The method of obtaining the latex diagnosticum

 

(57) Abstract:

The invention relates to medicine, and more specifically to Microbiology. The aim is to increase the information content of diagnosticum and sorption capacity of the latex. This objective is achieved in that get polygroove serum by simultaneous immunization of rabbits with a mixture of antigens of the causative agents of plague, glanders, melioidosis, tularemia and brucellosis in the ratio 0,1 : 1 : 1 : 6 : 4, as sensitin used immunoglobulins from polygrapholog serum. The latex sensitization was carried out at the ratio of 10 mg of carrier 0.1 mg globulins, at a PH of 7.0 to 7.4, for 4 h at room temperature, then for 12 h at 4C with constant uniform stirring. The method provides an increase in globulin load per unit of media and allows you to cut at least 2 times the time of analysis, 4 times to reduce the cost of drugs is relatively prototype using the received diagnosticum. table 1.

The invention relates to medicine, and more specifically to Microbiology, and for laboratory diagnosis of especially dangerous infections.

There are various ways to obtain diagnostics using artificial HV effect. cholerae serogroup 01 with sensitivity 5106-1107CFU/ml complicated and requires expensive equipment and chemicals.

Known also diagnosticum polymer microspheres with a diameter of 4-5 microns as carriers, covalently bound to immunoglobulins from plague commercial agglutinating sera. The activity of diagnosticum with typical strains of the plague pathogen grown at 37aboutWith a made 3 to 105MT /ml, but the sensitivity with respect to the microorganism cultivated in the 28aboutWith much lower - 5 107MT /ml.

The closest analogue (prototype) is a method of obtaining or antibody-based test diagnosticum, in which as the carrier used suspension polyacrolein latex with a diameter of spheres of 1.8 μm, activated casein and tannin. Latex particles were senzibilizirani immunoglobulins plague serum in a ratio of 0.15 mg immunoglobulin 50 mg media exposure 3 h at room temperature and constant stirring. Then sensitized suspension was besieged and twice washed with 0.15 M NaCl. The washed suspension sensitised polyacrolein media di. liofilizirovannoe the contents of capsules suspended in 4 ml of 0.05 M FSB pH 7.2 and used for setting the reaction of agglutination of latex.

Was marked by a high specificity and sensitivity obtained diagnosticum. However, if you need sharp increase in the volume of discovering the unknown pathogen dangerous infections of bacterial origin, for example in terms of epidural, commercial immunoglobulin nanodiagnostics not suitable. Each received sample should be investigated at least four different drugs, which is very cumbersome and unacceptable, especially in the field.

Offer polygrapholog latex diagnosticum four times reduces the number of analyses and saves backpropagate.

The aim of the invention is a method for polygrapholog immunoglobulin diagnosticum to five species of bacterial pathogens of especially dangerous infections that can increase the number of adsorbed immunoglobulins on the unit carrier and to reduce the time of analysis using the obtained diagnosticum.

This objective is achieved in that get polygroove savoryi and brucellosis in the ratio 0,1: 1: 1: 6: 4. Immunoglobulins from sera allocate Caprylic acid by well-known methods. Polyacrolein particles with microspheres of 1.8 μm sensibiliser immunoglobulins polygrapholog serum according to the following procedure. Poured in the tube is equal to the volume of 1.25% latex suspension and the suspension of immunoglobulins to protein concentration of 50-100 μg/ml and stirred on a magnetic stirrer at a temperature of 20-23aboutC for 4 h Then the process continues at a temperature of 4-5aboutWith 12 hours the Mixture is washed 0.15 M solution of the FSB and the precipitate resuspended in 1 ml of medium drying, consisting of a mixture of 2% solution of bovine serum albumin and 5% sucrose solution. Diagnosticum poured into ampoules of 0.5 ml and freeze-dried. Before setting reaction of agglutination of the latex content of the ampoule is dissolved in 5 ml of 0.05 M FSB pH of 7.2.

Thus, the application of the proposed combination of techniques (getting polygrapholog serum, sensitization of latex polypropolyne immunoglobulins) allowed to give way to a new quality to get polygrapholog diagnosticum, which allows you to detect any of the five bacterial pathogens of especially dangerous infections in contaminated samples, which significantly tx2">

P R I m e R 1. Prepare buffered 0.15 M NaCl pH 7.2 and use it as a planting liquid. 1 ml of 1.25% suspension polyacrolein latex poured to 1 ml suspension poligraphic immunoglobulins with a protein concentration of 100 μg/ml and stirred on a magnetic stirrer at 20aboutC for 4 h and Then stirring is continued at 4aboutFrom 12 o'clock Sensitized latex precipitated by centrifugation at about 3 thousand. /min, washed 0.05 M solution of the FSB and the precipitate resuspended in 10 ml of the same solution. Received diagnosticum put the reaction of agglutination of latex. Controls: 1) diagnosticum with distributing fluid; 2) diagnosticum with suspension heterologous microorganisms. See a positive reaction (umbrellas) in the wells containing the plague, glanders, melioidosis, tularemia brucellosis in concentration 6105MT /ml In controls-compact discs on the bottom of the hole.

P R I m m e R 2. Sensitization as in example 1, but as the distribution of the liquid used buffered 0.15 M NaCl pH of 7.0. When setting the reaction of agglutination of latex see "buttons" on the bottom of the hole in the controls, i.e., the result is doubtful.

P R I m e R 3. Sensitization of latex as worthwhile in the background, i.e. the result is doubtful.

P R I m e R 4. Serves 4 diagnosticum with protein concentrations of 50 and 100 µg/ml (1 pair) and 50 and 100 µg/ml (2 pair), as in example 1, with the first 2 sensibiliser only 4 h, and the other two additional 12 h at 4aboutC.

Received diagnosticums put agglutination reaction latex, using suspensions of killed microorganisms.

The results RAHL shown in the table.

Thus, as can be seen from the table, increasing the load globulin per unit of media leads to spontaneous agglutination in the control, hindering polygrapholog of diagnosticum known method, and the proposed method allows to achieve a specified target.

P R I m e R 5. For the study enrolled 20 field samples suspected to be infected with bacterial pathogens of especially dangerous infections. The analysis was carried out using a commercial nanodiagnostics plague, Sanogo, melioidosis, tularemia, brucellosis. Just put 80 reactions. Simultaneously examined the samples by diagnosticum Polygraphy latex. Conducted 20 tests. The results for all the samples obtained in the first case, after 6 h (3 min n 24 ml (6 ml x 4) nanodiagnostics and 6 ml polygrapholog of diagnosticum, respectively, in the 1-m and 2-m cases.

As you can see by the results, the time saved, diagnostic preparations, tablets, reduced labor costs for the study samples. Moreover, the effect of the application polygrapholog latex diagnosticum will increase with increasing amounts received for analysis of samples.

Thus, the proposed method managed to get polygrapholog diagnosticum, allowing to reduce in 2 times the time of analysis, 4 times to reduce the amount of consumption of drugs relative to the prototype and to increase globulin load per unit of media.

(56) USSR Author's certificate N 1596926, CL G 01 N 33/53, 1989.

The METHOD of OBTAINING the LATEX DIAGNOSTICUM by sensitization polyacrolein latex immunoglobulins plague serum, characterized in that, to increase the information content of diagnosticum and enhance the sorption capacity of the latex particles, sensitization is carried immunoglobulin antisera containing additional immunoglobulins to Sapa, melioidosis, tularemia and brucellosis and obtained by immunization of rabbits with the corresponding antibodies in the ratio 0,1 : 1 : 1 : 6 : 4, moreover, sensitization take 0.1 mg globulin 10 mg of latex particles, ingibuvannya.

 

Same patents:

The invention relates to biotechnology and can be used to obtain stable forms of immunobiological preparations

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.

2 cl

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.

3 cl

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.

17 cl

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.

2 tbl

Up!