Pseudotuberculous erythrocytic monoclonal diagnosticum
SUBSTANCE: invention can be used to identify a pseudotuberculosis agent in bacterial cultures, a biological material and environmental objects by applying the indirect hemagglutination test. Substance of the invention consists in development of a new diagnosticum that represents formalinised sheep's erythrocytes sensitised with monoclonal antibodies to lipopolysaccharide antigen of cold version Yersinia pseudotuberculosis serotype I (strain 164/84 serovariant I) and frozen-dried in a protective medium. Shelf life of the preparation is 2 years.
EFFECT: diagnosticum provides high sensitivity, specificity to the UHAT in detecting Yersinia pseudotuberculosis serotype I.
The invention relates to the field of medical and veterinary Microbiology, in particular to the problem of obtaining erythrocytic diagnosticums, and can be used for identification of the causative agent of pseudotuberculosis in bacterial cultures, biological material and the objects of the external environment by means of the reaction of indirect haemagglutination (rnga).
Family composition and (or) the purpose of the claimed solution is diagnosticums: erythrocyte sapnay and melioidosis monoclonal produced Volgograd NPCI, Volgograd [sapina L.V., Kasina IV, Malahaeva A.N. and other Evaluation of effectiveness of new erythrocytic spnego monoclonal diagnosticum // Sat. scientific works dedicated to the 75th anniversary of the Institute of Microbiology of the Ministry of defense. - Kirov, 2003. - P.53-54]; erythrocyte pseudotuberculosis immunoglobulin laboratory manufacturing [Dulatov M.V., Antonov, V.S., Golovacheva S.N. and other experience in the use of immunoglobulin erythrocyte preparation for early laboratory diagnosis of pseudotuberculosis // Ukr. microbiol. - 1992. No. 4. - P.55-57] and diagnosticum latex pseudotuberculosis immunoglobulin production research Institute of vaccines and sera, St. Petersburg [Shpilyuk GF, Golovashkin T.N., Volodin I. and other Immunoglobulin latex preparations for the rapid diagnosis of yersiniosis // Ukr. microbiol. - 1993. No. 6. - Pp.92-93].
Known diagnosticum erythrocyte sapnay and melioidosis monoclonal, preparation which comprises the steps: obtaining monoclonal antibodies to the antigens of the causative agents of glanders and melioidosis, formalnosci erythrocyte surface modification of erythrocytes tannin, sensitization their monoclonal antibodies (MCAT), lyophilization erythrocytic diagnosticum. The drug is designed to identify the causative agents of glanders and melioidosis in RNA.
Known also diagnosticum pseudotuberculosis erythrocyte immunoglobulin, which as sensitive includes immunoglobulins of class G of polyvalent pseudotuberculosis agglutinins serum. The drug was intended for the determination of Y. pseudotuberculosis antigens in the serum of patients. Laboratory testing showed that with the help of the developed erythrocytic diagnosticum can be identified 800 thousand M.K./ml in the absence of cross-reactions with antigens of pathogens dysentery, Salmonella major groups and intestinal yersiniosis [Dulatov M.V., Antonov, V.S., Golovacheva S.N. and other experience in the use of immunoglobulin erythrocyte preparation for early laboratory diagnosis of pseudotuberculosis // Ukr. microbiol. - 1992. No. 4. - P.55-57]. When the test diagnosticum in the clinic, the share of non-specific reactions amounted to 15% (that is the number of patients with intestinal yersiniosis). In addition, the drug has not been tested for specificity against the causative agent of plague, which, as you know, is the closest relative of Yersinia pseudotuberculosis microbe and often gives a cross-reaction. It is possible that the lack of specificity of diagnosticum is determined by the fact that sensitiva are polyclonal antibodies that cause cross-reactions with microorganisms other taxa.
Due to the fact that this drug was prepared as a laboratory sample, did not pass the state certification and, accordingly, has not been utilised to conduct a comparative analysis of diagnostic properties named and claimed pseudotuberculosis erythrocytic diagnosticums not possible.
Closest to the claimed solution is diagnosticum pseudotuberculosis latex immunoglobulin liquid production research Institute of vaccines and sera, St. Petersburg [Shpilyuk GF, Golovashkin T.N., Volodin I. and other Immunoglobulin latex preparations for the rapid diagnosis of yersiniosis // Ukr. microbiol. - 1993. No. 6. - Pp.92-93]. Today it is the only certified and authorized to use the drug to identify the causative agent of pseudotuberculosis (along with immunoglobulins of Yersinia pseudotuberculosis adsorbed fluorescent horse is tion) [sapina L.V., Malahaeva AV, Barulin I.S. State of production and the introduction of new drugs for the diagnosis of Yersinia // Infection caused by Yersinia: proceedings of the II all-Russian scientific-practical conference with international participation. - SPb. Of epidemiology and Microbiology them. Pasteur, 2006. S-122]. The drug made using polyvalent serum to Y. pseudotuberculosis serotype I, is intended to indicate this pathogen in coprofilia by setting the reaction of agglutination of latex (RAF) on glass [Shpilyuk GF, Golovashkin T.N., Volodin I. and other Immunoglobulin latex preparations for the rapid diagnosis of yersiniosis // Ukr. microbiol. - 1993. No. 6. - Pp.92-93].
Preparation of latex immunoglobulin diagnosticum includes the steps of: receiving immunoglobulin G from polyvalent pseudotuberculosis agglutinins serum, sensitization of latex particles immunoglobulins, conservation of diagnosticum. In common with the claimed diagnosticum is the stage of sensitization specific immunoglobulins solid media.
Along with such positive qualities as speed of analysis and simplicity of statement, this drug also has disadvantages: low sensitivity and relative specificity due to the use in the preparation of polyvalent serum, the high cost of anal is for, as well as the complexity quantification of the pathogen in the analyzed sample. In addition, the drug is available in liquid form, which limits its validity.
The most promising approach to improving the means and methods immunochemical detection of the causative agent of pseudotuberculosis is the identification of species-specific antigens of Y. pseudotuberculosis, obtaining MCAT to them and to develop a diagnostic test systems [drobkov VI, Dharma IV Immunochemical methods for diagnosis of pseudotuberculosis (review) // Clinical laboratory diagnostics. - 1993. No. 5. - P.3-7]. At the moment there is no information about the development and certification of products based on these principles [sapina L.V., Malahaeva A.N., Barulin I.S. State of production and the introduction of new drugs for the diagnosis of Yersinia // Infection caused by Yersinia: proceedings of the II all-Russian scientific-practical conference with international participation. - SPb. Of epidemiology and Microbiology them. Pasteur, 2006. S-122].
The objective of the invention is to develop a highly sensitive, specific and stable during storage pseudotuberculosis erythrocyte monoclonal diagnosticum, suitable for detection of Y. pseudotuberculosis using RGA.
The problem is solved due to the fact that in the present diagnosticum there is differences, associated with the use as a carrier of a specific component formalisation sheep red blood cells; as sensitive solid phase - MCAT received on lipopolysaccharide antigen "cold" version of Yersinia pseudotuberculosis microbe strain 164/84 I serovariants; in addition, diagnosticum subjected to lyophilization.
The claimed diagnosticum prepared according to the following scheme:
1. obtain ascitic fluid;
2. the selection of MCAT of ascitic fluid;
3. preparation formalisation sheep erythrocytes;
4. sensitization formalisation erythrocytes, MCAT;
5. lyophilization prepared diagnosticum.
1. Obtain ascitic fluid.
Culture of hybrid cells YP-105C5 (received and deposited in 48 Central research Institute of the Ministry of defense of Russia) is removed from the Dewar vessel with liquid nitrogen and thawed in a water bath at a temperature of 37°C. the Contents of the bottle poured into a centrifuge Cup with medium RPMI-1640 and centrifuged at 1100 g for 10 min. the Supernatant liquid is drained and the sediment resuspended in 10.0 ml of RPMI-1640 medium and select a sample for evaluation of the quality of the working culture. All work is done in compliance with the rules of asepsis in a laminar flow Cabinet. The working culture must meet the following requirements: percentage of viable cells is not less than 80%; the concentration of living cells is to - not less than 500 thousand cells/ml
Prepared a working culture injected intraperitoneally white mice BALB/c mice at a dose of 0.5-1.0 million cells. 7-10 day control engraftment hybrid. The mice developed ascites killed by cervical dislocation, wash the abdominal cavity of 5.0 ml phosphate buffer solution (FBI). Ascitic fluid is transferred into a centrifuge tube and incubated for 2-4 h at room temperature and 18 hours at a temperature of 2-6°C, then centrifuged for 10 min at 1100 g. The supernatant liquid is filtered through a filter paper moistened with the FBI in a bottle and take a sample for assessing the quality of ascitic fluid.
Ascitic fluid is controlled by the rate of specific activity by asking enzyme-linked immunosorbent assay (ELISA) with lipopolysaccharide antigen of Yersinia pseudotuberculosis microbe I serovariants. Ascitic fluid is considered conforming if the titer of MCAT in ELISA is not less than 1:80000.
2. Selection of monoclonal antibodies from ascitic fluid.
Received ascitic fluid precipitated with a saturated solution of ammonium sulfate in the ratio of 1:1, incubated at a temperature of from + 2 to + 6°C for 18-20 h, after which the mixture is centrifuged for 20 min at 7000 g. The supernatant decanted, and the residue is dissolved in a volume SFR, is equal to the original volume of ascitic liquids is I. Operation is repeated again, but with a volume ratio of 1.5:1. After the second resultant deposition rates immunoglobulins dissolved in a minimum volume of 0.1 m carbonate buffer solution (CBD) with a pH of 9.5 sufficient to fully dissolve the precipitate.
The solution of immunoglobulins cialiswhat against 1.0 l of 0.1 M KBR with a pH of 9.5. The dialysis is carried out at a temperature from + 2 to + 6°C for 18-24 hours Shift CBD carried out after 2 and 8 hours Otvetsvennyy the solution is centrifuged for 30 min at 7000 g. The supernatant is decanted, the precipitate is discarded.
Otvetsvennyy solution of immunoglobulin injected into the chromatographic column with DEAE-separate CL-4B at a rate of not more than 20 mg of protein per 1 ml of gel. Immunoglobulins elute from the column 0.05 M FBI with a pH of 7.2±0.1, the controlling output of protein extinction at a wavelength of 280 nm through a flow-through UV absorptiometry and potentiometric recorder. Immunoglobulins G suiryudan one broad peak.
The obtained preparations of MCAT preserved by the addition thereto of a solution of sodium azide to a final concentration of 0.2% and stored at a temperature from + 2°to + 6°C not more than 7 days or no more than two months at a temperature not higher than minus 20°C.
The resulting purified preparations of MCAT control indicators serological activity and protein concentration. Serologies the th activity determined in ELISA, the concentration of protein by determining the optical density of the solution in the spectrophotometer (MR-580) at a wavelength of 280 nm. The protein concentration in the resulting purified preparations of MCAT should be not less than 10.0 mg/ml Purified MCAT considered conforming if the concentration of protein 1.0 mg/ml they react in ELISA with lipopolysaccharide antigen of Yersinia pseudotuberculosis microbe in the credits of not less than 1:100000 (Preparation of lipopolysaccharide antigen isolated from cell cultures of Y. pseudotuberculosis grown on solid nutrient medium at a temperature from + 4 to + 6°C by hot aqueous phenol" [Westphal O., Jan K. Bacterial lipopolysaccharides. Methods Carbohydr. Chem. 1965, 6: 83-91]).
3. Preparation formalisation sheep erythrocytes.
Blood from intact rams selected in 500 ml glass flask placed in them sterile beads. After blood collection flask continuously stir for 20 min for adhesion of fibrin on the beads. Defibrinating blood is filtered through a nylon cloth; from whey proteins erythrocytes washed 20-minute centrifugation at 1100 g and + 4°C 0,9%solution of sodium chloride (pH 6.8), cooled to the same temperature. The supernatant is removed, the operation laundering erythrocytes repeat 3-5 times.
To get 8%suspension dense precipitate washed erythrocytes diluted in 12.5 times with 0.9%solution of soda which I chloride, warmed to + 37°C. In a solution of formalin (GOST 1625-75) determine the percentage of formaldehyde in accordance with ANGUISH 4.1/4.2.588-96, p.100-102. Needed to formalisation erythrocyte volume of formalin solution calculated by the formula: X=3V/C, where
X - search volume,
V - volume of the prepared 8%suspension of erythrocytes,
With the percentage of formaldehyde in the formalin solution.
To obtain a 3%aqueous solution of formaldehyde to X ml of formalin solution is poured (V-X) ml of 0.9%sodium chloride solution.
In a prepared 8%erythrocyte suspension with constant stirring poured 3%formaldehyde solution, warmed to + 37°C. the Bottle with the mixture is placed in the original apparatus for formalisation of red blood cells, maintains the water temperature of the water bath to ensure a continuous mixing of the water in the bath and erythrocytes inside the bottle. Formalisation spend at plus 37°C for 18 hours Then formalisation erythrocytes washed four times in 10-fold volume of 0.9%sodium chloride by 15-min centrifugation at 1100 g. A dense precipitate is diluted 10-fold with 0.9%sodium chloride solution. a 10%suspension formalisation erythrocytes preserved by adding formaldehyde to a final concentration of 0.8%. Stored at + 4°C for 3-5 years. For you need a kitchen is of erythrocytic diagnosticum optimal are the red blood cells after storage during the year.
Before using erythrocytes of control on the homogeneity and lack of susceptibility to spontaneous agglutination. To control the homogeneity of the erythrocytes diluted in 0.9%solution of sodium chloride and one drop of a 0.5%suspension of erythrocytes put on a glass slide, cover with a cover glass and examine under low magnification microscope. Red blood cells should be isolated, Pets 2-3 clusters containing not more than 10 cells in 10 fields of view. The lack of susceptibility to spontaneous bonding: 0.4 ml of 0.9%sodium chloride solution in a polystyrene plate, add 0.05 ml of a 2.5%suspension of erythrocytes, homogenize and leave at room temperature for 2-3 hours Erythrocytes should precipitate in the form of "buttons" or a small ring with a smooth edge. In case of occurrence of spontaneous agglutination series of red blood cells is discarded.
4. Sensitization formalisation erythrocytes, MCAT.
The required amount of 10% formalisation of washed erythrocytes from formalin in 0.9% sodium chloride (pH 7,2) by 4-fold 15-minute centrifugation at 1100 g. Then the erythrocytes bred warmed to + 37°C in 0.9%sodium chloride solution (pH of 7.2) to a concentration of 5%. To the resulting suspension with constant stirring add an equal volume of a solution of tannin in a dilution of 1:20000. Anizatio about the W ill result in a period of 15 minutes at a temperature of suspension plus 37°C.
Suspension washed with 0.9% sodium chloride solution (pH 6.4) by 3-fold to 20-minute centrifugation at 1100 g. Then the erythrocytes diluted with 0.9% sodium chloride solution (pH 6.4) to a concentration of 5%.
Before cooking the main party of diagnosticum for this series formalisation erythrocytes and this series of MCAT determine the optimum sensitizing dose of the latter. For this 16 ml erythrocytes obtained is poured into 4 ml of 4 test tubes. An aliquot of the prepared immunoglobulin diluted with 0.9% sodium chloride solution (pH 6.4) to 10, 20, 40, 80 μg/ml and added to 4 ml of each concentration in a test tube with erythrocytes (one concentration of immunoglobulin per tube). After 60-minute incubation at 45°C sensitin fixed for 30 min at 45°C by addition of glutaraldehyde to a final concentration of 0.4%. Tubes with sensitized erythrocytes 4x washed with 0.9% sodium chloride solution (pH of 7.2) by 10-minute centrifugation at 1100 g.
The resulting erythrocytes sensitized with MCAT in various concentrations, check in rnga with killed cultures of Y. pseudotuberculosis serotype I sensitivity, as well as with inactivated cultures of Y. pestis, Y. enterocolitica, E. coli specificity. Setting rnga carried out according to the instructions for use of the drug. Optimal sensibilities the second dose of immunoglobulin consider that, where get diagnosticum with the highest sensitivity and does not reveal the culture of the above heterologous microorganisms at a concentration of 100 million M.K./ml
For the preparation of the main part of diagnosticum to 5% mist Anisimovna erythrocytes with constant stirring add an equal volume containing 1.5 optimal dose immunoglobulin in 0.9% sodium chloride solution (pH 6.4). After 60-minute incubation at 45°C sensitin fixed for 30 min at 45°C by addition of glutaraldehyde to a final concentration of 0.4%. The drug 4 times washed with 0.9% sodium chloride solution (pH of 7.2) by 20 minutes centrifugation at 1100 g and the same solution was adjusted to 10% concentration.
5. Lyophilization prepared diagnosticum.
The mixture is precipitated by 10-minute centrifugation at 1100 g sediment resuspended in 10%sucrose solution, and centrifuged for 30 min at 1100 g.
To prepare the environment drying in 650,0 ml of distilled water is dissolved in 100.0 g sucrose 10.0 g of bovine serum albumin (BSA), 10.0 g tetraborate sodium and 1.0 g of sodium azide. To the obtained solution under pH control and with constant stirring poured (100,0±20,0) ml of 3%aqueous solution of succinic acid to obtain a pH value of 7.0±0,1). The amount of lead distilleria the Oh water to 900,0 ml, the solution is thoroughly mixed and stored at + 4°C not longer than one year.
The precipitate was washed 10%sucrose solution erythrocytes diluted with medium drying to 10%concentration and poured into vials (3,0±0,5) ml.
Vials diagnosticum placed on the shelves, pre-cooled to a temperature of minus 30°C, sublimation chamber freeze-drying "of the MASSES-5". The chamber is hermetically closed. Diagnosticum freeze for 2-3 h until the material temperature not higher than minus 25°C, and then create the vacuum chamber supported throughout the process of freeze-drying on the level of 1.1-1Torr (13,33 n/m2). After 1.5-2.5 hours include heating shelves. After 1 h the shelves are heated to temperature plus 28°C, supported until the end of drying, the temperature in vials with diagnosticum 12-14 h also reaches plus 28°C, after which the lyophilization continue for 5.5-6 hours Duration lyophilization of diagnosticum - 20-22 hours
At the end of the process, the vials sealed with rubber stoppers, closing, sealing, aluminum caps and control the quality of the dry diagnosticum for the following:
5.1. Solubility - adding in a bottle of 11.0 ml of 0.9% solution of sodium chloride, the drug must dissolve for 1 min;
5.2. the pH should be in the range (7,0±0,2);
5.3. The loss in weight on drying not more than 5%;
5.4. Homogeneity resuspending drug under low magnification - not more than 3 clusters 10 and less red blood cells in 10 fields of view;
5.5. The absence of spontaneous haemagglutination - when added to 0.4 ml of 0.9% sodium chloride, 0.05 ml of a 2.5% suspension of red blood cells after 3 h should form button or a narrow ring;
5.6. The number of red blood cells in 1.0 ml of 2.5% diagnosticum 5,4·105±0,6·105M.K./ml;
5.7. Sensitivity - should reveal Y. pseudotuberculosis serotype I at a concentration of 500 thousand M.K./ml micromethods in rnga;
5.8. Specificity should not be identified in RNA formalin inactivated cultures of Y. pestis, Y. enterocolitica, E. coli at a concentration of 100 million M.K./ml;
5.9. The quality of sealing bottles in accordance with ANGUISH 4.1/4.2.588-96, p.64.
Warranty period of storage of diagnosticum at a temperature of from + 2 to 8°C is 2 years.
Comparative evaluation of specificity and sensitivity RAHL, RNA using question-based assays were performed with cultures of Y. pseudotuberculosis serotype I (strains 164/84, 147, 149, 847, 881, 66, 432, p, ± 5, 59, 282, 1179, 497, I, as well as with the culture of the strain 275 attached to the kit latex diagnosticum), Y. pestis (strain EV), Y. nterocolitica (serogroups O:3, O:5, O:9), E. coli (strain HB-101). All strains, except for Y. pseudotuberculosis St deposited in the collection of microbial the cultures 48 Central research Institute of the Ministry of defense of Russia. Setting RAHL and RIGA (micromethod) was carried out according to the instructions on the use of drugs.
The specificity of RNA tested by its inhibition (production RTHA serum pseudotuberculosis agglutinins in a dilution of 1:1000 added to each well in a volume of 0.025 ml). Rnga is considered specific if RTHA agglutination is absent or occurs in fewer holes than rnga (3-4 wells less). In the experience of inhibition was 4 wells.
When investigating the specificity of diagnostics found that none of the studied heterologous strains of microorganisms at a concentration of 100 million M.K./ml test diagnostics have not yielded positive results, indicating that their specificity.
The purpose of a comparative study of the sensitivity of Yersinia pseudotuberculosis erythrocyte monoclonal and latex-based assays was tested 15 strains of Y. pseudotuberculosis serotype I. The results obtained are presented in table 1.
Analysis of the obtained data allows to conclude that diagnosticum pseudotuberculosis latex immunoglobulin inferior in sensitivity and range of detection cultures of Y. pseudotuberculosis serotype I claimed design. So, of the 15 studied strains only 6 were identified by RAHL installed NTD sensitivity, 3 strain reveals the ü in a concentration of 125 million M.K./ml and above, 6, when the concentration of microbial cells in the sample 500 million, were not found at all. Diagnosticum pseudotuberculosis erythrocyte monoclonal allowed to identify in rnga 12 out of 15 strains of Y. pseudotuberculosis serotype I at a concentration of 250-500 thousand M.K./ml, 2 strain at a concentration of 62.5-125,0 million M.K./ml and 1 strain at a concentration of more than 125 million M.K. in 1 ml of sample.
Within this work it was studied the influence of different temperature-time storage mode on the specific activity of the claimed diagnosticum. The study was conducted by setting rnga with three series of dry drug, kept various periods of time at two different temperatures. Each production rnga both methods was carried out in four replications, independent of each other.
The results of the evaluation of the persistence of the developed erythrocytic diagnosticum pseudotuberculosis monoclonal during storage at different temperatures are shown in table 2.
As follows from the data presented in table 2, a marked decline in the specific activity of diagnosticum was observed after 6 months of storage at temperature from + 18 to + 22°C. to reduce the sensitivity of the listed drug was not observed in the case of permanent storage at temperature from + 2 to + 8°C within 2 years. Thus, the claimed dia is noctium has the advantage over the closest analogue in the retention period (one year for latex immunoglobulin liquid diagnosticum), which significantly extends the capabilities of its application.
The calculations of experts on the profitability of production of diagnostic products based on MCAT showed that in the process of their production the most expensive stage of the experiments are to obtain hybridomas producing specific immunoglobulins. After the selection of hybrid clones, stably secreting MCAT to diagnostically relevant antigens of a particular microorganism, the cost of monoclonal diagnostic products falls below the similar indices of products manufactured on the basis of polyclonal immunoglobulins. Thus, the advantages of the first due to not only their exceptional specific activity in relation to "critical"
The sensitivity of RNA with the declared diagnosticum and RAHL with the closest analogue
|Type, serotype||The number of strains||The percentage of detected regulated NTD* sensitivity of the cultures of Y. pseudotuberculosis in...|
|Y. pseudotuberculosis, I serotype||15||80||40|
|Note: * - approved documentation of diagnosticum pseudotuberculosis latex immunoglobulin sets the sensitivity RAHL not lower than 1.6 million M.K./ml, project documentation on erythrocyte monoclonal diagnosticum - not less than 500 thousand M.K./ml|
The results of the study the persistence of diagnosticum
|Temperature storage diagnosticum||The series number||The specific activity of diagnosticum in the study of cultures of Y. pseudotuberculosis strain 164/84, mn M.K./ml|
|immediately after production (n=4)||after storage for...|
|21 days (n=4)||3 months (n=4)||6 months (n=4)||12 months (n=4)||18 months (n=4)||24 months (n=4)|
|Constantly at the temperature from + 18 to + 22°C||1||0,5||0,5||1,0||2,0||-||-||-|
|Constantly at a temperature of from + 2 to + 8°C||1||0,5||-||0,5||0,5||0,5||0,5||0,5|
|Notes: 1 n is the total number of definitions|
2 - " definitions are not held
Comparative cost of one analysis using the proposed diagnosticum and closest analogue
|Diagnosticum pseudotuberculosis...||The cost of the kit, RR||The number of analyses||The cost per analysis, RR|
|Notes: * under one analysis see: for RNA - titration of antigen on 1 row dvenadcatiletnego tablet, RAHL - analysis with one bussing antigen|
**- presents the estimated cost of one set of erythrocyte monoclonal diagnosticum
antigenic determinants, by standardization of quality parameters, but also economic sense.
Comparative characteristics of the cost of a single analysis using a question-based assays are presented in table 3.
Thus, the claimed diagnosticum allows a greater number of determinations for the difference in the cost of one analysis is approximately 10 times compared to diagnosticum pseudotuberculosis latex immunoglobulin. In addition, it is technically much easier and with greater material savings can be the determination of the titer of the antigen (concentration) in the sample using pseudotuberculosis erythrocyte monoclonal diagnosticum.
The results showed that IP is itemy the drug has advantages over the closest analogue on the following parameters:
the sensitivity and range of detection cultures of Y. pseudotuberculosis serotype I: the proposed model allows us to identify RNA Y. pseudotuberculosis at a concentration of 500 thousand M.K./ml in cultures 80% of the studied strains, whereas the closest analogue was found in RAHL Y. pseudotuberculosis culture at a concentration of 2-4 million M.K./ml only in 40% of cases;
lyophilization of diagnosticum pseudotuberculosis erythrocyte monoclonal allowed to provide a shelf life of 2 years at a storage temperature from + 2 to + 8°C, while the shelf life of the nearest analogue produced in liquid form, 1 year;
the estimated cost of one analysis, performed using the proposed diagnosticum 10 times less than the cost analysis using the nearest equivalent.
Thus, the proposed diagnosticum provides high sensitivity, specificity, RNA in the detection of Yersinia pseudotuberculosis microbe serotype I, with a shelf life - 2 years. The relatively low cost of the drug makes the analysis using the developed diagnosticum more accessible to medical practice.
Diagnosticum pseudotuberculosis erythrocyte monoclonal representing formalisation the sheep erythrocytes sensitized with monoclonal antibodies to the lipopolysaccharide antigen of Yersinia pseudotubercuosis I serotype and lyophilized in a protective environment.
FIELD: biotechnology, in particular production of vaccines and diagnosis objects.
SUBSTANCE: bacterium Lawsonia intracellularis is cultivated in Hep-2, Me Coys or IEC-18 cells in suspension at oxygen concentration from more than 0 to about 18 %. Vaccines, containing bacterium L. intracellularis obtained by claimed method, is useful in inducing of immune response to said bacteria in animals.
EFFECT: improved method for large-scale cultivation of bacterium L. intracellularis.
21 cl, 5 tbl, 6 ex
SUBSTANCE: strain 5A10 of hybridomal line of cells of mouse Mus. museums, producing monoclonal antibodies to immunoglobulin IgG of cattle (C) is permanent line of cells and is suitable for biotechnology in elaboration of preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 71. Antibody titers in native culture liquid constitute 1:32-1:64, in ascitic liquid 1:640-1:5120 in immuno-enzymatic analysys. Monoclonal antibodiesproduced by strain are specific to immunoglobulin IgG of cattle and do not react with immunoglobulins of sheep. Peroxydase-marked monoclonal antibodies ensure high sensitivity and specificity of IEA for detection of antibodies to C leucosis virus in biological material. Strain 5A10 - producent of monoclonal antibodies to immunoglobulin IgG of cattle can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis.
EFFECT: application of said test-system will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.
SUBSTANCE: obtained is strain 1H8 of hybridomal line of cells of mouse Mus. musculus - producent of monoclonal antibodies to IgG of sheep, suitable for biotechnology in elaboration of diagnostic preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 73. When determined by method of immuno-enzymatic analysys (IEA) antibodies titers in native culture liquid constituted in IEA 1:32-1:64, in ascitic liquid 1:640-1:5120. Monoclonal antibodies are specific to immunoglobulin IgG of sheep and do not react with immunoglobulin of cattle. When used for fixation on solid phase of glycoproteidal antigen of cattle (C) leucosis virus (in composition of complex glycoproteidal antigen- monoclonal antibodies of sheep to glycoproteidal antigen) in IEA, they ensure strength of fixation and optimal availability of antigen for antibodies in testes samples.
EFFECT: strain 1H8 can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.
1 tbl, 4 ex
SUBSTANCE: obtained is strain 8C12 of inter-species hybrid cells of mouse Mus musculus and sheep Ovis aries - producent of monoclonal antibodies of sheep to glycoproteidal antigen of virus of cattle (C) leucosis. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 72. Strain is permanent hybrid line of cells and possesses high level of production of monoclonal antibodies of sheep. Antibody titers in native culture liquid constitute 1:32-1:64 in immuno-enzymatic analysys (IEA). Monoclonal antibodies are specific to general antigen determinant of glycoproteids of C leucosis - external gp51 and transmembranous gp30. When used in IEA for detection of antibodies in blood serum and milk of C infected with leucosis virus, antibodies provide strong selective binding with solid-phase carrier and optimal space orientation of glycoproteidal antigen.
EFFECT: strain 8C12 can be used in production of immuno-enzymatic test-system for diagnostics of cattle leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms and, as a result, reduce incidence of leucosis in cattle.
1 tbl, 4 ex
SUBSTANCE: present invention refers to immunology and biotechnology. There are antibody-antagonist to CD40 with their variable areas derived from an antibody produced of hybridoma 4D11 (FERM BP-7758). The constant areas of antibodies are derived from human IgG4 with mutations S228P and L235E. There are described related coding polynucleotides and the based expression vector. There is disclosed host-cell containing said vector. There is described method for preparing monoclonal antibody and application thereof in the pharmaceutical composition.
EFFECT: application of the invention provides reduced ADCC and CDC activity that can find application in therapy of autoimmune diseases and graft rejection.
10 cl, 26 dwg, 2 tbl, 22 ex
SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.
EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.
7 cl, 13 dwg, 4 tbl, 7 ex
SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.
EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.
16 cl, 34 dwg, 7 tbl, 20 ex
SUBSTANCE: strain A-4A7 is prepared by fusion of mouse myeloma cells of line SP2/0.Agl4 with mouse lymphocytes of line Balb/c immunised by introduction in pads of a purified preparation AMGF (alpha2-microglobulin of fertility) separated from amniotic fluid, and deposited in the transplantable mammal cell culture collection of the Research Institute of Human morphology of the Russian Academy of Medical Science numbered 131/2002. Strain A-4A7 synthesises monoclonal antibodies (MCA) of IgGI class specifically reacting in solid-phase immune-enzyme analysis (IEA) with AMGF isoforms of endometrial, follicular and sperm nature. Activity of the strain: cultural supernatant contains MCA 3-5 mkg/ml, while ascetic fluid contains MCA 2-5 mg/ml. The antibody titre in cultural fluid is 1:500-1:1000, in ascetic fluid up to 1:1×107. A-4A7 bonds various AMGF protein glycoforms produced in male and female reproductive organs. Application of MCA A-4A7 as an immunodiagnosis test systems allows for high-specific and high-sensitive (1 ng/ml) quantitative analysis of various AMGF/glykodeline isoforms in biological liquids.
EFFECT: new compounds are characterised with valuable biological properties.
SUBSTANCE: according to the present invention, there are disclosed disease diagnostic techniques and sets characterised by nonphysiological content of protein Hepsidin in mammals. Substance of the invention lies in binding antibody and polypeptide, where antibody or its fragment is specifically bound with one or more average epitopes or epitopes of carboxyl termination of sequence SEQ ID NO: 2. Additionally, there are disclosed relevant antibodies.
EFFECT: disease screening characterised by nonphysiological content of protein Hepsidin in organism.
19 cl, 2 ex, 16 dwg, 2 tbl
SUBSTANCE: method for making polyclonal Nogo protein antibodies by protein immunisation of an animal, with at least 6 amino-acid residues of active area Nogo A, free from any other myelin material of central nervous system whereto bound in natural conditions. Said active area covers position 1-171, 260-974, 1163-1178 of the corresponding amino acid sequence of protein Nogo A. There is disclosed separated antiserum based on polyclonal antibodies made under said method. There are disclosed methods for immunisation of a non-human animal to make polyclonal antibodies, as well as method for making monoclonal antibody, and the specified monoclonal antibody.
EFFECT: making active protein Nogo A antibodies to be applied in medicine for neurite growth activation.
26 cl, 18 dwg, 2 tbl, 8 ex
SUBSTANCE: present invention relates to biotechnology. An A-5D1 strain is obtained from fusing mouse myeloma cell line SP2/0.Ag14 with mouse lymphocyte line Balb/c, immunised by introduction into pouches of a purified preparation of alpha 2-fertility microglobulin, extracted from amniotic fluid, and deposited in the collection of passaged culture of mammal cells of the state research institute of human morphology (Russian Academy of Medical Sciences) under the number 144/2002. The strain of hybredome A-5D1 synthesises the IgG1 class monoclonal antibody, which specifically interacts in enzyme-linked immunosorbent assay and immunohistochemical assay with isoforms of alpha 2-fertility microglobulin of endometrial or special origin. Monoclonal antibodies do not have cross reaction with placental α 1-microglobulin, trophoblastic β1-globulin, human chorionic gonadotropin, α-fetoprotein, bovine serum albumin, and C-reactive protein. Monoclonal antibodies detect alpha 2-fertility microglobulin/glycodelin in cells and tissue of female and male reproductive organs.
EFFECT: use of monoclonal antibody A-5D1 as an immunodiagnostic system allows for quantitative analysis of isoforms of alpha 2-fertility microglobulin/glycodelin in body fluids with high sensitivity (1 ng/ml) and specificity.
FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex
FIELD: biology, biotechnology, medicine.
SUBSTANCE: strain of murine hybridoma cells is prepared by fusion of murine splenocytes immunized with cell lysates of lymphoblastoid line RAMOS with cells of murine myeloma. Hybridoma secrets monoclonal antibodies directed to antigen of molecular mass 110 kDa located on microtubules in cell cytoplasm and detected in nuclear cells of different species. Using the invention allows studying pathogenesis of cell dividing and state of mitotic activity of health and malignant cells, and evaluation of anti-mitotic (anti-tumor) effect of different chemopreparations and substances. Invention can be used for identifying phase of mitotic activity of cells.
EFFECT: valuable properties of strain.
FIELD: biology, hybridoma technology.
SUBSTANCE: invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 103 ID50 x cm-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology.
EFFECT: improved method preparing, valuable properties of strain.
3 tbl, 1 dwg, 1 ex
FIELD: medicine, immunobiology, pharmacy.
SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.
EFFECT: valuable medicinal properties of antibody.
75 cl, 78 dwg, 14 ex
FIELD: immunology; treatment of mediated diseases IL-1 and failures.
SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.
EFFECT: enhanced efficiency.
15 cl, 3 dwg, 5 ex
FIELD: biotechnology, hybridoma technology.
SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.
EFFECT: valuable properties of strain.
1 tbl, 2 ex