RU2595429C1 - STRAIN OF HYBRID ANIMAL CELLS Mus musculus L. 5H6 - PRODUCER OF MONOCLONAL ANTIBODIES FOR DETECTING GLYCOPROTEIN E OF WEST NILE VIRUS, 5H6 MONOCLONAL ANTIBODIES PRODUCED BY SAID STRAIN OF HYBRID CELLS, AND IMMUNOENZYMOMETRIC KIT FOR DETECTING GLYCOPROTEIN E OF WEST NILE VIRUS USING SAID MONOCLONAL ANTIBODIES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and immunology. Described is a strain of hybrid animal cells Mus musculus L.Strain produces monoclonal antibody 5H6, specific to conformation epitope of glycoprotein E of West Nile virus. Also described is a kit for detecting glycoprotein E of West Nile virus by enzyme immunoassay of "sandwich" format containing said antibody.

EFFECT: disclosed group of inventions can be used in medicine, immunology and biotechnology for detection of West Nile virus antigen.

3 cl, 4 dwg, 3 tbl, 8 ex

Description

The invention relates to a strain of hybrid cells of an animal Mus musculus L., a producer of monoclonal antibodies for detecting West Nile virus glycoprotein E; to monoclonal antibodies produced by the indicated hybrid cell strain and enzyme immunoassay for the detection of West Nile virus glycoprotein E using these monoclonal antibodies and can be used in medicine, immunology and biotechnology to detect West Nile virus antigen (WNV) in field (mosquitoes, ticks) and clinical (in homogenates of human and animal organs) materials.

The causative agent of West Nile fever (LZN) - West Nile virus (WNV) belongs to the Flaviviridae family, the genus Flavivirus and is part of the antigenic complex of Japanese encephalitis (Lvov D.K. West Nile fever. // Virusol. - 2000. - No. 2. - S. 4-9). An extensive outbreak among people in 1999 in the Volgograd Region and Krasnodar Territory of LZN, which was previously considered endemic for tropical and subtropical countries, drew attention to the problem of diagnosing this infection in Russia (Lvov D.K., Butenko AM, Gaidamovich S.Ya . et al. Epidemiological outbreak of meningoencephalitis in the Krasnodar Territory and Volgograd Region caused by West Nile Fever virus (preliminary report) // Virusol. - 2000. - No. 1. - P. 37-38). Moreover, later there were reports of WNV circulation in birds, mosquitoes and ticks from Belarus to the Primorsky Territory, in the Caucasus, the Caspian Sea basin, the republics of Central Asia, and Siberia (Ternovoi V.A., Shchelkanov M.Yu., Shestopalov AM et al. Detection of West Nile virus in birds on the territory of the Baraba and Kulunda lowlands (West Siberian flyway) in the summer-autumn period of 2002 // Virusol. - 2004. - No. 3. - S. 52-56). In 2014, antibodies to WNV were registered in the population in 25 regions of the Russian Federation, and the incidence of the population was registered in 8 regions of the Russian Federation [http://ecdc.europa.eu/en/healthtopics/west_nile_fever/West-Nile-fever-maps/ pages / index.aspx].

Distribution of LZN has also been reported in at least 10 countries in Europe. Long-term observations indicate a constant circulation of WNV in Eurasia, including in Russia. Peaks in the incidence rate in the Russian Federation were recorded in 1999, 2010 and 2012, which indicates the constant circulation of WNV in natural foci in the Russian Federation and an unfavorable epidemiological prognosis for LWD in the Russian Federation (On the epidemiological situation of LWD in 2014 and the forecast for 2015, letter Head of Rospotrebnadzor No. 01 / 2030-15-32 dated 03/02/2015).

WNV virions have a spherical shape typical of flaviviruses and sizes from 40 to 60 nm in diameter, contain an electron-dense core of about 30 nm in diameter, surrounded by a lipid bilayer. The WNV genome is represented by single-stranded infectious RNA with a size of about 11 thousand nucleotide residues. The WNV genome encodes a single polyprotein, which is processed by viral and cellular proteases to form three structural proteins (envelope E, membrane M, capsid C) and seven non-structural viral proteins - NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (Liu J , Liu B, Cao Z et al. Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein. J Virol Methods. - 2008. - Vol. 154. - No. 1-2. - P. 20-26). Shell glycoprotein E (53 kDa) is the dominant antigen in hemagglutination and neutralization reactions, determines the tropism of the virus. The largest number of immunological studies of WNV is focused on this protein, since it induces the synthesis of neutralizing antibodies and is the main target for them (Beasley DW, Barrett AD Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. // Virol. - 2002. - Vol. 76. - No. 24. - P. 13097-13100). WNV genotyping is carried out by RT-PCR using specific primers for the protein E gene (Ternovoi VA, Protopopova EV, Surmach S.G. et al. Genotyping of West Nile virus detected in birds in the south of Primorsky Krai during 2002 -2004. // Molecular Genetics, Microbiology and Virology. - 2006. - No. 4. - S. 30-34). To detect the antigen, an immunohistological study using specific antibodies is used (Pisarev VB, Lvov D.K., Smirnov A.V. et al. Morphological changes in the medulla oblongata of mice during experimental infection with West Nile virus (strain 986). // Questions virusol. - 2007. - No. 3 - P. 23-25) and ELISA in the sandwich format based on polyclonal antibodies (Lvov D.K., Butenko AM, Vyshemirsky OI, etc. Isolation of West Nile Fever Virus from Patients people during an epidemic outbreak in the Volgograd and Astrakhan regions. // Vir. salting. - 2000. - No. 3. - S. 9-12).

At present, one commercial ELISA test system for detecting WNV antigen based on polyclonal rabbit antibodies is being produced in Russia (CJSC Bioservice, registration number of the FSR registration certificate 2012/13840 dated 09/10/2012).

The main advantage of hybridoma MCAs is the possibility of obtaining purified preparations in unlimited quantities with physico-chemical characteristics identical from batch to batch, which makes it possible to create standard diagnostic reagents based on them. All MCAs produced by a hybrid cell line originating from one clone of one cell belong to the same class and subclass of immunoglobulins and have exactly the same specificity and affinity. They interact with only one antigen, with one antigenic determinant - an epitope consisting of approximately 3-10 amino acid residues (Roy I. Immunology. // M .: Mir, 2000. - 581 p.)

The first closest analogue (the prototype of the hybridoma cell line) is the mouse hybridoma cell line 9E2, producing MCA 9E2, specific for the linear epitope 260-466 aa III domain of WNV glycoprotein E (Volgograd strain) and virus neutralizing agents on Vero cell culture (RF Patent No. 2265658, IPC C12N 5/18, A61K 39/42, publ. 10.12.2005).

A well-known laboratory ELISA test system based on two types of MCAs obtained for the recombinant III domain of WNV protein E (strain 5810 avian) (Liu J, Liu B, Cao Z et al. Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein . // J Virol Methods. - 2008. - Vol. 154. - No. 1-2. - P. 20-26). Detecting MCAs are conjugated to biotin and the system involves an additional step of sorption of the streptavidin conjugate with peroxidase. This test system has a low detection sensitivity for the recombinant protein - 100 ng / ml.

The second closest analogue (prototype test system) is the laboratory ELISA test system described in the scientific literature on the basis of two types of MCA obtained for native WNV (strain Egypt-101) and having specificity for the III domain of protein E and having neutralizing activity (Lelli D, Moreno A, Brocchi E et al. West Nile virus: characterization and diagnostic applications of monoclonal antibodies. // Virol J. - 2012 .-- Vol. 13. - P. 9-81). Detecting MCAs are conjugated to peroxidase. However, this test system has a low sensitivity to detect an infectious virus - 10 ^3.8 TTCPD50 / 100 μl.

The technical result of the proposed technical solution is to obtain a strain of hybrid cells of Mus musculus L. producing specific MABs to the WNV antigen (Volgograd strain 27889), not competing with MKA 9E2 for antigenic glycoprotein E epitopes, to use a pair of MKA (5H6 and 9E2) in the enzyme immunoassay sandwich format test system for detecting WNV glycoprotein E (Volgograd strain and Egypt strain), which is more sensitive than known analogues.

The specified technical result is achieved by the creation of a strain of hybrid cells of the animal Mus musculus L. 5H6 deposited in the Cotection of cell cultures FBSI SSC WB ″ Vector ″ under registration No. 311 of 02.032015, which is a producer of monoclonal antibodies specific for the West Nile virus (Volgograd strain - 27889 )

The indicated technical result is also achieved by obtaining 5H6 monoclonal antibodies produced by the strain of hybrid animal cells of the mus Mus musculus L. 5H6 subclass of IgGl immunoglobulins having a heavy 55 kDa and a light 25 kDa chain specific for the conformational (nonlinear) epitope of West Nile virus glycoprotein E (strain Volgograd - 27 , strain Egypt - 101) and used as indicators in the enzyme immunoassay to detect West Nile virus antigen in infected cells and tissues.

The indicated technical result is also achieved by creating a kit for detecting West Nile virus glycoprotein E by enzyme-linked immunosorbent assay of the “sandwich” format, comprising: 100-fold conjugate of 5A6 MCA labeled with horseradish peroxidase; positive control sample (purified inactivated West Nile virus antigen (Volgograd strain 27889); immunosorbent with MCA 9E2; negative control sample; solution for diluting test samples (RGO); conjugate dilution solution (RRM); substrate buffer solution with hydrogen peroxide ( CEP); 25-fold concentrate of phosphate-saline buffer solution with tween (FSB-T); 025% solution of tetramethylbenzidine (HMB); stop reagent (05 M sulfuric acid).

The pedigree of the strain. The inventive strain of hybrid cells was obtained by fusion of mouse p3-X63 / Ag8.653 (NS / 1) myeloma cells with spleen cells of BALB / c mice immunized with purified, concentrated, inactivated antigen with Freund's complete adjuvant (during the first immunization) and purified native infectious (for second and booster immunizations with Freund's incomplete adjuvant) WNV preparation obtained from the brain tissue of infected sucker mice. A 45% polyethylene glycol solution of Sigma company with a molecular weight of 1300-1600 kDa was used as a merging agent according to the method (Gefter ML, Margulies DH, Schcoff MD. A simple method for polyethylene glycol promoted hybridization of mouse myeloma cells. // Somat. Cell Genet. - 1977. - Vol. 3. - P. 231-236). The cells after fusion were grown in GAT selective medium (10 ^-4 M hypoxanthine, 4 × 10 ^-5 M thymidine, 10 ^-5 M aminopterin) in 96-well culture plates. Peritoneal macrophages of outbred mice were used as feeder cells. Hybridomas stably producing specific MABs were cloned 3 times. The yield of positive clones in the last cloning was 100%.

The inventive strain was deposited on 03/02/2015 in the Collection of cell cultures of the Federal State Institution of Science "State Scientific Center of Virology and Biotechnology" Vector "under registration No. 311. (certificate of deposit of the strain is attached).

The number of passages at the time of deposit: 7-10 passages.

Marker signs and methods for their assessment. The strain secretes murine monoclonal immunoglobulins of the IgGl subclass (having a heavy 55 kDa and a light 25 kDa chain) that specifically interact with WNV antigen in ELISA. Analysis of murine immunoglobulins was carried out by the method of solid-phase ELISA (TIFA) using inactivated WNV (Volgograd strain 27889) as an antigen at a concentration of 2 μg / ml and anti-mouse IgG antibodies labeled with horseradish peroxidase (Sigma) in 1/5000 dilution. The titer of purified MCA 5N6 in TIFA is 1/656100. By means of immunoblotting, MCA 5H6 did not detect a linear epitope on protein E in a viral preparation and did not interact in ELISA with recombinant fragments of protein E of WNV. The ability of MCA 5H6 in ELISA of the sandwich format to “capture” viral antigen paired with MCA 9E2 specific strictly to the linear epitope of 260-466 aa The III domain of protein E allows us to suggest the specificity of 5H6 mAb for the nonlinear (conformational) epitope of protein E.

Contamination by bacteria and fungi was not detected.

Cultural properties. Cultivation medium DMEM / F12 with glutamine, pyridoxine, Hepes (FBUN SSC WB "Vector" Rospotrebnadzor). The content of fetal serum optimized for hybridomas (HyClone, USA) in the growth medium is 10%. 80-160 μg / ml gentamicin sulfate is also added to the growth medium.

The strain is a monolayer-suspension culture in which up to 20% of the cells are in suspension, without attaching to the surface of the dishes for cultivation. Cells from the surface of the culture dishes are removed with a trypsin / versene solution in a ratio of 1/3 (volume / volume). Sowing dose of 200 thousand cells / ml. Passaging frequency after 3-4 days. Proliferation index - not less than 5.

The cultivation of hybridomas in the body of the animal. Female BALB / c mice (vivarium of the State National Center for Seventeenth Century "Vector") are pre-injected intraperitoneally with 0.3-0.5 ml pristan (Sigma). After 2-4 weeks, animals are inoculated intraperitoneally with 10 million / mouse hybrid cells. Ascitic tumor forms on the 7-10th day. Hybridomas are vaccinated in 100% of cases. 3-5 ml of ascitic fluid containing MCA can be obtained from one animal.

The characteristic of a useful product. Typing of hybridoma immunoglobulins was performed using the ELISA method using monospecific murine antibodies (Sigma, USA). MCA 5H6 belong to the IgGl subclass and specifically interact in TIFA with the native infectious and inactivated VZN antigen. In TIFA, the titer of MCA in ascites is 1/729000, in the purified preparation 1/656100.

Stable production of MCA is maintained for at least 10 passages in vitro with continuous cultivation. From one milliliter of ascitic fluid, 3-6 mg of purified MCA can be obtained.

Cryopreservation. The medium for freezing hybrid cells is DMEM (M) medium - 50%, fetal serum - 40%, dimethyl sulfoxide - 10%. 1-1.5 ml of the cell suspension is transferred into plastic cryovials and placed in a foam container with a wall thickness of 1 cm. The container is placed in a pair of liquid nitrogen and after 18-24 hours the tubes are transferred into liquid nitrogen. Defrosting is carried out by lowering the tubes into water at a temperature of 37-41 ° C. Cells are diluted with DMEM (M) and centrifuged at 1000 rpm. The sediment is resuspended in a growth medium and the cells are transferred into culture bottles at a concentration of 200-300 thousand per milliliter. Cell viability after thawing is 60-80% (according to the results of staining with 0.25% trypan blue). Each ampoule contains at least 1 million / ml cells.

The application is illustrated by the following graphic materials. In FIG. 1 shows an electrophoregram to assess the cleaning efficiency of the MCA. Notes: 1 - protein markers of molecular weight in kDa (BioLabs); 2 - antibodies of polyclonal mouse serum immunized with WNV antigen precipitated with 50% ammonium sulfate solution; 3 - MCA 5H6 precipitated from ascites with a 50% solution of ammonium sulfate; 4, 5 - MCA 5H6 and 9E2 (as a control), purified from ascites with caprylic acid, followed by precipitation with a 50% solution of ammonium sulfate. In FIG. Figure 2 shows a titration schedule for purified WNV viral antigen in TIFA. Notes: purified MCA preparations and murine sera were used in 1/1000 dilutions; angivid horseradish peroxidase-labeled anti-mouse IgG antibodies (Sigma) were used in 1/5000 dilutions. For each titration point, the result is the arithmetic mean (n = 3); error bars are displayed using standard errors. In FIG. Figure 3 shows a titration schedule for typhus of purified 5A6 MCA and serum of mice immunized with purified infectious antigen. Notes: WNV antigen is adsorbed onto plastic at a concentration of 2 μg / ml; antibody titration was carried out in a 3-fold step (12 points) with a 1/300 dilution. For each titration point, the result is the arithmetic mean (n = 3); error bars are displayed using standard errors. In FIG. Figure 4 shows a graph of titration of the lysate and purified antigen in ELISA of the sandwich format using a pair of MKA 9E2 and 5H6 *. Notes 1: purified antigen strain LEIV-VLG99-27889 used at a concentration of 0.5 mg / ml; MCA 9E2 (immunosorbent) for sorption in the cells of the tablet used at a concentration of 4 μg / ml; Peroxidase-labeled MKA 5H6 * (conjugate) was used at a concentration of 1 μg / ml. For each titration point, the result is the arithmetic mean (n = 3); error bars are displayed using standard errors. Notes 2: ELISA of a sandwich format with a pair of MCA 9E2 and 5H6 * does not detect purified tick-borne and Japanese encephalitis viruses (data not shown), detects a viral antigen in a purified preparation of 2.4 ng / ml (titration schedule). RT PCR test system for detecting VZN RNA (production of InterLabService, Cat. No. R-V53, RG, iQ, MX) detects the purified LEIV-VLG99-27889 antigen at a concentration of 1.2 mg / ml (data not shown).

The following examples detail the achievement of the technical result of the objects of the invention.

Example 1. Cultivation of hybrid cells of the strain Mus musculus L. FBSI SSC VB "Vector" - 5H6, producing MCA to protein E of WNV, in animals.

BALB / c mice (vivarium of the SSC VB "Vector"), weighing 20-22 g, were injected intraperitoneally with 0.3-0.5 ml pristan not less than 10 days before the vaccination of hybridoma cells. The cultured cells located in the logarithmic phase of growth were sterile centrifuged for 5-10 minutes at 1000 rpm. The supernatant was removed and the cell pellet was suspended in a sterile Earl or Hanks solution. Mice were intraperitoneally injected each with 1 ml containing at least 10 million hybridoma cells. After 7-10 days, the animals were euthanized and 3-5 ml of ascitic fluid were removed from the abdominal cavity under aseptic conditions. Cells from ascitic fluid were separated by centrifugation, and the titer of MCA was determined in the supernatant using TIFA, as described below (example 4).

Example 2. Isolation of purified monoclonal bodies produced by hybrid cultured cells of the strain Mus musculus L. SSC VB "Vector" 5H6.

One volume of ascitic fluid containing MCA was diluted with 4 volumes of 0.6 M acetate buffer (0.004 M citric acid; 0.2 M sodium acetate, pH 4.0) and adjusted to pH 4.5 with 0.1 N caustic solution soda. Caprylic acid was added dropwise to the diluted sample, with constant stirring, at a rate of 25 μl per 1 ml of solution and incubated overnight at 4 ° C. Then it was centrifuged for 30 min at 8000 rpm and the precipitate was removed, and the supernatant was mixed with 10-fold phosphate-saline buffer (PBS) and pH 7.4 was adjusted with a solution of 1.0 N sodium hydroxide. An equal volume of a cold saturated solution of ammonium sulfate was added to this solution, shaken and kept overnight at 4 ° C or 30 min at minus 18-20 ° C. Centrifuged for 15 min at 5000 rpm. The supernatant was carefully drained and the precipitate was dissolved in PBS (pH 7.4). Ammonium sulfate residues were removed by dialysis against 50-100 volumes of PBS (pH 7.4).

Electrophoresis of purified MCA preparations was carried out according to the method described in the work (Osterman A.A. Methods for the study of proteins and nucleic acids. Electrophoresis and ultracentrifugation. // Moscow, 1981. - S. 37-64). in a discontinuous buffer system using 10% polyacrid gel (PAG) with 0.1% sodium dodecyl sulfate (SDS) in Tris-glycine buffer. Separating gel contains 0.0625 Tris-HCl (pH 8.8); 0.1% SDS; 10-15% acrylamide; 1% N, N-methylene bisacrylamide. MCA preparations (1 μl each) were applied to the lane in a volume of 30 μl of buffer containing 0.0625 M Tris-HCl (pH 6.8), 2% SDS, 5% mercaptoethanol, 10% glycerol, 0.01% bromophenol blue. Before application, the buffer containing the MCA was heated for 3 min at 95 ° C. Electrophoresis was performed at 10 V / cm. The gel was stained using Coomassie G-250. The purified preparation MCA 5H6 in the form of an electrophoregram is shown in Fig. 1. Determination of protein concentration in purified MCA preparations was performed using the Protein Assay Kit (Bio-Rad, USA) on a UV mini 1240 CE spectrophotometer (Shimadzu Corporation) at a wavelength of 595 nm. The data obtained were evaluated by a calibration curve. The tiger of purified MCAs was determined using TIFA as described below (Example 4).

Example 3. Obtaining antigen. The WNV strains used in this work were obtained from the State collection of viral strains of the D.I. Ivanovsky. The strain LEIV-Vlg99-27889-human (Vlg99-27889, Volgograd - 27889) was isolated in 1999 from the brain of a deceased person in the infectious diseases hospital of Volgograd (Lvov D.K., Butenko AM, Vyshemirsky O.I. et al. Isolation of West Nile fever virus from sick people during an epidemic outbreak in the Volgograd and Astrakhan regions // Voprosy virusol. - 2000. - No. 3. - P. 9-12) and strain Eg-101, which was isolated in 1951 from the blood serum of a clinically healthy child in the vicinity of Cairo, Egypt (Anishchenko M, Shchelkanov M.Yu., Alekseev V.V. et al. Molecular markers of the pathogenicity of West Nile virus A. // Questions of virusol. - 2010. - No. 1. - S. 4-10). To obtain a purified and concentrated viral preparation, a cell lysate was prepared as follows: Vero cells were grown in glass 1-liter culture vessels and infected with the virus according to standard methods. The support medium was removed from the monolayer of infected cells (before the obvious cytopathic effect of the virus in the form of lysis of infected cells), and cell debris in a small volume of serum-free medium was precipitated by low-speed centrifugation at 1000 rpm. Cell pellet in a small volume of STE buffer solution (0.001 M ethylenediaminetetraacetic acid; 0.1 M NaCl; 0.01 M Tris HCl, pH 8.0) was sonicated at 50 W for 30 seconds on melting ice. The cell lysate was freed from the cell membrane pellet by centrifugation at 1000 rpm for 10 minutes and applied to a linear sucrose gradient (13.5 ml of 60% sucrose and 13.5 ml of 20% sucrose) in STE buffer solution. The purified viral preparation was homogenized in 0.05 M disubstituted Na-phosphate buffer (pH 7.4) for freezing in aliquots at minus 20 ° C. Protein concentration in purified viral preparations was determined using the Protein Assay Kit (Bio-Rad, USA) on a UV mini 1240 CE spectrophotometer (Shimadzu Corporation) at a wavelength of 595 nm. The data obtained were evaluated by a calibration curve. In fig. 2 is a graph of titration of purified antigen into TIFA (as described in Example 4). Purified, concentrated virus was inactivated in Tris-HCl lysis buffer containing Nonidet-40 detergent and formalin. A positive control sample of viral antigen for the test system was prepared as a working antigen solution (3 μg / ml) with stabilizers, preservatives, protease inhibitors and a red dye.

Example 4. Determination by the method of solid-phase ELISA (TIFA) of the specific interaction of MCA produced by hybrid cells of the strains of Mus musculus L. SSC VB "Vector" 5H6 with purified antigen WNV. TIFA was performed on polystyrene tablets (Testiks). The purified WNV antigen in working dilution (concentration 2 μg / ml) or in the form of stepwise titration was sorbed in FSB (pH 7.4) in a volume of 100 μl / well. Non-specific binding sites were saturated for 45 min at 37 ° C with a 0.5% casein solution in TSB-T buffer (0.145 M sodium chloride (AppliChem), 20 mM Tris-HCl (Sigma), 0.1% Tween-20 (Serva ), pH 7.4) and then incubated with ascitic fluid or purified MCA for 45 min at 37 ° C. Specific binding of MCA to the antigen was detected by antispecies horseradish peroxidase-labeled antibodies against mouse IgG. Then added a chromogen, (0.1% O-phenylenediamine in citrate phosphate buffer containing 0.2 M citric acid, 0.5 M Na ₂ HPO ₃ , pH 5.0) with 0.03% hydrogen peroxide. The reaction was stopped by adding 100 μl per well of 1N HCl and the optical density (OD) of the samples was measured on a Multiscan spectrophotometer using a light filter with a transmission maximum of 492 nm. As a negative and positive control, homologous normal (non-immune) and hyperimmune sera were used, respectively. In fig. Figure 3 shows a titration schedule for purified 5A6 mAbs and hyperimmune mouse serum.

Example 5. Preparation of conjugate MCA 5H6 with horseradish peroxidase. The preparation of conjugates of MKA 5N6 with horseradish peroxidase was carried out according to the periodic method of Nakane described in the manual (Egorov AM, Osipov A.P., Dzantiev B.B. Theory and practice of enzyme-linked immunosorbent assay. // M .: Higher school, - 1991. - 287 from). To stabilize the conjugate, bovine serum albumin, merthiolate and glycerin (50% by volume) were added for storage at minus 20 ° С.

Example 6. The composition of the reagent kit "Test enzyme-linked immunosorbent assay for the detection of West Nile virus antigen using monoclonal antibodies" ("Vector - West Nile Antigen"). The composition of the reagent kit is presented in table. one.

Example 7. Detection of viral antigen in the enzyme-linked immunosorbent assay system "Vector - West Nile Antigen".

Test design - IFA "sandwich" based on two types of MCA. If there is WNV in the test sample, the viral antigen (protein E) binds to specific antiviral MCAs adsorbed in the wells of the plate. The resulting complex (antibody-antigen) then binds to the conjugate (the second antiviral MCA labeled with horseradish peroxidase) and is detected in the reaction with an indicator solution, which changes the color (optical density) of the reaction mixture in the wells of the plate. OD is recorded spectrophotometrically.

In fig. 4 is a graph of antigen titration.

The sensitization of cells of highly sorption polystyrene plates (Testiks or Nunc) was carried out from a volume of 100 μl of purified MCA 9E2 (RF Patent No. 2265658) in 0.5 M carbonate buffer solution (pH 9.0) with a concentration in the range of 0.4-8 μg / ml for 18 hours at 22 ° C. Additional blocking of the cell surface was carried out with a 0.5% casein solution for 30 minutes at 37 ° C. 50 μl of control samples (K ⁺ and K ^- ), 50 μl of the panel samples (containing 4 positive and 4 negative samples) (Table 3), and 50 μl of 50 μl MCA 5H6 conjugate solution were added to the wells of the strip (with a concentration in the range of 0.1-1.0 μg / ml) with horseradish peroxidase. They were incubated for 2.5 hours at a temperature of 37 ° C on a thermal shaker (type ST-3 Elmi, Latvia), with a stirring intensity of 100 rpm or 16-18 hours at a temperature of 4 ° C. The strips were washed 6 times with FSB-T washing solution (Table 1) prepared from FSB-T concentrate (28 ml) diluted with purified water to 700 ml. In case of precipitation of salts in the concentrate, it is necessary to warm it up at 30-40 ° C until the precipitate is completely dissolved. Storage: up to 4 weeks at 2-8 ° C). At the same time, at least 250 μl of the solution was added to each well during one washing. Residual fluid from the wells was carefully removed by tapping an inverted strip on filter paper. Then, in each well of the strip, 100 μl of a freshly prepared solution of TMB in SBR was added (Table 1, Table 2) and the strips were kept for 15–25 min at a temperature of 18–25 ° C in the dark. The reaction was stopped by adding 100 μl stop reagent (0.5 M sulfuric acid) to the wells. The magnitude of the OD of the solutions in the wells of the immunosorbent was measured on a Multiscari spectrophotometer at a wavelength of 450 nm. The quality of the test system was considered satisfactory if the average optical density in the wells with negative control (OD _cf K ^- ) does not exceed 0.25 pu, and the average OD value of the positive control sample (OD _cf K ⁺ ) is not less than 4 times higher than OP _cf. K ^- . Samples with an optical density equal to or higher than the value of OD _crit were considered positive, which was calculated by the formula: OD _crit. = 2 × OD _cp K ^- .

Example 8. Preparation of control samples of the enterprise, containing and not containing viral antigen

In the table. 3 shows methods for preparing samples, their inactivation and concentration of viral antigen.

The investigated properties of the hybrid strain of mus musculus L. cells of the SSC VB “Vector” 5H6 (the author's name is the 5H6 cell line) allow us to conclude that for the first time based on mouse myeloma, hybridomas of mus musculus L. were obtained. specific to the non-linear (conformational) epitope of West Nile glycoprotein E. The titer MKA 5H6 detected by the TIFA method (when using inactivated WNV at a concentration of 2 μg / ml and antibodies against mouse IgG labeled with horseradish peroxidase (1/5000 dilution) is 1/656100. 5A6 MKA do not compete for antigenic epitopes with previously described MCA 9E2 (RF Patent No. 2265658), which allows them to be used in pairs in ELISA format "sandwich" to detect viral antigen.

In 2013, on the basis of FKUZ “Microbe” (Saratov), technical tests of 3 series of a medical device were carried out: reagent kit “Immunoenzyme test system for detecting West Nile virus antigen using monoclonal antibodies” (“Vector - West Nile - antigen"). Registration of a medical device TU-9398-020-0566012-2013 in the Federal Service for Supervision in the Sphere of Health CODE OKP 939817 from 10.16.2013. Validity of registration until 16.10.2018. Permission from the Federal Service for Supervision in Healthcare obtained (ROSZDRAVNADZOR) dated January 23, 2014 No. 28/2014 for conducting clinical trials of a medical device: a set of "Vector - West Nile Antigen". In 2014, on the basis of FBUN SSC VB "Vector" (pp. Koltsovo, NSO), in 2014 clinical trials of 3 series of a medical device were carried out: a set of "Vector - West Nile Antigen" using a set of enterprise SPP-ZN-2013 " Standard panel of samples containing and not containing West Nile virus antigen ", consisting of 8 samples and a set of clinical samples (270 pcs.) Containing West Nile virus antigen (including: purified antigens of Volgograd strains - 27889 and Egypt - 101; lysates Vero cells infected with Volgograd strains - 27889 (Vlg99-27889-human), Volgograd - 27924 (Vl g00-27924-human), Egypt - 101 (Eg-101), Tur73-2914-ticks, Az70-72-ticks, Az67-1640-nuthatch, Ast63-94-ticks; supernatants from infected Vero cells; 20% suspensions of the brain, liver, lung, spleen, heart, kidney of white b / n mice infected with Volgograd strains - 27889 and Egypt - 101; tick pools Ixodes persulcatus, with the addition of purified antigens Volgograd - 27889 and Egypt - 101; 20% brain suspensions of a deceased person (bridge, medulla oblongata, cerebellar cerebellum, optic tubercle, caudate nucleus), with the addition of purified antigens Volgograd - 27889 and Egypt - 101) and not containing West antigen Nile (including: non-purified tick-borne encephalitis antigen, strain 205; Vero uninfected cell lysate; 20% suspensions of organs of pure mice; tick pools Ixodes persulcatus; 20% brain suspensions of a deceased person (bridge, medulla oblongata, cerebellar cerebellum, optic tubercle, caudate nucleus).

In 2014, on the basis of FKUZ "Microbe" (Saratov), clinical trials of 3 series of a medical device were carried out: a set of "Vector - West Nile Antigen" using a set of enterprise SPP-ZN-2013 "Standard panel of samples containing and not containing West Nile virus antigen, "consisting of 8 samples and a set of clinical samples (270 pcs.), containing and not containing West Nile virus antigen (listed above).

Conclusion on the medical test reports of 2014 on the basis of the microbial health care facility Microbe (Saratov) and on the basis of the Federal State Budget Scientific Center State Research Center of the Vektor Vector (Koltsovo village, NSO): Medical product reagent kit “Vector - West Nile Antigen », Russia, intended for the qualitative and quantitative determination of West Nile virus antigen in field (ticks) and clinical (in homogenates of animal and human organs) samples by indirect enzyme-linked immunosorbent assay (ELISA) on a solid-phase carrier in clinical and epidemiological studies, as a result s medical tests may be recommended for use in medical practice, medical and sanitation facilities for clinical laboratory diagnostics in the Russian Federation. By the order of Roszdravnadzor dated 09.09.2014, the medical device “Vector - West Nile Antigen” was registered and a registration certificate No. RZN 2014/1944 was issued.

Claims (3)

1. The strain of hybrid cells of the animal Mus musculus L. 5H6, which is a producer of monoclonal antibodies specific for West Nile virus (strain Volgograd - 27889), and deposited in the Collection of cell cultures FBSI SSC WB ″ Vector ″ under registration number 311. 2. Monoclonal antibody 5H6 produced by the strain of hybrid cells of the animal Mus musculus L. 5H6 subclass of IgG1 immunoglobulins, having a heavy 55 kDa and light 25 kDa chain specific for the conformational (nonlinear) epitope of the West Nile virus glycoprotein E (strain Volgograd - 27889 and - 101), used as indicators in the enzyme immunoassay for the detection of West Nile virus antigen in infected cells and tissues. 3. A kit for the detection of West Nile virus E glycoprotein E by enzyme-linked immunosorbent assay of the “sandwich” format, comprising 100-fold conjugate of MKA 5H6 labeled with horseradish peroxidase; positive control sample (purified inactivated West Nile virus antigen (Volgograd strain 27889); immunosorbent with MCA 9E2; negative control sample; solution for diluting test samples (PPO); conjugate dilution solution (PPK); substrate buffer solution with hydrogen peroxide ( SBR); 25-fold concentrate of phosphate-saline buffer solution with tween (FSB-T); 0.25% solution of tetramethylbenzidine (TMB); stop reagent (0.5 M sulfuric acid).

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