Tool for the differential diagnosis of brucellosis and method thereof
(57) Abstract:The tool is designed for the differential diagnosis of brucellosis by means of the formulation of the reaction between the antigen-antibody. Tool for the differential diagnosis of brucellosis contains cells of Brucella in physiological solution, phenol, and optionally sulfuric acid. These components the product contains the following ratio (per 100 ml of physiological solution with the content of dead cells 13 to 14 billion/ml): 10% solution of phenol 0.5 to 0.6 ml, 8 to 12% sulfuric acid solution of 0.6 - 0.8 ml To obtain a tool for the differential diagnosis of brucellosis spend the concentration of dead cells to content 13 - 14 billion in 1 ml of physiological solution. Next, 100 ml of the resulting solution make of 0.5 - 0.6 ml of 10% solution of phenol. The mixture was shaken and incubated for 5 to 6 hours Then to the mixture is added dropwise 0.6 to 0.8 ml of 8 - 12%-aqueous solution of sulfuric acid, shaken for 4 to 5 min and incubated for 4 to 5 days at room temperature. The resulting tool allows differential diagnosis of infected animals, can be used for the given reaction in a sample of whole blood. 2 S. p. f-crystals. Isobutane in medicine and veterinary medicine. For the diagnosis of brucellosis in humans and animals there are a number of antigens. For example, in medicine use of Hudelson, which tends to nonspecific reactions. In animal brucellosis is a single antigen for RA, RSK and GSK (in vitro method) and rose Bengal antigen (drip method). All existing antigens identify vaccinated and spontaneously infected with brucellosis, differentiating them, they are malespecific, slightly sensitive, labor-intensive, and are intended for the production of the reaction only in the serum.Closest to the claimed method is a diagnostic tool for the detection of brucellosis, containing killed Brucella cells in finalizando physiological solution .Given all the shortcomings of existing diagnostics, as well as lack of funds for differential diagnosis of vaccinated and infected animals, we have developed a tool and a method thereof, which would meet modern requirements practical Virology. We have developed a diagnostic tool can be successfully used as in blood serum and whole blood, and milk.Osnovivaite. The initial level of microbial bodies contained in the specified antigen,lead up to number 13 - 14 billion/cm3, then make a 10% solution of phenol, shake the mixture and leave for 5-6 hours then the mixture was added dropwise concentrated sulfuric acid, previously diluted with distilled water in the ratio 1 : 10, shaken for 4 to 5 minutes and leave the mixture for 4 to 5 days, after this time the product is ready to use.The drug, used for diagnostics, contains a final concentration of the following components (per 100 ml antigen):
Phenol 10% - 0,5 - 0,6 ml
Concentrated sulphuric acid is 0.6 - 0.8 ml
This diagnostic tool used for the detection of brucellosis plate (drip) method).Diagnostics using the funds obtained as follows.On the glass cause of 0.03 ml of serum and the top of 0.025 ml, mix thoroughly with a glass rod. Then shake the rotational movements for 5 minutes and take into account the result of the reaction. To accelerate the reaction better after mixing, the mixture was heated to 37 - 40oC. In this case, the result is detected for 3 minutes is athelney - the mixture remains homogeneous, including for vaccinated animals.Ways of placing reactions in the blood and milk is similar to the method of production of serum.Control is biofabrics positive hyperimmune serum, diluted 1 : 1 with saline. To verify the specificity and sensitivity of the antigen is taken as 0.03 ml hyperimmune serum and 0.05 ml of antigen, after mixing and rotational movement within 3 min takes into account the result of the reaction. When positive responses are large or small agglutinin in the form of flakes.The proposed antigen by their sensitivity and specificity allows to determine the antibody titer in the serum drip method. For this purpose, the glass is applied 0,03; 0,02; 0,01; 0,005; 0,0025 ml, brucellosis positive serum, and from above they applied 0,025 ml of antigen. After thorough mixing (which start with a small caption) heated over the flame of a spirit lamp until 37-40oC and by rotational movement in a period of 5 min take into account the results of the reaction. Agglutination in a mixture of 0,03 0,025 corresponds to the title 1 : 100, 0,02 - 0,025 - 1 : 200 and so onThe main advantage is the prob is that when using only whey about 30 40% of the antibodies remain in the blood clot, thus do not allow to identify the disease at an early stage.This method gives the opportunity by revaccinate to get rid of diseases-human and animal brucellosis, which brings moral and material damage to mankind and livestock.The preparation method of differential antigen simple, does not require additional investments and changes in production technology biofactories. For setting reactions do not require thermostats and refrigerators. 1. Tool for the differential diagnosis of brucellosis by means of the formulation of the reaction between the antigen antibody containing killed cells of Brucella in physiological solution and phenol, wherein the tool further comprises sulfuric acid in the following ratio of components (per 100 ml of physiological solution with the content of dead cells 13-14 billion/m): a 10% solution of phenol 0,5 0,6 ml, 8-12% solution of sulfuric acid of 0.6 to 0.8 ml.2. The method of obtaining funds for the differential diagnosis of brucellosis, including the addition of phenol in saline containing killed cells of Brucella, characterized in that conduct kontsentrirovaniem 0,5 0,6 ml of 10% aqueous solution of phenol, the mixture was shaken and incubated for 5 to 6 h, and then to the mixture is added dropwise 0.6 to 0.8 ml 8 12%-aqueous solution of sulfuric acid, shaken for 4 to 5 min and incubated for 4 to 5 days at room temperature.
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.