RU2570638C1 - Strain of cultivated hybrid animal cells mus musculus l bpm vd-10 - producer of monoclonal antibody 5h11/e5 to antigen kda 200 of melioidosis pathogen - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of cultivated hybrid animal cells Mus musculus is claimed - producer of monoclonal antibodies to an epitope as part of the antigen 200 kDa, exposed on the surface of microbial cells of the melioidosis pathogen (B. pseudomallei). The strain is deposited in the State collection of the pathogenic microorganisms and cell cultures "GKPM-Obolensk" under number H-40.

EFFECT: invention enables to obtain highly specific monoclonal antibodies suitable for the manufacture on their basis of the highly active diagnostic preparation for the MFA, specifically staining the microbial cells of virulent strains of melioidosis pathogen, which does not have the cross activity against the closely related opportunistic Burkholderia.

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Description

The invention relates to biotechnology for producing hybridomas producing monoclonal antibodies (MAB) of a given specificity. Bpm Vd-10 (5H ₁₁ / E ₅₎ strain producing monoclonal antibodies to an antigen of 200 kDa named. The strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number H-40.

The invention can be used in research work to study the properties of collection strains of pathogenic and non-pathogenic burkholderia, as well as for the manufacture of diagnostic fluorescent monoclonal immunoglobulin preparation for the method of fluorescent antibodies (MFA), which can detect and identify virulent strains of the pathogen melioidosis, but not possessing reactivity against closely related burkholderii III-IV pathogenicity group.

The use of MCA in immunodiagnostic reactions provides high sensitivity and specificity of tests aimed at detecting pathogens of dangerous and socially significant diseases [1, 2, 3]. A significant advantage of working with MCA is the presence of a constant source of their production - producer hybridomas. The use of MCAs in immunofluorescence analysis, as a rule, allows one to increase the probability of detecting microorganisms in the test material, eliminate background reactions, and increase the specificity of immunoassay [4, 5].

In the production of immunobiological agents for the detection of pathogenic microorganisms, MCA has long been given priority as the most sensitive component of modern drugs and test systems used for the rapid and reliable detection of various pathogens.

The first publications on the use of melioidosis and glomerular MCAs as ingredients in various immunodiagnostic tests designed to detect, identify the causative agents of melioidosis and glanders and their differentiation from closely related species of burkholderii date back to the late 80s - early 90s of the last century [6, 7, 8, 9].

Abroad, MCAs for B. pseudomallei antigens are used in a number of traditional immunodiagnostic tests, used mainly in endemic regions where the infection spreads, to identify the secreted burkhhold cultures and their differentiation from other representatives of the genus Burkholderia [10].

Domestic experience in the use of MCA in the production of drugs for MFAs confirmed the significant advantages of monoclonal raw materials [11, 12].

To differentiate two species of microorganisms close in antigenicity, B. mallei and B. pseudomallei, MCAs were obtained for surface-localized antigens of these bacteria [5, 11]. It was found that when checking the spectrum of specific activity "Immunoglobulins diagnostic fluorescent melioid monoclonal" interact with 75-80% of the collection strains of this microorganism. The basis of the drug is MCA (1F ₄ ) against one of the epitopes in the composition of the cell wall of B. pseudomallei. It was later found that this drug has cross-activity against B. thailandensis, a closely related non-pathogenic bacterium.

It should be noted that the creation of immunodiagnostic agents for differentiation of B. pseudomallei and B. thailandensis does not lose relevance. Data on drugs of this type, put into practice, are not available.

Further improvement of the laboratory diagnosis of melioidosis is focused on the development of immunodiagnostic drugs and test systems based on MCAs for the 200 kDa antigen (capsule glycoprotein) pseudomallei for the detection and identification of virulent strains of this pathogen. It is known that the 200 kDa antigen is a marker of virulent strains of the causative agent of melioidosis [13, 14].

The purpose of the invention is to obtain a strain of a hybridoma producer of a highly specific MAB against an epitope in the composition of a 200 kDa antigen exposed on the surface of microbial cells of the melioidosis pathogen (B. pseudomallei), suitable for use as a basis for the preparation of fluorescent immunoglobulins designed to detect virulent B. pseudomallei strains.

Obtaining a hybridoma is achieved by hybridization of two types of cells: splenocytes of an inbred mouse of the BALB / c line, immunized with the 200 kDa antigen of the causative agent of melioidosis, and mouse myeloma cells.

The hybridoma producer MCA 5H ₁₁ / E _{5 was} obtained by the fusion of BALB / c immune mouse splenocytes with mouse P3-X63.Ag8.653 mouse myeloma cells in the presence of polyethylene glycol (PEG-4000) as a conjugating agent, sieving the cell suspension into wells 96 -well culture plates, growing in selective media for three weeks with a gradual transition to a complete culture medium for hybrid cells, selection of the primary clone in terms of production of specific antibodies to the 200 kDa antigen of the melioidosis pathogen, its cloning and using the method of limiting dilution. The strain is named Bpm Vd-10 (5H ₁₁ / E ₅ ), deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number H-40, has the following characteristics.

In vitro hybridoma culturing conditions. The optimal medium for growing an in vitro hybridoma is RPMI-1640 medium with 15% fetal calf serum (ETF), 2 mM L-glutamine, 10 mM Hepes, 4 mM sodium pyruvate, pH 7.1-7.2.

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

In vivo hybridoma culturing conditions in a syngeneic animal. Hybridoma cells are injected intraperitoneally into previously primed BALB / c mice, 2 · 10 ⁶ - 4 · 10 ⁶ cells per mouse. Ascites is formed after 2-4 weeks.

Cultural signs. The growth pattern on the nutrient medium is half-suspension; in vivo, the grafting of hybrid cells in the abdominal cavity reaches 80%.

Strain productivity. MCA production in the cultivation medium is 0.27 μg / ml, in ascitic fluid - 10-12 μg / ml. The volume of ascitic fluid on average is 4.5-5.0 ml.

Characteristics of the resulting product. Antibodies belong to the IgM class. They specifically interact with the 200 kDa antigen of the melioidosis pathogen. The specificity of the interaction is determined using the enzyme-linked immunosorbent assay (TIFM).

Cryopreservation. To transfer the cells of the hybridoma producer MCA 5H ₁₁ / E ₅ to a cryopreserved state, a suspension of cells with a concentration of 4 · 10 ⁶ cells per 1 ml is prepared in a protective medium consisting of RPMI-1640 medium with 20% ETS and 7% dimethyl sulfoxide. 1 ml of suspension is transferred into plastic ampoules, which are placed in a device for controlled freezing of biological material under the mode of gradual programmable lowering of temperature. Then the ampoules are immersed in a bio-storage with liquid nitrogen and stored until use. Thawing of hybridoma cells is carried out at 37 ° C in a water bath for 2-3 minutes. Cell viability after thawing reaches 75-80%.

Example 1. Obtaining a strain of hybridoma producer

Obtaining a producer hybridoma strain consists of sequential steps for preparing partner cells (BALB / c mouse B lymphocytes immunized with 200 kDa antigen and P3-X63.Ag8.653 mouse myeloma cells) and somatic hybridization, followed by hybrid cell growth selective media, cloning of primary hybridomas by the method of limiting dilution and selection of monoclones secreting immunoglobulins directed against epitopes of the antigen used to stimulate lymphocytes.

All manipulations are performed in a laminar flow cabinet with a horizontal air flow (type "product protection").

1. Preparation of splenocytes. Immunization of a mouse donor of B-lymphocytes stimulated with a 200 kDa B. pseudomallei antigen is performed according to the scheme described in [15]. The antigen for immunization of mice is obtained by the method of extraction of glycoprotein from B. pseudomallei 100 cells according to Fuller’s modification of N. N. Piven’s, which consists in changing the temperature regime of biopolymer extraction: the stage must be carried out under milder temperature conditions (extraction at 20 ° C instead of 150 ° C recommended by Fuller) [16].

2. Preparation of myeloma cells. As a malignant partner in experiments on the hybridization of cell lines, cells of one of the most common murine myeloma lines P3-X63-Ag 8.653 are used.

By the time of hybridization, myeloma cells should be in an exponential growth phase. This is achieved by sequentially performing the following steps: culturing myeloma cells in RPMI-1640 medium with 10% fetal calf serum and 8-azaguanine for 2 weeks, then the next 2 weeks in the main medium without 8-azaguanine. Regular change of environment - every 3-4 days.

3. Hybridization of splenocytes and mouse myeloma cells. The procedure for somatic hybridization of splenocytes and myeloma cells is performed in the presence of a conjugating agent - PEG-4000 - according to the following scheme:

- a suspension of immune splenocytes (1 · 10 ⁸ cells) and myeloma cells (1 · 10 ⁷ ) are centrifuged at 800-1000 rpm for 10 min, the supernatant is decanted;

- 1 ml of 50% PEG solution is added slowly (over 2 minutes) to the cell pellet, then 1, 2, 4 and 8 ml of serum-free medium are added, 2 minutes each application. During the addition of reagents, the centrifuge cup is continuously rotated;

- after completion of this manipulation, the cell suspension is centrifuged, the supernatant is decanted, the cell pellet is resuspended in 20 ml of selective NAT medium with 15% fetal calf serum;

- a suspension of cells, 2 drops each, contribute to the wells of two 96-well plates with a pre-prepared layer of feeder cells.

All stages of the subsequent cultivation of hybrid cells, selection of growing clones secreting specific immunoglobulins, cloning, replication of cultures and accumulation of MCA in vivo are performed according to the traditional protocol [17].

For screening antibody production using the enzyme-linked immunosorbent assay [18]. 100 μl of a 200 kDa B. pseudomallei antigen solution (10 μg / ml protein) in 0.05 M carbonate-bicarbonate buffer, pH 9.5, is added to the wells of the polystyrene plate for enzyme immunoassay. The plate was kept at 4-8 ° C overnight, then washed with phosphate buffered saline with 0.05% Tween-20 (FBI-T) three times and blocked the free sections of the plastic with a 1% solution of neutral protein (bovine serum albumin), adding 30 minutes to each well, 100 μl of this solution. The plate is again washed three times. Then, for 1 h at 37 ° C, 100 μl of various dilutions (1:10, 1:20, 1:40) of the medium for growing hybrid cells from the wells of culture plates prepared in 0.1 M phosphate buffer, pH 7 are added to the wells , 4, with 0.05% Tween-20, followed by three times washing and introducing an anti-species conjugate in working dilution of 100 μl (commercial preparation of diagnostic antibodies against white mouse IgG (H + L) labeled with peroxidase). The plate is incubated at 37 ° C for 1 h, washed three times. At the end of the reaction, for 15 minutes, 100 μl of a mixture of hydrogen peroxide and chromogen are added to the wells. The plate is placed in a dark place for 15-20 minutes, then the reaction is stopped by a stop reagent. The reaction results are recorded on a tablet photometer at a wavelength corresponding to the characteristic of the chromogen used.

Antibody-producing hybrid clones are screened from 7-10 days after the sowing of the hybrid cells into 96-well plates. When viewing the wells register those of them in which there is a characteristic growth of cell groups. From these wells, quotas of the culture medium are selected 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 over the course of a month are cloned and recloned using the limiting dilution method. 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 productive clones secreting monoclonal immunoglobulins against various epitopes of antigen (Ag) 200 kDa of B. pseudomallei, the clone producer Bpm Vd-10 (5H ₁₁ / E ₅ ) was selected.

Example 2. The accumulation of MCA 5H ₁₁ / E ₅ in preparative quantities

The ampoule with hybridoma cells is taken out of the bio-storage with liquid nitrogen and quickly thawed according to the generally accepted method [19]. Next, a cell viability check is performed. In vitro culture conditions are similar to those described previously. With the consistent fulfillment of these conditions, the production of MCA 5H ₁₁ / E _{5 is} restored to the level of 0.26-0.28 μg / ml. Monoclonal immunoglobulins are isolated from culture medium samples.

In vivo replication of hybridoma cells in the abdominal cavity of a syngeneic animal to accumulate high concentrations of MCA. The 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 (At) 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 they are used as the basis of the preparation for MFA. Before amplification, part of the solution is selected to determine (1) the protein concentration in it spectrophotometrically at a wavelength of 280 nm, (2) the activity of immunoglobulins in TIFM and the indirect method of fluorescent antibodies (NMFA).

Example 3. Control of the specific activity of MCA 5H ₁₁ / E ₅ in the indirect method of fluorescent antibodies

All samples of MCA 5H ₁₁ / E ₅ prepared for subsequent use as a basis for a diagnostic preparation for MFAs must be checked in the indirect method of fluorescent antibodies to confirm that they “recognize” their epitopes homologous to them on the surface of microbial cells of the virulent strain of the melioidosis pathogen [20 , 21]. The reaction is carried out according to the scheme: test object + MCA + immunoglobulins diagnostic fluorescent anti-virus against white mouse immunoglobulins (pr-in NIIEM them. Gamalei RAS).

A suspension of microbial cells of B. pseudomallei 100 with a concentration of 5 · 10 ⁸ - 1 · 10 ⁹ is applied to thoroughly defatted glass slides (10 smears per glass). The preparations are dried at room temperature and fixed with 96% ethanol for 30 minutes. On the surface of each smear, 30 μl of MCA 5H ₁₁ / E ₅ with various concentrations are applied. Contact time Ar + At - 20-25 minutes at room temperature in a humid chamber. Then the glass is washed with buffered saline twice for 10 minutes and once with distilled water. The glasses are dried in an upright position, then stained with an antispecific conjugate in working dilution for 20 minutes, washed again and dried according to the above scheme.

Microscopy of smears is carried out using a luminescent microscope. To assess the intensity of a specific luminescence of a bacterial cell, the generally accepted four-cross system is used. For a positive result, a specific glow with a brightness of 4 or 3 crosses is taken. A negative result (1-2 crosses) indicates the absence of the desired target on the surface of the bacterial cell.

Based on the results of viewing smears, the minimum concentration of MCA is determined, which ensures a positive result in NMFA. For MCA 5H ₁₁ / E _{5, the} minimum concentration of immunoglobulins in the solution applied to the smear is 30-36 μg / ml, which indicates a high specific activity of immunoglobulins.

Example 4. Determination of the diagnostic capabilities of MCA 5H ₁₁ / E ₅ in the method of fluorescent antibodies

In the manufacture of a diagnostic drug for MFA, monoclonal immunoglobulins are labeled with fluorochrome - fluorescein isothiocyanate (FITC) [20]. For the conjugation procedure, the protein concentration in the MCA solution should be 20-25 mg / ml. The labeling procedure is carried out at room temperature and with constant stirring of the reagents. The FITC load should not exceed 25 mg per 1 g of immunoglobulins. The introduction of a fluorochrome solution is fractional during the first 30 minutes. The total conjugation time is 2.5 hours. All of the above steps are carried out with constant monitoring of the pH of the solution, avoiding exceeding this indicator by 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 0.1 M phosphate buffer, pH 7.2, for elution. When calculating the indicators of protein concentration and molar ratio M _FITC / M _protein, The T.N. and Feltkamp T.E. [22]. To determine the working dilution of the MKA-FITC conjugate, it is necessary to prepare disinfected smears-preparations from a suspension of B. pseudomallei 100 with a concentration of 5-10 m.k. / ml. After determining the working dilution, the preparation is ready for the stage of checking the spectrum of specific activity on the set of homologous strains of the causative agent of melioidosis and the stage of checking the specificity on the set of strains of heterologous burkholder. The finished and characterized preparation is ampouled, lyophilized or stored at minus 20 ° C. The intended purpose of the drug is the detection of the causative agent of melioidosis in samples from environmental objects and biological material using the MFA method.

The diagnostic capabilities of the experimental preparation of fluorescent monoclonal immunoglobulins prepared on the basis of 5H ₁₁ / E ₅ immunoglobulins are evaluated by the results of MFA. For this, we used fixed and disinfected smears of pure cultures of collection strains of burkholdery of the second pathogenicity group (B. pseudomallei - 31 strains) and conditionally pathogenic burkholdery of the III-IV pathogenicity group (B. thailandensis - 5 strains, B. cepacia - 7 strains , B. allicola, B. gladioli, B. marginata, P. putida - one strain each, P. aeruginosa - 3 strains) (table).

It was established that immunoglobulins diagnostic fluorescent monoclonal 5H ₁₁ / E ₅ specifically stain microbial cells in 71% of collection strains of the causative agent of melioidosis, but do not interact with m.k. closely related burkholderii III-IV group of pathogenicity.

Thus, the above properties of the hybridoma producer MCA 5H ₁₁ / E ₅ allowed us to draw the following conclusion. The strain of cultured hybrid cells of the animal Mus musculus L. (the author’s name is Bpm Vd-10 cell line) is intended to produce a monoclonal antibody 5H ₁₁ / E ₅ against the epitope of the 200 kDa antigen exposed on the surface of microbial cells of the pathogenic B. pseudomallei burkholdery suitable for use as a basis for obtaining a highly active diagnostic preparation for MPA that specifically stains microbial cells of virulent strains of the melioidosis causative agent, which does not have cross activity against lizkorodstvennyh opportunistic Burkholderia.

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Claims (1)

The strain of cultured hybrid cells of the animal Mus musculus L. Bpm Vd-10, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number H-40, is a producer of monoclonal antibody 5H ₁₁ / E ₅ to the epitope in the composition of the antigen 200 kDa exposed on the surface of microbial cells of the causative agent of melioidosis, suitable for the manufacture on its basis of a diagnostic preparation for MPA, designed to detect and identify virulent B. pseudomallei strains and their differentiation from strains closely related burkholderii B. thailandensis.