Method for noncovalent immobilisation of lipopolysaccharide shigella flexneri 2a on solid hydrophobic carrier
SUBSTANCE: method involves passing a solution of lipopolysaccharide Shigella flexneri 2a in a 0.05 M triethylammonium acetate buffer at pH 4.7, which contains 10% n-propanol, through a column with a hydrophobic carrier which is Octyl Sepharose CL-4B, and then washing the column with the same buffer solution.
EFFECT: method can be used to produce carriers which enable extraction and purification of specific antibodies to lipopolysaccharides using affinity chromatography.
The SCOPE of the INVENTION
The invention relates to the field of immunopharmacology and can be used to create media for affinity chromatography.
The LEVEL of TECHNOLOGY
Affinity chromatography is one of the most effective methods of isolation and purification of specific immunoglobulins (antibodies) . For this purpose usually carry out the following sequence of operations: activation of the solid carrier, chemical immobilization on it antigenic ligand, the processing of the thus obtained carrier mixture containing the specific antibodies, formation of a strong immune complex immobilized ligand with specific immunoglobulin complexes (antigen-antibody), delete all not contacting the ligand of the components of the mixture, the dissociation of the complex antigen-antibody, accompanied by the release of specific immunoglobulins purified.
One of the few drawbacks of this method is the need to create "links" (often apparently numerous) when immobilization of the antigen molecule on activated carrier that can lead to distortion of its spatial structure. When using affinity chromatography for isolation of antibodies against lipopolysaccharides (LPS) of gram-negative microorganisms often have extra for is rudnany, due to the formation of a strong micelles, which may include up to several thousand LPS molecules .
LPS is the major polysaccharide antigen of gram-negative bacteria, localized on the outer surface of the outer membrane of the cell wall and is a family of structurally related amphiphilic macromolecules. Polysaccharide fragment FSC presents the O-specific polysaccharide (OPS), built of oligosaccharide repeating units, the number of which may vary from 1 to several dozen. OPS is attached to the oligosaccharide bark, which, in turn, is associated with the lipid component of the lipid A. Lipid And serves as an "anchor"that holds the LPS molecule in the membrane of the bacterial cell, and the polysaccharide fragment is directed outward toward the environment.
In the LPS micelles formed in aqueous solutions, lipid And is located inside the micelles, and OPS, built in most cases of hydrophilic monosaccharide residues, as in bacterial cells, pointing towards the aqueous phase. When immobilization of LPS on the activated carrier the formation of covalent bonds ("links") can occur only with spatial affordable LPS molecules in the micelles, as well as with the individual, not part of the micelles LPS molecules, the number of which in aqueous solution is x small. Thus, despite the fact that quantification can show the desired level of immobilized LPS, the number of LPS molecules irreversibly covalently coupled to a carrier, may be low. So often at the stage of immobilization and subsequent stages of affinity chromatography in the eluates there is a noticeable level of LPS, which leads to contamination of the target product.
DISCLOSURE of INVENTIONS
We propose a method of immobilization of the LPS of gram-negative bacteria on solid hydrophobic matrix without the formation of covalent bonds between the matrix and the LPS molecules. Received thus activated by lipopolysaccharide media can be used for isolation and purification of specific antibodies to LPS using affinity chromatography. A strong Association between LPS and hydrophobic matrix fixation of LPS on the surface of the matrix as in the formation of an immune complex, and the process of dissociation. The proposed method includes processing a hydrophobic matrix solution chemicalromance LPS in aqueous buffer with a pH of 3.0 to 8.0, containing the required number n-propanol. The introduction of n-propanol is achieved domiciliaria LPS that provides for effective adsorption of individual molecules of LPS on the surface of the matrix. The tendency LPS to domiciliario depends on solargeneration components in OPS (N-acetylamino-, deoxy-, detoxifier, O-acetyl groups and the like), as well as the charge OPS. So OPS, part of the FSC can be both neutral and contain acid fragments (residues of uronic acids, phosphate), positively charged components (residues of amino sugars, ethanolamine), as well as to have zwitter-ionic character, i.e. it may bear, for example, alternating carboxyl and amino groups. The degree of domicilliary LPS is significantly influenced by the composition, ionic strength and pH of the buffer. Therefore, when immobilization of specific FSC is necessary to determine the optimal qualitative and quantitative composition of the buffer, providing a full digitalnirvana LPS. Control of the completeness of domicilliary can be carried out using penetrating gel-chromatography on a column of Separate CL-4B or other similar characteristics of the separation gel with elution buffer intended for immobilization. Retention time chemicalromance form LPS is almost identical to the retention time of the OPS, which can be selected from the given LPS using mild acid hydrolysis (1%acetic acid, 100°C, 1 hour)accompanied by cleavage of lipid A.
The IMPLEMENTATION of the INVENTION
As a solid carrier for immobilization of LPS can be used phenyl-, methyl-, ethyl-, butyl-, amyl-, hexyl-, ecil or domiciliaries (4% crosslinking), and octyl-. or phenyl-Sepharose CL-4B, and the column Supersoy 4 or 6 (Fast Flow) column with Toyopearl Resins butyl-650 and phenyl-650. The concentration of n-propanol in the buffer is determined experimentally, but usually does not exceed 10-15%. The salt concentration of the buffer components does not exceed 0.2 M Temperature in the process of immobilization is maintained in the range of 10-20°C, the temperature at which is affinity chromatography, typically no greater than the temperature maintained in the process of immobilization of LPS on the media. The immobilization time is usually 0.5 to 20 hours. The hydrophobic carrier is mixed with a solution of LPS in the buffer at a speed of 20-50 rpm using an orbital shaker and then washed with buffer for affinity chromatography, to lack of components immobilization buffer and LPS. All stages of immobilization can be carried out directly in the column for affinity chromatography. To determine the number sorbirovannoe on the media FSC measured volume of the gel is washed 2-3-fold volume of immobilization buffer containing 40-45% n-propanol, followed by dialysis against distilled water and lyophilization.
The following example is given to detail and illustrate the invention, is not intended to limit the claims of the present invention.
Through a column (1.5 x 1cm) with 10 ml octyl-Sepharose CL-4B miss 30 ml aqueous 0.05 M triethylammonium buffer, pH of 4.7, containing 45% n-propanol, at a speed of 5 ml/hour, and then balance column with the same buffer containing 10% n-propanol (about./about.) (buffer A). Next to the column attached to a flow-through UV detector (wavelength 226 and 276 nm), put a solution of 10 mg LPS Shigella flexneri 2a (obtained water-phenol extraction method of Westphal and Desription) in 3 ml of buffer A. After washing with 30 ml of buffer A. the column was washed with 30 ml fishriver and put on her 1 ml of hyperimmune rabbit serum. The column was washed with fistarol to absorption at 226 nm of less than 0.05 AU and then a 3.5 m solution of magnesium chloride for the destruction of the immune complex. The fractions containing (according to UV-detector) protein components, unite, dialist against fishriver (4 sessions, 1 hour) at a temperature of 8-10°C and lyophilized. A comparative analysis of the original serum and the resulting product using a response inhibition passive haemagglutination assays, enzyme immunoassay and Western blot turns showed that the affinity chromatography quantitatively selected mixture of specific antibodies against LPS Shigella flexneri 2a.
Sources of information
1. Affinity chromatography. Methods. Editors Pdin, Ugandan, Fmil. TRANS. from English. Kida. chem. Sciences Bagladeshi. M.: Mir, 1988.
2. High-molecular Weight Components in Lipopolysaccharides of Salmonella typhimurium, Salmonella minesota, and Escherichia coli. Peterson, A.A., and E.J.Mcgroarty. J.Bacterioloy, 1985, 162:738-745.
The method of immobilization of the lipopolysaccharide of Shigella exneri 2a on a hydrophobic solid media due to the formation of non-covalent bonds between the carrier and the lipopolysaccharide molecules, characterized in that through a column of hydrophobic media, representing octyl-Sepharose CL-4B miss the solution of lipopolysaccharide of Shigella flexneri 2a in 0.05 M triethylammonium buffer, pH 4,7, containing 10% n-propanol, followed by washing the column with the same buffer.
SUBSTANCE: methods involve obtaining a bacterial cellulose product through fermentation, possible lysis of bacterial cells from the bacterial cellulose product, mixing it with at least one polymeric thickener or precipitating agent and coprecipitation of the obtained mixture with alcohol. The bacterial cell lysis step can be performed after mixing with the precipitating agent. The polymeric thickener used can be at least one charged cellulose ester, at least one precipitating agent or combination thereof. The composition obtained using said method, having improved rheological properties and containing bacterial cellulose, has viscosity of at least 300 cP (300 mPa·s) and tensile yield 1.0 dine/cm2 (0.1 N/m) when more than 0.36 wt % of it is put into a 500 ml water sample and after application of not more than two passages in an extensional homogeniser at pressure 1500 lb/in2 (10342.5 kPa).
EFFECT: improved rheological properties of the composition.
31 cl, 20 ex
SUBSTANCE: what is offered is a method of making a construct containing crystal cellulose. Cellulose-forming organisms are grown at least partially in a hollow template made by means of a three-dimensional printer. What is offered is a method of making the hollow template by means of the three-dimensional printer which grows portions of the hollow template in layers from a modelling material.
EFFECT: invention can be used for making the construct which can be successfully used as an implant or for cultivation of mammal or human living cells.
16 cl, 7 dwg
SUBSTANCE: starting high-molecular chitosan is dissolved in acid solution. The chitosan dissolved in the acid is then precipitated by adding alkali solution. The re-precipitated high-molecular chitosan is washed from the formed salt and excess alkali using a coarse-porous filter. The re-precipitated chitosan is dissolved in acid solution until achieving pH 5.5. An enzyme preparation is then added and hydrolysis is carried out. The reaction is stopped after formation of low-molecular chitosan.
EFFECT: method is characterised by avoiding the need to remove salts from the enzymatic mixture and the end product, as well as low level of loss of material.
2 dwg, 7 tbl, 6 ex
SUBSTANCE: polysaccharide contains galactose, glucose, ramnose as monosaccharide components and pyruvic acid bound with any of the monosaccaride components. Molar content of galactose, glucose and ramnose in polysaccharide is in ratio 4:2:1, quantity of pyruvic acid being from 4 to 7% in weight. Said polysaccharide has structure, represented by formula (I): Polysaccharide is obtained by cultivation in anaerobic conditions of strain Bifidobacterium longum JBL05 (NITE BP-82) - polysaccharide producent. Also claimed are immunostimulator and moisturising means representing said polypeptide.
EFFECT: increase of moisturising and immunostimulating activity.
5 cl, 6 dwg, 6 ex
SUBSTANCE: according to the method, wood raw material undergoes coarse grinding first and resinous substances are then extracted, after which the obtained product undergoes fine grinding and mechanical activation. The installation has for coarse grinding wood raw material, apparatus for extracting resinous substances and apparatus for fine grinding and mechanical activation arranged in series.
EFFECT: invention simplifies the method of preparing wood raw material and increases output of sugars from said material at the fermentolysis step.
8 cl, 1 dwg, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: there is offered a method for preparing low-molecular heparins by enzymatic degradation. For enzymatic depolymerisation, immobilised lysozyme is used.
EFFECT: prepared low-molecular heparin shows higher inhibitor activity with respect to blood coagulation factor Xa and lower inhibitor activity with respect to thrombin in comparison with initial heparin.
2 tbl, 3 ex
FIELD: food industry.
SUBSTANCE: method provides for depth cultivation of basidium fungi Pleurotus ostreatus on nutrient medium of the following composition (g/l): soya flour - 21, KH2PO4 - 3.5, MgSO4 - 0.4, milk whey - 200 ml, water - up to 1 litre. Mycelium is exposed to deep freezing with further defrosting. Then its disintegration is carried out. Food fibres are cleaned by alternating treatment with cold and hot water, mix of 1 M sodium carbonate or sodium hydroxide with ethanol in the ratio of 1:2 and 0.5 M solution of citric acid. Yield of food fibres makes 16-18% of dry biomass, content of chitin - 16-19%, glucan - 40-50% of dry mass of food fibres.
EFFECT: food fibres with high content of chitin.
SUBSTANCE: invention refers to immunology and biotechnology. There is offered method of cleaning bacterial capsular polysaccharide Neisseria meningitidis or Haemophilus influenzae. Polysaccharide is precipitated with using a cationic detergent, gathered, solubilised with pure ethanol or mixed ethanol:water with final concentration of ethanol 50% to 95%, filtered and then precipitated with calcium or sodium salts. There are described versions of the method of preparing the vaccines with using cleaning capsular polysaccharide. There is disclosed the method for solubilisation of capsular polysaccharide.
EFFECT: application of the invention ensures simple and fast method of cleaning capsular polysaccharide that can find application in medicine for preparing the vaccine.
26 cl, 10 dwg, 24 tbl
SUBSTANCE: method for production of agarose includes preparation of dry sea algae Gracilaria spp., preferably, Gracilaria dura from natural sources or cultivated in polyethylene bags or on rafts, treatment of dry algae with alkali. Treated algae are washed with water till pH of washing waters makes from 7 to 8, water is added. Autoclaving and further homogenisation are carried out with production of extract. Extract is treated with activated carbon and celite at the temperature of 85-95°C to prepare hot extract. Vacuum filtration of extract is carried out through a layer of celite. Filtrate is frozen with preparation of mass, and produced mass is thawed. In order to remove thawed liquid, product produced at the stage of freezing and thawing is compressed and further squeezed. Agarose produced as a result of this process is dissolved in water by means of heating in autoclave. Stage of freezing and thawing, as well as product compressing and squeezing are repeated. Produced product is dissolved in water and dried by means of spraying to produce finely dispersed powder. Agarose is also suggested, which is produced by specified method.
EFFECT: invention makes it possible to produce agarose with high strength of gel and low temperature of gel formation Strength of 1% gel makes ≥1900g/cm2, and gel formation temperature makes 35-35,5°C.
6 cl, 6 tbl, 8 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention pertains to biotechnology and can be used in biomedicine for producing hyaluronan. Proposal is given of a method of producing hyaluronan, involving culturing Bacillus host cells in conditions, suitable for obtaining hyaluronan, and extraction of the target product from the culture medium. The Bacillus host cell contains a genetic structure, comprising a promoter, functionally active in the given cell, and encoding a region, consisting of a nucleotide sequence, encoding streptococcal hyaluronansynthase (hasA); sequence encoding UDP-glucose-6-dehydrogenase Bacillus (tuaD) or a similar enzyme of streptococcal origin (hasB), and a sequence, encoding bacterial or streptococcal UDP-glucose pyrophosphorylase (gtaB and hasC respectively). Use of the invented method provides for production of considerable quantities of hyaluronan with good examined, nonpathogenic cellular system.
EFFECT: obtaining considerable of hyaluronan with good examined, nonpathogenic cellular system.
15 cl, 45 dwg, 2 tbl, 20 ex
FIELD: industrial organic synthesis.
SUBSTANCE: isopropyl alcohol production process comprises hydrogenation of starting acetone including from 0.01 to 10000 ppm benzene in presence of hydrogen and catalyst to give isopropyl alcohol and benzene hydrogenation products, acetone and benzene contained in feedstock being hydrogenated simultaneously. In its second embodiment, isopropyl alcohol production process comprises product separation stage. Process of producing phenol and isopropyl alcohol containing benzene hydrogenation products comprises stages: alkylation of benzene with isopropyl alcohol and/or propylene to form cumene, oxidation of resulting cumene into cumene hydroperoxide, acid cleavage of cumene hydroperoxide to produce phenol and acetone including from 0.01 to 10000 ppm benzene, preferably concentration of produced benzene-polluted acetone, and catalytic hydrogenation of benzene-polluted acetone into isopropyl alcohol containing benzene hydrogenation products, hydrogenation of benzene and acetone proceeding simultaneously.
EFFECT: enhanced process efficiency.
3 cl, 1 dwg, 1 tbl
FIELD: chemical technology.
SUBSTANCE: invention relates to a method for hydrogenation of acetone to yield isopropyl alcohol as an intermediate compound widely used in organic synthesis and an important industrial solvent. Method involves the hydrogenation reaction that is carried out in multitube reactor wherein nickel-base catalyst is used and reactor works in small reagents flow feeding into reactor. Based on using multitube reactor the method provides carrying out the regulated and controlled elimination of heat formed in the reaction carrying out. Method shows economy as it doesn't require recycle of the valuable end reaction product.
EFFECT: improved method of hydrogenation.
9 cl, 1 tbl, 1 dwg
FIELD: industrial organic synthesis and chemical engineering .
SUBSTANCE: invention relates to a process of producing liquid oxygenates, including methanol, C2-C4-alcohols, formaldehyde, lower organic acids, or mixtures thereof, and to installation for implementation the process. Process comprises successively supplying natural gas from complex gas preparation plant to a series of "gas-gas" heat exchangers and into annular space of at least one tubular reaction zone of reactor, wherein natural gas is heated to temperature of the beginning of reaction, whereupon heated gas is passed to the entry of the tubular reaction zone mixer, into which compressed air or oxygen is also injected to provide gas-phase oxidation in reaction zone of reactor. Resulting reaction mixture is discharged from reactor into a series of "gas-liquid" and "gas-gas" heat exchangers, wherein reaction mixture is cooled to ambient temperature and sent to separator, wherefrom liquid phase is passed through lower carboxylic acid recovery vessel to the system of rectification columns to isolate the rest of mixture components, whereas leaving gas is recycled to complex gas preparation plant. More specifically, oxidation is carried out within temperature range 240 to 450°C and pressure from 2 to 10 MPa at residence time of reaction mixture in reactor 2-6 sec and oxidant concentration 2 to 15 wt %. In reactor having mixers hollow and at least one tubular reaction zones, required temperature is maintained constant throughout all length of tubular reaction zone and at entries for compressed air or oxygen in mixers of each of tubular reaction zones and hollow reaction zone. Liquid oxygenate production plant is composed of aforesaid complex gas preparation plant, a series of "gas-gas" heat exchanger to heat natural gas, reactor, a series of "gas-liquid" and "gas-gas" heat exchangers to cool reaction mixture obtained in reactor, gas-liquid separator, lower carboxylic acid recovery vessel, and system of rectification columns to isolate the rest of products.
EFFECT: enabled implementation of the process directly near gas and gas condensate deposits, increased conversion of methane per one passage through reactor, and increased yield of oxygenates due to improved design of plant.
6 cl, 1 dwg, 1 tbl
FIELD: organic chemistry, chemical technology.
SUBSTANCE: invention relates to a method for synthesis of isopropanol of high purity by hydrogenation reaction of acetone. Hydrogenation of acetone is carried out in liquid phase for at least two stages at temperature from 60oC to 140oC and under pressure from 20 to 50 bars. In hydrogenation nickel-containing catalyst on neutral carrier, preferably on a carrier made of α-form of aluminum oxide is used. The molar ratio hydrogen : acetone = (1.5-1):1. Reactor of the first stage can be circulation reaction, and at the second stage - tube reactor. For hydrogenation reaction acetone containing 1.0 wt.-% of water, not less, is used. Invention provides carrying out the process with high selectivity to yield isopropanol containing impurities in the concentration less 300 mole fr., preferably, less 100 mole fr.
EFFECT: improved hydrogenation method.
6 cl, 2 dwg, 1 ex
SUBSTANCE: method involves specific immobilisation of fragments of the specified bacteria from a solution on an affine surface consisting of a protein G layer coated with a layer of antibodies specific to the identified bacterial cells. Two-stage washing of a sample from a non-specifically bound material implies washing in a buffer pH 8.5-9.5 and washing in deionised water. The atomic-force microscopy is used as a visualisation tool of the bound bacterial fragments and enables direct count of a bound analyte amount per a surface unit.
EFFECT: offered invention allows identifying the minute cell concentration in the analysed solution.