RU2535982C1 - Strain of cultivated hybrid animal cells mus musculus l cchfv vd-1-producer of monoclonal antibody 4g4/b6 to virus of crimean-congo hemorrhagic fever (cchf) - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention describes a strain of cultivated hybrid animal cells Mus musculus L. CCHFV Vd-1. The strain is obtained by hybridisation of two types of cells: splenocytes of an inbred mouse of BALB/c line, which is immunised with an antigene of Crimean-Congo hemorrhagic fever virus (CCHF), and cells of mouse myeloma Sp 2/0. The strain - IgG1 MCA producer to CCHF virus is called CCHFV Vd-1 (4G4/B6). The strain is deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-OBOLENSK" and is provided with number H-25. The strain is a producer of raw material for preparation of a diagnostic product for a method of fluorescent antibodies, as well as a source for obtaining one of the components of a set of monoclonal reagents for immunoenzymometric detection of antigens of CCHF virus.

EFFECT: invention can be used in medical practice to detect Crimean-Congo hemorrhagic fever virus in clinic or sectional specimens to specify a diagnosis and to solve scientific and research tasks on study of properties of CCHF virus and on creation of diagnostic preparations against Crimean hemorrhagic fever.

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Description

The invention relates to biotechnology, in particular to a method for producing hybridomas producing monoclonal antibodies (MCAs) of a given specificity, and can be used in medical practice to detect the Crimean Congo virus hemorrhagic fever (CCHF) in clinical or sectional samples to clarify the diagnosis, and to solve research problems to study the properties of the CCHF virus, the creation of diagnostic drugs against the Crimean hemorrhagic fever (CFS), a particularly dangerous natural focal infection. The strain producing monoclonal antibodies to the CCHF virus is named CCHFV Vd-1 (4G ₄ / B ₆ ). The strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-25.

When performing work on the detection of the pathogen in samples of clinical and field material, they are guided by the principles for organizing studies of material suspected of being infected with CCHF virus in institutions by the Practical Guides [1, 2], as well as in MUK 4.2.3007-12 [3] Rospotrebnadzor. According to these documents, the regulated immunodiagnostic methods for detecting CCHF virus are the sandwich variant enzyme-linked immunosorbent assay (ELISA) and the method of fluorescent antibodies (MFA).

To reproduce the first of these two methods, the “VeskoKrym-KGL-antigen” enzyme-linked immunosorbent assay for detecting the Crimean-Congo virus hemorrhagic fever is produced (Vector-Best CJSC, Russia; registration number RK-IMN-5 No. 007764, date registration 12/14/2010), made on the basis of polyclonal antibodies to the CCHF virus.

The use of MFAs in practical laboratories has a number of difficulties associated with the lack of the medical immunobiological preparations “Immunoglobulins diagnostic fluorescent for detecting CCHF virus” in the domestic market.

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.

The possibilities of applying MCA to the CCHF virus were reported by domestic and foreign authors [4, 5, 6, 7]. They obtained experimental samples of drugs for use in enzyme-linked immunosorbent and immunofluorescence methods for the detection of CCHF virus. Despite the fact that these authors studied the possibility of manufacturing immunodiagnostic drugs with high quality indicators based on MCA for the CCHF virus to detect this pathogen, the production of commercial drugs in our country has not been mastered.

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. ФСР 2010/07327). Due to the lack of standard drugs for quality control of laboratory diagnosis of CCHF, the relative sensitivity and specificity of this kit is unknown.

There are no registered preparations of fluorescent antibodies for the detection of CCHF.

The purpose of the invention is to obtain a strain of a hybridoma producer of highly specific monoclonal antibodies, the basis for the manufacture of immunodiagnostic means for detecting CCHF virus.

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.

The hybridoma producer MCA 4G ₄ / B _{6 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, Merck, Germany), followed by screening of the cell suspension in wells of 96-well culture plates, cultivation in selective medium, selection of the primary clone, its cloning and reclone by limiting dilution method. The resulting strain, named CCHFV Vd-1 (4G ₄ / B ₆ ), 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 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 at 1 · 10 ⁶ -2 · 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 0.64 μg / ml, in ascitic fluid - 10-12 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 amounts of MCA 4G ₄ / B ₆ , 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 then at a speed of 5 ° C per minute - from minus 40 ° C up 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 75% 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 intrasplenicly (20 μg of antigen in 0.3 ml of FR). 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 gentle stirring. Then, with constant stirring, 1, 2, 4, and 8 ml of serum-free medium are 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 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. 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 CCHF virus antigens, the clone producer CCHFV Vd-1 (4G ₄ / B ₆ ) 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 over large areas with the obligatory control of antibody production. In vitro cultivation conditions, tactics for selecting and replacing the culture medium, sieving the breeding cell population are the same as described above (example 1). If these conditions are correctly met, MCA production is restored to the level of 0.56-0.64 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. The animals are pre-administered intraperitoneally with 0.4 ml of sterile mineral oil, pristan (2,6,10,14-tetramethyl-pentadecane) or incomplete Freund's adjuvant. After 5-7 days, mice are injected intraperitoneally with 1 · 10 ⁶ -5 · 10 ⁶ hybridoma cells. After the accumulation of ascitic fluid, it is collected. Cells are pelleted by centrifugation, the supernatant is separated and used to isolate the antibodies by sulfate triple protein reprecipitation at 50% saturation with ammonium sulfate. The loose precipitate is centrifuged at 10000-15000 rpm for 20 minutes, the supernatant is 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 checked using TIFM. The day before the reaction is set up, 100 μl of antigen solution (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 enzyme-linked immunosorbent assay. The plate was 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 was performed with a 1% solution of bovine serum albumin, adding to each well 150 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 step 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 , washing three times and adding 100 μl of antispecific conjugate in working dilution. The conjugate is a diagnostic antibody against white mouse IgG (H + L) labeled with horseradish peroxidase. The plate is incubated at 37 ° C for 1 h, then 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 4G ₄ / B ₆ , isolated from ascitic fluid, are active in dilutions of 1:10 ⁵ -1: 10 ⁶ .

The average volume of ascitic fluid obtained from one mouse when culturing a CCHFV Vd-1 (4G ₄ / B ₆ ) hybridoma in vivo is 3-4 ml, and the concentration of MCA is 10-12 mg / ml.

Example 3. Verification of the specific activity of MCA 4G ₄ / B ₆ in ELISA.

To assess the effectiveness of the use of the obtained MCAs in solving the problem of improving the means of immunological diagnostics of CHF, the specific activity of MCA 4G ₄ / B ₆ in the reaction with a number of CCHF virus antigens was studied. As a reference panel, weak dilutions of three samples containing CCHF virus antigens and one concentrated sample that did not contain 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 State collection of causative agents of viral infections and rickettsioses FBUN SSC WB "Vector" under the number VK 28848.

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

The diagnostic design is based on a direct three-step enzyme-linked immunosorbent assay for the double sandwich variety. Its basis is made up of two components: an immunosorbent plate with immobilized on the surface of the MCA wells to the CCHF virus proteins and a MKA-biotin conjugate that detects the formed immune complexes of the immunosorbent antibodies with CCHF virus antigens. To increase the sensitivity of the diagnostic test system, two conjugates are 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 choice of a variant of MCA suitable for performing the function of “capturing” the antigen, and the selection of its optimal concentration during the preparation of immunosorbent was performed as a result of a comparative analysis of the data obtained in the experiment with three separately cultivated MCAs (1E ₂ / E ₅ , 3H ₆ / F ₂ , 4G ₄ / B ₆ ) from the working panel of monoclonal immunoglobulins to CCHF virus antigens. The most effective antibody adsorbed on the plate was MCA 3H ₆ / F ₂ , used at a concentration of 1.5 μg / ml.

After determining the variant of MCA for use at the stage of preparing the solid phase for ELISA (a tablet with antibodies adsorbed on the surface of its wells with the function of "capturing" the desired antigen), it was necessary to prepare a conjugate of a "detecting" antibody with biotin.

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 conjugate MCA 4G ₄ / B ₆ -biotin obtained according to this method. The optimal concentration was determined in ELISA by using four consecutive dilutions of 10 times different biotinylated MCA separately, starting with a maximum of 150 μg / ml.

The initial samples of the antigens of the reference panel were diluted 100 times with Tris-EDTA solution with the addition of Triton X-100 and incubated for 1 h at 37 ° C. After washing FSB-T five times, the biotinylated conjugate MCA 4G ₄ / B ₆ -bin 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 of three samples containing CCHF antigens (No. 1, No. 2, and No. 3), a 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 the MCA of the immunosorbent and biotinylated conjugate.

The data for determining the optimal concentration of the conjugate MCA 4G ₄ / B ₆ -biotin to perform a sandwich ELISA are presented in table 1.

Table 1 Determination of the optimal dilution of the conjugate MCA 4G ₄ / B ₆ -biotin, which provides effective detection of CCHF virus antigens in the studied samples using the ELISA sandwich Conjugate Conjugate Dilution (μg / 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 MCA 4G ₄ / B ₆ -bin 150 1,1 1,1 1,1 fifteen 0.9 1,0 0.9 1,5 12.9 13.0 13.0 0.15 0.5 0.9 1,2 Notes: - as first-order antibodies sorbed on an ELISA plate that perform the function of “capturing” the desired antigen, MCA 3H ₆ / F ₂ at a concentration of 1.5 μg / ml was used; - 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 untreated CCHF virus; - positivity coefficient (Cpos) is an indicator of the ratio of the OD of the test sample containing CCHF antigens to the negative control OD at each dilution point of the 4G ₄ / B ₆ biotinylated conjugate; - indicators Kpos selected in the table correspond to the positive results of the analysis.

The detection efficiency of CCHF virus antigens was assessed by the maximum Kpos index.

As can be seen from the table, the most effective detection of antigen in three samples containing antigens of the CCHF virus (No. 1, No. 2 and No. 3) occurs when conjugate MCA 4G ₄ / B ₆ -bibin in a final dilution of 1.5 μg / ml .

Example 4. The use of MCA 4G ₄ / B ₆ for the detection of CCHF virus by the method of fluorescent antibodies. When using MCAs as raw materials for the manufacture of a diagnostic preparation for MFAs, monoclonal immunoglobulins are labeled with fluorochrome - fluorescein isothiocyanate (FITC) [8]. Labeled with fluorochrome are MCAs accumulated in vivo. The conjugation procedure of MCA with fluorochrome is carried out under the following conditions: room temperature, constant mixing of the reagents, FITC load - 25 mg per 1 g of immunoglobulins, fractional introduction of fluorochrome solution during the first 30 minutes, total conjugation time - 2.5 hours. All of these steps perform with constant monitoring of the pH of the solution, avoiding exceeding this indicator more than 9.0. Subsequent purification of the conjugate from unlabeled protein and unbound dye was carried out on a Sephadex G-25 column using phosphate buffer, pH 7.2, to elute ODM. When calculating the indicators of protein concentration and molar ratio M _FITC / M _protein , The T.N. and Feltkamp T.E. [9]. After determining the working dilution of the drug, it is amplified, lyophilized or stored at minus 20 ° C. The purpose of the drug is the detection of CCHF virus in samples of biological material by the method of MFA. The diagnostic capabilities of the experimental preparation of fluorescent monoclonal immunoglobulins prepared on the basis of 4G ₄ / B ₆ immunoglobulins were tested in MFAs using fixed and disinfected smears of the organs of six deceased patients with a diagnosis of fever of unknown origin. When viewing smears-prints of the organs of five patients, positive results were obtained and in one - negative. In the same five patients, the diagnosis of Crimean hemorrhagic fever was subsequently confirmed in the laboratory. In a patient with a negative result in MFA, the diagnosis of CHF by other methods was also not confirmed.

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-1) is designed to obtain a monoclonal antibody 4G ₄ / B ₆ to the Crimean-Congo hemorrhagic fever virus belonging to the class IgG ₁ . The concentration of immunoglobulins in the culture medium is 0.64 μg / ml. The strain is a producer of raw materials for the manufacture of diagnostic tools for the detection of the Crimean Congo virus hemorrhagic fever in samples of biological material.

Information sources

1. 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.

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

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

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

5. Vyshemirsky OI Analysis of the antigenic structure of Crimean-Congo virus hemorrhagic fever strains using monoclonal antibodies // Abstract of dissertation for the degree of Cand. honey. sciences. - M., 1993.

6. 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, N2. - P.392-397.

7. Saijo M., Tang Q., Shimayi B. et al. Antigen-capture enzyme-linked immunosorbent assay for the diagnosis of crimean-congo hemorrhagic fever using a novel monoclonal antibody // J. Med. Virol. - 2005. - V.77, N1. - P.83-88.

8. Goding J.W. Monoclonal antibodies: principles and practice. - Acad. Press Inc., London Ltd., 1986. - 2nd ed. - P.59-103.

9. The T.N., Feltkamp T.E. Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation // Immunology. - 1970. - N.18. - P.865-873.

Claims (1)

The strain of cultured hybrid cells of the animal Mus musculus L. CCHFV Vd-1, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-OBOLENSK" under the number H-25, which is a producer of monoclonal antibody 4G ₄ / B ₆ to the Crimean-Congo virus hemorrhagic fever suitable for the manufacture on its basis of regulated means for the detection of CCHF virus antigens in samples of biological material using enzyme-linked immunosorbent assay and the method of fluorescent antibodies.