RU2158760C2 - Strain of hybrid cultured animal cells of mus musculus l used for production of monoclonal antibodies to chlamydia - Google Patents

Abstract

FIELD: veterinary biotechnology. SUBSTANCE: strain Chla-K-1 is obtained by fusing mouse myeloma cells NSO with splenocyte cells of BALB/c mouse line immunized by elementary corpuscles Chlamydia psittaci prepared from strain "K-1" feline chlamydiosis conjunctivitis exciter. Hybridoma Chla-K-1 produces genus-specific Gl isotype monoclonal antibodies reacting with different agents exciting feline, cheep, fur-bearing animal, and human chlamydiosis. Activity of antibodies in cultural fluid ranges from 1:2000 to 1:6000, in ascitic fluid from 1:200000 to 1:1000000. Antibodies are revealed in immunoenzyme test involving immuno-fluorescence. EFFECT: enabled preparation of express diagnosticum for detection of Chlamydia genus detection. 1 tbl, 4 ex

Description

 The invention relates to the field of veterinary biotechnology, in particular to a strain of hybrid cultured cells producing monoclonal antibodies that can be used to create diagnostic drugs for the identification of C. psittaci. Known strains of hybrid cultured animal cells producing monoclonal antibodies (M-AT) Chla-62, Chla-154, Chla-159, Chla-175, Chla-178, Chla-366 and others against Ch.psittaci antigens (1). The resulting hybridoma strains are less active and specific and less suitable for creating diagnostic preparations for the rapid diagnosis of Ch.psittaci.

 The aim of the invention is to obtain a highly active strain of hybrid cells producing monoclonal antibodies to C.psittaci antigens for subsequent construction on their basis of highly specific and standard domestic test systems for the rapid diagnosis of chlamydial infections by direct immunofluorescence in animals and birds.

The strain is obtained as follows. To obtain a hybridoma, the strain C.psittaci "K-1" of the causative agent of chlamydial conjunctivitis of cats is used. Female BALB / c mice weighing 10-12 g were immunized with antigen with complete Freund's adjuvant intraperitoneally, twice, with an interval of 14 days, by suspension of elementary chlamydia cells inactivated by formalin and destroyed by Triton X-100. Booster immunization is carried out after 1 month, 3 days before the merger, using the same dose of antigen in saline. The antigen is injected into the intraorbital venous sinus (100 μg) and intraperitoneally (200 μg). The fusion of mouse NSO line mouse myeloma cells with peripheral lymph node cells (inguinal and popliteal) of BALB / c mice taken in a 1: 3 ratio is carried out 3 days after the last injection according to the standard method using 50% polyethylene glycol (PEG) solution with m. m 1000 and distributed in a selective HAT medium on 96-well plates (Flow company) with a thymocyte-fed layer. Cell tablets were cultured in an atmosphere with 5% CO ₂ . The cultivation temperature is 36.5 ^o C. Every three days, the selective medium in the tablets is replaced with fresh. Starting from the 20th day from the moment of fusion, the hybrid cells are subcultured into 24-well plates with a thymocyte-fed layer. Clones are grown to form a continuous layer, and specific antibodies are detected in the culture fluid.

Antibody-producing clones are screened by direct solid-phase ELISA using antiviral immunoperoxidase conjugates, where elementary bodies of chlamydia inactivated by formalin (15-20 μg per well) are used as the solid phase, and antigen prepared from uninfected vitelline membranes is used as a nonspecific antigen. cooked like a specific antigen. Antibody clones were grown in plastic flasks of 25 cm ² with a layer of Reticulated-thymocyte followed by evaluation of activity and specificity M-AT in the culture fluid, and collecting monoclonals with the highest accumulation in mass culture.

 A hybridoma is selected representing a monoclone with group specificity for Chlamydia psittaci antigens - Chl "K-1". Monoclonal antibodies produced by Chla "K-1" are able to react with all investigated strains of Ch. psittaci and ch. trachomatis in two serological reactions of ELISA and NRIF. M-AT Hl "K-1" in RSK do not activate complement.

 The strain is deposited in a specialized collection of transplantable somatic vertebrate cells of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines under the number Hla-K-1 DEPT.

 The resulting strain Hla-K-1-DEPT has the following characteristics.

 Morphology of Hla-K-1 hybridoma.

 The cells of the hybrid line are round in shape, differ from the parent line in somewhat larger sizes.

 Cultural properties.

The Hla-K-1 hybridoma is grown in RPMI-1640 nutrient medium with 0.1% NaHCO ₃ , glucose 4 g / l, 10% serum of cow embryos; sodium pyruvate - 0.05 mg / cm ³ , insulin - 0.2 u / cm ³ , HEPES - 10 mm. The cultivation temperature is 36.5 ^o C. Hybridoma cells grow in suspension, have a weak adhesive ability to the surface of plastic or glass. The sowing dose is 250-300 • 10 ³ cells / cm ³ , the sieving ratio is 1: 3-1: 4 twice a week.

The Chla-K-1 hybridoma is malignant in 100% of cases and in the body of syngeneic BALB / c mice older than 2-2.5 months old, sensitized by intraperitoneal administration of 0.5 cm ³ pristan, it forms an ascites tumor. Ascites tumors form in mice on days 8-14 after administration of 2-5 x 10 ⁶ hybrid cells.

The titers in immune ascitic fluids (in inverse quantities) are in ELISA from 2 • 10 ⁶ to 1 • 10 ⁷ , in RNIF 1: 640-1: 1280.

 The stability of the hybridoma Hla-K-1.

 Stability of antibody production by hybrid cells is maintained for 70 passages in cell culture and 5 passages in BALB / c mice (observation time).

 Storage and viability of the Hla-K-1 hybridoma.

The Hla-K-1 hybridoma is stored cryopreserved in liquid nitrogen. The freezing medium is 90% of the fetal serum of cattle, 10% of dimethyl sulfoxide. Freezing mode: up to minus 70-80 ^o C, then liquid nitrogen (-196 ^o C). Recovery after thawing: rapid thawing at 37 - 39 ^o C, diluted 10 times with serum of cow embryos, precipitated by centrifugation at 500 rpm for 10 minutes, resuspended in a growth medium at a concentration of 3-4 • 10 ⁵ cells / cm ³ . The viability of the restored cells is 70-80% and is determined by the differential staining of the cells using a 0.25% trypan blue solution, pH = 7.2-7.3.

 The Hla-K-1 hybrid cell population is free of mold, yeast, bacterial and mycoplasma contaminants.

 The invention is illustrated by the following examples.

 Example 1

 The activity and specificity of monoclonal antibodies (M-AT) obtained from the ascitic fluid of BALB / c mice immunized with a Chl-K-1 hybridoma culture is determined.

 The activity and specificity of M-AT is determined in ELISA and NRIF. For ELISA, M-AT peroxidase conjugates are used, which are prepared by the periodic method according to M. Wilson and P. Nakane.

 For the production of ELISA, a corpuscular antigen from elementary bodies (ETs) C.psittaci, strain K-1, Lori, Rostinovo-70, C.trachomatis serovar L2 and strain CM, purified and concentrated by differential centrifugation, are used and in a stepwise gradient of sucrose and formalin inactivated. ELISA results are taken into account on a multi-channel spectrophotometer "Titertek Multiskan" company "Flow" at a wavelength of 492 nm.

An indirect reaction of microimmunofluorescence (NRIF) is carried out on the defatted surface of the slots of the slide (company "Flow"). As antigens, a suspension of the shells of yolk sacs infected with the previously indicated strains is used. Fixation of antigenic slides is carried out with cold acetone (4 ^o C) for 20 minutes Ready slides are stored at minus 20 ^o C.

For each group of antigens, 0.6 cm ³ dilutions of the test materials (culture and ascitic fluids) prepared in phosphate-buffered saline (PBS) pH 7.4 are applied. Incubation is carried out in a humid chamber at 37 ^o C for 30 minutes Then the slides are rinsed with FSB and washed 2 times for 5 minutes in a washing chamber. The slides are slightly dried and luminescent rabbit serum is applied against white mouse immunoglobulins (manufactured by the Gamalei Scientific Research Institute of Electro-Chemical Medicine) in working dilution.

To color the background, Evans blue is added to the luminescent serum at a final dilution of 1: 10000. The slides are incubated for 30 min at 37 ^° C in a humid chamber, washed 3 times in FSB pH 7.4 for 5 min, and then dried in air with 1- 2 drops of buffered glycerin pH 8.5 and cover with a coverslip.

 The stained preparations are examined in a LUMAM I-3 luminescent microscope with a combination of SZS 24-4, FS 1-4, BS 8-3 light filters and an increase of 600-1000X for the presence of a specific glow and morphological features of chlamydia in the studied samples.

 The results for the determination of the specificity and activity of monoclonal antibodies of the Chla-K-1 hybridoma in ELISA and NRIF are shown in the table.

 These tables illustrate the high activity and specificity of M-ATs contained in ascitic fluid produced by the Hla-K-1 hybridoma with respect to both C.psittaci and C.trachomatis.

 Example 2

Monoclonal antibodies obtained from ascitic fluid by double precipitation with a solution of ammonium sulfate 50% saturation, are used as fixing antibodies in the sandwich ELISA. For immobilization on micropanels, monoclonal antibodies are taken at a concentration of 50 mg / cm ³ , and polyclonal antiserum is used as detecting antibodies. For detection, purified ET chlamydia, a culture fluid containing lipopolysaccharide (YPS), and blood serum from experimentally infected and spontaneously infected animals are used. The sensitivity of the method is 25 ng / cm ³ .

 Example 3

20 cm ^{3 of the} obtained ascitic fluid (AF) is thawed in a water bath at a temperature of 18-25 ^o C and centrifuged at 1000 rpm for 15 minutes. After centrifugation and lipid separation, an acetate buffer solution and caprylic acid are added dropwise to the AF to a final concentration of 1.1%. The mixture is subjected to stirring on a magnetic stirrer for 30 minutes, followed by centrifugation at 1000 rpm for 30 minutes at 5 ^o C. A caprylic acid clot is separated, and the liquid containing M-AT is filtered, dialyzed against phosphate-buffered solution, and then M-AT from ascites is precipitated with a saturated solution of ammonium sulfate. Precipitated precipitate is precipitated by centrifugation at 1000 rpm. The precipitate is dissolved in a phosphate-buffered solution pH = 7.2 and put on dialysis against the same buffer solution. The yield of purified M-AT is 3 cm ³ . The protein concentration is 15 mg / cm ³ .

 Establish in the ELISA activity of purified M-AT, which should not be lower than a dilution of 1: 50,000.

Chlamydial M-AT buffer solutions are purified by passing through Sephadex gel chromatography columns. M-AT fractions were collected under control of protein content using 10% sulfosalicylic acid. Protein concentration is measured by Lowry, which is more than 10 mg / cm ³ .

To the purified M-AT protein pool, add isothiocyanate-fluorescein (FITC) prepared immediately before mixing at the rate of 0.3 cm ³ of fluorochrome solution (concentration of 1 mg FITC in 1 cm ^{3 of} buffer solution) per 1 cm ³ of M-AT solution. The mixture of components is maintained at a temperature of 18-25 ^o C for 2 hours. The stained M-AT solution is purified from unbound FITC by passing Sephadex gel chromatography columns. The purified conjugate has a yellowish orange color.

 The degree of conjugation of the stained FITC M-AT with the fluorochrome / protein ratio is determined by the optical density of the reagent at 280 nm and 495 nm. The fluorochrome / protein ratio should be at least 1.0.

Serum albumin as a stabilizer, sodium azide for preservation and an equal volume of Evans blue solution are added to the prepared conjugate. The finished preparation of fluorescent M-AT is a clear blue liquid. When stored at a temperature of 4 ^o C liquid diagnosticum retains its biological properties for 2 months. The activity of fluorescent M-ATs is determined by their coloring titer. The coloring titer is determined by staining preparations from infected and fixed cells with a prepared preparation (0.3 cm ³ ) diluted from 1: 8 to 1: 256. Fluorescent M-ATs should have a coloring titer of not less than 1:32 in the absence of a specific glow in control preparations prepared from uninfected cell cultures.

 The control of the specificity of fluorescent M-ATs is carried out on preparations from infected and uninfected cell cultures with the application of anti-Chlamydia M-ATs on one and the control non-immune ascitic fluid on the other. Specific intra- and extracellular luminescence was detected in preparations from infected cell cultures stained after pretreatment with a control non-immune AF, and in preparations pretreated with chlamydial M-AT, there was no luminescence.

Fluorescent immunoglobulins that meet the requirements for activity, specificity and sterility are packed in ШП-3 ampoules or in FI-5 vials of 0.1 ± 0.02 cm ³ and lyophilized. The ampoules are sealed, the bottles are corked with rubber stoppers and crimped with aluminum caps.

Lyophilically dried diagnosticum is in an ampoule or vial a dry, homogeneous, amorphous mass of bluish-lilac color. When stored in a dry, dark place at a temperature of 2-8 ^o C, the drug retains its biological properties for 18 months.

 Example 4

The lyophilized M-AT prepared according to Example 3 is mixed with a solvent. The dissolved reagent is stored in the dark at a temperature of 2-8 ^o C for 6 weeks. Imprints are prepared after taking material from the mucous membranes of the respiratory or urogenital tract from 12 sick animals onto glass slides with holes. Dried smears are fixed in 96% ethanol for 5 minutes, or applied to smears of 0.1 cm ^{3 of} acetone until it is completely evaporated. 0.3 cm ³ labeled antibodies are applied to the fixed smears using a micropipette and placed in a humid chamber for 30 min at 37 ^° C, followed by washing the preparations with distilled water and drying them. On the dried preparations, 0.3 cm ^{3 of} liquid is applied for conclusion under coverslips with their subsequent microscopy in a luminescent microscope.

 In 10 studied preparations, a bright green or yellowish-green glow was recorded in the form of cytoplasmic inclusions, as well as in the form of regular round formations with smooth edges about 300 microns in size (1/100 of the size of the intact cell) located extracellularly. Epithelial cells in the preparations are colored bright red. The diagnosis is positive for chlamydia. However, in two preparations, a specific glow was not found. The result of the study on chlamydia is negative.

 The strain of hybrid cultured mouse cells produces monoclonal antibodies that identify strains of chlamydia, pathogens of ornithosis (psittacosis), pneumonia, abortion, polyarthritis, encephalitis, keratoconjunctivitis of domestic and farm animals and birds. The prepared monoclonal antibody preparation in the direct immunofluorescence reaction for a short period of time (35 min) allows a diagnosis of chlamydia to be established with a high degree of certainty.

Sources of information
Nagieva F.G., Shafikova R.A., Nikulin V.G. and others. Monoclonal antibodies to Ch. psittaci: preparation, characterization and application. J. Microbiology, Epidemiology, and Immunobiology, 1991, 10, 58-61.

Claims (1)

 The strain of hybrid cultured cells VGNKI Hla-K-1 DEPT animal Mus. musculus L. used to produce monoclonal antibodies to Chlamydia.

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