Method for assessing local humoral anticholeraic immunity in vitro simulated in experimental animals
SUBSTANCE: method involves immunising with 5×107 microbial cells Vibrio cholerae 5879; 10 days later, a common pool of anticholeraic Ig is recovered from enteric washing liquid. That is combined with producing the primary enterocyte culture from intact mice. Thereafter, the doubly-diluted anticholeraic Ig are applied in an amount of 3 ml on a monolayer of erythrocytes formed in wells of culture panels. The latter are added with alive choleraic vibrions of the strain Vibrio cholerae 5879, 40 m.c. per 1 erythrocyte and incubated for 3 hours. Then, the panels are washed three times, fixed with ethanol for 20 minutes and stained by Romanowsky-Giemsa. Local humoral anticholeraic immunity is assessed under the microscope by the number of vibrions stained in dark blue and attached to one erythrocyte.
EFFECT: method enables assessing specific local humoral immunity and intensifying the anti-adhesive activity of the common pool of anticholeraic immunoglobulin.
4 cl, 2 ex, 2 tbl
The invention relates to medicine, in particular for the study of anti-adhesive activity of cholera immunoglobulins and enhance it to improve specific prophylaxis of cholera.
Cholera is anthroponosis disease, so the development of models of infection in animals is always met serious difficulties. To date, the experimenters take numerous attempts to develop a model of infection in mice. Models are generalized forms of cholera in mice, sealed mouse. Given that the disease cholera develops and runs in the small intestine, were developed several experimental models that can be used in animals pathogenetic picture of cholera, similar to that in humans: a model tied loops of the small intestine of an adult rabbit, rabbit-suckers.
However, currently available methods and models to assess the effect of various drugs, such as immunomodulators, for forming a local cholera immunity, in particular the increase in the antibody productions and anti-adhesive activity of cholera immunoglobulins (Ig). The development of such experimental methods and models is very important as the first obstacle to many infections, including Ho is a career, are protective factors acting on the surface of mucous epithelial cells, various antibacterial agents, secretory IgA and other) and preventing the adhesion of pathogens [1, 2]. It is shown that the prevailing value of the antibacterial component, preventing the attachment of V. cholerae to the mucous membrane of the intestine in the early stages of infection of the organism . One of the main indicators of antibacterial immunity is anti-adhesive activity of cholera Ig, which, along with antitoxic antibodies, indicating the adequacy of the formation of local humoral immunity [4, 5].
A prototype of the chosen method of evaluation of the effectiveness of immunomodulator imunofana in experimental cholera (see patent RU No. 2481791, CL AV 10/00, A61K 38/08, A61P 43/00, G09B 23/28), namely, that prior infection with animals in an isolated loop of the small intestine virulent strain they impose immunomodulator Imunofan dose of 0.2 µg dissolved in 0.3 ml of novocaine in the amount of 10 injections. Evaluating the effectiveness of imunofana carried out according to the presence of experienced tied the loops of the small intestine enteropathogenic effect, the severity of which is estimated at crosses, and the presence of choleragenic effect, which is calculated by the formula K=V/NP, where K is the coefficient of expansion loops, V - is the volume of the liquid, NP is the length of the loop, if the experimental loops enteropathogenic effect is absent and K<1,0, consider the use of imunofana effective.
However, the problem of the known method consisted in the evaluation of the action of immunomodulators on the development and severity of pathogenetic picture of the experimental cholera and does not give the possibility of studying in vitro the formation of cholera immunity, in particular the actions of various drugs, including immunomodulators, anti-adhesive activity total pool cholera Ig of the small bowel wash fluids of mice.
The purpose of the present invention was to develop ways to assess in vitro site-specific humoral immunity in experimental cholera, as well as to strengthen the anti-adhesive activity total pool cholera Ig of the small bowel washing liquids of white mice in the formation of cholera immunity.
This objective is achieved in that in the known method to assess local humoral cholera immunity in the experiment of white mice subjected to immunization with 5×107microbial cells of Vibrio cholerae 5879, after 10 days, allocate the total pool of cholera Ig of the small bowel wash fluid of mice additionally receive a primary culture of small intestine enterocytes from intechnica, then cholera Ig in a twofold dilution is applied in a volume of 3 ml on a monolayer of enterocytes, formed in the wells of the culture of the panels, and then at last put a live cholera Vibrio cholerae strain of Vibrio cholerae 5879, 40 M. K. 1 enterocyte and incubated for 3 hours, after panel washed three times, fixed with ethanol for 20 min and stained by Romanovsky - Institute, evaluation of local humoral cholera immunity are under the microscope by the number of vibrios, painted in dark blue color and adherent to one enterocyte.
The total pool of cholera Ig obtained from the wash fluid of the small intestine, this impose on the last two ligatures at a distance of 5 cm from each other and injected into the isolated section 5 ml of physiological solution, massaged and syringe take the liquid, which is precipitated by 50% ammonium sulfate, and formed overnight at 4°C the precipitate centrifuged at 1000 rpm for 10 minutes
In addition, primary cultures of enterocytes of the small intestine receives from intact white mice in the lumen flushed the small intestine of animals injected with a syringe and 5 ml of 0.2% ethylenediaminetetraacetic acid, massage 30 min and suck the contents of the pipette, the received cells are washed three times with saline, suspended in culture medium with 10% AMB the optional calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×105M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar, after the panel incubated in CO2the incubator during the day for the formation of a sparse monolayer, the cells are then washed three times with fresh portions of medium without serum.
And for immunization of animals strain of Vibrio cholerae 5879 last grown at 37°C for 24 h on agar Martin (pH 7,6) with subsequent inactivation by boiling in a water bath for 30 min and checking for specific sterility.
The method is carried out in two stages:
Phase I: produce a common pool cholera Ig of the small bowel wash liquids white mice;
Phase II - evaluate the anti-adhesive activity obtained under the first stage of cholera Ig on the monolayer of enterocytes.
For conducting the first stage of the two groups of experimental mice of 12 pieces in each oral subjected to immunization killed microbial cells (M. K.) Vibrio cholerae 5879 (5×107M. K.). Strain 5879 V. cholerae O1 of serovar Inaba receive from the collection of live cultures of Rostopchin. The last pre-grown at 37°C for 24 h on agar Martin (pH 7,6). Inactivation of bacterial culture performed by boiling in a water bath for 30 minutes After checking on specific sterilized is of a suspension of bacterial cells used for immunization of animals.
Then the second experimental group of animals intramuscularly, daily for 10 days, injected with 0.2 ml of 0.0005% solution imunofana. The first experimental group immunized mice, immunomodulator, it will not.
Then allocate the total pool of cholera Ig from the wash fluid of the small intestine of dead mice of each experimental group. To do this, white mice in the small intestine impose 2 ligatures at a distance of 5 cm from each other, is introduced into the isolated section of the small intestine 5 ml of physiological solution, gently massage a few minutes and take the contents with a syringe. The washing liquid is precipitated by 50% ammonium sulfate. Formed overnight at 4°C the precipitate was separated by centrifugation at 1000 rpm for 10 min. the Precipitated proteins cialiswhat against distilled water (three times the change of diluent used), was diluted to the original volume and sterilized by filtration through membrane filters with a pore diameter of 0.22 μm Schleicher&Schuell.
Stage II is: a) in the allocation of primary cultures of enterocytes; b) in the evaluation of anti-adhesive activity of cholera Ig on the monolayer of enterocytes
a) primary culture of enterocytes of the small intestine to evaluate the adhesive activity of living V. cholerae get 10 intact white mice. To do this in the lumen flushed the small intestine of animals injected with syringe 5 ml,2% ethylenediaminetetraacetic acid (EDTA) (Germany), gently massaged for 30 min and suck the contents of the pipette. Cells are washed three times with saline, suspended in a nutrient medium, DMEM (Germany) with 10% fetal calf serum, 2 mm L-glutamine (Russia), 10 mm HEPES (Switzerland), 5×105M 2-mercaptoethanol (USA), 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar (the Netherlands). Panel incubated in CO2the incubator during the day for the formation of a sparse monolayer, after which the cells washed three times with fresh portions of medium without serum.
b) to assess anti-adhesive activity of cholera Ig last two-fold dilution in a volume of 3 ml is applied to the panel by a monolayer of enterocytes. In some wells contribute cholera Ig obtained from animals of the experimental group, where he was held only immunization strain of V. cholerae 5879, and other common pool Ig selected from the group of animals immunized and treated Imunofan. In the control wells to enterocytes add saline solution. Then all wells make a living cholera Vibrio cholerae strain of Vibrio cholerae 5879 (1 enterocyte - 40 M. K.) and incubated for 3 hours in CO2-incubator. Panel washed three times with fresh portions of saline, fixed with ethanol for 20 min and paint what about the Romanovsky - From the Institute. For this purpose, 3.8 g of anhydrous Romanovsky dissolved in 250 ml of pure ethyl alcohol. Within 4 days the solution is shaken out, then add 250 ml of glycerol and again shaken for 4 days. Panel dye working solution of the dye (1 ml of ink : 10 ml distilled water) for 20 minutes After exposure, the wells washed three times with distilled water, dried, and counted under a microscope (ocular 10, the lens 90) number painted in dark blue color, adherent to 200 enterocytes vibrios and determine their average value.
About the presence of expression of anti-adhesive activity is judged by the decrease in the number of adherent to one enterocyte vibrios not less than 2 times in comparison with the control.
After immunization, the number of adherent cholerae is reduced in comparison with control 2 times at 14 day postimmunization period, and the addition of immunotherapy reduces this period to 7 days, indicating an increasing anti-adhesive activity of cholera antibodies under the influence of immunomodulator imunofana.
Thus, this method allows you to determine the anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice at various times after immunization, and to assess the impact immunomodulator is s, in particular imunofana, to increase the ability of these antibodies to inhibit adhesion of V. cholerae to the enterocytes in vitro.
Example 1. Evaluation of anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice.
Studied anti-adhesive activity on the technology of the proposed method (see "How is...") cholera Ig isolated from the small intestine immunized M. K. strain V. cholerae 5879 mice. At various times after immunization (see table 1) revealed a decrease in the average number of adherent to one enterocyte vibrios on the 5th day, testified to the beginning of the synthesis of specific antibodies. 14 day anti-adhesive activity of these Ig significantly increased in comparison with the beginning of the immunization and continued to increase until the end of the observed period.
|Day after immunization||The number of Vibrio cholerae attached to one enterocyte|
Thus, the example shows that at 14 days after immunization, the average number attached to enterocytes vibrios decreased compared with control 2 times, indicating the presence of a sufficient amount of a specific cholera antibodies. The proposed method gives the opportunity to evaluate their anti-adhesive activity and allows you to judge the adequacy of the formation of local humoral cholera immunity.
Example 2. Study of the effect of the immunomodulator imunofana on the anti-adhesive activity total pool cholera Ig obtained from the small bowel wash liquids immunized mice in the formation of local cholera immunity.
Study of the effect of the immunomodulator imunofana on the ability of cholera Ig to inhibit adhesion of Vibrio cholerae in different terms postimmunization period showed (see table 2) that the anti-adhesive activity of the antibodies isolated from swabs of the mucosa of the small intestine immunized and treated with immunomodulator mice, manifested in a greater extent and and earlier, than in immune animals. On day 7, the mean number of adherent to one enterocyte vibrios in the samples containing the washing liquid derived from animals after immunization and immune disorders was significantly lower than in the wells with Ig isolated from animals immunized and not receiving Imunofan. This trend was maintained during all periods of observation (up to 28 days). Decrease 2 times the average number attached to enterocytes vibrios in comparison with the control already on day 7 postimmunization period testified about the increase in anti-adhesive activity of cholera Ig under the influence of immunomodulator.
|Day after immunization||The number of Vibrio cholerae attached to one enterocyte|
Thus, using the proposed method to model in vitro revealed a stimulating effect of immunodulatory imunofana products cholera Ig and to increase their anti-adhesive activity. Anti-adhesive activity of immunoglobulin prevents adhesion of V. cholerae to the enterocytes, providing the most significant level of protection of the mucous membrane of the small intestine.
The use of the present invention allows for the possibility of studying the anti-adhesive activity total pool cholera Ig of the small bowel wash liquids immunized mice to evaluate the in vitro site-specific humoral immunity in experimental cholera.
In addition, this method allows to study the increased synthesis cholera antibodies and strengthening their anti-adhesive activity under the influence of immunomodulators directly into the intestine, where the interaction of the microorganism with the causative agent of cholera.
Thus, the proposed method allows to assess the adequacy of the formation of local cholera immunity for anti-adhesive activity of specific antibodies, as well as the influence of various drugs on the immune response to the causative agent of cholera, which opens up new approaches to improving immunization e is th disease.
Sources of information
1. Kostyukova N. N. The initial stage of infection is colonization and ways of its prevention, Zh. microbiol., Epidemiol., immunobiol. - 1989. No. 9. - S. 103-110.
2. Williamson E., G. M. Westrich, and I. L. Viney Modulating dendritic cells to optimize mucosal immunization protocols // J. Immunol. - 1999. - Vol.163, No. 7. - P. 3668-3675.
3. Yamamoto T., Fibert M. J., Bradley, S. R. et al. Adherence to human small intestines of capsulated Vibrio cholerae 0139 // FEMS Environ. Lett. - 1994. - Vol.119, No. 1-2. - P. 229-235.
4. Chelyadinova A. Century, the Study of the biological properties of Vibrio cholerae 0139 serogroup: author. dis... Kida. the honey. Sciences. - 2000. - 18 S.
5. Jerborn M., Nordstrom I., Kilander A. et al. Local and systemic immune responses to rectal administration of recombinant cholera toxin subunit In in humans // Infect. Immun. - 2001. - Vol.69, No. 6. - P. 4125-4128.
1. The method of estimating local humoral cholera immunity in the experiment, characterized in that the white mice subjected to immunization with 5×107microbial cells of Vibrio cholerae 5879, after 10 days, allocate the total pool of cholera Ig of the small bowel wash fluid of mice additionally receive a primary culture of small intestine enterocytes from intact mice, then cholera Ig in a twofold dilution is applied in a volume of 3 ml on a monolayer of enterocytes, formed in the wells of the culture of the panels, and then at last put a live cholera Vibrio cholerae strain of Vibrio cholerae 5879, 40 M. K. 1 enterocyte and incubated for 3 hours, after panel washed three times, fixed with ethanol in ECENA 20 min and stained by Romanovsky - The Institute, evaluation of local humoral cholera immunity are under the microscope by the number of vibrios, painted in dark blue color and adherent to one enterocyte.
2. The method according to p. 1, characterized in that the total pool of cholera Ig obtained from the wash fluid of the small intestine, this impose on the last two ligatures at a distance of 5 cm from each other and injected into the isolated section 5 ml of physiological solution, massaged and syringe take the liquid, which is precipitated by 50% ammonium sulfate, and formed overnight at 4°C the precipitate centrifuged at 1000 rpm for 10 minutes
3. The method according to p. 1, characterized in that the primary culture of small intestine enterocytes get from intact white mice in the lumen flushed the small intestine of animals injected with a syringe and 5 ml of 0.2% ethylenediaminetetraacetic acid, massage 30 min and suck the contents of the pipette, the received cells are washed three times with saline, suspended in culture medium with 10% fetal calf serum, 2 mm L-glutamine, 10 mm HEPES, 5×105M 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin to a concentration of 150,000 cells/ml and seeded in wells of a 6-hole culture panels Costar, after the panel incubated in CO2the incubator during the day for the formation of represenromania, the cells are then washed three times with fresh portions of medium without serum.
4. The method according to p. 1, characterized in that for the immunization of animals strain of Vibrio cholerae 5879 last grown at 37°C for 24 h on agar Martin (pH 7,6) with subsequent inactivation by boiling in a water bath for 30 min and checking for specific sterility.
SUBSTANCE: invention refers to medicine, particularly to experimental nephrology and can be used for simulating generalised amyloidosis in animals for studying pathogenesis, prevention and treatment of amyloidosis. The method involves administering native sheep plasma into golden hamsters subcutaneously at 0.025 mg/g of body weight. Administering is performed for 30 experimental days.
EFFECT: method is easily reproducible, provides reducing the time of the simulation of the above pathology by greater antigenic strength of the selected protein preparation for the above animal series; the method enables studying the functional state of the renal system, mineral exchange and other body characteristics successfully.
1 ex, 2 tbl, 4 dwg
SUBSTANCE: invention refers to medical equipment. A device comprises a shock mechanism in the form of a pendulum with adjusted impact strength comprising two crossbars with fastener holes with two impact floors, with a long slot, which accommodates a movable load platform fixed at a preset radius and having a rod bearing replaceable loads, a movable carriage for positioning an injured object, a friction brake coupled with the movable carriage and presented in the form of a flexible split sleeve enclosing a guiding axis along which the carriage moves.
EFFECT: using the presented device enables extending the range of technical products, namely the device for simulating the impact whiplash exposure of the upper spine.
2 cl, 5 dwg
SUBSTANCE: single-step testing of white rats is carried out in an open field facility. The total length of locomotion, sniffing, and sitting actions as well as the number of upright postures and freezing acts are recorded. Prognostic coefficients F1 and F2 are determined by formulas and compared. If F2 is more than F1, cognitive disorders are stated in experimental animals, and if F1 is more than F2, the above are stated to be absent. That is followed by calculating a prognostic index (PI), which is used to determine an intensity of the cognitive disorders: low, moderate and high.
EFFECT: method enables detecting the presence of the cognitive disorders in the rats by using the values of motor and investigatory activity and anxiety.
2 cl, 2 tbl, 4 ex
SUBSTANCE: device comprises a 3-litre flat-bottom cylindrical glass vessel (1), a neck of which is slightly elongated and narrowed, and has a rounded hole of 90 mm in a diameter. There is a screw thread for the tight fixation of the lid (2) on an outer surface of the elongated portion. What is also provided is a semi-sphere cup-shaped metal container (3) having a volume of 0.03±0.01 l, a thickness of 0.5 mm, on the bottom perimeter of which there are five holes of the diameter of 2 mm at a distance of 5 mm from each other; a cylindrical metal base (4) having a thickness of 0.5 mm, a height of 115 mm, a base diameter of 75 mm, and an apex diameter of 75 mm; on a side surface of the metal base, there are two parallel rectangular holes 75×45 mm. The container (3), wherein the combustion process takes place, is fixed on the base apex. What is also provided is a rectangular wooden base (5) 200 mm long, 15 mm wide and 3 mm thick, the front surface of which is provided with a rigidly fixed semi-circle metal collar (6) of the diameter of 150 mm with the glass container (1) tightly fixed.
EFFECT: invention provides the development of a biomodel poisoning with gas products of wood combustion.
3 dwg, 1 ex
SUBSTANCE: invention relates to medicine and biology and can be used for studying physiological functions of absorption of various substances and motility in an isolated section of the small intestine by Thiry-Vella in experiment. The abdominal cavity is opened. A purse-string suture in the form of a circle is made on the anterior surface of the stomach. In the centre of the purse-string suture the stomach wall is dissected. A guide catheter with a ball at the end is introduced into the small intestine lumen through the stomach cavity to the place of an anastomosis formation. The small intestine with the mesentery is intersected. After isolation of an isolated section of the small intestine a bolus-tube is fixed on the guide catheter with the ball at the end. An enteroenteroanastomosis between proximal and distal ends of the small intestine on the bolus-tube is formed. Integrity of the intestine and its impermeability are recovered by the application of single-row interrupted serous-muscular-subcutaneous sutures. The guide catheter with the ball at the end and the bolus-tube are removed through an opening in the stomach limited by the purse-string suture. Impermeability and consistency of the enteroenteroanastomosis are checked. Fistula tubes are fixed on the proximal and distal ends of the isolated section of the small intestine.
EFFECT: method makes it possible to preserve the normal lumen of the small intestine in the formation of the enteroenteroanastomosis "end-to-end" in small laboratory animals and check consistency and impermeability of the formed anastomosis intraoperationally, as well as to preserve the created model for a long time for studying small intestinal functions due to the application of the purse-string suture on the stomach, application of the guide-catheter with the ball at the end and the bolus-tube, and reliable intestinal sutures.
1 ex, 5 dwg
SUBSTANCE: method involves administering insulin-producing cells in a dose of 500 thousand cells in a solution of isotropic gel 0.5 ml, 15% ascorbic acid 10 mcl and 0.1% hydrogen peroxide 10 mcl. The preparation is administered subcutaneously into theanterior abdominal wall in a projection of the pancreas.
EFFECT: method reduces the number of complications related to developing tissue incompatibility.
SUBSTANCE: anthracosilicosis is simulated by a 4-hour inhalation exposure of coal-rock dust on animals 5 times a week. Gas-oil coal of the concentration of 50 mg/m3 at a dust particle size of 5 mcm is used. The disease respective to a 3-year miners' experience is simulated by 3-week coal poisoning of the animals. The disease respective to a 5-10-year experience is simulated by 6-week poisoning. The disease respective to more than 10 years of experience is simulated by 12-week poisoning.
EFFECT: creating the most reliable environment simulating the miners' working conditions, and enabling providing the experimental early changes specific for anthrasilicosis for the further determination of the time and methods for prevention thereof.
1 tbl, 1 ex
SUBSTANCE: invention can be used for studying pathogenesis mechanisms of combined radiation injuries (CRIs) including the mutual burdening phenomenon, as well as testing new methods and aids for preventing and treating CRIs. That is ensured by the CRI simulation by the sequential radiation exposure on rats. Total γ-radiation is first provided. That is followed by local beta-radiation covering a hair-free area of the animal's skin area isolated by a screen. A degree III-a burns are simulated by the use of beta-radiation in doses 30 and 60 Gy generated by a contact ionising radiation source having an activity of 24.3 mKi (900 MBq), an average radiant energy of Eav=1.4 MeV at a dose rate at the skin surface of 2.1 Gy/min triple attenuated through the skin thickness.
EFFECT: method enables the experimental reproduction of the radiation disease and the degree III-a superficial radiation burns equal in severity, and studying the effect of superficial skin injuries on the clinical course and outcomes of CRIs depending on the total radiation doses selected.
1 ex, 1 dwg
SUBSTANCE: invention refers to medicine. Implementing the method involves presenting an audio signal in the form of a superposition of separate tone components of input multiple-modulated vibration generated by an overlap of several acoustic vibrations. The vibration is processed on a signal processing model in the outer, middle and inner ear. The signal processing model in the outer ear is presented by a broad-band amplifier with an average amplification frequency of 3 kHz. The signal processing model in the middle ear is presented by a parametric system, wherein one of its reactive elements changes with time synchronously with the parameter variations of the multiple-modulated vibration. The signal processing model in the inner ear is presented by a dispersive delay line, the principle of action of which is based on the elastic audio wave velocity and frequency relation.
EFFECT: invention enables providing the more accurate detection of the biophysical processes implementing the hearing mechanism of the periphery portion of the acoustic system by interpreting it into an electronic analogue.
SUBSTANCE: invention refers to experimental medicine and can be used for examining diabetes mellitus, as well as in developing new methods of treating changes caused by type I diabetes mellitus. That is ensured by simulating alloxan diabetes in white outbread rats. Developing subcompensated diabetes mellitus is ensured by administering an alloxan solution in the rats intermittently intraperitoneally on an empty stomach, successively in doses of 5 mg/100 g, 7 mg/100 g and 5 mg/100 g of the animal's weight every 7 days, and developing decompensated diabetes mellitus is provided by three administrations of the alloxan solution in a dose of 10 mg/100 g every second day.
EFFECT: method provides higher probability of successful repetition and predictability of stimulating this disease by modifying the model corresponding to its subcompensated and decompensated forms.
SUBSTANCE: invention concerns a method for producing a reference panel for serum samples to assess a specificity of serodiagnostic results of socially significant diseases, including: examining donors' and patients' samples for the presence of an infectious material, taking samples containing no hepatitis C virus (HCV) antigen, hepatitis B virus (HBV) surface antigen (HBsAg) antibodies, human immunodeficiency virus (HIV) early protein p24 and antibodies, as well as Treponema pallidum antibodies for a negative side of the reference panel, taking samples containing HIV, HCV, HBsAg and Treponema pallidum antibodies for a positive side of the reference panel, and introducing a stabilising compound into the samples.
EFFECT: invention provides creating such method for producing the reference panel for blood serums that makes it possible to prepare the negatives serums of the panel containing no HBsAg, as well as Treponema pallidum, HCV and HIV antibodies, and allows one to keep the specific characteristics of all the serum samples of the panel for a long period of time.
4 cl, 4 ex, 2 tbl
SUBSTANCE: group of inventions concerns kallikrein, a version or fragment thereof having common epitopes recognized by antibodies with wild-type kallikrein used to prepare a diagnostic composition for in vitro diagnosing of type 1 allergy, wherein kallikrein is taken from a dog; a method for preparing a composition of allergens involving the stage of adding dog's kallikrein or version or fragment thereof; a method of kallikrein recovery from dog's urine.
EFFECT: higher sensitivity in diagnostic tests.
16 cl, 11 ex, 13 dwg, 5 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention discloses using the monoclonal antibodies (MCAs) produced by the hybridomas B/4H1 and 4H7 and directed to variable determinants as a part of influenza B virus proteins. The hybridoma B/4H1 and 4H7 strains are deposited in the Russian Collection of Vertebral Cell Cultures (RKKK P) under Nos. 719 D and 720 D, respectively. The two current influenza B viral evolution lines are identified with using the MCAs B/4H1 and MCAs 4H7 directed to variable determinants of Victorian-type and Yamagata-type influenza B hemagglutinin, respectively.
EFFECT: using the prepared MCA kit enables identifying the newly recovered influenza B viruses Besides, the MCAs under the invention may be used to study the antigenic structure and variability of this agent, to identify them as belonging to a specific evolution line; the MCAs may be also used to construct the diagnostic test systems.
3 cl, 12 dwg, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention describes a chemically modified peptide that is a fragment of human protein S100β consisting of a sequence of 15 amino acids corresponding to the amino acids in position 18-32 of the amino acid sequence of protein S100β. The peptide is covalently bound to a spacer (Ahx) consisting of 2-aminoheptane acid or a similar compound, and covalently bound to the spacer by a biotin molecule (Bio). The peptide is described by general formula: A-Tyr1-Ser2-Gly3-Arg4-Glu5-Gly6-Asp7-Lys8-His9-Lys10-Leu11-Lys12-Lys13-Ser14-Glu15-E, wherein A represents Bio-Ahx, Bio-Acp, Bio or 0, and B represents 0 or -NH2. Bio means biotin; Ahx means a residue of 2-aminoheptane acid; Acp means a residue of 6-aminocapronic acid, 0 means the absence of any amino acid. A diagnostic technique and a method for prediction of melanoma provides measuring blood natural antibodies (nAB) to the peptide according to the invention with the stages of melanoma diagnosed by the antibody concentration as compared to the reference. The clinical outcome and therapeutic effectiveness in a craniocereberal injury are predicted by measuring the nAB to the peptide according to the invention. If observing the blood nAB level in the patients lower as compared to the standard reference, the favourable clinical outcome is diagnosed, and the therapy is stated to be effective.
EFFECT: invention may be used effectively as instant diagnosing of the disturbed blood-brain barrier and monitoring of the clinical effectiveness in CCIs and some cancers.
3 cl, 9 dwg, 1 tbl, 5 ex
SUBSTANCE: invention relates to the method to produce a brucellosis antigen for the rose bengal test (RBT). The method to produce the brucellosis antigen includes growing of the strain B. abortus 104M on the solid nutrient medium, washing of the culture with 0.5% phenolized solution, inactivation of biomass in a water bath at the temperature of 88±1°C for 30 minutes, performance of inspection for sterility, whirling of the bacterial suspension at 6-7 K rpm for 40 minutes, slurrying of the brucelli residue in 0.7% water solution of rose bengal at the ratio of 1:1 and dyeing of microbial cells at 4°C, re-slurrying of the microbial suspension in the buffer dissolvent and performance of standardisation.
EFFECT: method makes it possible to produce an antigen of higher activity and may be used in production of preparations for brucellosis diagnostics.
SUBSTANCE: invention refers to veterinary science and concerns a method for preparing a Setaria antigen. The method comprises homogenising the mature helminthes Setaria labiato-papillosa manually in the cold, extracting homogenated proteins in phosphate-buffered saline pH 7.2-7.4 in ratio 1:10 for 48 hours at +4°C in a magnetic mixer, centrifuging the homogenate and collecting a supernatant at 15 thousand rpm and +4°C for 20 minutes; the extracted supernatant is additionally purified by gel chromatography in a Superose 12 Prep Grade column at flow rate of sodium phosphate buffer (pH 6.0) - 30 ml/h; the antigen-active fractions are collected by ELISA assay with using dog's dirofilariasis positive and negative reference serums.
EFFECT: fractions containing the antigen-active protein components and having shown the best results with reference serums are combined, measured for the protein content and used as an antigen for ELISA assay for diagnosis of dog's dirofilariasis.
SUBSTANCE: method of determination includes: sorption in microboard holes of IgAl-protease,introduction into microboard holes of samples, which contain functionally determinable active C1 inhibitor, carrying out incubation, during which covalent binding of C1 inhibitor with IgAl-protease takes place, determination of amount of bound in holes C1 inhibitor by means of conjugate of antibodies to C1 inhibitor with enzyme and chromogenic substrate for said enzyme. Method is based on ability of functionally active C1 inhibitor to bind covalently with IgAl-protease. Kit contains flat-bottomed microboard with sorbed IgAl-protease preparation, conjugate of enzyme with antibodies to human C1 inhibitor, substrate buffer of said enzyme and standard for active C1 inhibitor.
EFFECT: group of inventions ensures elaboration of simple method and kit for determination of functional activity of C1 inhibitor and studying its inhibition.
2 cl, 1 dwg
SUBSTANCE: invention represents a method for preparing weakly positive reference serum containing AD and AY subtypes of HBsAg involving donor serum sampling with ELISA, titration of the positive serum containing AD & AY subtypes of HBsAg in a dissolving stabilised solution, genotyping, thermal degradation test for establishing panel shelf life, differing by the fact that the positive reference serums containing AY and AD subtypes of HBsAg are dissolved to the range of the concentrations of 0.05 to 0.025 IU/ml and viscosity equal to that of normal human serum; and the dissolving stabilised solution is presented by negative defibrinated and lipid-free serum containing no markers of HBV infection which is to be detected by the test system of interest.
EFFECT: invention provides detection of HBV infection at the earlier stage of the disease and enables creating and controlling the test system quality.
5 cl, 2 tbl, 1 ex
SUBSTANCE: what is presented is a method for making a panel of serums for the purpose of quality control of diagnosing hepatitis B with the certified low concentration of HBsAg subtypes AD and AY which involves the analysis of donor serums for the presence of anti-HIV, anti-HCV and anti-HBs antibodies and the nonspecific HBsAg binding; the selection of donor serums with said parameters not found; the use of the selected serums to prepare diluting solutions with a stabilising additive introduced; the certification of the monopreparations of HBsAg subtypes AD and AY by titration dilutions of international standards and the preparation of a reference panel from the certified monopreparations of HBsAg subtypes AD and AY within the range of 0.01 to 0.5 IU/ml; the lyophilisation of the serums followed by thermal degradation panel testing to determine a shelf life. The use of a new formulation of the stabilising additive containing proline 200-250 mM and benzoic acid 0.05-0.08 wt %, and the selection of an optimum temperature storage conditions promote the prolonged activity preservation at annual loss (at +4°C) making less than 0.5%.
EFFECT: higher shelf life.
3 cl, 5 tbl, 1 dwg, 1 ex
SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.
EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.
53 cl, 28 dwg, 5 tbl, 10 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to new bacitracin compounds with antibiotic activity
wherein at least one of R1, R2 and R3 represents -CH=CH2 and wherein R1, R2 and R3 independently represent -H, -CH3 or -CH=CH2.
EFFECT: preparing the new bacitracin compounds.
13 cl, 8 dwg, 7 tbl, 5 ex