RU2116344C1 - Strain of cultured hybrid cells of animal mus musculus - a producer of monoclonal antibody raised to thermostable surface antigen of meliodosis pathogen - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: antibodies relate to isotype IgG_2b. Concentration of immunoglobulins in cultural medium is 0.35-0.52 mg/ml. Antibodies interact with meliodosis pathogen surface antigen, it does not interact with cells of glanders pathogen cells and therefore this type of monoclonal immunoglobulins is selected for using as raw for production of species-specific preparation of fluorescent meliodosis immunoglobulins. EFFECT: cell strain indicated above, increased specificity of interaction. 3 tbl

Description

 The invention relates to hybridoma technology and for the production of monoclonal antibodies. The strain is named MVd - 2 (1F4). The strain is deposited in a specialized collection of transplantable somatic cells of vertebrates of the All-Russian collection of cell cultures under the number VSCK (P) 648D.

 Until recently, immunodiagnostic preparations for the detection and identification of glanders and melioidosis using the method of fluorescent antibodies were made on the basis of immune specific sera of various animal species. Technological features of the production of such drugs make it difficult to obtain standard diagnostic tools by properties. At the same time, the creation of species-specific preparations based on polyclonal immunoglobulins presents certain difficulties due to the close relationship of glanders and melioidosis pathogens [1].

 However, the process of obtaining specific specific hyperimmune sera from animal producers is long and complex. In the manufacture of the drug, a stage of additional adsorption of heterologous antibodies by glanders cells is provided, which, as a rule, reduces the activity of immunoglobulin raw materials and does not ensure the absolute specificity of fluorescent drugs. These difficulties are a serious obstacle to the full reproduction of all stages of the production technology, affect the quality of individual series and make standardization of drugs difficult.

 The aim of the invention is to obtain a strain of a hybridoma producer of specific homogeneous in composition and properties of monoclonal antibodies MCA, interacting with an antigen localized on the surface of the cell wall of the melioid bacterium, a raw material for the production of immunoglobulins diagnostic fluorescent melioid species-specific monoclonal.

Hybridoma production is achieved by immunization of BALB / c mice with whole Pseudomonas pseudomallei cells, followed by hybridization of spleen lymphocytes of immunized mice with mouse myeloma cells. The concentration of immunoglobulins (IgG _2b ) in the medium is on average 0.6-1.2 mg / ml, the activity of immunoglobulins in the culture medium in TIFA 1: 640, in ascitic fluid 1: 1 • 10 ⁷ . Stability of production is maintained for 20 passages in the culture. Antibodies interact with the surface antigen of the causative agent of melioidosis, do not interact with the closely related pathogen of glanders, as a result of which this type of monoclonal immunoglobulin is selected for use as a raw material for the production of a species-specific preparation of fluorescent melioid immunoglobulins.

 The hybridoma was obtained by fusion of the myeloma of mice PZ-X63-Ag 8.653 and mouse splenocytes of the BALB / c line. The merger was carried out using polyethylene glycol followed by cloning of the producer strain by the method of limiting dilutions. The resulting strain is named MVd-2 (1F4) and is characterized by the following features.

 Cultural signs.

 In vitro cultivation. Cultivation medium is RPM1-1640 medium with the addition of 15% fetal calf serum, 2 mM L - glutamine, 10 mM HEPES, 4 mM sodium pyruvate.

Cells are cultured at 37 ^o C in an atmosphere of 5% CO ₂ and 70-80% humidity. Cells are passaged once every 3-4 days, controlling microscopically the growth rate. Multiplicity of sieving 1: 4-1: 10. Suspension culture.

Cultivation in the body of the animal. To accumulate ascites, hybridoma cells are administered to BALB / c primed mice at a dose of 1 • 10 ⁵ -1 • 10 ⁶ cells per mouse. Ascites is formed after 2-4 weeks.

 Strain productivity. The production of MCA in the culture medium is 0.6-1.2 mg / ml, in ascitic fluid - 25-30 mg / ml. MCA production in the culture fluid is maintained for 20 passages.

Characteristics of the resulting product. Antibodies belong to the IgG class _2b . They specifically interact with a specific epitope of the thermostable surface antigen of the causative agent of melioidosis. The specificity of the interaction is determined using TIFA and NMFA.

Cryopreservation. 2-4 • 10 ⁶ cells of the strain are preserved in 1 ml of RPM1-1640 medium with 20% fetal calf serum and 7% dimethyl sulfoxide in plastic ampoules. Freezing is carried out at 4 ^o C (30 min), and then placed in a complex of cryopreservation of biomaterials designed for controlled freezing of cell tissue cultures in a given mode: up to a temperature of 20 ^o C at a rate of 1 ^o C / min, up to -40 ^o C a speed of 1.5 ^o C / min, up to -70 ^o C with a speed of 5 ^o C / min. Then the ampoules are immersed in liquid nitrogen and stored in it until use. Defrosting is carried out quickly at -37 ^o C in a water bath. Viability after thawing is 65-75%.

 Example 1. The strain is obtained as follows.

BALB / c mice are immunized with ultrasonic disintegrates of cells, P. pseudomallei C-141. The first two injections with an interval of one week are performed subcutaneously at a dose of 5 • 10 ⁸ mk. with incomplete Freund's adjuvant (1: 1) in a volume of 0.3 ml and intraperitoneally at a dose of 1 • 10 ⁹ mk in a volume of 0.5 ml of physiological saline. The boosting administration of antigen is performed on 21-22 per day after the start of immunization intrasplenicly at a dose of 5 • 10 ⁸ mk cells. in 0.1 ml of physiological saline. 3 days after that, 1 • 10 ⁸ splenocytes of immune mice hybridize with 1 • 10 ⁷ mouse P3-X63-Ag8.653 mouse myeloma cells in the presence of 1 ml of 50% polyethylene glycol per mol.m. 4000 for 2 minutes 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 1000 rpm for 10 minutes The precipitate was resuspended in a selective medium GAT medium with 15% fetal calf serum, 2 mm L - glutamine, 4 mm sodium pyruvate. Cells are seeded in a 96-well plate with a feeder of 2.5-5 x 10 ⁵ cells per well. Change of environment is carried out after 3-4 days. The selection of hybrid producers in the wells of the experimental plate is determined by the presence of specific immunoglobulins in the culture medium using TIFA. Identified producer hybridomas are cloned at least twice by limiting dilution on RPMI-1640 medium with 15% fetal calf serum supplemented in 96-well plateaus with feeder - mouse peritoneal macrophages (4 • 10 ⁴ cells per well). After the second cloning, almost all 100% of the subclones produce MCA.

 Among the productive clones secreting MCAs against antigenic determinants of glanders and melioidosis, the clone producer MVd-2 (1F4) was selected.

 Example 2. The accumulation of MCA.

Ampoules with hybridoma cells are removed from a vessel with liquid nitrogen and quickly thawed in a water bath at 37 ^° C. Thawed cells are transferred to a centrifuge tube with 20 ml of serum-free RPMI-1640 medium and the cell suspension is carefully layered onto the medium. Cells are pelleted by centrifugation and, after removal of the washing medium, plated on a 25 cm ² mattress on RPMI-1640 medium with 15% fetal calf serum. The mattress is incubated in a humid atmosphere with 5% CO ₂ at 37 ^o C. When the cell colony covers more than a third of the bottom of the mattress, culture medium samples are taken to determine the titer of MCA using TIFA. When the bottom of the mattress is completely covered by hybrid cells, they are sieved into several mattresses. In this way, in vitro hybridoma cells are replicated.

To obtain a more concentrated antibody preparation, producer hybridomas accumulate BALB / c mice in the ascitic fluid. Animals are pre-sensitized by intraperitoneal administration of pristan - 2,6,10,14-tetramethylpentadecane (0.3 ml per mouse) or incomplete Freund's adjuvant (0.5 ml per mouse). After a week, mice were injected intraperitoneally with 1 • 10 ⁵ -1 • 10 ⁶ hybridoma cells. After the appearance of ascitic fluid and solid tumors in mice, the contents of the abdominal cavity are collected. Cells are pelleted by centrifugation, ammonium sulfate is added to the supernatant to its 50% saturation. The loose precipitate was centrifuged at 10,000 rpm for 20 minutes, the supernatant was removed. A dense precipitate is dissolved in physiological saline to the original volume, dialyzed against the same solution for 24 hours at 4 ^o C until ammonium sulfate is completely removed. The deposition of MCA with ammonium sulfate to 50% saturation is repeated three times, as described above. In the resulting solution of purified MCAs, the protein concentration is determined spectrophotometrically. A 1% bovine serum albumin solution is used as standard.

The specific activity of MCA is determined using TIFA and NMFA. When setting TIFA, polystyrene plates are treated overnight at 4 ^° C. with a water-salt extract of P. pseudomallei 56830 or P.mallei 10230 diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.5, to a concentration of 1-10 μg / ml of protein. Then the plates are washed three times with buffered saline with 0.05% Tween-20. At the same solution, titrated MCA samples were titrated in a volume of 100 μl, which was kept in the plates for 1 h at 37 ^° C. After the next three times washing of the plates, an antispecies conjugate was added in working dilution prepared using EGF with tween-20. The conjugate is rabbit anti-mouse IgG labeled with horseradish peroxidase. An incubation time of 1 h at 37 ^o C, a volume of 100 μl per well. Repeat washing again. In conclusion, a mixture is introduced - hydrogen peroxide with 5-aminosalicylic acid. After incubation at room temperature for 40 min - 1 h, the optical density of the liquid in each well was measured at a wavelength of 405 nm. MCAs isolated from ascitic fluid in TIFA are active at a dilution of 1: 5 • 10 ⁵ -1 • 10 ⁷ .

The specific activity of MCA is evaluated in an indirect method of fluorescent antibodies with a commercial antiviral conjugate of luminescent immunoglobulins against mouse globulins. MCAs isolated from mouse ascitic fluid in NMFAs are active at a dilution of 1: 1 • 10 ³ -1: 10 ⁴ .

 The average volume of ascitic fluid obtained by culturing the hybridoma MVd-2 (1F4) in animals is 5.6 + 0.14 ml; in vivo producing activity is 25-30 mg of monoclonal immunoglobulins per ml of ascitic fluid.

 Example 3. The use of MVA MVd-2 (1F4) for the manufacture of immunoglobulins for diagnostic fluorescent melioid species-specific monoclonal.

To obtain immunodiagnostic drugs, MVA-2 (1F4) MCA samples with a titer of at least 1: 5 • 10 ³ -1 • 10 ⁴ in NMFA are used. Conjugation of immunoglobulins with fluorochrome is carried out with constant stirring, a temperature of +4 ^o C and monitoring the pH of the solution. At the same time, fluoresceinisothiocyanate (FITC) dissolved in 1 ml of 0.1 M NaHCO ₃ , pH 9.5 at the rate of 25 mg per 1 g of protein is fractionally added to the protein solution for 30-40 minutes. During this time, the pH of the solution was maintained at 8.5-9 with 0.1 M NaHCO ₃ . After stabilization of the pH of the solution, conjugation is continued for 2 hours. Purification of the conjugate from unbound fluorochrome is carried out on columns with Sephadex G-25 eluting with 0.1 M phosphate buffer, pH 7.2 at a rate of 40-50 ml / h. The quality of the prepared conjugates is evaluated by the protein concentration index and the FITC / protein molar ratio. The calculations were carried out according to the formulas: M _fitts / M _protein = 2.87 OD ₄₉₅ / OD ₂₈₀ - (0.35 OD ₄₉₅ ); Sat = OD ₂₈₀ - (0.35 OD ₄₉₅ ) / 1.4. Spectrophotometrically determine the optical density of the whole conjugate and its dilutions at a wavelength of 280 and 495 nm.

The protein concentration in the finished product should be equal to 5 + 1, the molar ratio - 7.0 + 3.0. Lyophilized preparations MKA-FITZ stored in ampoules at +4 ^o C for 2 years.

 Example 4. Evaluation of the activity, specific activity and specificity of immunoglobulins diagnostic fluorescent melioid species-specific monoclonal.

Working dilution of the preparations was determined on fixed smears prepared from bacterial suspensions of 5 typical P. pseudomallei strains with a concentration of 5 • 10 ⁸ mk / ml.

 The results of determining the titer and working dilution of the conjugates are presented in table. one.

 The data presented in Table 1 indicate that fluorescent preparations based on MVA-2 (1F4) MCAs exceed the capabilities of commercial polyclonal analogs in terms of activity parameters. Working dilution of MCA MVd-2 (1F4) conjugated to FITC, 1: 64-1: 128 and higher. The minimum concentrations of MKA-FITZ, providing staining of bacteria by 3-4 + for various series of preparations, ranged from 1 to 4 mg / ml for commercial preparations: 0.16-0.8 mg / ml for commercial preparations ml

The specific activity and specificity of MCA 1F4 conjugated to FITC is evaluated in MPA on fixed swabs from living cultures of Pseudomonas mallei, closely related (Pseudomonas cepacia, P.putida, P.aeruginosa, P. fluorescens) and heterologous species of microorganisms prepared from suspensions with 1 • 10 ⁷ mk / ml (tab. 2).

 As a comparison drug (prototype), a commercial preparation manufactured by VolgNIPCHI is used: immunogloulins diagnostic melioid adsorbed species-specific luminescent.

 Thus, the data given in table. 1, 2, indicate that the developed diagnostic monoclonal LIG for MFAs are highly active and specific.

It should be noted that the sensitivity of MFAs when staining smear preparations with monoclonal preparations remained at the level of 1 • 10 ⁴ - 5 • 10 ⁴ m.k. / ml. During this stage of the study, smear preparations used suspensions of 5 P.mallei strains and 5 P.pseudomallei strains prepared in concentrations of 5 • 10 ⁵ , 1 • 10 ⁵ , 5 • 10 ⁴ , 1 • 10 ⁴ , 5 • 10 ³ m .k./ml. The results of the experiments are presented in table. 3.

 Thus, as a result of the studies, methods were developed for the preparation of standard fluorescent melioid monoclonal species-specific preparations, and their high sensitivity and specificity in MPA were established when P. pseudomallei was detected and identified. In terms of activity parameters, the diagnostic preparation for MFA obtained on the basis of MVA MVd-2 (1F4) surpasses the commercial polyclonal analogue.

 The advantages of using monoclonal antibodies as the basis for the construction of diagnostic fluorescent melioid preparations are stability, homogeneity of immunoglobulin raw materials, higher specific activity per 1 mg of protein, in which reliable indication of the causative agent of melioidosis is possible, and almost 100% reproducibility of experimental results. The use of stable hybrid cell lines deposited in the All-Russian Specialized Collection of Cell Cultures of the Russian Academy of Sciences (St. Petersburg) provides specific immunoglobulins in the required quantities for the production of standard immunodiagnostic preparations for MPA.

Claims (1)

 The strain of cultured animal hybrid cells Mus. musculus VSCK (P) 648 D - producer of monoclonal antibodies to the thermostable surface antigen of the causative agent of melioidosis.

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