RU2117042C1 - Strain of cultured hybrid cells of animal mus musculus - producer of monoclonal antibodies to thermostable component of capsule-like substance of meliodosis pathogen - Google Patents

Abstract

FIELD: molecular biology, medicine. SUBSTANCE: antibodies are related to isotype Ig-G₃. Immunoglobulins concentration in cultural medium is 0.46-0.62 mg/ml. Antibodies interact with epitopes localized on the site of antigenic complex 8 (glycoprotein of molecular mass 800 kDa) that is one of the main factors of meliodosis pathogen pathogenicity. Proposed strain ensures to construct test-system using the obtained antibodies. EFFECT: enhanced level of sensitivity of test-system. 2 tbl, 4 ex

Description

 The invention relates to hybridoma technology and for the production of monoclonal antibodies. The strain is named MVd-4 (2A6). The strain is deposited in a specialized collection of transplantable somatic vertebrate cells of the All-Russian collection of cell cultures under the number BCKK (P) 649D.

 The use of monoclonal antibodies as the basis for the construction of enzyme-linked immunosorbent assay systems opens up new prospects for improving TIFM in all its variants.

 To detect glanders and medioidosis pathogens and their antigens in various research objects based on immunoglobulins of hyperimmune sera, a test system for TIFM was developed (Yakovlev A.T., Rybkin V.S., Akhmedov A.N. Particularly dangerous infectious diseases: immunology, genetics, and biology, collection of scientific works (Volgograd, 1989, pp. 111-115). However, the experience of various teams of authors indicates that the number of immunodiagnostic agents presented for the researcher's choice should include preparations for general and video-specific antigens (antigenic markers) of glanders and melioidosis pathogens.

 An enzyme-linked immunosorbent assay system using MCA (Ismail G., Embi MN, Omar O. A competitive immunosorbent assay for detection of Pseudomonas pseudomallei extoxin. - J. Med. Microbiol, 1987, Vol. 23 was developed and tested to detect exotoxin Pseudomonas pseudomallei. , N 4, p. 353 - 357). MCAs proved to be highly specific reagents in TIFM for the detection of P. pseudomallei exotoxin in extremely low concentrations in the blood of sick people. The proposed test system for TIFM with MCA was highly sensitive (16 ng / ml) and was recommended as a reliable test for the early diagnosis of melioidosis.

 The purpose of the invention is to obtain a strain of a hybridoma producer of specific monoclonal antibodies homogeneous in composition and properties, which interact with the thermostable component of the capsule-like substance of the melioidosis pathogen, the basis for the construction of an enzyme-linked immunosorbent assay system.

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 ₃ ) in the medium is on average 0.46 - 0.62 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 epitopes localized at the site of antigenic complex 8 (glycoprotein with a molecular mass of 800 kD), one of the main pathogenicity factors of the causative agent of melioidosis exposed extracellularly in a capsule-like substance surrounding the cell of the melioid bacteria (Piven N. N., Smirnova V. I., Viktorov D.V. Immunogenicity and heterogeneity of surface antigen 8 P. pseudomallei. - Journal of Microbiology, Epidemiology and Immunobiology, 1996, N4, p. 75 - 78).

 The hybridoma was obtained by fusion of P3-X63-Ag 8.653 mouse myeloma and BALB / c mouse splenocytes. The merger was carried out using polyethylene glycol followed by cloning of the producer strain by the method of limiting dilutions. The resulting strain was named MVd-4 (2A6) and is characterized by the following features.

 Cultural signs. In vitro cultivation. Cultivation medium is RPMI-1640 medium supplemented with 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 to 4 days, controlling microscopically the growth rate. The ratio of sieving 1: 4 - 1: 10. The 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 to 4 weeks.

 Strain productivity. MCA production in the cultivation medium is 0.46–0.62 mg / ml, in ascitic fluid 35–40 mg / ml. MCA production in the culture fluid is maintained for 20 passages.

Characteristics of the resulting product. Antibodies belong to the class of IgG ₃ . They specifically interact with a specific epitope of the thermostable antigen of the melodosis pathogen. The specificity of the interaction is determined using TIFM.

Cryopreservation. 2 - 4 • 10 ⁶ cells of the strain are preserved in 1 ml of RPMI-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: to a temperature of -20 ^o C at a rate of 1 ^o C / min, up to -40 ^o C at a speed of 1.5 deg / min, up to -70 ^o C at a speed of 5 deg / 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 70 - 75%.

Example 1. The strain is obtained as follows. BALB / c mice are immunized with ultrasonic disintegrates of P. pseudomallei C-141 cells. 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 in a volume of 0.3 ml and intraperitoneally in a dose of 1 • 10 ⁹ m. to. in a volume of 0.5 ml of physiological saline. The boosting administration of antigen is carried out on the 21st-22nd day after the start of immunization intrasplenicly at a dose of cells of 5 • 10 ⁸ mk. in 0.1 ml of physiological saline. 3 days after this, 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 • 10 ⁵ cells per well. The change of environment is carried out after 3 to 4 days. The selection of producer hydride in the wells of the experimental plate is determined by the presence of specific immunoglobulins in the culture medium using TIFM. 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-4 (2A6) 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 ^o C. Thawed cells are transferred to a centrifuge tube with a 20 ml serum-free medium RPMI-1640 carefully layered cell suspension 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 Freund's incomplete adjuvant (0.5 ml per mouse). After a week, 1 • 10 ⁵ - 1 • 10 ⁶ hybridoma cells are intraperitoneally injected into mice. 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 50% of its 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 the MCA is determined using TIFM. When setting TIFA, polystyrene plates are treated overnight at 4 ^° C. with 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 anti-species conjugate was added in working dilution prepared on PBS 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, make a mixture of hydrogen peroxide with 5-aminosalicylic acid. After incubation at room temperature for 40 minutes 1 hour, 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: 1 • 10 ⁵ - 1 • 10 ⁷ .

 The average volume of ascitic fluid obtained by culturing the hybridoma MVd-4 (2A6) in animals is 3.5 + 0.12 ml, producing an in vivo activity of 35-40 mg of immunoclonal immunoglobulins per ml of ascitic fluid.

Example 3. Construction of a test system for TIFM with MCA MVd-4 (2A6) for the detection of antigen 8 P. pseudomallei in various objects of study. Detection of antigen 8 in various antigenic mixtures, fractions, and cell suspensions of P. mallei and P. pseudomallei was carried out using the TIFM sandwich variant with MCA. MKA-based MVd-4 (2A6) immunoperoxidase conjugate was prepared according to the method of Mathisen et al. (Enzyme-linked immunosorbent assay for detection of hepatitis A antigen in sera: comparison with solid phase radioimmunoassay, immune electron microscopy and immune adherence hemagglutination assay / Mathiesen LR, Feinstone SM, Wong DS et al. // J. Clin. Microbiol, 1987 , N7, p. 184 - 193). In this case, 5 mg of horseradish peroxidase is dissolved in 1 ml of 0.3 M NaHCO ₃ and 0.25 ml of 0.32% formalin solution is added. Then, 1 ml of 0.04 M NaIO ₄ , 1 ml of 0.16 M ethylene glycol are fractionally added to the solution and dialyzed against 0.01 M carbonate-bicarbonate buffer, pH 9.5 at 4 ^° C for 18 hours. Conjugation of immunoglobulins (5 mg / ml in 0.01 M carbonate-bicarbonate buffer, pH 9.5) with the enzyme is carried out for 2 hours at room temperature. Then, 5 mg of NaBH _{4 was} fractionally added to the resulting solution and dialyzed against 0.01 M phosphate buffer, pH 7.4 at 4 ^° C for 18 hours. The conjugate was purified on G-100 columns eluting with 0.1 M phosphate buffer pH 7.4. The optical density of the conjugate fractions is determined spectrophotometrically at a wavelength of 403 and 280 nm. The fractions with the ratio of optical densities (OD ₄₀₃ : OD ₂₈₀ ) 0.4 - 0.5 are combined. The concentration of protein in the conjugate ranges from 0.2 - 0.3 mg / ml, RZ = 0.45 - 0.5, working dilution 1: 150 - 1: 200. The activity of the conjugate is evaluated in TIFM with water-salt extract of P. pseudomallei 56830 by the method of chess titration. The conjugate is used in the TIFM sandwich variant to detect P. pseudomallei antigen 8.

As first-order antibodies for sensitization of the plates, use: 1) samples of MAB 2 A6 interacting with antigen 8 (AG8); 2) additive mixtures of MCA 2 - 4 types. MCA diluted in carbonate-bicarbonate buffer, pH 9.5, to a concentration of 10 μg / ml, applied to a plate of 100 μl, incubated at 4 ^o C for 18 h. Then the plates are washed three times with buffered saline with 0.05% tween -twenty. Additionally, the plate wells are treated with a 1% solution of bovine serum albumin (100 μl each). After 30 minutes of exposure, the wells are washed with buffered saline with 0.05% Tween-20. The same solution is used for titration of samples of the test material containing the antigen in a volume of 100 μl, which are kept in the plates for 1 h at 37 ^o C. After the next three times washing of the plates, the immunoperoxidase conjugate is added in working dilution prepared on PBS with tween-20 . An incubation time of 1 hour at 37 ^o C, a volume of 100 μl per well. Repeat washing again. In conclusion, make a mixture of hydrogen peroxide with 5-aminosalicylic acid. After incubation at room temperature for 40-60 minutes, the optical density of the liquid in each well is measured at a wavelength of 405 nm. The sensitivity of the developed enzyme-linked immunosorbent assay system with MCA is presented in table 1.

 The high efficiency of the use of MCA compared with traditionally used polyclonal immunoglobulins is shown in column 5 horizontally (Table 1). When using globulins isolated from hyperimmune melioid goat serum (analogue) as a “substrate”, the sensitivity did not exceed 50 ng / ml protein in 1 ml, i.e. was 1 - 100 times lower.

 The maximum sensitivity of TIFM was noted in cases of using MCA pairs (2A6 + 2H7 or 2A6 + 1E5) for sensitizing MCA plates with an additivity index of more than 40%. The use of a pair of MCA or more monoclonal ingredients that are similar in specific direction, but interacting with different AG8 epitopes, increased the likelihood of antigen detection from test samples of experimental material.

 The use of MCA mixtures in preparing the tablet for the reaction provided detection of 0.37 - 3.7 ng / ml PS and 0.5 - 5 ng / ml protein (2A6 + 1E5 and 2A6 + 1E5 + 1G2 + 2H7). The specificity of the developed enzyme-linked immunosorbent assay system was confirmed by the negative results of TIFM when using a first-order antibody mixture of MAB to antigen 6 epitopes (protein-lipopolysaccharide complex localized in the cell wall of glanders and melioidosis pathogens).

 Thus, the maximum sensitivity of the method with respect to AG8 was noted when using monoclonal ingredients as first-order antibodies of the first order plastic layers: 1) a mixture of four types of MCA; 2) MCA pairs (2A6 + 1E5 and 2A6 + 1G2); 3) MCA 2A6.

 Example 4. The use of an enzyme-linked immunosorbent assay system with MCA for the detection of AG8 in extracellular antigenic mixtures from cultivation media of Pseudomonas pseudomallei and Pseudomonas mallei.

 Antigen 8, one of the pathogenicity factors of the causative agent of melioidosis, has also been identified in a number of glanders pathogen strains. To study the dynamics of accumulation of this antigen and evaluate its total content in liquid nutrient media of microorganism cultivation, the developed test system for TIFM with MCA can be used.

 Presented in the table. 2 data of the research results indicate the effectiveness of the proposed enzyme-linked immunosorbent assay system based on MCA for detecting the minimum concentrations of AG8 in extracellular antigenic mixtures from cultivation media of P. pseudomallei and P. mallei.

 The data table. 2 indicate that the levels of glycoprotein (AGS) in the growing medium are subject to wide fluctuations and depend on the species and strain affiliation of the microorganism cultivated in a liquid medium.

 In addition, this test system for TIFM with MCA can be used to identify the desired antigen in the study of extracellular antigen fractions obtained by gel chromatography in antigenic mixtures and bacterial suspensions of P. pseudomallei and P. mallei.

 Thus, based on MCA 2A6 (a strain of the hybrid cell line MVd-4 (2A6)), a highly sensitive enzyme-linked immunosorbent assay system designed to detect 0.5 - 5.0 ng / mg protein (according to Lowry) was developed for AG8 epitopes. 37 - 3.7 ng / ml PS (anthrone method) of this antigen in extracellular antigenic mixtures and cultivation media of P. pseudomallei and P. mallei.

 The advantage of using the melioid monoclonal antibodies MVd-4 (2A6) as the basis for constructing an enzyme immunoassay for detecting antigen 8, one of the main pathogenicity factors of P. pseudomallei, is the stability of the properties of monoclonal immunoglobulin raw materials, in the presence of a producer clone of almost 100% - the apparent reproducibility of the experimental results, which favorably distinguishes this basis from polyclonal immunoglobulins in the production of diagnostic tools.

Claims (1)

 The strain of cultured hybrid cells of the animal Mus musculus BCCK (P) 649D is a producer of a monoclonal antibody to the thermostable component of the capsule-like substance of the causative agent of melioidosis.

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