SU1666532A1 - Strain of hybridous cultured mammalian cells, mus musculcs l. producing monoclonal antibodies for nucleocapside protein or cattle virus - Google Patents

Abstract

This invention relates to hybridoma technology and can be used to diagnose gastrointestinal diseases of animals caused by a coronavirus. The aim of the invention is to obtain a strain of hybrid cells producing monoclonal antibodies (MoH / AT / k nucleocapsid protein / NP-53 / cattle coronavirus / Bov /. The strain is obtained by hybridization of myeloma cells of the X-63 AG 86 line. 5. 3 with splenocytes of mice immunized with bovine coronavirus. The resulting strain 3 G ₆ D ₅ secretes and MonAT JG2B, specifically interacting with the protein NP - 53 of the coronavirus of cattle. The strain was deposited as VSKK / P / 375 D and is characterized by cultural characteristics typical of hybrids: Cultivation medium R PMJ - 1640 with 10% fetal calf serum, crop concentration - 2 ^. 10 ⁵ cells in 1 ml, the frequency of passage of 2 - 3 days. The productivity of the strain is 10 μg / ml MonAT in culture medium and 4 mg / ml in ascites.

Description

gene with incomplete Freund's adjuvant. The last immunization was carried out with 50 µg of antigen in 0.15 M phosphate-buffer solution, pH 7.2, and after 3 days spleen cells were used for hybridization. Hybridization is carried out by adding to the mixture of 4,106 myeloma cells 28 × O6 splenocytes 1 ml of solution containing 45% PEGg-4000, 10% sulfoxide and 45% of RPMI-1640 medium. The pouring agent is applied for 15 seconds, then the cells are slowly mixed. 10 ml of RPMI-1640r medium is added for 60 seconds and for 15 seconds. Then they are left to incubate for 10 min. After that, the cells are precipitated by centrifugation at 1000 rpm for 10 minutes. The cell pellet is suspended in RPMI-1640 medium with a pressure of 10% by volume of fetal calf serum and seeded into well plates, 100 μl per well. On the second day after the fusion, selective medium with hypoxanthine, aminopterin and thymidine (GAT) is added. After 1СЫ4 days from the wells, where cell clones have grown, a culture liquid is selected for testing for activity, i.e. for antibody production. , synthesized by hybrid cells, is determined by immunofermental analysis (ELISA) using rabbit antibodies to immunoglobulins of mice conjugated with horseradish sidase and coronavirus anthrax, purified in a density gradient Carose Hybrid cells that produce MonAT are cloned twice by the limiting dilution method. After the second cloning, 100% of the subclones produce antibodies to the tested antigen. The strain of cells secretes the antibodies into the culture medium or ascites fluid.

Ytamma of 3GgDs hybrid cells of nitts in the specialized collection of transplanted somatic vertebrate cells of the Institute of Cytology, USSR Academy of Sciences under the number BCKK (II) 375Dt

The strain is characterized by the following properties.

Morphological features The culture consists of round cells that are weakly attached to the substrate and are the same size as the original myeloma cell. The nucleus occupies a large part of the cell. Tsito - ppasm has the appearance of a thin rim "

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Cultural properties Culture medium: RPMI-1640 medium with the addition of the following components: 10% by volume of fetal calf serum; 20 ml / l 200 mM L-glutamine; 3.375 g / l HEPES; 0.5 ml / l 0.1 M 2-mercaptoethanol; sodium pyruvate; 100 U / ml penicillin, 10 µg / mp streptomycin; 3.7 g / l sodium bicarbonate. The cells are cultured at 37 ° C in an atmosphere with 5% С0Ј Growth pattern - stationary suspension Seeding cell concentration of 2-10 cells in 1 ml „The frequency of passaging through SPS (U) and intraperitoneal inoculation of syenogenic mice the hybridoma strain forms a tumor in ascites form on day 10-14 o Cell dose on grafting 2-10 o 7-14 days prior to the introduction of hybridoma, 2,6,10,14 tetramethylpent need to be injected into animals in a dose of 0.5 ml per head Strain retains the ability to produce specific monoclonal bodies for 5 passages on gene mice x (time of observation).

The productivity of the strain on the day of cultivation of the hybridoma concentration of monAT reaches 10 µg / ml in the culture medium. In purified as5-citric fluid, the concentration of immuno-globulins is 4 mg / ml

The characteristic of a useful product MonAT refers to immunoglobulins of the subclass G2b, MonAT specifically interact with the nucleocapsid protein of molecular weight 53 KDa. Specificity is determined by the method of microblotting „

gContamination of Contaminants in cells, including bacteria, fungi, yeast and mycoplasma, not detected

Cryopreservation, fetal calf serum is added to the sediment of hybrid cells, followed by dimethyl sulfoxide to a final concentration of 10%. The cell suspension is poured into sterile plastic ampoules with screw caps of 2 each. 10b current in a volume of 1 ml Ampoules are placed in a foam box and left for 1 day at -70 ° C, then transferred to liquid nitrogen

The defrosting condition, the Frozen vial, is quickly placed in a 37 ° C bath. As soon as the last pieces of ice have melted, the cell suspension is transferred with a sterile pi to a centrifuge tube containing 10 ml of cold serum free the medium, washed once and inoculated - watered in 24-well plates with a peritoneal macrophage feed layer. Number of viable cells after thawing at least 70%

Strain hybridoma use as follows

Example 1 Hybrid cells are placed in plastic mattresses with an area of 25 cm2, 5105 qls in 5 ml of nutrient medium, incubated at 37 ° C in an atmosphere of 5% CO2 and the culture medium is collected and centrifuged for 10 minutes at 800 g. separating cells from the supernatant - a supernatant containing antibodies. When hybridoma is cultured in mice, immunoglobulins from ascitic fluid are obtained by precipitating ammonium sulfate to 50% saturation. To do this, an equal volume of 0.15 M phosphate-buffer solution (FBI), pH 7.2, is added to the ascites fluid an equal volume of saturated ammonium sulfate solution and left under stirring overnight at 4 ° C. Immunoglobulins are separated by centrifugation at 5000 g for 30 min at 4 ° C. The precipitated antibodies are resuspended in a minimum amount of PBS. Next, the antibodies are purified on a column with protein A Sefaro In order to balance the binding buffer with the column (1.5 M glycine, 3 M NaCl adjusted to pH 8.95 M NaOH) 4 mg / ml precipitated with ammonium sulfate preparations of immunoglobulins were added, the column was washed with binding buffer and the antibodies were eluted with 0 , 1 M citric acid pH 4.00 The eluate is neutralized with 1 M HC1 buffer, pH 8.1 o. The purified antibody preparation is concentrated by ultrafiltration through a pM 100 membrane. The resulting preparations — the supernatant and purified ascites fluid — are used as reagents to study the specificity of the interaction. Monat with tiruemogo antigen using the ELISA method about

Example 2. On a polystyrene 96 - unit tablet, absorb the puppy coronavirus antigen at a concentration of 1 µg in 100 µl of 0.1 M carbonate buffer, pH 9.5, and incubate at 4 ° C overnight. The tablet is washed 3 times with 0, J5 M FBI and, 3 times 0.15 FBI with 0.1% Tween-20 (FBI-TW), then the cells of the tablet are multiplied with cultured or purified ascites fluid in the amount of 100 µl and incubated for 90 min at 37 ° Co The tablet is washed as described above and mouse anti-mouse immunoglobulin antibodies conjugated with horseradish peroxidase are added to the wells. After 1 hr of incubation: - repeat the procedure at 37 ° C.

5 washing in order to remove the unrelated products of the reaction S the conjugant turned out, and therefore the antibodies against the coronavirus are quantified by the breakdown of the substrate

0 in the presence of orthophenylene diamine. The result is taken of the intensity of the staining of the reaction mixture in a yellow-brown color, read on a spectrophotometer with vertical

5 by the flow of light at a wavelength of 492 nm. The results of the ELISA show that if the antibody titer in the culture medium on the first day of substitution of the hybridoma is 1: 8, then after 3 days the titer

0 rises to 1: 256. The titer of antibodies in the purified ascitic fluid equals "- from 1: 512000. This indicates that the hybridoma produces pyphonic MonAT with cattle coronavirus

An example is given in the variant of quantitative determination of the corona - viral antigen. To do this, by 0, a 96 mm cleavage tablet adsorb the purified monomet monomer with fathamum ammonium monomer at a concentration of 1 µg in 100 µl of 0.1 M bicarbonate buffer pH 9.5 and incubate 4 ° C in

5 overnight The tablet was washed as described in example 2. Double-fold dilutions with a concentration of 10 µg / ml of 100 µl of the coronavirus antigen were added to the plate cells and the cell was washed for 90 min at 37 ° C. t second MonAT to coronoviruses of cattle conjugated with peroxidase On this, on the polystyrene surface of the plates forms the complex "- lex: MonAT - coronavirus antigen" second MonAT conjugated with horseradish peroxidase After 1 h, and # 5

3. MonAT ZSB using an ELISA sandwich

cubes at 37 ° C repeat the procedure.

716665328

washing to remove unattached - i show that using

Claims (1)

their reaction products Forming -MonAt in a sandwich variant reveals a complex to it, and therefore a coronavirus — up to 10 ng / ml of viral protein, a ny antigen, is determined quantitatively by the decomposition of the H402 substrate in the Formula of the invention of orthophenylene diamine Taking into account the Strain hybrid cultivated yut- result on the intensity of the occlusion cells of animals Musmusculus L. shivan the reaction mixture in iron-BCKK (II) 375D, producing mono brown color on a spectrophotometer jgklonapny antibodies to nucleocapsid with a vertical stream of light to the receiving protein of the large-sized coronavirus wavelength of 492 nm Results IFAh cattle