Dragoon strain of parotitis virus for obtaining antigene component of test system, and immunoenzyme test system for parotitis virus antibody diagnostics
FIELD: chemistry; biochemistry.
SUBSTANCE: invention concerns medical virology and microbiology. Strain is deposited in culture collection of Federal State Research Institution State Research Centre of Virology and Microbiology "Vektor" of Rospotrebnadzor, under registration No VB-05. Strain features higher productivity. More sensitive immunoenzyme test system for hepatitis virus antibody diagnostics is created on the basis of this strain. Invention can be applied in virology.
EFFECT: production of more sensitive immunoenzyme test system for hepatitis virus antibody diagnostics.
2 cl, 1 dwg, 1 tbl, 4 ex
The invention relates to medical Virology and Microbiology, and in particular to identification of a new strain of mumps virus Dragoons to retrieve antigen, a component of the diagnostic test system and the test system for the diagnosis of antibodies to mumps virus.
A known strain of mumps virus L-3, the productivity of which is not more than 8×105PFU/ml (.G.Andzhaparidzel, Yu.S.Boriskini, N.N.Bogomoloval and V.D.Lotte Accumulation of altered viral nucleocapsids in mumps virus - Persistently infected cell cultures // JOURNAL ARCHIVES OF VIROLOGY PUBLISHER SPRINGER WIEN, 1983, vol.75, N 4, 283-289).
The closest analogue (prototype) is a strain of mumps virus Enders, the productivity of which is not more than 106PFU/ml (Fleisner Century, Kreth H.W. Mumps Virus Replication in Human Lymphoid Cell Lines and in Peripheral Blood Lymphocytes: Preference for T Cells. // INFECTION AND IMMUNITY, 1982, vol.35, No.1, p.25-31).
The disadvantages of the above analogues is their low productivity in obtaining antigen mumps virus.
Famous native enzyme immunoassay system for the detection of antibodies to mumps virus "Mumps screen" (developer and manufacturer of test systems "Mumps-screen" CJSC Biotechnology company BIOSERVICE (instructions for use of test kits ELISA for the detection of antibodies to mumps virus "Mumps screen"; Moscow, 2001). The test system contains:
- immunosorbent - polystyrene tablets for immunological what reactions the holes are adsorbed antigen mumps virus (SHGA). SHGA - in the ranks of the tablet a, C, E, G and control antigen (CAg) in rows b, D, F, H. SHGA is a infected with the mumps virus Vero cells, containing not less than 80% vaccinated cells; CAg represents uninfected Vero cells;
- positive control sample (+) serum human blood containing antibodies to mumps virus;
negative control sample ( -) serum human blood or serum albumin human, not containing antibodies to mumps virus;
- conjugate - diagnostic antibodies against human immunoglobulins labeled with horseradish peroxidase;
- substrate buffer mixture with hydroperiod (AAH) - for preparation of a solution of a Chromogen;
the Chromogen - orthophenylphenol (OFD);
- stabilizer - bovine serum albumin (BSA) or casein;
concentrate of phosphate-saline buffer to launder tablets and prepare a dilution of sera and conjugate.
However, this test-system (analogue) has low sensitivity because it uses SHGA, representing the crude antigen - infected with the mumps virus Vero cells (infected with at least 80% of the cells).
The closest test system (prototype) is an ELISA test system for the phenomenon of antibodies to mumps virus firm Wampole Laboratories (Wampole Laboratories mumps IgG ELISA // P/N 6000-29W REV C. No. 4, 1996; Jeremy M. et al. Mumps Virus-Specific Antibody Titers from Pre-Vaccine Era Sera: Comparison of the Plaque Reduction Neutralization Assay and Enzyme Immunoassays // J. Clin. Environ, 2005, No.9, vol.43, p.4847-4851). Specified ELISA test system contains a tablet with immobilized inaktivirovannye antigen mumps virus, antibodies against human immunoglobulins labeled with horseradish peroxidase (conjugate) in liquid form, the positive control (K+), negative control ( -), calibrators, sample diluent; the Chromogen - tetramethylbenzidine (TMB)liquid stop reagent, wash buffer concentrate.
However, in the specified test system (prototype) as the antigen used untreated NP-protein of mumps virus, resulting in the test system has insufficient sensitivity (does not detect in the blood serum of antibodies to other proteins, such as hemagglutinin-neuraminidase - HN, the fusion protein F, the matrix protein M).
The technical result of the invention is the creation of producer strain of mumps virus, with higher productivity and enzyme immunoassay system on its basis for the diagnosis of antibodies to mumps virus with higher sensitivity.
This technical result is achieved by creating a new strain of mumps virus Dragoon to obtain antigen-component enzyme immunoassay with the system for the diagnosis of antibodies to mumps virus, deposited in the collection of cultures of microorganisms of the Federal state institution of science "State research center of Virology and biotechnology "Vector" of Rospotrebnadzor under registration number №VB-05.
This technical result is also achieved by the fact that in ELISA test system for the diagnosis of antibodies to mumps virus, including tablet with immobilized purified antigen mumps virus, vaccine positive control sample (+), inactivated negative control sample (K-), the conjugate solution (RK), Chromogen solution (PX), 25x concentrate of phosphate-saline buffer (25x FSB-T), the solution for dilution of sera (PC), a stop reagent, according to the invention as inactivated immobilized antigen mumps virus test system contains highly purified antigen prepared on the basis of the strain of the mumps virus Dragoon according to claim 1 claims, registration No. VB-05.
The drawing shows the analysis of the complete nucleotide sequence of the inventive strain of mumps virus and represented graphically in the form of a phylogenetic tree. Black rectangle marked strains isolated in Russia.
Note: strains, which were compared (CRU94Dra=s-genotype, EN-the country in which the isolate is selected, 94 the selection Dra-called strain-Dragoon).
Description of the proposed strain.
The strain of the mumps virus Dragoon belongs to the Paramyxoviridae family of viruses of the genus Rubulavirus. The inventive strain obtained at the State research center of Virology and biotechnology "Vector", the Ministry of health of the Russian Federation, to Koltsovo, Novosibirsk region
The strain is deposited in the collection of cultures of microorganisms of the Federal state institution of science "State research center of Virology and biotechnology "Vector" of Rospotrebnadzor under registration number №VB-05, registration date may 16, 2002 (certificate of Deposit of strain No. 0502 attached to the application) and used for the production of antigen in production test systems immunoassay for the detection of antibodies to mumps virus "Anti-mumps ELISA".
The source of the receipt.
The strain isolated from patient D. during an outbreak in 1994 GV repalone, Novosibirsk region. Patient Doctor had close contact with infectious patients who had serologically confirmed diagnosis of "mumps". The course of infection in patient Days was typical: the illness began with a temperature increase to 37.4°With the appearance of swelling of the salivary glands first one side and then the other. After 2 days the inflammation of the salivary glands disappeared. On the basis of the Kli is practical manifestations and increased titers protivoprostudnyh antibodies in paired sera, defined using rcga and ELISA, the patient D. was diagnosed with mumps. On day 3 after increasing the temperature of the patient Days were taken blood samples and nasopharyngeal wash. Cotton swab, which was done nasopharyngeal wash with mucous nasopharynx of the patient D., was placed in 1 ml of medium DMEM containing antibiotics and after sterilization through the filters of 0.45 μm was placed in a culture vessel (Kovarik, 75 cm3) with a monolayer of Vero cells. Vero cells were obtained from the Institute of cell cultures fsri SRC VB "Vector". As the nutrient medium used environment DMEM (production fsri SRC VB "Vector") with the addition of 2% fetal bovine serum (FSRS), 2 mmol/l glutamine and antibiotics: 100 µg/ml penicillin and 100 IU/ml streptomycin. By visual inspection under a microscope on a 3 day incubation at plus 37°was detected change in cell morphology. On the 4th day it was registered the appearance of simpleton. 5-6 day observed the destruction of simpleton (JRC). On the 6 day culture vessel was placed on a minus 20°With, then made the defrosting cultural vaccinated liquid (KWG). The resulting mixture CVG and lysate of Vero cells were Packed up and one piece flow, liofilizirovanny, and the other was stored at minus 70°C.
The authenticity of the strain.
From the sample hoco the tray flush patient D with a set RNaidTM PLUS (F. "BIO 101, USA) was isolated total RNA. Using a set of AmpliTaq (F. "Perkin Elmer, USA) conducted polymerase chain reaction (PCR). For the reaction primers were used on the SH gene of mumps virus (SH1F - AGT AGT GTC GAT GAT CTC AT SH2R - GCT CAA GCC TTG ATC ATT GA). PCR was positive, indicating the presence in the sample of washout from the nasopharynx of the patient D.
Conducted the full sequence of the genome of the selected isolate. Analysis of the complete nucleotide sequence of the inventive strain of mumps virus were conducted with all available the complete nucleotide sequences of strains of mumps virus placed in GebBank (http://www.ncbi.nlia.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar) and shown in figure 1 in the form of a phylogenetic tree. From the drawing it is seen that the inventive strain differs genetically from known strains of mumps virus.
The genotype of strain.
The genotype With [the Number of the deposited sequences in GebBank(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar) - AY 669145].
Cultural properties. Strain Dragoons under cultivation on the monolayer of Vero cells causes the formation of simpleton for 4-6 days (temperature cultivation 37°). The maximum titres were reached in 5-6 days after infection of a monolayer of Vero cells and was 8.6×107PFU/ml Yield: 3 liters QUI obtained 24 mg of antigen for ELISA. IP is an application derived from a strain of Dragoon antigen - 0.2-0.4 μg/well allows you to identify protivoprostudnye antibodies in the sera of vaccinated patients and mumps people by ELISA.
Detects antibodies to mumps virus in the sera of patients and vaccinated people. The selected strain has serological intersection with the reference strain Enders and vaccine strain L-3.
Pathogenicity for humans. Clinical signs of disease in a patient, which was isolated strain of Dragoon (temperature of 37.8°C, inflammation of the salivary glands disappeared for 2 days after emergence, after the disease complications not registered), we can assume that the strain of Dragoon is not highly pathogenic. Strain Dragoon is not phyto - and zoopathogenic.
For long-term storage strain lyophilizer using as a protective environment solution of gelatin and sucrose.
For the propagation of strain using the following nutrient medium: Needle mA'am, Needle MEM ×AVC, DMEM containing 2-5% fetal cattle serum, 2 mmol/l glutamine and antibiotics: 100 µg/ml penicillin and 100 IU/ml streptomycin.
The study of the serological properties of the resulting strain (Dragoon) revealed significant similarity to the reference strain Enders (prototype) mumps virus and vaccine strain L-3. Obtained from immunized mice of BALB/c antisera to Ref the Rense-strain Enders, to the vaccine strain L-3 and to the claimed strain Dragoon isolated from the patient, were investigated in the reaction of inhibition of haemagglutination (rtga) by a standard method using erythrocytes monkeys Macaco, mulatta, and in reaction enzyme-linked immunosorbent assay (ELISA). The results of the study of the intersection between the obtained sera are presented in table 1. As can be seen from the results of table 1, serum from mice immunized with mumps virus strain Enders, reacted with mumps virus strain Enders and L-3, and with the strain of Dragoon. On the other hand, sera from all mice immunized with the claimed strain Dragoons that were selected during the outbreak of mumps in 1994, reacted with the virus itself, and the reference strain Enders and vaccine strain L-3. Proceeding from the obtained results it can be concluded that the claimed strain on serological properties similar to the reference strain Enders and vaccine strain L-3.
The study of the antigenic properties of mumps virus strain Dragoon
|The specificity of serum||N serum||Antigen/response/antibody titer|
|the strain L-3||strain Dragoon||strain Enders||the strain L-3||Strain Dragoon|
Example 1. A method of producing antigen mumps virus
Purified and inactivated antigen mumps virus used for production of reaction enzyme immunoassay.
Getting vaccinated liquid.
The original Vero cells stored in liquid nitrogen and grown by serial subcultures. As the growth environment of the use of the solution Needle MEM with 8-10% of fetal serum of cattle.
The cell culture is grown for 3-4 days at sowing cell concentration of 5-10×105cells/ml of medium and subcultured accepted way.
The mumps virus (strain Dragoon) infect 1-liter mattresses monolayer culture of Vero cells (multiplicity of infection of 1:10). After incubation at 20-25°C for 40 minutes in a 1-liter culture vessel poured 200 ml of culture DMEM with the addition of (4,0±0,1) ml bovine serum, 0.001%trypsin, 100 u/ml benzylpenicillin and 100 ng/ml streptomycin, and after incubation for 5-7 days at (36±1)°merge With nutrient medium. The titer of virus in CVG not less than 108PFU/ml
Obtaining a lysate of Vero cells.
Under cultivation the transfer of the virus from cell to cell is due to the formation of simpleton without departing from the principal amount of virus in the culture vaccinated liquid (CVG), therefore, the accumulation of the virus occurs in CVG and inside cells. In a 1-liter culture vessel add 5-7 ml of 0.01 M Tris HCl, pH=9.0. Cells destroy two freezing-thawing to release the virus from the cells. Cell free lysate from cell debris by low-speed centrifugation in the rotor JA-10 centrifuge J2-21 (f. "Beckman, USA) at 1500 rpm for 10 min at a temperature of+4°Star of the virus in the cell lysate is from 6×1043×106PFU/ml
Concentration and purification of mumps virus from CVG performed on membranes "Vladipor" type UAM-100 (Russia) or RTK 0005 (F. "Millipore, USA). Linear feed rate KVG the cell must be equal to 0.6 l/min outlet Pressure should be no more than 2.5 kg/cm2the inlet pressure must not exceed the outlet pressure more than 0.4 kg/cm2. The final product is QUI concentrated in 20-25 times, freed from low molecular weight compounds and cell detritus. The titer of mumps virus Dragoon in concentrate not less than 109PFU/ml, which is higher by 2-3 orders of magnitude in comparison with the known analogues (strains Enders and L-3).
The ultracentrifugation vaccinated material.
The deposition of the virus. The antigen for ELISA obtained from two sources: from concentrate CVG and from the lysate of the cells of th is defrost.
In the first stage, the virus is precipitated through a "cushion" 10%sucrose in the rotor SW 28 centrifuge L-8-70 (F. "Beckman, USA). For this purpose, the tubes of the rotor carefully add 5 ml of 10%sucrose and the top layer 30 ml concentrate CVG or lysate of cells. The deposition of the virus is carried out at 25000 rpm, 4°S, 10 hours. The supernatant carefully pipetted, and the residue dissolved in 0.5-1.0 ml of NTE buffer (0.1 M NaCl, 0.01 M Tris HCl, 0.001 M EDTA, pH=7.6).
Purification of antigen mumps virus. The antigen for ELISA clear of foreign proteins by ultracentrifugation in a density gradient of sucrose. Precipitation of all tubes (precipitation from concentrate CVG and lysate of cells) are combined to carry out processing on the ultrasonic disintegrator at 6000 Hz 3 times for 10 sec and layered on the gradient of the solution of 20-60%sucrose. For the preparation of a linear gradient of sucrose solution using a mixer consisting of two chambers, which can be isolated by closing the valve between them. One of the cameras (first) connect the flexible hose with a peristaltic pump. The hose coming from the pump, put in a test tube with a capacity of 12 ml so that it touched the wall of the tube in its upper part. The mixer is mounted on a magnetic stirrer. In the first chamber is placed anchors. The mixer is filled with a closed valve as follows: in a first chamber filled 4,5 ml%sucrose solution, the second of 4.5 ml of 20%sucrose solution. Then include the magnetic stirrer, the peristaltic pump and carefully open the valve between the chambers. The solution from the second chamber flows into the first, mixed, and through the pump enters the tube. Total prepared with a linear gradient (20-60%) is 9 ml in each tube.
Precipitated antigen mumps virus gently layer by using a syringe with a thick needle on a linear sucrose gradient 20-60% in the glasses of the rotor SW-41 centrifuge Beckman L-8-70. Mode centrifugation - 37000 rpm at 4-6°C for 8 hours. The bar on the level 42-45%sucrose carefully selected syringe with a thick needle and after determining the purity of the antigen by the method of electrophoresis and determination of the concentration of the antigen protein is used for ELISA.
The output is suitable for ELISA antigen with 3 l KUR and lysate of cells is 24 mg protein.
Stage virus inactivation: purified in the density gradient of sucrose antigen mumps virus inactivate by heating at 56°C for 30 min (Zhdanov V.M., Russian, SJ Virology. M.: Medicine, 1966, s).
Example 2. The composition of the test system
The components of the test system ELISA for the detection of antibodies to mumps virus (Anti-mumps ELISA), which is a set of reagents:
- immunosorbent tablet 96-hole is for immunological reactions in THE 64-2-375-86 or tablet collapsible type "Nunc", Denmark or similar with immobilized purified antigen mumps virus;
- inactivated positive control sample (+)* serum human blood containing antibodies to mumps virus, inactivated by heating, and the dye neutral red on THE 6-09-3070-84;
- inactivated negative control sample ()* blood serum of healthy donors do not contain antibodies to mumps virus;
the conjugate solution (RK) - mouse monoclonal antibodies against human immunoglobulins labeled with horseradish peroxidase in THE 6-09-10-1408-79;
- Chromogen solution (PX) - tetramethylbenzidine (TMB);
- 25x concentrate of phosphate-saline buffer (25x FSB-T) **;
the solution for dilution of sera (PC) - a single solution FSB-T containing concentrate blocking solution (CBD)*** in a dilution of 1:100;
- stop reagent sulfuric acid according to GOST 4204-77 with a concentration of 0.9 mol/l;
The set is designed to hold 96 assays, including controls.
*Preservative is thimerosal sodium Sigma (USA) is contained in K+, K - and RK, at a concentration of 0.01%; K+and K - containing antibiotic is gentamicin sulfate for VFS 42-2626-89 at a concentration of 1 mg/ml
** The 1 l 25x FSB-T
|Sodium phosphate disubstituted 12-water|
|GOST 4172-76||90.0 g|
|Sodium phosphate one-deputizing 2-water|
|according to GOST 245-76||4,75 g|
|Sodium chloride GOST 245-76||22,0 g|
|Tween-20, Sigma, USA||12.5 ml|
Salt successively dissolved with stirring in 0.9 l of distilled water at a temperature of 40-50°C, pH 7.5±0,3, correction is performed as described above. The solution volume was adjusted to 1 l, add tween-20 and mix.
*** The 100 ml CBD
|Cleared from debris by centrifugation|
|dissolved in a nutrient medium at a concentration|
|5.0 mg/ml protein cell culture|
|used for infection with mumps virus|
|during its life||10.0 ml|
|Thimerosal sodium Sigma (USA)||10 mg|
|Phosphate-saline without twin||90 ml|
Example 3. The composition and methods of using commercial test systems "Anti-mumps ELISA"
1. Part of a set
1. Immunosorbent device with immobilized purified antigen mumps virus - 1 tablet (folding is);
2. Inactivated positive control sample (+), liquid, red - 1 vial with a volume of 2.0 ml;
3. Inactivated negative control sample (K-), liquid, yellow - 1 vial volume of 3.0 ml;
4. The conjugate solution (RK), liquid, green - 1 vial of 12 ml;
5. The Chromogen solution (PX) - tetramethylbenzidine (TMB)liquid, colourless, clear solution - 1 bottle with a volume of 13 ml;
6. 25x concentrate of phosphate-saline buffer with tween (25x FSB-T), liquid, colorless, transparent, slightly opalescent solution - 1 bottle with a volume of 26 ml;
7. The solution for dilution of sera (PC), liquid, colorless, transparent, slightly opalescent solution 1 vial of 12 ml;
8. Stop reagent, transparent colorless liquid 1 vial with the volume of 6 ml
2. Conducting immunoassay
The purified antigen (mumps virus strain Dragoon) diluted in carbonate buffer (pH=9,7) to a concentration of 8 μg/ml and contribute 100 μl of antigen solution into the wells of 96 well plate. The adsorption of the antigen is carried out at 20±1°C for 16-20 hours. After incubation of the tablet is removed neemalirovannym antigen and make a blocking solution (5% sucrose, 2.5% peptone from casein and 0.5% sodium azide in purified water), in each well of the tablet make the volume of the solution is not less than 200 μl. Lock the tablet wire is t at 27± 1°C for 2-3 hours. Then the contents of the wells are removed by vytryahivanie and Ethiopianism tablet. Drying is effected at 27±1°C for 3 hours. Tablet with adsorbed antigen mumps virus (immunosorbent) sealed in package under reduced pressure or with silica gel. To determine the titers of measles antibodies in the serum raschitajutsja in the holes vertically in the auxiliary 96-hole tablet. All wells of the subsidiary tablet (except the 1st number - wells a-1 through A-11) add 130 ál PC. To all wells of row a, since wells "a-1" (wells 1-11), contribute 225 ál R-RA PC. The last (12th row) tablet is used for controls. Then all wells of row a, since wells "a-1" (wells 1-11), contribute to 25 μl of test sera and retitrement them each on their own row with step dilutions of 1:2. For example, from wells a-1 selected 130 μl of the test serum in a dilution of 1:10 and placed in the hole "B-1", thoroughly pipeinput selected 130 ál and transferred into hole "C-1". The procedure is repeated and transferred 130 µl in the hole "D-1" and so on until the hole "N-1". Similarly in the wells of row 2 (from hole "a-2" to the hole "H-2") rasterbilete the following test serum and so on, until the holes 11 in the second row. The last (12th row) tablet is used for controls. Thus, the tablet can be rasterbate to 11 sera. All breeding saw the pigs (100 µl) of the auxiliary tablet is transferred to the corresponding wells with immobilized antigen mumps virus. In wells A-12 and b-12 contribute To the+volume of 100 μl in the wells, C-12 and D-12 by making OFS volume of 100 μl in the wells F-12 and F-12 contribute To a volume of 100 μl in the wells G-12 and H-12 contribute FSB-T volume of 100 μl for control conjugate. Then the tablet is covered with a lid or with adhesive tape and incubated for 30 min at a temperature of (37±1)°C. After five times washing FSB-T and remove moisture in every hole making RK volume of 100 μl. The tablet is closed and incubated for 30 min at a temperature of (37±1)°C. after incubation the unbound washed five times the FSB-T and remove any traces of moisture from the tablet. Next, each well of the tablet make PX volume of 100 μl. The tablet is covered with a lid and placed for 20 min in the dark place at a temperature of 20-25°C. the Reaction is stopped by the introduction of stop solution 50 ál to each well of the plate.
3. Profitability analysis
The ELISA results recorded on the spectrophotometer. The optical density (OD) was measured at a wavelength of 450 nm. The results take into account only if the mean OD value in the wells with the control conjugate (Kg) not more than 0,100 in the holes with K - mean OD value(OP - Wed) not more than 0,200, and in the holes with K+ the average value of OP(OP+ cf) not less than 4 times OP - Ms.
The smallest positive value of Opcrit. calculated by the formula:
Opcrit.=0,2+OP - cf
The results are on oitelnye, if OP test sera more Opcrate the determination of the titer of antibodies to mumps virus in serum titers should be considered as the last dilution of the test serum, the OD value of which exceeds the value of Opcrit.
Test-system "Anti-mumps ELISA" is released in the form of set, Packed in polystyrene or cardboard box. One set designed to hold 96 assays, including controls.
Examples of use of the test system Anti-mumps ELISA in clinical practice
Example 4. While sporadic cases in the incidence of mumps in the Novosibirsk region from patients with clinical diagnosis of mumps in 5-8 days and after 3 weeks they took blood and received the serum. Since 2001, this work was carried out under the orders of the health Department of the Novosibirsk region. Serum was analyzed by three different methods - enzyme linked immunosorbent assay (ELISA), response inhibition of haemagglutination (rcga) and the reaction of neutralization in cell culture Vero (PH). ELISA was performed using test systems CJSC bioservice (Moscow), Wampole (USA), Boehringer Mannheim (Germany), Human (Germany) and tested the test system Anti-Mumps ELISA", produced by CJSC "MBS" Novosibirsk, the main component of which is a purified antigen mumps virus strain Dragoons.
Just per the od 1997-2005 period these methods were analysed 18 paired sera from people with clinical diagnosis of mumps with the aim of serological confirmation of clinical diagnosis. The final diagnosis of mumps was staged after PCR with specific primers on the SH gene of mumps virus.
All 18 patients noted at least 4-fold increase of specific antibodies, specific methods rtha and PH. It should be noted that when determining the titer of antibodies in the blood sera of patients with clinical diagnosis of mumps method PH 4 sera was not registered 4-fold increase of specific antibodies.
All 18 patients in the collected samples using ELISA test systems Wampole (USA), Boehringer Mannheim (Germany), Human (Germany) recorded a growth of antibodies to mumps virus.
When determining the titer of antibodies in the blood sera of patients with clinical diagnosis of mumps" using the test system manufactured by bioservice (Moscow) in 3 sera was not registered 4-fold increase of specific antibodies.
All 18 patients using the test system Anti-mumps ELISA was registered at least 4-fold rise in antibodies to mumps virus.
All 18 patients by PCR was detected RNA virus of mumps.Thus, the test system "Anti-mumps ELISA can be used to detect specific antibodies in paired sera obtained from patients, and thus to identify, remove or confirm the diagnosis in the presence of a disease with clinical manifestations characteristic of mumps.
Example 4.2. Between 1996 and 2004 in Novosibirsk region was carried out to study the titer of specific antibodies to mumps virus in the blood serum of children age 6 years prior to the second vaccination against mumps. For this purpose in children before vaccination took 5 ml of blood and received the serum. Serum antibody to mumps virus were analyzed by three different methods - enzyme linked immunosorbent assay (ELISA), response inhibition of haemagglutination (rcga) and the reaction of neutralization in cell culture Vero (PH). ELISA was performed using test systems CJSC bioservice (Moscow), Wampole (USA), Boehringer Mannheim (Germany), Human (Germany) and tested the test system Anti-Mumps ELISA", produced by CJSC "MBS" Novosibirsk, the main component of which is a purified antigen mumps virus strain Dragoons. Total for the period was studied 254 serum. Of the 254 12 sera sera had titers of antibodies against mumps virus <1:4 in rtga and <1:4 in PH and can be considered as a negative is, the remaining 242 sera were positive in the study in rtga and PH. These same 12 negative sera gave negative results in ELISA test systems for all of these manufacturers.
The comparison results show that using the method enzyme immunoassay using the test system manufactured by bioservice (Moscow) specific antibodies were determined in 240 sera from the total number of sera (242 serum), which were found protivoprostudnye antibodies using methods rtha and PH.
Using ELISA test system production Wampole (USA) - specific antibodies were determined in 240 sera from the total number of sera (242 serum), which were found protivoprostudnye antibodies using methods rtha and PH.
Using ELISA test system production Boehringer Mannheim (Germany) specific antibodies were determined in 241 serum of the total number of sera (242 serum), which were found protivoprostudnye antibodies using methods rtha and PH.
Using ELISA test system for the production of Human (Germany) specific antibodies were determined in 240 sera from the total number of sera (242 serum), which were found protivoprostudnye antibodies using methods rtha and PH.
Using the proposed ELISA test system "Anti-Mumps ELISA" production company "MBS" New is Sibirsk specific antibodies were detected in all 242, in which were found protivoprostudnye antibodies using methods rtha and PH.
Thus, the greatest percentage of coincidence detection of positive and negative sera was obtained using the ELISA test systems Boehringer Mannheim (Germany) and Anti-Mumps ELISA" production company "MBS" Novosibirsk (99.5% and 100%, respectively).
Simple averages of antibody titers when using test kits based on ELISA method (including the investigated test systems "Anti-Mumps ELISA" production company "MBS" Novosibirsk) were also higher than in rtga and PH (1:146, 1:37 and 1:52, respectively).
Thus, the title of the proposed strain of mumps virus Dragoon higher by 2-3 orders of magnitude in comparison with the known analogues and, thus, on the steps of obtaining antigen higher the productivity of the antigen.
In addition, the claimed ELISA test system "Anti-mumps ELISA has a higher sensitivity compared to many known analogues and is recommended for use to determine the strength of individual immunity to the disease "epidemic parotitis".
1. The strain of the mumps virus Mumps virus for obtaining antigen - component enzyme immunoassay system for the diagnosis of antibodies to mumps virus deposited in the collection of cultures of microorganisms of the Federal state in which the institution of science "State research center of Virology and biotechnology "Vector" of Rospotrebnadzor under registration number №VB-05.
2. ELISA test system for the diagnosis of antibodies to mumps virus, including tablet with immobilized purified antigen mumps virus, vaccine positive control sample (+), inactivated negative control sample (K-), the conjugate solution (RK), Chromogen solution (PX), 25x concentrate of phosphate-saline buffer (25x FSB-T), the solution for dilution of sera (PC), a stop reagent, characterized in that the immobilized as inactivated virus antigen mumps test system contains highly purified antigen, prepared on the basis of the strain according to claim 1.
FIELD: medicine; ophthalmology.
SUBSTANCE: invention is intended for simultaneous verification of uveal melanoma and forecast of metastasis development. Immunohistochemical analysis of protein S-100 and melanin-A expression within tumour cell is accompanied with simultaneous estimation of S-100-positive cells number within visual field. Uveal melanoma is verified if reaction of S-100 and melanin-A is positive with forecasted high possibility of tumour dissemination in case number of S-100-positive cells number is less than 50.
EFFECT: simplified examination associated with high specificity and sensibility of simultaneously verified uveal melanoma and forecasted metastasis.
1 tbl, 1 dwg, 2 ex
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
SUBSTANCE: method involves determining total IgA quantity using immunoenzyme method based on polyclonal IgA antibodies as binding antibodies on the micropanel and the same antibodies in conjugate with peroxidase for detecting bound IgA in sample before and after treatment with specific IgA1-protease. Total IgA1 and IgA2 content is determined before being treated with enzyme. IgA2 content only is determined after being treated with enzyme. IgA1 is not determined after being disintegrated with IgA1-protease. Kit has microplate containing absorbed polyclonal IgA antibodies, conjugate of the same antibodies with peroxidase, reference sample having given IgA concentration, IgA1-protease and substrate buffer.
EFFECT: simplified method and kit for determining IgA1 and IgA2; improved results reproducibility; no specific monoclonal antibodies being used.
2 cl, 1 tbl
SUBSTANCE: method involves absorbing myeloma IgA1 on micro panel, incubating solutions containing IgA1-protease and determining enzyme activity from IgA1 sorbate quantity reduction determined by conjugate with polyclonal IgA antibodies peroxidase. Determining IgA1-protease activity with inhibitors available in various concentrations enables one to calculate its inhibition constants. Kit has microplate containing absorbed myeloma IgA1 preparation, peroxidase conjugate with antibodies against human IgA and substrate buffer.
EFFECT: simplified method and kit for determining IgA1-protease activity and studying its inhibition process.
2 cl, 1 dwg, 1tbl
FIELD: veterinary virology.
SUBSTANCE: the present innovation deals with interaction of antibodies with an antigen, with antibodies labeled with horseradish peroxidase, addition of substrate mixture and registration of reaction results. Moreover, one should apply plotting boards with presorbed antibodies and general immunoenzymatic conjugate. The innovation enables to shorten terms for diagnosis and obtain more significant results of diagnostics.
EFFECT: higher efficiency.
FIELD: biotechnology, analytical chemistry.
SUBSTANCE: claimed method includes providing of complexes between antigen molecules and specific antibodies on carrier surface, wherein said complexes are disclosed by addition of enzyme label thereto followed detection thereof based on formation of enzyme reaction product. Enzyme label addition is carried out by two protein interaction, namely bacterial ribonucleaze barnase and barstare, which is inhibitor thereof, wherein either of the two is added to immunoreagent, and the other one is added to enzyme label. Abovementioned complexes have high affinity, specificity, and binding constant of 1014 M-1.
EFFECT: new method for antigen detection.
6 dwg, 2 ex
FIELD: veterinary and medicine.
SUBSTANCE: invention, in particular, relates to production and use of biological preparations intended for differential diagnostics of brucellosis and to a method of differentially diagnosing brucellosis. Method involves serologic analysis of sera using antigenic "IFK", which is horse-radish peroxidase-labeled electrophoretically purified polypeptide fraction of virulent strain B.arborus 54. Diagnosis of brucellosis is stated when anti-brucellosis antibodies in sick animal sera diluted to 1/100 and higher are revealed at CSP reaction intensity 2.1 and higher.
EFFECT: elaborated method increasing immuno-enzymatic test specificity and allowing performing differentiation of postvaccinal immunological reactions and postinfectious reactions induced by microorganisms having antigenic affinity with brucellas, especially Yersinia enteriocolitica.
2 cl, 4 ex
FIELD: medicine, biotechnology.
SUBSTANCE: the present innovation deals with elaborating diagnostic reagents for testing prionic protein in mammalian cerebral tissue due to IEA technique and refers to antibodies specifically reacting with prionic protein PrP or its fragment. The innovation includes polyclonal antibodies obtained to synthetic peptides including amino acid sequences of bovine prionic protein 143-168, 101-134 and 211-241, their conjugates with horseradish peroxidase and diagnostic reagents obtained upon their basis that enable to detect prionic protein in mammalian cerebral tissues.
EFFECT: higher specificity of detection.
15 cl, 8 ex, 8 tbl
FIELD: medicine, obstetrics.
SUBSTANCE: in pregnant women at pregnancy terms being after 39 wk in average portion of morning urine one should detect due to a solid-phase immunoenzymatic assay the concentration of pregnancy-associated protein-A (PAPP-A). At the level of PAPP-A being 768.2 ng/ml and more it is possible to conclude upon physiological mature pregnancy. The innovation is noninvasive and enables to increase accuracy in predicting true over-mature pregnancy.
EFFECT: higher efficiency and accuracy of diagnostics.
2 ex, 1 tbl
SUBSTANCE: one should test blood serumal samples in immunoenzymatic assay by applying a test system ELI-N-1, the components of which are being antigens of nervous tissue or their immunochemical analogs specifically binding antibodies with tendency to antigens of nervous tissue, and, also, idiotypical antibodies to them, or their variable parts. Prediction should be performed according to the level of idiotypical and anti-idiotypical autoantibodies to antigens of nervous tissue and, also, according to their ratio. Thus, a new test system has been elaborated that enables to predict the flow of available nervous-psychic diseases.
EFFECT: higher accuracy of prediction.
2 cl, 7 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention concerns veterinarian virology and biotechnology. Invention claims new industrial strain of Asia-1 type murrain virus deposited in microbe strain collection of Federal State Institution Russian State Centre for Quality And Standardisation of animal medicines and feed under registration number Amur No1987-DEP.
EFFECT: extended range of industrial strains of Asia-1 type murrain virus with high infection, antigenic and immunogenic performance, suitable for antigenic and immunogenic performance control of vaccines and for medicine production for diagnostics and specific prevention of Asia-1 type murrain.
1 dwg, 7 tbl, 12 ex
FIELD: medicine; bioengineering.
SUBSTANCE: combined vaccine contains inactivated Teotropine culture virus-containing raw epizootic isolate of pig's enzootic encephalomyelitis (Teschen disease) virus of initial biological activity 107.5-109.0 lg "ТЦД"50/cm3 and inactivated Teotropine culture virus-containing raw vaccine strain of Aujeszky's disease virus of initial biological activity 107.0-108.0 lg "ТЦД"50/cm3, and oily adjuvant in the following ratio, wt %: inactivated Teotropine culture virus-containing raw epizootic isolate of pig's enzootic encephalomyelitis (Teschen disease) virus - 23-27, inactivated Teotropine culture virus-containing raw vaccine strain of Aujeszky's disease virus - 23-27, Teotropine - 0.03-0.05, oily adjuvant - the rest.
EFFECT: high immunogenicity, areactogenicity, harmlessness and storage stability.
2 tbl, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention refers to veterinarian virology. Strain of bird influenza A of subtype H5N1 deposited in microorganism collection of Russian Scientific Research Institute of Veterinarial Virology and Microbiology of Russian Academy of Agricultural Sciences under No. 2633 has been obtained. Invention may be used at research and production laboratories and bioindustry facilities for development of biopreparations, in particular bird influenza A vaccines of the said subtype, and monitoring of their potency, as well as for development of antigens for diagnostic sets.
EFFECT: production of bird influenza A strain.
FIELD: chemistry; biochemistry.
SUBSTANCE: invention refers to biotechnology and can be used in production of biological preparations for prevention and treatment of salmonellosis. Bacteriophogum SalmonellaL IBP-1 bacteriophage strain lytic for S. enteritidis can be used for production of medical bacteriophage preparation.
EFFECT: extended spectrum of lytic action to bacterium Salmonella enteritidis.
FIELD: medicine; virology.
SUBSTANCE: strain expresses the VP2 protein which contacts a monoclonal antibody moab B69 and moab 67, cosecreted by the monoclonal antibody cellular lines HB-9437 and HB-11122. Contains a mutation of coding area of the classical VP2 in the position 222, coding series or threonine, and the nucleotide sequence coding amino-acid sequence, presented in any of SEQ ID No. 1-5 in position 318-323.
EFFECT: effective utilisation for bacterination against infectious bursal disease.
24 cl, 1 dwg, 5 tbl, 2 ex
FIELD: medicine; virology.
SUBSTANCE: strain of a virus of flu of the new antigenic version, deposited in the State collection of viruses of scientific research institute of virology of D.I.Ivanovo under № 2413 is obtained.
EFFECT: expansion of a spectrum of strains for vaccinating against the virus of flu A and an effective utilisation in public prevention health services.
2 tbl, 2 ex
FIELD: medicine, microbiology.
SUBSTANCE: invention concerns area of biotechnology and medical virology. The diagnostic test system for revealing of a virus of bird flu A/H5N1 is offered at enzyme immunoassay carrying out on a solidphase carrier with use of peroxidase conjugate. As a solidphase carrier the activated aluminosilicate matrix with a magnetic material with immobilized immunoglobulins against a virus of bird flu is used. The invention can be used for diagnostics of a virus of bird flu A/H5N1.
EFFECT: obtaining of diagnostic test system for revealing if virus of bird flu A/H5N1 at enzyme immunoassay carrying out on solidphase carrier with use of peroxidase conjugate.
SUBSTANCE: Vibrio metschnikovii bacteria strain KM-185 is recovered from water during vibrioflora analysis. The strain is sensitive to a specific PK-85 phage recovered from Vibrio metschnikovii bacteria strain 15818. Phage titre is 108- 109 particles per ml.
EFFECT: maintaining phage reproduction as a mentor culture.
2 tbl, 1 ex
SUBSTANCE: invention refers to biotechnology. Method includes cultivation of transplantable cells MDCK in nutrient medium, production of virus-containing substances by cultivation vaccinating influenza virus strains in MDCK cells culture on the same nutrient medium, virus substance purification from ballast impurities, stabilising additives introduction to purified substance and drying of the finished vaccine form. Thus inoculation dose of transplantable cells MDCK is 100-600 thousand cells per 1 ml of environment. Vaccinating cold-adapted reasortants of influenza virus strains are used as virus strains with inoculation dose of infection multiplicity 0.01-0.0001 "ЭИД50/КЛ-". Serum-free nutrient medium added with soya hydrolysate concentrated 0.01-10% and proteolytic enzyme in amount 0.25-50.0 mkg/ml prior to introduction of influenza virus strains inoculation dose is used as nutrient medium for cultivation of MDCK cells and influenza virus strains on specified cells. Cultivation of MDCK cells cultures and cold-adapted influenza virus strains in specified cell culture is performed in suspensions on microcarriers brought in serum-free nutrient medium in concentration 1-6 g/l. Collection of virus-containing liquid is carried out after specific activity of influenza virus is not less than 8.0 lg "ЭИД"50/ml. Saccharose and soya peptone in final concentration (5-10) and (3-8) mass % respectively are used as stabilising additives introduced into purified substance before drying.
EFFECT: method enables to produce vaccine with lower content of foreign animal protein components.
7 cl, 3 tbl, 7 ex
SUBSTANCE: method of obtaining a continuous cell line capable of maintaining growth of cell-associated alpha herpes viruses involves the infection or transfection of a cell with the nucleic acid or fragment of the herpes virus, the selection of cells expressing the said nucleic acid or its fragment, and the cultivation of the said infected or transfected cell or its progeny in conditions suitable for the expression of the said nucleic acid and the multiplication of the said cell or its progeny. The said nucleic acid includes the herpes virus glycoprotein gene gE or its functional fragment. The expression of the said gE gene or its functional fragment takes place in a sustainable manner. The cell of the continuous cell line capable of maintaining growth of cell-associated alpha herpes viruses includes the herpes virus nucleic acid or its fragment. The said nucleic acid includes the herpes virus glycoprotein gene gE or its functional fragment, the expression of the said gE gene or its functional fragment takes place in a sustainable manner. The said cell can go through at least 10 passages. The invention group includes the application of the said cell for obtaining a vaccine capable of inducing protection against disease in vertebrates, and for obtaining and/or maintaining and/or isolation of a hypervirulent and/or very virulent Marek's disease virus strain, as well as for obtaining the Marek's disease diagnostic antigen. The invention group also includes a method of obtaining and/or isolation and/or maintaining the Marek's disease virus strain. The method involves the infection of the above cell with the said strain and the cultivation of the said cell in conditions suitable for the multiplication of the said cell and obtaining, maintaining, and isolation of the Marek's disease virus.
EFFECT: invention group provides a method of obtaining a vaccine capable of inducing protection against the disease; allows to satisfy the need in new cell systems allowing to maintain new properties acquired by the cell.
35 cl, 6 dwg, 6 ex.
FIELD: biotechnology, veterinary medicine.
SUBSTANCE: invention relates to the development of biological preparation for prophylaxis and treatment of colibacillosis (escherichiosis) and for control of carriage of escherichious infections pathogens in animals and poultries also. Also, invention can be used in producing curative fodders and ecologically pure human foodstuffs. Biopreparation for prophylaxis and treatment of escherichiosis in animals and poultries comprises strains of bacteriophages Phagum Escherichia coli Ec022-DEP and/or Phagum Escherichia coli Ec021-DEP, and/or Phagum Escherichia coli Ex0782-DEP, and/or Phagum Escherichia coli Ec0781-DEP, and/or Phagum Escherichia coli EPZ-1-DEP, and/or Phagum Escherichia coli EPZ-2-DEP, and/or Phagum Escherichia coli EG-5-DEP, and/or Phagum Escherichia coli BC-1-DEP, and/or Phagum Escherichia coli M78-DEP, and/or Phagum Escherichia coli Sheksna 2k-DEP taken in the effective amount. The biopreparation comprises also antiseptic, for example, quinosol and a stabilizing agent. Protein (for example, soybean protein), vegetable meal, organic polymer, milk, serum, albumin can be used as a stabilizing agent. Among organic polymers can be used: dextran, polyglucin, starch, polyvinylpyrrolidone. The biopreparation can be dried by lyophilization, granulated and placed in polymeric matrix. The biopreparation has no toxic properties on animals, it shows good hygroscopicity and can be good dispersed in water. The biopreparation can be used in liquid and dry prescription formulations and in different methods of its administrations: both by subcutaneous, intraperitoneal, intramuscular injections and as an aerosol, by administration of phage particles into lung compartments including applying as curative fodder and supplement to fodder, and by applying on surface of cutaneous integuments. Invention provides enhancing the effectiveness of treatment of animals and poultries with gastroenteric infections due to reducing treatment period, expanding spectrum of lytic effect of the biopreparation, resistance to effect of digestive tract enzymes and convenience in using.
EFFECT: valuable veterinary properties of biopreparation.
9 cl, 5 tbl, 7 ex