RU2528868C1 - STRAIN OF CULTIVATED HYBRID CELLS OF ANIMAL MUS MUSCULUS L CCHFV Vd-2-PRODUCER OF MONOCLONAL ANTIBODY 1E2/E5 TO VIRUS OF CRIMEAN-CONGO HAEMORRHAGIC FEVER - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a strain of cultured hybrid cells of animal Mus musculus L. CCHFV Vd-2 deposited in the State collection of pathogenic microorganisms and cell cultures "SCPM-OBOLENSK" under number H-26, which is a producer of monoclonal antibody 1E₂/E₅ to virus of Crimean Congo haemorrhagic fever (CCHF), suitable for the manufacture on its base of one of the components of the set of reagents for immunoenzymometric detection of antigens of CCHF virus in samples of biological material.

EFFECT: invention enables to obtain highly specific monoclonal antibodies suitable for use as one of the components of the set of reagents for immunoenzymometric detection of antigens of CCHF virus.

1 tbl, 3 ex

Description

The invention relates to biotechnology for producing hybridomas producing monoclonal antibodies (MCAs) of a given specificity and can be used in virological and immunological studies to create an enzyme-linked immunosorbent assay designed to detect antigens of the Crimean Congo virus hemorrhagic fever (CCHF) - a causative agent of dangerous transmissible natural focal disease of a person included in the list of infectious (parasitic) diseases requiring measures for the sanitary protection of territories Theories of the Russian Federation [1, 2]. The strain producing monoclonal antibodies to the CCHF virus is named CCHFV Vd-2 (1E ₂ / E ₅ ). The strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-26.

When carrying out work on the detection of the pathogen in samples of clinical and field material, they are guided by the principles of organizing studies of material suspected of being infected with the CCHF virus in the institutions of Rospotrebnadzor described in the "Practical Guides" [3, 4], as well as in MUK 4.2.3007-12 [ 5]. According to these documents, one of the regulated immunodiagnostic methods for detecting CCHF virus is the sandwich variant of enzyme-linked immunosorbent assay (ELISA).

Currently, for the enzyme immunoassay for detecting CCHF virus antigens, the “Test enzyme immunoassay for detecting the Crimean-Congo hemorrhagic fever virus“ Vecto Crimea-KGL-antigen ”(CJSC Vector-Best, Russia; Registration number RK-IMN-5№ is being produced 007764, registration date 12/14/2010), made on the basis of polyclonal antibodies to the CCHF virus.

It is known that the use of MCA as the basis for the detection of pathogens of dangerous infectious diseases, including the CCHF virus, opens up new prospects for the creation of highly effective standardized drugs for rapid detection and identification of the desired pathogens. When creating immunoenzymatic test systems for identifying one or another pathogen, the components of which are designed to perform a sandwich variant of a solid-phase ELISA, as a rule, two variants of MCAs of different epitope orientation are used, one of which serves as a “capture” of antigen, and the second - functions detection of the formed immune complex. To avoid errors in evaluating the results of the reaction, the selection of the most effective pairs of monoclonal ingredients is carried out from among the options included in the MCA panel that recognize epitopes on diagnostically significant antigenic targets of a specific pathogen.

The possibilities of applying MCA to the CCHF virus were reported by domestic and foreign authors [6, 7, 8, 9, 10, 11, 12].

They obtained experimental samples of drugs for use in enzyme-linked immunosorbent and immunofluorescence methods for detecting CCHF virus, tested in laboratory conditions, confirmed the main advantages of working with MCA: the ability to work with raw materials homogeneous in composition for the manufacture of highly active immunodiagnostic reagents of a given specificity. Data on the implementation of these diagnostic tools in practical health care are not available. Currently, there is only one kit registered as a medical device designed to detect CCHF virus in biological liquid samples (enzyme-linked immunosorbent assay kit manufactured by Vector Best CJSC, Novosibirsk, cat. No. D-5056, VektoKrym-KGL-antigen) , RU No. FSR 2010/07327). Due to the lack of standard drugs for quality control of laboratory diagnostics of the Crimean hemorrhagic fever (CHF), the relative sensitivity and specificity of this kit is unknown.

The purpose of the invention is to obtain a strain of a hybridoma producer of a highly specific, monoclonal antibody - a component for the manufacture of an enzyme immunoassay system designed to detect CCHF virus antigens in samples of biological material.

The production of hybridoma is achieved by hybridization of two types of cells: splenocytes of an inbred mouse of the BALB / c line immunized with the CCHF virus antigen, and mouse myeloma cells.

Hybridoma producer MCA 1E ₂ / E _{5 was} obtained by fusion of BALB / c immune mouse splenocytes with murine Sp 2/0 myeloma cells in the presence of a conjugating agent - polyethylene glycol (PEG-4000), followed by sieving of cell suspension into wells of 96-well culture plates, cultivation in a selective medium, the selection of the primary clone, its cloning and reclone by limiting dilution method. The resulting strain, named CCHFV Vd-2 (1E ₂ / E ₅ ), is characterized by the following features.

Cultural signs. For in vitro hybridoma culturing, RPMI-1640 medium with 15% fetal calf serum (ETS), 2 mM L-glutamine, 10 mM Hepes, 4 mM sodium pyruvate is used.

Cells are cultured at 37 ° C in an atmosphere of 5-7% CO ₂ and humidity 70-80%. Reseeding is done every 3-4 days, the growth rate of the cell population is monitored by viewing the plate wells in an inverted microscope. Multiplicity of sieving 1: 4-1: 8. The nature of the growth of the culture is semi-suspension.

Cultivation in the body of a syngeneic animal. To accumulate ascites in vivo, hybridoma cells are injected intraperitoneally into pre-primed BALB / c mice, 2 · 10 ⁶ -4 · 10 ⁶ cells per mouse. Ascites is formed after 2-4 weeks.

Strain productivity. MCA production in the cultivation medium is 0.53 μg / ml, in ascitic fluid - 8-10 mg / ml. The volume of ascitic fluid is on average 3-4 ml.

Characteristics of the resulting product. Antibodies belong to the IgG class ₁ . They specifically interact with the CCHF virus antigen. The specificity of the interaction is determined using the enzyme-linked immunosorbent assay (TIFM).

Cryopreservation. Outside the period of experiments on the accumulation of preparative quantities of MCA 1E ₂ / E ₅ , the hybridoma cells are kept in a cryopreserved state. To translate them into such a state, a suspension of hybridoma cells in an amount of 4 · 10 ⁶ cells in 1 ml of a protective medium consisting of RPMI-1640 medium with 20% ETS and 7% dimethyl sulfoxide is transferred into plastic ampoules. Ampoules are placed in the apparatus for cryopreservation of biological material. Use the programmable gradual decrease in temperature: within 20 minutes - lower the sample temperature to 0 ° C, then at a speed of 1 ° C per minute to a temperature of minus 40 ° C and at a speed of 5 ° C per minute from minus 40 ° C to minus 70 ° C. Then the ampoules are immersed in a bio-storage with liquid nitrogen and stored until use. Defrosting is carried out at 37 ° C in a water bath for 2-3 minutes. Cell viability after thawing is 80% or more.

Example 1. Obtaining a strain of hybridoma producer. BALB / c mice are immunized with the inactivated CCHF virus antigen. To stimulate mouse B-lymphocytes, a fractional administration of minimal doses of antigen for a relatively short period of time (5 weeks) is used. The first injection (day 0) is performed subcutaneously in the back with a mixture of antigen (15 μg of antigen in 0.3 ml of physiological saline (RF) with an equal volume of incomplete Freund's adjuvant). The second, third and fourth injections (7, 14 and 21 days) - intraperitoneally 15 μg of antigen in 0.3 ml of FR, the fifth boosting injection - after 2 weeks intrasplenic (20 μg). The total dose of antigen should not exceed 80 μg of protein according to Bradford. 3 days after this, hybridization of 1 · 10 ⁸ immune mouse splenocytes with 1 · 10 ⁷ mouse myeloma cells is performed in the presence of 1 ml of 50% PEG-4000 for 2 minutes at room temperature with constant stirring. Then, with constant stirring, 1, 2, 4, 8 ml of serum-free medium is added to this mixture, each portion for 2 minutes. After that, the cells are centrifuged at 800-1000 rpm for 10 minutes. The precipitate was resuspended in a selective HAT medium with 15% ETF, 2 mM L-glutamine, 10 mM Hepes, 4 mM sodium pyruvate. Cells are seeded in a 96-well plate with a feeder, 2.5 · 10 ⁵ -5 · 10 ⁵ cells per well. A change of environment is performed every 3-4 days. Screening of antibody-producing hybrid clones is performed no earlier than 7-10 days after sieving in 96-well plates. When viewing the wells, register those in which there is a characteristic growth of cell groups, then no more than 50 μl of culture medium is taken from them for research in the indirect variant of TIFM. The positive results of this screening method, obtained with a sample from the same well at least three times, are the basis for the selection of the primary producer clone of the target product. All primary clones selected during the month are cloned and cloned by limiting dilution, plating the wells of a pre-prepared 96-well plate with a feeder - peritoneal macrophages of a syngenic intact mouse (4-6 × 10 ⁴ cells in each well). At this stage, RPMI-1640 medium with 15% ETS and the necessary additives is used. After the second cloning of each of the primary variants of the clones, a working collection of hybridoma producers of MABs of a given specificity is formed. Among the productive clones secreting monoclonal immunoglobulins against the CCHF virus antigen, the clone producer CCHFV Vd-2 (1E ₂ / E ₅ ) was selected.

Example 2. The accumulation of MCA. The ampoule with hybridoma cells is removed from the bio-storage with liquid nitrogen and quickly thawed in a water bath at 37 ° C. To wash the reconstituted cell suspension from impurities of the protective medium, it is transferred to a centrifuge tube with 10 ml of serum-free medium RPMI-1640 and carefully layered on the surface of the liquid. Cells are pelleted by centrifugation at 800-1000 rpm, the supernatant is removed, and the settled cells are resuspended in complete hybridoma culture medium (RPMI-1640 with 15% ETS and necessary additives). Cells are seeded in 5-6 holes of a 12-well plate or in a mattress with an area of 25 cm ² . When the colony is enlarged to sizes covering half the area or more, screening is carried out on large areas with the obligatory control of antibody production. In vitro cultivation conditions, selection and replacement tactics of the culture medium, sieving of the breeding cell population are performed as described above (example 1). With the correct fulfillment of these conditions, MCA production is restored to the level of 0.52-0.54 μg / ml. All samples of the culture medium taken at the time of its replacement with equal volumes of fresh medium are collected in a vial for subsequent isolation of monoclonal immunoglobulins from it.

In vivo replication of hybridoma cells in the abdominal cavity of a syngeneic animal is the most effective way to accumulate high concentrations of MCA. Animals are pre-intraperitoneally injected with 0.4 ml of sterile mineral oil, pristan - 2,6,10,14-tetramethyl-pentadecane or incomplete Freund's adjuvant. After 5-7 days, 2 · 10 ⁶ -4 · 10 ⁶ hybridoma cells are intraperitoneally injected into mice. After the accumulation of ascitic fluid, it is collected. Cells are pelleted by centrifugation, the supernatant is separated and used to isolate antibodies by sulfate triple protein reprecipitation at 50% saturation of ammonium sulfate. The loose precipitate was centrifuged at 12,000 rpm for 20 minutes, the supernatant was removed. The precipitate is dissolved in 0.01 M phosphate buffer, dialyzed to completely remove ammonium sulfate ions. The resulting solution of monoclonal immunoglobulins is sterilized by membrane filtration, ampouled and stored at minus 20 ° C until use. The protein concentration in the MCA solution is determined spectrophotometrically at a wavelength of 280 nm.

The specific activity of the MCA is determined using TIFM. The day before the reaction is set up, 100 μl of a solution of antigen (with a concentration of 7-10 μg / ml per protein) in 0.05 M carbonate-bicarbonate buffer, pH 9.5, is added to the wells of the polystyrene plate for ELISA. The plate is kept at 4-8 ° C overnight, then washed three times with buffered saline with 0.05% Tween-20 (PBS-T) and the blocking step of plastic is performed with a 1% solution of bovine serum albumin, adding 100 to each well μl of this solution. The plate is incubated at 37 ° C in a thermostat for 30 minutes and again washed three times with PBS-T. The next stage is the introduction into the wells of a plate of 100 μl of various dilutions of MCA prepared on 0.1 M phosphate buffer, pH 7.4, with 0.05% Tween-20 (PBS-T). The incubation time was 1 h at 37 ° C, washing three times and introducing an anti-conjugate in working dilution of 100 μl. The conjugate is a diagnostic antibody against white mouse IgG (H + L) labeled with peroxidase (commercial preparation). The plate is incubated at 37 ° C for 1 h, washed three times. In conclusion, 100 μl of a mixture of hydrogen peroxide with a chromogen are added to the wells. The plate is placed in a dark place. The enzyme-substrate reaction lasts no more than 20-25 minutes at room temperature. To stop it, use a stop reagent. The results of the reaction (optical density (OD) of the contents of the wells) are recorded on a tablet photometer at a wavelength corresponding to the characteristic of the chromogen used. MCA 1E ₂ / E ₅ , isolated from ascitic fluid, are active at dilutions of 1:10 ⁵ -5: 10 ⁶ .

The average volume of ascitic fluid obtained from one mouse during cultivation of the CCHFV Vd-2 (1E2 / E5) hybridoma in vivo is 3-4 ml, the concentration of MCA is 8-10 mg / ml.

Example 3. Verification of the claimed beneficial properties of MCA 1E ₂ / E ₅ in ELISA.

To assess the feasibility of using the obtained MAB as one of the reagents in the kit for detecting CCHF virus antigens, the specific activity of MAB 1E _{2 /} / E ₅ was studied.

As a reference preparation, weak dilutions of a sample containing CCHF virus antigens and one concentrated sample without them were used.

Sample 1. Recombinant protein containing amino acid sequences similar to CCHF virus proteins isolate "1-2";

Sample 2. Negative control - a mixture of lysate proteins of the transplantable line of human adrenal adenocarcinoma cells SV-13 and E. coli (probable impurities of sample No. 1).

The diagnostic design is based on the direct solid-phase three-stage ELISA of a double sandwich variety. It includes two monoclonal components: an immunosorbent plate with MKA immobilized on the surface of the wells — to CCHF virus proteins and a MKA-biotin conjugate that detects the formed immune complexes of immunosorbent antibodies with CCHF virus antigens. To increase the sensitivity of the detection reaction for the desired antigen, two conjugates were sequentially used: MKA-biotin and horseradish streptavidin-polyperoxidase forming a strong bond with it (SPH, STD GmbH, Germany). The latter consists of one streptavidin molecule and 800 peroxidase molecules; its use increases the sensitivity of ELISA by 200-300 times relative to the classical protein-enzyme conjugate. Visualization of the results of the analysis is carried out by adding to the formed immune complexes a solution of chromogen 3,3 ′, 5,5′-tetramethylenebenzidine (Moss Inc, USA), followed by fixation of the color with a solution of sulfuric acid and recording on a plate photometer in units of OD.

To activate the immunosorbent (96-well plate of modified polystyrene, international classification "Maxisorp"), the composition of the sorption solution was selected. To reduce the background in the analysis results, as well as to give the immunosorbent stability during storage, the most effective was the use of a carbonate buffer solution with subsequent blocking of free plastic sections with a solution of casein and sucrose.

The beneficial properties of MKA 1E _{2 /} / E ₅ have been studied both in relation to their activation of the immunosorbent and to its use as a component of the MKA-biotin conjugate.

To select the optimal concentration of MCA in the immunosorbent in order to identify a specific signal at low concentrations of CCHF virus antigen for MCA 1E _{2 /} / E ₅ , four 10-fold dilutions were prepared, starting from 0.15 μg per well. The optimal concentration was established in ELISA.

To obtain the MKA-biotin conjugate, the routine biotinylation procedure was used: adding sulfo-NHS-biotin to the dialyzed anti-bicarbonate MCA solution, followed by purification of the unbound biotin from the conjugate by gel filtration on a Sephadex G-25 column.

The optimal concentration of MCA 1E _{2 /} / E ₅ -biotin conjugate obtained according to this procedure was established in ELISA by using four consecutive dilutions of 10 times different biotinylated MCAs separately, starting with a maximum of 150 μg / ml.

At the first stage, ELISA, the initial samples were diluted 100 times with Tris-EDTA solution with the addition of X-100 triton and incubated in immunosorbent wells based on various concentrations of 1A _{2 /} / E ₅ mA for 1 h at 37 ° C. After washing FSB-T five times, the MKA 1E _{2 /} / E ₅ -bin conjugate was added to the plate wells and incubated for 1 h at 37 ° C. Then, after a similar washing, an SPX solution was added to the wells of the plate and incubated for 1 h at 37 ° C. At the stage of imaging of immune complexes, a chromogen solution was added to the wells of the plate and incubated for 15 min at 37 ° C. The results were taken into account at an absorption wavelength of 450 nm. For each OD value of sample No. 1 containing the CCHF virus antigen, the positivity coefficient (Kpos) was determined as the ratio of its OD to the negative control OD of No. 2 at each dilution point of the MCA of the immunosorbent and conjugate.

The data for determining the optimal concentrations are presented in Table 1. The efficiency of detecting CCHF virus antigen in sample No. 1 was evaluated by the maximum Cfc value of MCA 1E ₂ / E ₅ -biotin (in Cfc units).

As can be seen from the table 1, the simultaneous use of MCA 1E ₂ / E ₅ to activate the immunosorbent in the concentration range of 0.15-1.5 μg / ml, and as a component of the conjugate in the working dilution of 15 μg / ml should be considered promising. The data obtained indicate a significant avidity of MCA 1E ₂ / E ₅ to epitopes of CCHF virus proteins.

Thus, the above properties of the hybridoma producer MCA allowed us to draw the following conclusion. The strain of cultured hybrid cells of the animal Mus musculus L. (the author's name is CCHFV Vd-2 cell line) is intended to produce a monoclonal antibody 1E ₂ / E ₅ against the Crimean-Congo hemorrhagic fever virus belonging to the IgG class ₁ . The concentration of immunoglobulins in the culture medium is 0.53 μg / ml. The proposed strain is a producer of monoclonal immunoglobulins - one of the components of the enzyme immunoassay system for the detection of the Crimean Congo virus hemorrhagic fever in samples of biological material.

List of sources used

1. International Health Regulations (2005). - 2-ed. - WHO, 2008. - 90 p.

2. Sanitary and epidemiological rules of the joint venture 3.4.2318-08 “Sanitary protection of the territory of the Russian Federation”.

3. Specific indication of pathogenic biological agents. Practical Guide / Ed. G.G. Onishchenko. - M., 2006 .-- 288 p.

4. Laboratory diagnosis of dangerous infectious diseases. Practical Guide / Ed. G.G. Onishchenko, V.V. Kutyreva. - M .: OJSC "Publishing house" Medicine ", publishing house" Shiko ", 2009. - 472 p.

5. MUK 4.2.3007-12 “The procedure for organizing and conducting laboratory diagnostics of the Crimean hemorrhagic fever for laboratories at the territorial, regional and federal levels”.

6. Vyshemirsky OI, Kostikov MA, Gordeeva Z.E., Melnikova E.E., Sveshnikova N.A., Gaidamovich S.L. / Isolation and identification of six strains of the Crimean hemorrhagic fever virus from patients in Tajikistan // VINITI, vol. 27, Virology, M., 1992. - pp. 53-57.

7. Vyshemirsky O.I. Analysis of the antigenic structure of Crimean-Congo virus hemorrhagic fever strains using monoclonal antibodies // Abstract of dissertation for the degree of candidate of medical science - M., 1993.

8. Gaidamovich S.Ya., Lvov A.I., Obukhova V.R. et al. Antibodies to arboviruses in ascites of immunized mice // Vopr. virusol. - 1969. - No. 6. - S.676-683.

9. Evchenko Yu.M., Lvov D.K., Sysolyatina G.V. et al. Tick infestation of Hyalomma marginatum Koch. 1844 Crimean Congo virus hemorrhagic fever in the Stavropol Territory in the epidemic season of 2000 // Vopr. virusol. - 2001. - No. 4. - S.18-20.

10. Lavrova N.A., Navolokin O.V. Enzyme-linked immunosorbent assay for the detection of arboviruses and antibodies to them // Sat: Arboviruses. - M., 1986. - S.146-153.

11. Antigen-capture enzyme-linked immunosorbent assay for the diagnosis of crimean-congo hemorrhagic fever using a novel monoclonal antibody / Saijo M., Tang Q., Shimayi B., Han L., Zhang Y., Asiguma M., Tianshu D., Maeda A., Kurane I., Morikawa S. // J. Med. Virol. - 2005. - V.77, No. 1. - P.83-88.

12. Blackburn N.K., Besselaar T.G., Shepherd A.J., Swanepoel R. Preparation and use of monoclonal antibodies for identifying Crimean-Congo hemorrhagic fever virus // Am. J. Trop Med. Hyg. - 1987. - V.37, No. 2. - P.392-397.

Table 1 Evaluation of the effectiveness of the detection of CCHF virus antigens in ELISA for different concentrations of MAB 1E ₂ / E ₅ immunosorbent and conjugate (in units of Cpos) Immunosorbent Dilution of MCA in immunosorbent, μg / ml Kpoz in the analysis of sample No. 1, containing the CCHF virus antigen, in ELISA for four concentrations of conjugate MCA 1E ₂ / E ₅ -biatin Dilution of conjugate MCA 1E ₂ / E ₅ -biotin 150 mcg / ml 15 mcg / ml 1.5 mcg / ml 0.15 mcg / ml MCA 1E ₂ / E ₅ 150 0.3 1,0 1,1 1,1 fifteen 0.8 1.3 1,0 0.9 1,5 1,0 3.0 0.9 0.9 0.15 1,0 4.1 1,0 0.9 Notes: - coefficient of positivity (Kpos) - an indicator of the ratio of the OD of the test sample No. 1 containing the CCHF virus antigens to the negative control OD No. 2; - tested antigens: No. 1 - a recombinant protein containing amino acid sequences similar to CCHF virus proteins isolate "1-2", No. 2 - negative control - a mixture of lysate proteins of the transplantable human adrenal adenocarcinoma cell line SV-13 and E. coli (probable impurities sample No. 1); - indicators Kpos selected in the table correspond to the positive results of the analysis.

Claims (1)

The strain of cultured hybrid cells of the animal Mus musculus L. CCHFV Vd-2, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-26, which is a producer of monoclonal antibody 1E ₂ / E ₅ to the Crimean-Congo virus hemorrhagic fever suitable for the manufacture of one of the components of a kit of reagents for enzyme immunoassay for identification of antigens of the Crimean Congo virus hemorrhagic fever in samples of biological material.