RU2528869C1 - STRAIN OF CULTIVATED HYBRID CELLS OF ANIMAL MUS MUSCULUS L CCHFV Vd-3-PRODUCER OF MONOCLONAL ANTIBODY 3H6/F2 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-3 deposited in the State collection of pathogenic microorganisms and cell cultures "SCPM-OBOLENSK" under number H-27, which is a producer of monoclonal antibody 3H6/F2 to virus of Crimean Congo haemorrhagic fever (CCHF), is suitable for manufacture on its base of one of the components of a 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 (MAB) of a given specificity. The strain producing monoclonal antibodies to the Crimean Congo virus hemorrhagic fever (CCHF) is called CCHFV Vd-3 (3H ₆ / F ₂ ). The strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-27.

The invention can be used in virological and immunological studies when creating reagent kits for enzyme immunoassay for detecting CCHF virus antigen, the causative agent of a particularly dangerous transmissible natural focal human disease, which is included in the list of infectious (parasitic) diseases requiring sanitary protection of the territory of the Russian Federation [1 , 2].

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).

To reproduce this method at the stages of express and accelerated analysis of samples submitted to the study in practical laboratories that carry out epidemiological surveillance of the causative agent of Crimean hemorrhagic fever (CHF), only one kit registered for the detection of CCHF virus is currently available as a medical device in biological liquid samples (enzyme-linked immunosorbent assay kit "VektoKrym-KGL-antigen" produced by CJSC Vector Best, Novosibirsk, cat. No. D-5056, RU No. FSR 2010/07327), made on the basis of polyclonal antibodies to this virus. Registration number RK-IMN-5№007764, registration date 12/14/2010. Due to the lack of standard drugs for quality control of laboratory diagnosis of CHF, the relative sensitivity and specificity of this kit is unknown.

At the same time, it is recognized that the implementation of a modern approach to the creation of diagnostic drugs based on monoclonal antibodies designed to detect pathogens of dangerous infectious diseases has several advantages due to the ability to work with a certified production strain of the hybridoma producer of the target product (MCA) with confirmed useful properties stably preserving antibody production and proliferative activity for a long period of time, provided that it is stored in cryopreserved condition. If it is necessary to start the next cycle of accumulation of preparative quantities of a specific MCA for production purposes, the corresponding hybridoma is again removed from the frozen state, the cells are replicated and receive the necessary quantities of raw materials for the manufacture of a serial sample of a diagnostic preparation with known properties, which is a significant technological advantage compared to working with polyclonal antibodies.

In recent years, more and more attention has been paid to introducing into practice modern methods and means of detecting dangerous pathogens, including standardized immunodiagnostic drugs based on monoclonal immunoglobulins that meet the requirements for rapid detection and identification of microorganisms.

The possibilities of obtaining and using MCA for CCHF virus were previously reported by domestic and foreign authors [6, 7, 8, 9, 10, 11]. They obtained experimental samples of monoclonal preparations for ELISA, tested in laboratory conditions for research purposes. There are no data on the drugs introduced into practice.

The purpose of the invention is to obtain a strain of a hybridoma producer of highly specific MABs suitable for use as one of the components of a kit of reagents for enzyme immunoassay for detecting CCHF virus antigens.

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-producing MCA 3H ₆ / F _{3 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 selective media for three weeks with a gradual transition to a complete hybrid cell culture medium, selection of the primary clone in terms of the production of specific antibodies that recognize the homologous antigen of the CCHF virus, its cloning and meth House limiting dilution. The strain is named CCHFV Vd-3 (3H ₆ / F ₂ ), deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-27, is characterized by the following features.

Cultural signs. Cells are cultured in vitro at 37 ° C in an atmosphere of 5-7% CO ₂ and humidity 70-80%. Hybridomas were cultured using RPMI-1640 medium with 15% fetal calf serum (ETS), 2 mM L-glutamine, 10 mM Hepes, 4 mM sodium pyruvate. The nature of the growth of the culture is semi-suspension. Reseeding is done every 3-4 days, the growth rate of the cell population is monitored daily when viewing the plate wells in an inverted microscope. Multiplicity of sieving 1: 4-1: 8.

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. Vaccination of hybrid cells in the abdominal cavity is good. Ascites is formed after 2-4 weeks.

Strain productivity. MCA production in the cultivation medium is 0.51 μg / ml, in ascitic fluid - 7-8 mg / ml. The volumes of ascitic fluid are 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 amounts of MCA 3H ₆ / F ₂ , 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 from 1 · 10 ⁷ mouse Sp 2/0 myeloma cells is performed in the presence of 1 ml of 50% PEG-4000 for 2 min at room temperature with constant stirring. Then, with constant stirring, 1, 2, 4, 8 ml of serum-free RPMI-1640 medium is added to this mixture, each portion over 2 minutes. After that, the cells are centrifuged at 800-1000 rpm for 10 minutes The precipitate was resuspended in selective NAT 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. Two weeks after the start of seeding the cells in a 96-well plate, the growth medium is replaced with an HT medium with the above additives. After this period and at all subsequent stages, RPMI-1640 medium with 15% ETS, 2 mM L-glutamine, 10 mM Hepes, 4 mM sodium pyruvate is used. 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 the latter for research in the indirect variant of TIFM. The antibody production test is repeated before each change of medium. 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-3 (3H ₆ / F ₂ ) was selected.

Example 2. The accumulation of MCA in preparative quantities. 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). A cell viability test is performed, then the cells are plated in 5-6 wells 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 over large areas with the obligatory control of antibody production. In vitro cultivation conditions, tactics for selecting and replacing the culture medium, and sieving the breeding cell population are the same as described above. With the correct fulfillment of these conditions, the production of MCA 3H ₆ / F _{2 is} restored to the level of 0.50-0.52 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. One day before the reaction is set up, 100 μl of a solution of antigen (with a concentration of 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 immunoassay. 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 (FSB-T), followed by incubation for 1 h at 37 ° C, three times washing and application of the antispecific 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 3H ₆ / F ₂ allocated from ascitic fluid, active in dilutions 1:10 ⁵ -1: 10 ⁶ .

The average volume of ascitic fluid obtained from one mouse during cultivation of the CCHFV Vd-3 (3H ₆ / F ₂ ) hybridoma in vivo is 3-4 ml, the concentration of MCA is 7-8 mg / ml.

Example 3. Verification of the claimed beneficial properties of MCA 3H ₆ / F ₂ in ELISA.

To assess the feasibility of using selective obtained 3H ICA ₆ / F ₂ in a set of monoclonal reagents for the detection CCHF virus antigens was studied for their specific activity in the ELISA. As a reference panel of antigens, weak dilutions of three samples containing CCHF virus antigens and one concentrated sample not containing them were used.

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

Sample 2. Recombinant protein containing amino acid sequences similar to CCHF virus proteins isolate "3".

Sample 3. Natural untreated CCHF virus, deposited in the collection of microorganisms FBSI SSC WB "Vector" under the number VK 28848.

Sample 4. Negative control - a mixture of lysate proteins of the transplanted line of human adrenal adenocarcinoma cells SV-13 and E. coli (probable impurities of samples No. 1-3).

The diagnostic design was built according to the scheme of a direct solid-phase three-stage ELISA of a double sandwich variety. The reaction was carried out according to the following scheme. The first stage is the preparation of an immunosorbent plate with MKA 3H ₆ / F ₂ immobilized on the surface of the wells, which performs the function of “capturing” the CCHF virus antigen. The second stage is the introduction of antigen samples into the wells of the plate and the formation of the immune complex. The third stage is the introduction into the wells of the tablet conjugate MKA-biotin, revealing the formed immune complexes of antibodies of the immunosorbent with antigens of the CCHF virus. To increase the sensitivity of the reaction, 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 fixing 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 the claimed MCA 3H ₆ / F ₂ have been studied in relation to their activation of the immunosorbent. 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 3H ₆ / F ₂ four 10-fold dilutions were prepared, starting from 0.15 μg per well. The optimal concentration was established in ELISA.

A routine biotinylation procedure was used to obtain the MKA-biotin conjugate: adding sulfo-NHS-biotin to the dialyzed anti-bicarbonate solution of the MCA, followed by purification of the unbound biotin by gel filtration on a Sephadex G-25 column. Four MCA-specific biotin conjugate samples of different MCA specificity were obtained, of which the most effective MCA 4G ₄ / B _{6 was} used in application at a working concentration of 1.5 μg / ml.

At the first stage of ELISA, the initial samples of the reference panel were diluted 100 times with Tris-EDTA solution with the addition of Triton X-100 and incubated in the wells of the immunosorbent based on various concentrations of MCA 3H ₆ / F ₂ 1 h at 37 ° C. After washing FSB-T five times, the MKA 4G ₄ / B ₆ - biotin conjugate was added to the wells of the plate 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 in reactions with three different samples of CCHF virus antigens (No. 1, No. 2, and No. 3), the positivity coefficient (Cpos) was determined as the ratio of the OD of the sample containing CCHF virus antigens to the negative control OD of No. 4 at each dilution point of MCA .

Data on determining the optimal concentration of MCA 3H ₆ / F ₂ included in the composition of the immunosorbent, ensuring effective capture of antigens, are summarized in table 1.

The efficiency of detection of antigens was evaluated by the maximum index of Kpos.

The optimal concentration of MCA 3H ₆ / F ₂ adsorbed on the tablet was found to be 1.5 μg / ml (see table 1).

The data presented in table 1 indicate that when MCA 3H ₆ / F ₂ is used as a first-order antibody in the test system, and MCA 4G ₄ / B ₆ - second-order antibodies, the most efficient detection of CCHF virus antigens is achieved in all reference panel samples (No. 1, No. 2 and No. 3).

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 the cell line CCHFV Vd-3) is intended to produce a monoclonal antibody 3H ₆ / F ₂ against the Crimean-Congo hemorrhagic fever virus belonging to the class IgG ₁ . The concentration of immunoglobulins in the culture medium is 0.51 μg / ml. The strain is a producer of raw materials for the manufacture of one of the components of a kit of reagents for enzyme immunoassay of the Crimean Congo virus hemorrhagic fever in samples of biological material.

Information sources

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. Gaidamovich S.Ia., Mel'nikova E.E., Shutkova T.M., Novokhatskil A.S., Turchinskaia A.L. Characteristics of the monoclonal antibodies induced by the Crimean hemorrhagic fever virus // Virusol. - 1989. - V.34, N 2. - P.201-204.

7. Shutkova T.M., Mel'nikova E.E., Gaidamovich S.Ia., Novokharskii A.S., Turchinskaia A.L. Hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus // Zh. Mikrobiol. Epidemiol. Immunobiol. - 1989. - N6. - P.102-107.

8. Vyshemirsky OI, Kostikov MA, Gordeeva Z.E., Melnikova E.E., Sveshnikova N.A., Gaydamovich S.Ya. Isolation and identification of six strains of the Crimean hemorrhagic fever virus from patients in Tajikistan // VINITI, v.27. Virology, M., 1992, p. 53-57.

9. 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.

10. 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.

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.

Table 1 Evaluation of the efficiency of sorption on a tablet of various concentrations of MCA 3H ₆ / F ₂ for the detection of CCHF virus antigens in the setting of a sandwich ELISA with conjugate MCA 4G ₄ / B ₆ - biotin Immunosorbent Dilution, mcg / ml The indicators of the coefficient of positivity (Kpos) in individual wells of the reaction The investigated antigen samples No. 1 Number 2 Number 3 ICA 150 1,1 1,1 1,1 3H ₆ / F ₂ fifteen 0.9 1,0 0.9 1,5 12.9 13.0 13.0 0.15 0.5 0.9 1,2 Notes: - tested antigens: No. 1 - recombinant protein containing amino acid sequences similar to CCHF virus proteins isolate "1-2", No. 2 - recombinant protein containing amino acid sequences similar to CCHF virus proteins isolate "3", No. 3 - natural unrefined virus CCHF; - conjugate MCA 4G ₄ / B ₆ - biotin in working dilution; - positivity coefficient (Cpos) - an indicator of the ratio of the OD of the test sample containing CCHF virus antigens to the negative control OD at each dilution point adsorbed on the MKA 3H ₆ / F ₂ plate; - 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-3, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-27, which is a producer of monoclonal antibody 3H ₆ / F ₂ against the Crimean-Congo hemorrhagic fever virus 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.