RU2348691C1 - Dragoon strain of parotitis virus for obtaining antigene component of test system, and immunoenzyme test system for parotitis virus antibody diagnostics - Google Patents

Abstract

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

Description

The invention relates to medical virology and microbiology, and in particular to the identification of a new strain of Mumps virus Dragoon to obtain antigen - a component of the diagnostic test system and a test system for the diagnosis of antibodies to mumps virus.

A known strain of mumps virus L-3, the productivity of which is no more than 8 × 10 ⁵ PFU / ml (O. G. Anzhaparidzel, Yu.S. Boriskini, NN Bogomoloval and VDLotte Accumulation of altered viral nucleocapsids in mumps virus - Persistently infected cell cultures / (JOURNAL ARCHIVES OF VIROLOGY PUBLISHER SPRINGER WIEN, 1983, vol. 75, No. 4, 283-289).

The closest analogue (prototype) is the strain of mumps virus Anders, the productivity of which is not more than 10 ⁶ PFU / ml (Fleisner B., Kreth HW 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 are their low productivity in obtaining the mumps virus antigen.

A well-known domestic enzyme immunoassay test system for detecting antibodies to the mumps virus "Mumps-screen" (the developer and manufacturer of the test system "Mumps-screen" - ZAO Biotechnological company "BIOSERVICE" (Instructions for the use of the immunoassay test system for detecting antibodies to mumps virus "Mumps-screen"; Moscow, 2001). The specified test system contains:

- immunosorbent - polystyrene tablets for immunological reactions in the wells of which the antigen of mumps virus (AgVP) is adsorbed. AgVP - in the rows of tablet A, C, E, G and control antigen (kAg) - in rows B, D, F, N. AgVP is mumps infected with mumps virus Vero cells containing at least 80% virus-containing cells; kAG are uninfected Vero cells;

- positive control sample (K +) - human serum containing antibodies to the mumps virus;

- negative control sample (K-) - human blood serum or human serum albumin that does not contain antibodies to the mumps virus;

- conjugate - diagnostic antibodies against human immunoglobulins labeled with horseradish peroxidase;

- substrate buffer mixture with hydroperite (BSH) - for the preparation of a chromogen solution;

- chromogen - orthophenylenediamine (OFD);

- stabilizer - bovine serum albumin (BSA) or casein;

- a concentrate of phosphate-saline buffer for washing tablets and preparing dilution of serums and conjugate.

However, this test system (analogue) has low sensitivity due to the fact that it uses AgVP, which is a crude antigen - mumps virus infected with mumps virus Vero (infected with at least 80% of the cells).

The closest test system (prototype) is an enzyme-linked immunosorbent assay for detecting antibodies to the mumps virus company Wampole Laboratories (Wampole Laboratories mumps IgG ELISA // P / N 6000-29W REV B. - 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. MicrobioL, 2005, No.9, vol. 43, p. 4847-4851). The specified enzyme immunoassay system contains a tablet with immobilized inactivated mumps antigen, antibodies against human immunoglobulins labeled with horseradish peroxidase (conjugate) in liquid form, positive control (K +), negative control (K-), calibrators, sample dilution solution; chromogen solution - tetramethylbenzidine (TMB), liquid stop reagent, wash buffer concentrate.

However, in the indicated test system (prototype), the crude NP-protein of mumps virus was used as an antigen, as a result of which the test system has insufficient sensitivity (it does not detect antibodies to other proteins in blood serum, for example, hemagglutinin-neuraminidase - HN, fusion protein - F matrix protein - M).

The technical result of the invention is the creation of a producer strain of mumps virus with higher productivity and an enzyme-linked immunosorbent assay system for diagnosing antibodies to mumps virus with higher sensitivity.

The specified technical result is achieved by creating a new strain of mumps virus Dragun to obtain an antigen component of an enzyme-linked immunosorbent assay for the diagnosis of antibodies to mumps virus deposited in the collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor under registration number No. VB-05.

The indicated technical result is also achieved by the fact that in an enzyme-linked immunosorbent assay for the diagnosis of antibodies to mumps virus, including a tablet with immobilized purified mumps antigen, inactivated positive control sample (K +), inactivated negative control sample (K-), conjugate solution ( PK), chromogen solution (PX), 25x phosphate-buffered saline concentrate (25x FSB-T), serum dilution solution (PC), stop reagent according to the invention as an immobilized inactive of the mumps virus antigen, the test system contains a highly purified antigen prepared on the basis of the Mumps virus strain Dragoon according to claim 1, registration No.VB-05.

The drawing shows an analysis of the complete nucleotide sequence of the inventive strain of mumps virus and is presented graphically in the form of a phylogenetic tree. The black rectangle marks the strains isolated in Russia.

Note: the strains with which the comparison was carried out (CRU94Dra = C-genotype, RU-country in which the isolate was isolated, 94-year of isolation, Dra-name of the strain-Dragoon).

Characterization of the claimed strain.

The strain of mumps virus Dragoon belongs to the family of viruses Paramyxoviridae genus Rubulavirus. The inventive strain obtained in the State scientific center of virology and biotechnology "Vector" Ministry of Health of the Russian Federation, Koltsovo, Novosibirsk region.

The strain is deposited in the culture collection of microorganisms of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "of Rospotrebnadzor under registration number No. VB-05, registration date May 16, 2002 (certificate of deposit of strain No. 0502 is attached to the application) and is used for antigen production during the release of an enzyme-linked immunosorbent assay for the detection of antibodies to the mumps virus "Anti-mumps ELISA".

Source of receipt.

The strain was isolated from patient D. during an outbreak in 1994 in the village of Koltsovo, Novosibirsk Region. Patient D. had close contact with an infectious patient who had a serologically confirmed diagnosis of mumps. The course of the infection in patient D. was typical: the disease began with an increase in temperature to 37.4 ° C, the appearance of swelling of the salivary glands, first on the one hand, and then on the other. After 2 days, the inflammation of the salivary glands disappeared. On the basis of clinical manifestations and an increase in titers of antipartite antibodies in paired sera determined using rtga and ELISA, patient D. was diagnosed with mumps. On the 3rd day after an increase in temperature, patient D. was taken blood samples and a nasopharyngeal flush. A cotton swab, which was used for nasopharyngeal rinsing from the nasopharyngeal mucosa of patient D., was placed in 1 ml of DMEM medium containing antibiotics and after sterilization through 0.45 μm filters was placed in a culture vessel (Kubarik, 75 cm ³ ) with a monolayer of Vero cells. Vero cells were obtained from the Institute of Cell Culture of the Federal State Institution of Science and Science of the World Bank "Vector". As a nutrient medium, we used DMEM medium (manufactured by the Federal State Institution of Science and Science, VB Vektor) with the addition of 2% fetal bovine serum (FSCRS), 2 mmol / L glutamine and antibiotics: 100 μg / ml penicillin and 100 IU / ml streptomycin. Visual inspection under a microscope on the 3rd day of incubation at plus 37 ° C revealed a change in cell morphology. On the 4th day - the appearance of symplasts was recorded. At 5-6 days, destruction of symplasts (CPP) was observed. On the 6th day, the culture vessel was placed at minus 20 ° C, then the thawed culture virus-containing liquid (LEL) was thawed. The resulting mixture of LEL and Vero cell lysate was packaged and one part of the drain was lyophilized and the other was stored at minus 70 ° С.

The authenticity of the strain.

The total RNA was isolated from a sample of nasopharyngeal washout of patient D using the RNaidTM PLUS kit (F. "BIO 101", USA). Using the AmpliTaq kit (F. "Elmer Perkin", USA), a polymerase chain reaction (PCR) was carried out. For the reaction, primers were used for the mumps virus SH gene (SH1F - AGT AGT GTC GAT GAT STS AT, SH2R - GCT CAA GCC TTG ATC ATT GA). PCR was positive, which indicates the presence in the test sample of flushing from the nasopharynx of patient D.

The sequence of the complete genome of the isolated isolate was carried out. The analysis of the complete nucleotide sequence of the inventive mumps virus strain was carried out with all available complete nucleotide sequences of the mumps virus strains placed in GebBank (http://www.ncbi.nlia.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar) and shown in figure 1 as a phylogenetic tree. From the drawing it can be seen that the inventive strain differs genetically from the known strains of mumps virus.

The genotype of the strain.

Genotype C [The number of the deposited sequence in GebBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar) - AY 669145].

Cultural properties. The strain Dragoon when cultured on a monolayer of Vero cells causes the formation of symplasts at 4-6 days (cultivation temperature 37 ° C). The maximum titers of the virus reached 5-6 days after infection with a monolayer of Vero cells and amounted to 8.6 × 10 ⁷ PFU / ml. Productivity: 24 mg of antigen for ELISA is obtained from 3 liters of fluids. The use of the antigen obtained from the Dragoon strain — 0.2–0.4 μg / well, makes it possible to detect antiparitic antibodies in the sera of people vaccinated and patients with mumps by ELISA.

Antigenic properties.

Detects antibodies to the mumps virus in the sera of patients and vaccinated people. The isolated strain has a serological cross-section with the Enders reference strain and the L-3 vaccine strain.

Pathogenicity for humans. According to the clinical signs of the disease, the patient from whom the Dragoon strain was isolated (temperature - 37.8 ° C, inflammation of the salivary glands disappeared 2 days after the onset, after the complications were not registered after the disease), we can assume that the Dragoon strain is not highly pathogenic. Strain Dragoon is not phyto- and zoopathogenic.

For long-term storage, the strain is lyophilized using a gelatin and sucrose solution as a protective medium.

The following nutrient media are used to propagate the strain: MEM Needle, MEM × 2AVK Needle, DMEM containing 2-5% fetal bovine serum, 2 mmol / L glutamine and antibiotics: 100 μg / ml penicillin and 100 IU / ml streptomycin.

A study of the serological properties of the obtained strain (Dragoon) revealed a significant similarity with the reference strain - Enders (prototype) of the mumps virus and with the vaccine strain L-3. The antisera obtained from immunized BALB / c mice to the Enders reference strain, to the L-3 vaccine strain and to the inventive Dragoon strain isolated from the patient were tested in hemagglutination inhibition reaction (RTGA) using a standard method using Macaco monkey mulatta erythrocytes , and in the reaction of enzyme immunoassay (ELISA). The results of the study of the cross between the obtained sera are presented in table 1. As can be seen from the results of table 1, the serum from all mice immunized with the mumps virus strain Anders strain reacted both with mumps viruses strain Anders and L-3, and with the strain Dragoon. On the other hand, the sera from all mice immunized with the claimed Dragoon strain isolated during the outbreak of mumps in 1994 reacted with both the virus itself and the reference strain of Anders and the vaccine strain L-3. Based on the results obtained, it can be concluded that the claimed strain by serological properties is close to the reference strain of Anders and the vaccine strain L-3.

Table 1.
The study of the antigenic properties of mumps virus strain Dragoon Serum specificity N serum Antigen / reaction / titer of antibodies strain anders RTGA IFA strain L-3 Dragoon strain strain anders strain L-3 Strain Dragoon Serum one 1: 256 1: 128 1: 128 1: 640 1: 320 1: 640 against the virus 2 1: 128 1:64 1: 128 1: 640 1: 160 1: 320 mumps strain 3 1: 128 1:64 1: 128 1: 640 1: 160 1: 320 Anders four 1: 128 1:64 1: 128 1: 640 1: 160 1: 320 5 1:64 1:64 1:64 1: 320 1:80 1: 320 6 1:64 1:64 1:32 1: 160 1:80 1: 160 Serum one 1:16 1:32 1:32 1: 160 1: 160 1: 320 against the virus 2 1:16 1:32 1:16 1:80 1: 160 1: 320 mumps strain 3 1:16 1:32 1:16 1:80 1: 160 1: 320 L-3 four 1:16 1:32 1:16 1:80 1: 160 1: 320 5 1: 8 1:32 1:16 1:80 1: 160 1: 320 6 1: 8 1:32 1: 8 1:40 1:80 1: 160 Serum one 1:64 1:64 1: 128 1: 320 1: 160 1: 1280 against highlighted 2 1:64 1:32 1: 128 1: 320 1: 160 1: 640 strain 3 1:64 1:16 1: 128 1: 320 1: 160 1: 640 Dragoon four 1:64 1:16 1: 128 1: 160 1:80 1: 640 5 1:32 1:16 1: 128 1: 160 1:80 1: 320 6 1:32 1:16 1:64 1: 160 1:40 1: 320

Example 1. The method of obtaining the antigen of mumps virus

The purified and inactivated mumps antigen is used to set up an enzyme-linked immunosorbent assay.

Obtaining a virus-containing fluid.

The original Vero cells are stored in liquid nitrogen and grown by serial passages. As a growth medium using a solution of MEM Needle with 8-10% fetal bovine serum.

The cell culture is grown for 3-4 days at an inoculum cell concentration of 5-10 × 10 ⁵ cells / ml of medium and subcultured in a conventional manner.

Mumps virus (Dragoon strain) infects 1-liter mattresses with a monolayer of Vero cell culture (multiplicity of infection 1:10). After incubation at 20-25 ° C for 40 minutes, 200 ml of nutrient DMEM are poured into a 1-liter culture vessel with the addition of (4.0 ± 0.1) ml of cattle serum, 0.001% trypsin, 100 units / ml of benzylpenicillin and 100 ng / ml streptomycin and after incubation for 5-7 days at (36 ± 1) ° C, the nutrient medium is drained. The titer of the virus in LEL is at least 10 ⁸ PFU / ml.

Obtaining a lysate of Vero cells.

During cultivation, the transition of the virus from cell to cell is carried out due to the formation of symplasts without leaving the main amount of the virus in the culture virus-containing fluid (LEL), therefore, the virus accumulates both in LEL and inside the cells. 5-7 ml of 0.01 M Tris HCl, pH = 9.0, are added to a 1-liter culture vessel. Cells are destroyed by double freezing-thawing to exit the virus from the cells. The cell lysate is freed from cell detritus by low-speed centrifugation in a JA-10 rotor of a J2-21 centrifuge (F. Beckman, USA) at 1500 rpm for 10 min at a temperature of + 4 ° C. The virus titer in the cell lysate is from 6 × 10 ⁴ 3 × 10 ⁶ PFU / ml.

Conducting ultrafiltration.

Concentration and purification of mumps virus from COL is carried out on Vladipor membranes of the UAM-100 type (Russia) or RTNK 0005 (F. Millipore, USA). The linear flow rate of the QOL to the cell should be equal to 0.6 l / min. The outlet pressure should not exceed 2.5 kg / cm ² , the inlet pressure should not exceed the outlet pressure by more than 0.4 kg / cm ² . The final product is an up to 20-25 times LEL concentrated free of low molecular weight compounds and cellular detritus. The titer of mumps virus Dragoon in a concentrate of at least 10 ⁹ PFU / ml, which is 2-3 orders of magnitude higher than the known analogues (strains of Enders and L-3).

Ultracentrifugation of virus-containing material.

The deposition of the virus. The antigen for ELISA is obtained from two sources: from a fluids concentrate and from a cell lysate after thawing.

At the first stage, the virus is precipitated through a “pillow” of 10% sucrose in the rotor SW 28 of the L-8-70 centrifuge (F. Beckman, USA). For this, 5 ml of 10% sucrose are carefully poured into the test tubes of the rotor and 30 ml of LH concentrate or cell lysate are layered on top. Virus precipitation is carried out at 25,000 rpm, 4 ° C, 10 hours. The supernatant was carefully pipetted and the precipitate was 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).

Mumps antigen purification. The antigen for ELISA is purified from foreign proteins by ultracentrifugation in a sucrose density gradient. Precipitation from all test tubes (precipitates from LWC concentrate and cell lysate) are combined, they are processed on an ultrasonic disintegrator at 6000 Hz 3 times for 10 seconds and layered on a gradient of a solution of 20-60% sucrose. To prepare a linear gradient of a sucrose solution, a mixer is used, consisting of two chambers that can be isolated by shutting off a tap between them. One of the chambers (first) is connected with a flexible hose to a peristaltic pump. The hose leaving the pump is lowered into a 12 ml tube so that it touches the tube wall in its upper part. The mixer is mounted on a magnetic stirrer. An anchor is placed in the first chamber. The mixer is filled with the valve closed as follows: 4.5 ml of a 60% sucrose solution are poured into the first chamber, and 4.5 ml of a 20% sucrose solution are poured into the second chamber. Then turn on the magnetic stirrer, peristaltic pump and carefully open the crane between the chambers. The solution from the second chamber enters the first, mixes and enters the tube through the pump. The total volume of the prepared linear gradient (20-60%) is 9 ml in each tube.

The precipitated mumps antigen is carefully layered using a syringe with a thick needle onto a linear sucrose gradient of 20-60% in a cup of a SW-41 rotor of a Beckman L-8-70 centrifuge. The centrifugation mode is 37,000 rpm at 4-6 ° C for 8 hours. A band at the level of 42-45% sucrose is carefully selected with a syringe with a thick needle, and after determining the purity of the antigen by electrophoresis and determining the concentration of antigen by protein is used for ELISA.

The yield of ELISA-suitable antigen with 3 L of HPLC and cell lysate is 24 mg per protein.

The stage of virus inactivation: mump virus antigen purified in a gradient of sucrose density is inactivated by heating at 56 ° C for 30 minutes (Zhdanov V.M., Gaidamovich S.Ya. Virology. M .: Medicine, 1966, p. 382).

Example 2. The composition of the test system

The composition of the components of the enzyme immunoassay system for detecting antibodies to the mumps virus (Anti-mumps ELISA), which is a set of reagents:

- immunosorbent — 96-well plate for immunological reactions in accordance with TU 64-2-375-86 or a collapsible plate of the “Nunc” type, Denmark or similar with immobilized purified mumps antigen;

- inactivated positive control sample (K +) * - human blood serum containing antibodies to the mumps virus, inactivated by heating, and a neutral red dye according to TU 6-09-3070-84;

- inactivated negative control sample (K -) * - the blood serum of healthy donors not containing antibodies to the mumps virus;

- conjugate solution (PK) - monoclonal mouse antibodies against human immunoglobulins labeled with horseradish peroxidase according to TU 6-09-10-1408-79;

- a solution of chromogen (PX) - tetramethylbenzidine (TMB);

- 25x phosphate-buffered saline concentrate (25x FSB-T) **;

- a solution for the dilution of serum (PC) - a single solution of FSB-T containing a concentrate of 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 kit is designed for 96 analyzes, including controls.

Note:

* Preservative - Sigma sodium merthiolate (USA) is found in K +, K- and RK, at a concentration of 0.01%; K + and K- contain the antibiotic gentamicin sulfate according to VFS 42-2626-89 at a concentration of 1 mg / ml.

** Composition 1 l 25x FSB-T

Sodium Phosphate Disubstituted 12-Aqueous GOST 4172-76 90.0 g Monosubstituted sodium phosphate 2-water according to GOST 245-76 4.75 g Sodium chloride GOST 245-76 22.0 g Twin 20 Sigma, USA 12.5 ml

The salts are successively dissolved with stirring in 0.9 l of distilled water at a temperature of 40-50 ° C, pH 7.5 ± 0.3, the correction is carried out as described above. The volume of the solution was adjusted to 1 L, tween-20 was added and mixed.

*** Composition 100 ml CBD

Centrifuged from detritus dissolved in culture medium in concentration 5.0 mg / ml cell culture proteins, used to infect mumps virus at its operating time 10.0 ml Sigma Sodium Merthiolate (USA) 10 mg Tween phosphate-saline 90 ml

Example 3. The composition and methodology of the commercial test system "Anti-mumps IFA"

1. The composition of the set

1. Immunosorbent - tablet with immobilized purified mumps antigen - 1 tablet (collapsible);

2. Inactivated positive control sample (K +), liquid, red - 1 vial with a volume of 2.0 ml;

3. Inactivated negative control sample (K-), liquid, yellow - 1 vial with a volume of 3.0 ml;

4. The conjugate solution (RK), liquid, green - 1 bottle of 12 ml;

5. A solution of chromogen (PX) - tetramethylbenzidine (TMB), a liquid, colorless, clear solution - 1 vial with a volume of 13 ml;

6. 25x concentrate of phosphate-buffered saline with tween (25x FSB-T), liquid, colorless, transparent, slightly opalescent solution - 1 bottle with a volume of 26 ml;

7. Solution for dilution of serums (PC), liquid, colorless, transparent, slightly opalescent solution - 1 bottle with a volume of 12 ml;

8. Stop reagent, clear, colorless liquid - 1 bottle of 6 ml.

2. Enzyme immunoassay

The purified antigen (mumps virus Dragoon strain) is diluted in carbonate buffer (pH = 9.7) to a concentration of 8 μg / ml and 100 μl of antigen solution are added to the wells of a 96 well plate. Antigen adsorption is carried out at 20 ± 1 ° C for 16-20 hours. After incubation, the non-immobilized antigen is removed from the plate and a blocking solution (5% sucrose, 2.5% peptone from casein and 0.5% sodium azide in purified water) is added, and a solution volume of at least 200 μl is added to each well of the tablet. The tablet is locked at 27 ± 1 ° C for 2-3 hours. Then, the contents of the wells are removed by shaking and slapping the tablet. Drying is carried out at 27 ± 1 ° C for 3 hours. A tablet with sorbed mumps antigen (immunosorbent) is sealed in a bag under reduced pressure or with silica gel. To determine the titer of measles anti-measles antibodies, serum is triturated in the wells vertically in an auxiliary 96-well plate. 130 μl of PC was added to all wells of the auxiliary plate (except the 1st row — wells A-1 through A-11). In all wells of row A, starting from well “A-1” (wells 1-11), 225 μl of PC-solution was added. The last (12th row) tablet is used for controls. Then, in all wells of row A, starting from well “A-1” (wells 1-11), 25 μl of the tested sera are added and each of them is triturated in its own row with a dilution step of 1: 2. For example, 130 μl of test serum in a 1:10 dilution is taken from well A-1 and transferred to well B-1, thoroughly pipetted, 130 µl is taken and transferred to well C-1. The procedure is repeated and transferred 130 μl into the hole "D-1" and so on to the hole "H-1". Similarly, in the wells of row 2 (from well “A-2” to well “H-2”) the next test serum is grown and so on, to the wells of the 11th row. The last (12th row) tablet is used for controls. Thus, up to 11 sera can be grown in a tablet. All dilutions of serum (100 μl) from the auxiliary tablet are transferred to the corresponding wells of the tablet with the immobilized mumps antigen. 100 μl of K + is added to wells A-12 and B-12, 100 μl of SOP is added to wells C-12 and D-12, 100 μl of K is added to wells E-12 and F-12, PBS-T with a volume of 100 μl was added to wells G-12 and H-12 to control the conjugate. Then the tablet is closed with a lid or duct tape and incubated for 30 min at a temperature of (37 ± 1) ° С. After washing five times with FSB-T and removing moisture, 100 microliters of RK are introduced into each well. The tablet is closed and incubated for 30 minutes at a temperature of (37 ± 1) ° C. At the end of the incubation, the plate is washed five times with FSB-T and residual moisture is removed from the plate. Next, in each well of the tablet make PX with a volume of 100 μl. The tablet is closed with a lid and placed for 20 minutes in a dark place at a temperature of 20-25 ° C. The reaction is stopped by introducing a stop reagent of 50 μl into each well of the tablet.

3. Profitability analysis

ELISA results are recorded on a spectrophotometer. Optical density (OD) is measured at a wavelength of 450 nm. The results are taken into account only if the average OD value in the wells with conjugate control (K Kg) does not exceed 0,100, in the wells with K the average OD value (OD K-cf) is not more than 0,200, and in the wells with K + the average value OP (OP K + sr) is at least 4 times higher than OP K-sr.

The smallest positive value of OPcrit. calculated by the formula:

OPcrit. = 0.2 + OD K- cf

The test results are considered positive if the OD of the analyzed serum is greater than OPcrit. When determining the titer of antibodies to mumps virus in serum, the titer should be considered the last dilution of the studied serum, the value of which exceeds the value of Opcrit.

The Anti-Mump IFA test system is produced in the form of a kit packed in a polystyrene or cardboard box. One set is designed for 96 analyzes, including controls.

Examples of the use of the test system "Anti-mumps ELISA" in clinical practice

Example 4. During sporadic cases of the incidence of mumps in the Novosibirsk region from patients with a clinical diagnosis of mumps on days 5-8, and after 3 weeks, blood was taken and serum was obtained. Since 2001, this work has been carried out as part of the orders of the Novosibirsk Region Health Department. Serum was analyzed by three different methods - enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition test (RTGA) and neutralization reaction on Vero cell culture (PH). ELISA was performed using test systems of Bioservice CJSC (Moscow), Wampole (USA), Boehringer Mannheim (Germany), Human (Germany) and the tested Anti-Mump IFA test system manufactured by CJSC MBS Novosibirsk, the main component of which is the purified antigen of mumps virus strain Dragoon.

In total for the period 1997-2005. by these methods, 18 paired sera from people with a clinical diagnosis of mumps were analyzed to serologically confirm the clinical diagnosis. The final diagnosis of “mumps” was made after PCR with specific primers for the mumps virus virus SH gene.

All 18 patients showed at least a 4-fold increase in specific antibodies, determined by methods of rtga and PH. It should be noted that when determining the titer of antibodies in the blood serum of patients with a clinical diagnosis of mumps by the method of PH in 4 sera, 4-fold increase in specific antibodies was not recorded.

In all 18 patients in the collected samples using ELISA test systems Wampole (USA), Boehringer Mannheim (Germany), Human (Germany), an increase in antibodies to mumps virus was recorded.

When determining the titer of antibodies in the blood serum of patients with a clinical diagnosis of "mumps" using a test system manufactured by ZAO Bioservice (Moscow), 4-fold increase in specific antibodies was not recorded in 3 sera.

In all 18 patients, using the Anti-Mump ELISA test system, at least a 4-fold increase in antibodies to mumps virus was recorded.

In all 18 patients, mumps virus RNA was detected by PCR.

Thus, the “Anti-Mump ELISA” test system can be used to determine specific antibodies in paired sera obtained from patients, and thus to determine, remove or confirm the diagnosis during the course of the disease with clinical manifestations characteristic of mumps.

Example 4.2 In the period from 1996 to 2004, work was carried out in the Novosibirsk Region to study the titer of specific antibodies to mumps virus in the blood serum of children aged 6 years before the second vaccination against mumps. For this, 5 ml of blood was taken from the children before vaccination and serum was obtained. Serum for the presence of antibodies to mumps virus was analyzed by three different methods - enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition test (RTGA) and neutralization reaction on Vero cell culture (PH). ELISA was performed using test systems of Bioservice CJSC (Moscow), Wampole (USA), Boehringer Mannheim (Germany), Human (Germany) and the tested Anti-Mump IFA test system manufactured by CJSC MBS Novosibirsk, the main component of which is the purified antigen of mumps virus strain Dragoon. In total, 254 serums were studied for the indicated period. Of the 254 sera, 12 sera had anti-mumps antibody titers <1: 4 in rtga and <1: 4 in rH and can be considered negative, the remaining 242 sera were positive when studied in rtga and rn. The same 12 negative sera gave a negative result in ELISA on test systems of all these manufacturers.

Comparison results show that using the enzyme-linked immunosorbent assay using a test system manufactured by Bioservice CJSC (Moscow), specific antibodies were determined in 240 sera from the total number of sera (242 sera) in which antiparotitic antibodies were found using rtga and pH methods .

Using the ELISA test system manufactured by Wampole (USA), specific antibodies were determined in 240 sera from the total number of sera (242 sera) in which antiparitic antibodies were found using rtga and pH methods.

Using an ELISA test system manufactured by Boehringer Mannheim (Germany), specific antibodies were determined in 241 sera from the total number of sera (242 sera) in which antiparitic antibodies were found using rtga and pH methods.

Using the ELISA test system manufactured by Human (Germany), specific antibodies were determined in 240 sera from the total number of sera (242 sera) in which antiparotitic antibodies were found using rtga and pH methods.

Using the claimed ELISA test system "Anti-Mumps ELISA" manufactured by JSC "MBS" Novosibirsk specific antibodies were determined in all 242 in which antiparitic antibodies were found using the methods of rtga and pH.

Thus, the highest percentage of coincidence of detection of positive and negative sera was obtained using ELISA test systems Boehringer Mannheim (Germany) and Anti-Mumps IFA manufactured by ZAO MBS Novosibirsk (99.5% and 100%, respectively).

The arithmetic mean values of antibody titers when using test systems based on the ELISA method (including the test Anti-Mump ELISA test system manufactured by ZAO MBS Novosibirsk) were also higher than in RTGA and PH (1: 146, 1:37 and 1:52, respectively).

Thus, the titer of the claimed strain of mumps virus Dragoon is higher by 2-3 orders of magnitude compared with known analogues and, thus, at the stages of antigen production, its antigen productivity is higher.

In addition, the claimed ELISA test system "Anti-mumps ELISA" has a higher sensitivity compared to many well-known analogues and is recommended for use to determine the intensity of individual immunity to the disease "mumps".

Claims (2)

1. The strain of mumps virus Mumps virus to obtain antigen - an enzyme-linked immunosorbent assay for diagnosing antibodies to the mumps virus, deposited in the collection of microorganism cultures of the Federal State Institution of Science "State Scientific Center for Virology and Biotechnology" Vector "Rospotrebnadzor under registration number No. VB-05 . 2. An enzyme-linked immunosorbent assay for the diagnosis of antibodies to mumps virus, including a tablet with immobilized purified mumps antigen, inactivated positive control sample (K +), inactivated negative control sample (K-), conjugate solution (PK), chromogen solution (PX) ), 25x phosphate-buffered saline concentrate (25x FSB-T), serum dilution solution (PC), stop reagent, characterized in that the test system contains immobilized inactivated mumps virus antigen ysokoochischenny antigen prepared based on the strain of claim 1.