The allocation method c3component of the human complement
(57) Abstract:The invention relates to medicine, in particular to biochemistry and immunochemistry, and can be used in the diagnosis of diseases associated with activation of the complement system. The invention consists in that an alcohol residue IV - I Stake precipitated 16% poly (ethylene glycol) m m 4000 - 6000. The resulting precipitate emit C3component of complement, using ion-exchange chromatography on DEAE-Toarea 1 and gelfiltration on sephacryl S-200. The proposed method of purification of C3component of the human complement of sediment IV - I in the line has advantages over the prototype, because the method does not require expensive reagents, raw materials, simple in implementation. Advantages of the invention is to simplify and reduce the cost allocation method C3component of complement, waste disposal industrial fractionation of plasma. The invention relates to medicine, in particular to biochemistry and immunochemistry, and can be used in the diagnosis of diseases associated with activation of the complement system: diseases of immune complexes, infectious diseases, respiratory distress syndrome of adults, hereditary defeo3component of the human complement. For example, the widely used method of selection3component of the complement of the plasma, proposed by R. A. Harrison and R. J. Lachmann in 1979 , involving pre-treatment of fresh plasma inhibitors (EDTA, sodium azide, phenylmethylsulfonyl) to prevent activation of proteolytic enzymes in blood plasma and starting a cascade of reactions in activation of the complement system with subsequent fractionation of plasma sodium hydrosulphate to 20% and presidenial precipitate formed 11% of sodium sulfate. The supernatant from the second deposition is divided into pseudoglobulin and euglobulin faction in dialysis low ionic strength and slightly acidic pH. WITH3component of complement extracted from sediment euglobulin by ion-exchange chromatography on DEAE-cellulose and chromatography on hydroxyl-Apatite. Finish the selection WITH3component of the complement dialysis3-containing fractions against solution inhibitors (EDTA, NaN3), ultrafiltration.The prototype of obtaining high-purity WITH3component of the complement is the method described Tach. B. F. and Prahly. W. (1976). It includes: a step fractionation of fresh plasma is matography on L-lysine-sepharose 4B to remove plasminogen, ion-exchange chromatography on DEAE-cellulose, gel filtration on sepharose CL-6B, chromatography on hydroxyapatite) with control hemolytic activity at each stage. The method is sequential, time-consuming, it is used of expensive reagents and sorbents for chromatography.The aim of the present invention is to simplify and reduce the cost allocation method WITH3component of the human complement, which is especially important for diagnostic products, and recycling of waste industrial fractionation of plasma.The objective is achieved by the fact that3component of complement allocate only 3 main stages, using as source3component waste sludge IV-I at Stake. According to Cohnetal. (1946) in the sediment IV-I, obtained by the fractionation of blood plasma in the cold, contains components of complement.The first stage of the selection WITH3from the precipitate IY-I is the precipitation of 20% sediment IV-I, dissolved in 0.1 M K-R, pH 7,1-7,3, 10 mm EDTA, 0.5 M NaCl poly(ethylene glycol) 4000-6000. 32% PEG 4000-6000 add a 20% solution of sediment IV-I to a final concentration of 16% PEG 4000-6000 under stirring. The formed precipitate was separated by centrifugation for 20 min at 6000 rpm, 4varaut in 0.01 M K-R, pH 7.5, 10 mm EDTA and chromatographic on the column with DEAE-Tooreal I, equilibrated with the same buffer, elution of bound peroxidase protein, using a linear concentration gradient of NaCl 0-0,3 M Identification of fractions containing3conduct immunohistochemistry. Further concentrated using ultrafiltration membrane PM-30 component WITH3additionally purified by gel-filtration on sephacryl S-200 in 0.01 M K-R, pH 7.5, 10 mm EDTA, 0.15 M NaCl, 0.02% Of NaN3.The proposed method of cleaning WITH3component of the complement of the bargain sediment IV-I in the line has advantages over the prototype, because the method does not require expensive reagents, simple, has only 3 main stages: I - precipitation PEG 4000-6000;
3 - gel-filtering.Cleared WITH3component of complement can be used: 1) as the immunogen for hyperimmunization animals in order to obtain monospecific anti-C3precipitating serum; 2) to remove unwanted anti-C3antibodies of the polyvalent sera; 3) to determine the titers of anti-C3precipitating serum by the method of REED Mancini. THE ALLOCATION METHOD C3COMPONENT of COMPLEMENT CHE the relevant clearing of sediment on DEAE-aminoalkanoic and gel-filtration, characterized in that is used as raw material sediment IV - I fractionation of blood plasma at Stake, before processing the PEG it is dissolved in a buffer solution of high ionic strength at neutral pH and gel-filtration is carried out with the help of sephacryl S - 200.
FIELD: microbiology and immunology, in particular immunodiagnosis.
SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.
EFFECT: immune serum with increased specific activity.
2 tbl, 2 ex
FIELD: molecular biology, biotechnology, gene engineering, in particular diagnosis of atypical pneumonias.
SUBSTANCE: invention relates to DNA based on identified protein antigen epitope SARS-CoV. Particularly disclosed are nucleotide sequences of synthetic genes encoding recombinant protein fragments containing coronavirus protein antigen determinants SARS-CoV, represented in SEQ ID NO:1 - SEQ ID NO:8; as well as SARS-CoV virus protein fragments encoded by DNA sequences with amino acid sequences represented in SEQ ID NO:10 - SEQ ID NO:17. Said fragments are isolated by using preliminary obtained fusion protein by attachment to its N-terminal end GST-S-transferase protein with sequence represented in SEQ ID NO:9. Fusion proteins are useful in manufacturing of preparations for diagnosis of acute respiratory virus SARS-CoV.
EFFECT: new diagnostic agents for diagnosis of atypical pneumonias.
FIELD: medicine, radiation biology.
SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.
EFFECT: improved preparing method, improved method for diagnosis.
1 tbl, 3 ex
FIELD: biotechnology, medicine, immunology.
SUBSTANCE: method involves preparing control samples by preparing solution of heterologous chimeric antibodies consisting of whole molecules or fragments of immunoglobulin isolated from immunized animal serum and associated with whole molecules or fragments of human immunoglobulin in phosphate-saline buffer, pH 6.0 followed by preparing different dilutions in indicated buffer, their control in IFA and selection of dilutions at optical density values 1.0, not less. Invention provides preparing control samples comprising specific antibodies that elicit high specificity and capacity for detection in combination with their infectious safety, standard indices and availability with respect to economy aspects.
EFFECT: improved preparing method.
FIELD: veterinary microbiology.
SUBSTANCE: claimed method includes providing of stable culture from B.abortus 19 strain in L-form followed by rabbit immunization. Culture is obtained in dense broth containing additionally 10-15 % of normal hoarse serum wherein broth is singly exposed to streptomycin action in dose of 2.5-5.0 U/ml of medium. Then slurry is prepared from obtained culture, inactivated at 85-90°C for 160 min and triply intravenously administrated to rabbits in increasing doses with 72 h intervals.
EFFECT: method for more exact estimation of brucelliasis epizootic situation on basis of typical, dissociated and deep-altered brucella forms.
6 tbl, 4 ex
FIELD: veterinary virology, in particular production of latex diagnosticum for diagnosis of horned cattle and poultry infections.
SUBSTANCE: claimed method includes centrifugation of polymeric suspension and adjusting of polymeric suspension with distilled water up to 0.5 % (calculated as polymer dry residue)/ Then equal volumes of viral antigen in infective titer of 6.5 lg TCD/50 ml (tissue cytopatic doses) and 0.5 %-1.0 % polymeric suspension are mixed and mixture is hold in thermostatic regulator at 36-38°C for 4-6 h. Further 0.07-0.15 % aqueous solution of human serum albumin is added into total suspension volume, mixture is incubated .at 4-8°C for 11-13 h; suspension is washed, and diagnosticum concentration is adjusted with phosphate buffered saline with pH 7.2-7.4 up to 0.1-0.3 %. Prepared diagnosticum is stored at 4°C not more than 1 year.
EFFECT: diagnosticum for before-the-fact detection of different infective diseases, prophylaxis and treatment.
7 ex, 7 tbl
FIELD: medical engineering.
SUBSTANCE: device has substrate having polymeric working layer on it, produced from copolymer based on methacrylic acid derivatives with biological macromolecules (probes) immobilized thereon. The substrate is manufactured from activated or not activated glass, metal or polymer material. The working layer has macroporous monolithic copolymer glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass with affine biological probes immobilized thereon. Probe-copolymer proportion is 2-10 mg/g of copolymer, for protein, 1-20 mg/g of copolymer for peptide and for oligonucleotide, nucleic acid - 0.5-3 mg/g of copolymer, pore radius of 0.4-1.5 mcm, it has thickness of 50-700 microns and is manufactured as continuous or discrete microcellular layer. The method for manufacturing biochip involves preparing substrate, producing working layer by monomer copolymerization on methacrylic acid derivatives base, immobilizing biological macromolecules - probes on forming copolymer, washing, drying the received biochip. Radical copolymerization of glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass is carried out for producing working layer with photo-or thermal initiation in poregenic solvent medium being applied. Proportion of the sum of monomer volumes to solvent volume being equal to 6:9, initiator concentration in reactionary medium being equal to 0.2-1.0% by weight, given reaction mixture is placed on substrate as continuous or discrete layer. Macroporous monolithic continuous or discrete microcellular layer is formed as a result of copolymerization on the substrate. Then, covalent immobilization of biological macromolecules is carried out in the layer pores or their direct synthesis on formed copolymer with its native or modified epoxy groups being used. Biological affine probe is produced. The probe is introduced into copolymer in quantity of 2-10 mg/g of copolymer for fiber, for peptide - 1-20 mg/g of copolymer and for oligonucleotide or nucleic acid - 0.5-3 mg/g of copolymer.
EFFECT: manufacturing reusable biochip with predetermined controllable and reproduced quality.
FIELD: medicine, veterinary.
SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.
EFFECT: method reduces the test time and economical costs.
2 tbl, 2 ex
FIELD: medicine; gastroenterology.
SUBSTANCE: cell lysate is received by double destruction of bacterial cells: first, treatment with 1% sodium desoxycholate solution at a rate of 0.5 ml solution to 0.2 ml suspension with concentration (5-7) × 1010 microbes/ml, with following stirring at magnetic agitator in thermostat at 40°С for 2 hours, second, by performing 5 per 45 sec cycles of ultrasound disintegration, after which, the obtained suspension is precipitated by centrifuging at 8000 rpm for 40 min, and the obtained cell lysate is used as a sensitin, which is immobilised at polymer carrier with the following lyophilisation. In sensitin, the protein concentration 1.5-2 mg/m with wide immunoglobulin spectrum is received. As sensitin carrier, the globular polymeric particles with diameter 1.5 mcm and containing 1.3 mmol/g aldehyde groups can be used, and the lysate-produced immobilisation of micro spheres is performed by use of 0.1 mol carbonate buffer with pH 9.2. Interlocking of free aldehyde groups is performed by adding 2.0 ml 0.5% gelatose on 0.9% sodium chloride to suspension with carrier and soluble antigen, and leave to stay at constant stirring at the agitator at 20°С fro 120 min.
EFFECT: method provides the quantitative determination of antibodies to HPylory and efficient application for controlling the eradicative therapy.
2 tbl, 2 ex, 4 cl
FIELD: medicine; veterinary science.
SUBSTANCE: produced plasma after it is separated from blood and refined from trypanosome deposition is processed with 20-25% polyethylene glycol solution taken in proportion equal to blood plasma volume. Then produced mixture is kept at room temperature for 12-15 min, recentrifuged at 6000 revolutions/min for 15-20 min, after that supernatant is removed, and produced deposition is used as trypanosome exoantigen for serological reaction. At that its activity should be 95-97%.
EFFECT: timely finding of sick animals and possibility to take emergency measures for invasion elimination.