Immunosorbent to obtain monospecific antisera to transthyretin person
(57) Abstract:Usage: in medicine, when creating the immune test systems to determine the transthyretin (TTR) of a person in biological fluids. The essence of the invention: immunosorbant to obtain monospecific antisera to TTR person includes fibrinoliticeski active blood plasma of a person with total protein content 155,8 mg/ml, free from transthyretin, ovalbumin at a concentration of 65-90 mg/ml and as cross-linking agent is glutaric aldehyde at a final concentration of 0.7-0.8 vol.%. Immunosorbent allows researchers to simultaneously remove all non-specific antibodies from the antisera to TTR person, reduces the adsorption time and can significantly reduce the cost of production of diagnosticum to TTR person. 2 Il., table 2. The invention relates to medicine and can be used in the medical industry when creating the immune test systems for determination of transthyretin (TTP) person in biological fluids.Study the content of the TTP in the serum of patients will be used to assess the degree of failure and monitoring the effectiveness of clinical nutrition, monitoring the development of premature infants. The change in the patients with thermal, traumatic brain injury, some therapeutic diseases.The main stage of the creation of a diagnostic test system is to obtain monospecific antisera. After immunization of animals with purified drug transthyretin get antisera containing, typically as a mixture of antibodies to proteins of human blood plasma. In this regard, there is a need to remove nonspecific antibodies from such antisera to TTP person. The adsorption of antisera is recommended for adequate sorbents containing proteins that interact with the impurity antibodies [1,2,3] However, universal sorbent does not exist.The objective of the invention is the creation of a universal immunosorbent assay, increasing the specific activity of the antisera to the TTP person.To create a universal sorbent must have a biological environment of man, containing all the proteins of blood plasma, except for the TTP. In the process of adsorption of these proteins will interact with non-specific antibodies and, consequently, to increase the activity of the antisera to the TTP person.Unique biological environment of the person we encountered. Ustanovki range of proteins of blood plasma (PL. 1). This specific transfusion medium is widely used in clinical practice as a thrombolytic tools 
The process of "exhaustion" of antisera most technologically advanced in the presence of solid immunosorbent assay, so it was used a crosslinking agent and a filler ovalbumin.The problem is solved by mixing PFAP and ovalbumin to the concentration of total protein 75-110 mg/ml, using a cross-linking agent is glutaraldehyde at a final concentration of 0.7-0.8. by washing the obtained immunosorbent in buffer solutions at different pH.Immunosorbent prepared as follows: to PPAP with a certain concentration of protein added to ovalbumin concentration 75-110 mg/ml of total protein. To this mixture is added 2.5% solution of glutaraldehyde to a final concentration of 0.7-0.8. dropwise, with constant stirring. After three hours at room temperature, a gel is formed, which is ground using a homogenizer and washed various buffer solutions."Exhaustion" of the antisera to the TTP person provide in a centrifugal tube mix anticigarette to TTP containing nonspecific antibodies and policendarlehen anticigarette filtered and subjected to immunochemical study.Example.Cooking immunosorbent:
To 50 ml PHAP (pH 7.0) with the concentration of total protein of 10.2 mg/ml (total protein pool was 510 mg) added 3500 mg of ovalbumin powder and mix thoroughly. After complete dissolution of the protein, dropwise, with constant stirring to the mixture is added 15 ml of 2.5% solution of glutaraldehyde. For 3 h at room temperature, a gel is formed, which is ground in a homogenizer and alternately washed in 0.2 M phosphate buffer (pH 7.3) and 0.1 M GLYCANS (pH of 2.8). The crushed gel (immunosorbent) store in a bottle with 0.02 sodium azide at +4-6oC until use.Adsorption of antisera to TTP person:
In the centrifuge tube, mix 5 ml of the antisera to the TTP man (series 8-664/2 from the source specific titer of 1:10) and 5 ml immunosorbent assay. The mixture is stirred at Shuttle for 35 min at room temperature. Adsorbed anticigarette separated from the solid adsorbent by filtration and subjected to immunochemical analysis. Title adsorbed antisera was 1:4, it is revealed antibodies only to the TTP person.Checking the activity of the hard immunosorbent was held with history is recipitation in the gel on Ouchterlony and through immunoelectrophoresis (Fig. 1,2). As a reference antisera were used serum against TTP "Sevac" (Czechoslovakia), and antigen TTP whole serum of human blood (a mixture of samples from 50 donors). The titer obtained monospecific antisera to the TTP was on average 1:6 (PL. 2).Immunodiffusion analysis of antisera after adsorption showed that all series contain only antibodies to the TTP person and standard antigen TTP person ("Sigma", USA). Immunoprecipitate proved identical antigenic structures TTP person. All adsorbed antisera was completely absent nonspecific antibodies.The efficiency of adsorption of non-specific antibodies further investigated through immunoelectrophoresis and cross-immunoelectrophoresis (Fig. 2). The titer of specific antibodies in adsorbed antisera to TTP ranged from 1:4 to 1:8.Thus, the obtained immunosorbent allows researchers to simultaneously remove all non-specific antibodies from the antisera to the TTP person, its use reduces the adsorption time and can significantly reduce the cost of production of diagnosticum to TTP person. Note that the obtained immunosorbent can be repeatedly and the 1968, S. 294.2. Immunological methods. Under the editorship of Primes, Moscow, "Medicine", 1987, S. 472.3. The antibodies. Methods. Edited by Katie D. Moscow, "Mir", 1991, 2 h, 382.4. C. B. Grips. "Drugs fibrinolytic and antiproteinase steps from plasma suddenly dead people". Summary of the doctoral thesis. M. 1984, 48 S. Immunosorbent to obtain monospecific antisera to transthyretin person, including leaching fibrinoloticheskaya active blood plasma of a person with total protein content of 15 to 5.8 mg/ml, free from transthyretin, ovalbumin at a concentration of 65 to 90 mg/ml and as cross-linking agent is glutaric aldehyde at a final concentration of 0.7 to 0.8.
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.