein, which includes cultivation of a host cell in a culture medium under conditions suitable for obtaining the binding protein, capable of binding with RGM, where the host cell contains an expression vector, containing the separated nucleic acid, coding the said antibody. Described is a pharmaceutical composition for treating a disease, in which the SGM A activity produces a negative impact, which contains a therapeutically efficient quantity of the said antibody and a pharmaceutically acceptable carrier. Claimed is an application of the said antibody for obtaining a medication, used for a) reduction of hRGM A binding with a patient's Neogenin receptor; or b) for reduction of hRGM A binding with BMP-2 and BMP-4 in the patient.
EFFECT: invention makes it possible to obtain antibodies against GRM, which are used for treating diseases, associated with excessive interaction of RGM with the Neogenin receptor, BMP-2 and BMP-4.
13 cl, 16 dwg, 10 tbl, 11 ex
SUBSTANCE: claimed invention relates to biotechnology and represents a polypeptide construction for treatment, prevention and relief of disorders, associated with an adhesion of platelets and platelet-mediated aggregation or its dysfunction, which includes one or more single-domain antibodies, aimed against the von Willebrand factor (vWF), and one or more single-domain antibodies aimed against serum albumen (SA). The invention also relates to nucleic acid, coding such polypeptide construction, to compositions, containing the said construction, and to its application for obtaining medications for prevention, treatment and relief of the said disorders.
EFFECT: claimed invention makes it possible to extend an assortment of medications for treatment, prevention and relief of disorders, associated with the platelet adhesion and platelet-associated aggregation or its dysfunction.
15 cl, 30 dwg, 32 tbl, 69 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to medicine and deals with a pharmaceutical composition for enhancing the efficiency of treatment of liver cancer by Sorafenib, with the said composition containing an anti-glypican 3-antibody as an active ingredient.
EFFECT: invention provides an improved anti-cancer effect and reduction of the side effect, in particular loss of the body weight.
14 cl, 9 dwg, 2 tbl, 6 ex
SUBSTANCE: invention refers to medicine, namely to gastroenterology and endocrinology, and concerns treating patients suffering from dyspepsia syndrome in a combination with overweight. That is ensured by therapy with preparations improving metabolism, promoting weight loss and fat absorption, as well as antidepressants. The therapy is differentiated taking into account an anxiety level (HARS) and a depression level (HDRS) according to Hamilton rating scales, nutritional status assessed by bioimpedancemetry, a degree of manifestation of sleep disorders, eating behaviour typing, eating regimen and daily rhythm determination , level evaluation of glucose, immunoreactive insulin, cholesterol, high-density lipoprotein (HDLP), triglyceride in venous blood, blood glucose tolerance, gustation, life quality assessment.
EFFECT: differentiated approach provides an effective treatment of dyspepsia in a combination with weight loss, correction of eating behaviour and metabolic processes.
2 ex, 2 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present group of inventions refers to medicine, namely to therapy, and concerns treating vegetovascular dystonia (VVD). That is ensured by administering a pharmaceutical composition containing activated potentiated angiotensin II receptor antibodies and activated potentiated endothelial NO-synthase antibodies.
EFFECT: method provides effective treatment of vegetovascular dystonia ensured by the synergetic action of the ingredients of the pharmaceutical composition.
1 ex, 1 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to medicine, specifically cardiology, and concerns treating chronic cardiac failure. That is ensured by administering a pharmaceutical composition containing an activated potentiated form of very-low-dose angiotensin II receptor antibodies and an activated potentiated form of very-low-dose endothelial NO-synthase antibodies.
EFFECT: method provides improving quality of life and higher tolerance to physical loads in the given group of patients.
11 cl, 2 ex, 2 tbl
FIELD: genetic engineering, immunology, medicine.
SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.
EFFECT: improved preparing methods, valuable medicinal properties of antibody.
33 cl, 5 dwg, 1 ex