RU2265051C1 - Strain of hybrid cultured mammalian cells mus musculus l, pkkk(p) 679d - producer of monoclonal antibody with specificity to protein-polysaccharide antigen from yersinia enterocolitica o3 and o9 serovars - Google Patents

Abstract

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

Description

The invention relates to biotechnology, in particular hybridoma biotechnology, and is a strain of hybrid cultured animal cells of Mus musculus L. RKKK (P) 679D producing monoclonal antibodies (MCA) to the pathogen of intestinal yersiniosis Y.enterocolitica O3 in culture media and the abdominal cavity of syngenic animals and O9 serovars, and can be used to create diagnostic test systems for identifying strains of Y.enterocolitica O3 and O9 serovars for laboratory diagnostics in healthcare, veterinary medicine, and for conducting scientific research.

Intestinal yersiniosis is characterized by polymorphism of clinical manifestations, as a result of which making a clinical diagnosis is very difficult. Due to the duration of pathogen isolation, the effectiveness of bacteriological research methods is also reduced, especially in the acute course of the disease. For rapid diagnosis of intestinal yersiniosis, it is most advisable to use immunological methods for identifying the pathogen using test systems based on MCA, which have a number of advantages over polyclonal diagnostic sera.

The majority of epidemically dangerous strains of Y.enterocolitica circulating in nature on the European territory belong to O3 and O9 serovars / 1 /.

Known strains of hybrid cells producing monoclonal antibodies to Y.enterocolitica O9 serovar / 2 /, on the basis of which immunofluorescence diagnostic test systems are designed to identify strains of Y.enterocolitica O9 serovar.

Immunoglobulin enteric diagnostic agents designed to identify both serovars, both polyclonal and monoclonal, are currently absent.

The objective of the invention is to obtain a hybridoma strain - a producer of monoclonal antibodies to the protein-polysaccharide antigen Y.enterocolitica O3 and O9 of serovars used to identify strains of Y.enterocolitica of these serovars.

The essence of the invention lies in the fact that as a result of the fusion of mouse plasmacytoma Sp2 / 0-Ag.8 and B-lymphocytes of the spleen of the mouse inbred line BALB / c, immunized with a protein-polysaccharide complex (BOD) isolated from the biomass of bacteria Y.enterocolitica 908 O3 serovar acetic hydrolysis according to RVWhite | 3 | as modified by E.E. Bahrakh et al. / 4 / in the presence of 0.1 n acetic acid for 1 h at 100 ° C, when 50% polyethylene glycol solution (PEG) (Merck) with a molecular weight (m.m.) of 4000 kDa and subsequent cloning and reclining is used as a merging agent the method of limiting dilutions obtained gabridoma Y.ent. O3.O9.C10 producing MCA for Y.enterocolitica O3 and O9 serovars. An experimental enzyme immunoassay system based on these MCAs allows the identification of Y.enterocolitica strains of two serovars. The resolution of the test system is (3.7 · 10 ⁴ -6.2 · 10 ⁴ ) mk / ml of pure culture in the sandwich version of the enzyme immunoassay (ELISA), (6.2 · 10 ⁴ -1.2 · 10 ⁵ ) mk / ml in dot immunoassay (DIA).

The hybridoma strain is obtained by fusion of myeloma cells with splenocytes from a mouse immunized with BOD. On the first day of immunization, 25 μg of BOD diluted in 0.25 ml of physiological saline is injected intraperitoneally with 0.25 ml of Freund's complete adjuvant (PAF) added; on days 14, 28, 42, BOD is administered in a dose of 50 μg without PAF. 3 days after the last immunization, the mouse spleen is removed and 1 · 10 ⁷ mouse splenocytes are hybridized with 3 · 10 ⁶ cells of the myeloma line in the presence of 1 ml of PEG 4000 for 1 min.

After washing the polyethylene glycol, the cells are plated on 96-well plates on a layer of feeder cells (lymphocytes) isolated from the spleen of an outbred mouse. Selection of hybrid cells is carried out on a selective medium containing hypoxanthine-aminopterin-thymidine (GAT). After 21 days, the cells were cultured in HT medium without aminopterin. Subsequently, the cultivation is carried out on RPMI-1640 medium that does not contain selective components. 2-fold cloning is carried out by the method of limiting dilutions in 96-well plates on a layer of feeder cells. The yield of positive clones is 100%.

For screening antibody-producing clones of hybridomas, enzyme-linked immunosorbent assay (TIFA), anti-species immunoperoxidase conjugates and cells of type 34 serovars of Y.enterocolitica and other microorganisms were used. A clone (Y.ent. O3.O9.C10) was selected that produces MCA for serovars Y.enterocolitica O3 and O9 antigens.

The strain is deposited in the Specialized collection of vertebrate cell cultures of the Russian collection of cell cultures under the number RKKK (P) 679D.

The inventive strain of hybridomas has the following characteristics:

1. Karyological characteristics.

The modal class for the parent myeloma line Sp2 / 0-Ag.8 is 72 chromosomes.

2. Morphological characteristics.

The hybrid cell culture consists of round cells weakly attached to the substrate, which are smaller in size from the initial myeloma cell.

3. Cultural properties.

The hybridoma strain is cultured at a temperature of (37.0 ± 0.1) ° C in an atmosphere containing (5.0 ± 0.1)% carbon dioxide. The cultivation medium is RPMI-1640 medium (Flow Laboratories) containing 10% fetal calf serum, 2% L-glutamine, 2% Hepes, 10% MAHCO3, 1% penicillin (5000 IU / ml) / streptomycin (5000 μg / ml) . Cells are maintained in the stationary phase of growth in plastic bottles (Costar), in 96-12 well plates.

Passage of hybridoma cells is carried out 2 times a week with a dilution ratio of 1: 2. Inoculum cell concentration is 2 · 10 ⁵ -3 · 10 ⁵ in 1 ml of medium. The hybrid clone does not lose the ability to synthesize antibodies when passaged in vitro for 5 months (observation period).

4. The cultivation of hybridomas in the body of the animal.

Animal species - inbred mice of the BALB / c line. The dose of cells during vaccination is 1.5 · 10 ⁶ per mouse. 10 days before the introduction of hybridoma cells, mice were intraperitoneally injected with 0.5 ml of 2, 6, 10, 14 - tetramethylene pentadecane (pristan). Immunoascitic fluid is formed on 10-12 days in a volume of 5-7 ml. Digestibility 100%. The hybrid clone retains the ability to produce antibodies at the same level when transplanting cells in animals (in vivo) for 6 passages (observation period).

The number of passages at the time of certification and deposit - 3.

5. The contamination of the strain.

Hybrid line contaminants, including bacteria, yeast, fungi, were not detected. Mycoplasmas were not determined.

6. Biotechnological characteristics of a useful product.

The strain produces monoclonal antibodies of the IgG class. They are specific to microbial cells of strains of Y.enterocolitica O3 and O9 serovars.

The geometric mean titer of MCA in ELISA in the culture fluid (QOL) is 1:80 ^x /: 2, in the immunoascetic fluid (IAG) - 1: 102400 ^x /: 2. The concentration of MCA in QOL is (28 ± 2) μg / ml, in IAI - (7 ± 2) mg / ml. The titer of MCA isolated from the IAH is 1: 512000 ^x /: 2.

The specificity of MCA is checked in TIFA with whole cells of heterogeneous microorganisms: Y.enterocolitica, Y. pseudotuberculosis, Y. pestis, E. coli, Shigella sp., Salmonella sp., Br.abortus, Fr.tularensis.

The results of the study are presented in table 1. Evaluation of the results is done visually.

The data in the table show that the MCA of the strain RKKK (P) 679D specifically interact in ELISA with bacterial cells of Y.enterocolitica O3 and O9 of serovars and do not interact with other microorganisms.

7. Immunochemical characteristics of a useful product.

By the method of immunoblotting, it was found that MCAs produced by the strain RKKK (P) 679D interact with the protein-polysaccharide antigen (glycoprotein) of Y.enterocolitica O3 and O9 of serovars m.m. (16 ± 2) kDa.

8. The method of cryopreservation.

The cryopreservation medium contains 90% fetal calf serum and 10% cryoprotectant - dimethyl sulfoxide (DMSO) (Serva).

The mode of freezing and heating. Cells in an amount of 2 · 10 ⁶ -3 · 10 ⁶ in a cryopreservation medium are poured into plastic ampoules of 1 cm ³ and placed for 3 hours in a pair of liquid nitrogen (minus 120 ° C), the freezing rate is ~ 1 ° per min then the tubes are dipped in liquid nitrogen (minus 196 ° C). Cells are frozen at passage 3-5 in vivo. Cell recovery after thawing: rapid thawing in a water bath at 37 ° C until completely thawed, the cells are diluted in 10 ml of RPMI-1640 medium with 1 ml of horse serum and precipitated by centrifugation at 150 g for 10 min. Cell viability after cryopreservation is (70 ± 5)%.

Example 1. The use of strain RKKK (P) 679D.

Strain RKKK (P) 679D is grown in vitro to a final concentration of 1 · 10 ⁶ -1.5 · 10 ⁶ cells in 1 ml of medium. Cells are pelleted by centrifugation at 150 g for 10 min and injected intraperitoneally to BALB / c mice pretreated with marin, 1.5 × 10 ⁶ cells in a volume of 0.5 ml. After the formation of an ascites tumor, immuno-ascitic fluid is sampled. Cells are precipitated from the IAH by centrifugation at 150 g for 10 min and used for subsequent passage in vitro and in vivo.

The supernatant containing MCA is used in ELISA to identify strains of Y.enterocolitica O3 and O9 of serovars, or is used as a semi-finished product for isolation of monoclonal antibodies by double precipitation with a solution of ammonium sulfate 45% and 40% saturation, more suitable for long-term storage at a temperature of minus 20 ° C, without losing specific activity for 2 years (observation period), which can be used in the development of immunoglobulin preparations and test systems for laboratory diagnosis of intestinal yersiniosis.

Example 2. The use of the product of the strain.

To perform an indirect ELISA, 100 μl of a suspension of isolated pure Y.enterocolitica culture at a concentration of 1 · 10 ⁹ m.k./ ml prepared in buffered physiological solution (PBS), pH 7.2, is added to the wells of a 96-well plate of domestic production and placed in a thermostat at a temperature of 37 ° C for 2 hours, or kept at 4 ° C for 18-20 hours. Then, 150 μl of a 0.5% solution of bovine serum albumin (BSA) in phosphate-saline are added to each well buffer solution (PBS), pH 7.2, and incubated at 37 ° C for 30 minutes. Wells are washed three times with 200 μl PBS. MCA (or immuno-ascitic fluid) produced by a strain of hybridoma RKKK (P) 679D is added to all wells of the plate in the established working dilution in a volume of 100 μl and incubated at 37 ° C for 2 hours. After this, the wells of the tablet are washed three times with PBS and in each antibodies are added to the well against mouse immunoglobulins labeled with horseradish peroxidase (NIIEM named after N.F. Gamalei) in working dilution. The tablet is kept at a temperature of 37 ° C for 1 h, then the wells of the tablet are washed 6 times with PBS. 100 μl of a chromogen solution (2,2'-azino-di- [3-ethylbenzothiazolin sulfonate] a (ABTS)) was added to all wells and incubated for 30 min at room temperature. The reaction is evaluated visually by the appearance of green staining of the decomposition products of H ₂ O ₂ in the presence of a chromogen. If necessary, the reaction is stopped by adding 3 M NaOH to the wells. A positive reaction indicates that the identified strain belongs to O3 or to O9 serovar Y.enterocolitica.

A distinctive feature of the inventive hybridoma strain is that it produces monoclonal antibodies to both O9 and O3 serovars of Y.enterocolitica.

Based on the MCA, an experimental diagnostic enzyme immunoassay test system was tested, tested on 55 strains of Y.enterocolitica of the State collection of pathogenic bacteria "Microbe" (Saratov). The research results confirmed the high specificity of the designed test system (table 1).

Thus, the strain of hybrid cultured mouse cells produces monoclonal antibodies to the protein polysaccharide antigen of Y.enterocolitica O3 and O9 of serovars, allowing identification of Y.enterocolitica strains of these serovars, most often isolated in Europe from sick people, farm animals, and environmental objects and representing the greatest epidemic hazard among enteric bacteria circulating in nature.

Sources of information

1. Tseneva G.Ya. Yersiniosis and pseudotuberculosis (Manual for doctors). - St. Petersburg. - 1992. - 58 p.

2. Alekseeva L.P., Matyash E.V., Chemisova O.S., Smolikova L.M. Monoclonal antibodies to Yersinia enterocolitica O9 and their diagnostic value // Diagnostics, treatment and prevention inf. diseases. Biotechnology. Veterinary Medicine Mat. anniversary scientific Conf. 50th anniversary of the Center for Military Technical. Biological problems. Defense Research Institute of Microbiology of the Ministry of Defense of the Russian Federation (April 30 - June 1, 1999). - Yekaterinburg, 1999. - P.5-6.

3. White P.V. Observation on the polisaccharide complex and variants of Vibrio cholerae // Brit. J. exp. Pathol. - 1936. - V.17. - P.229-234.

4. Bahrakh E.E., Korobkova E.I., Pavlova L.P. Polysaccharide-containing fraction of the plague microbe as an allergen for detecting active immunity in guinea pigs // Tr. Inst "Microbe". - Saratov, 1960 .-- Issue 4. - S. 63-65.

Table 1 Interaction of MCA strain RKKK (P) 679D in TIFA with bacterial strains Types of microorganisms The number of strains The specific interaction of the MCA strain of hybridoma RKKK (P) 679D Y.enterocolitica O3 Serovar eleven + Y.enterocolitica O9 Serovar 10 + Y.enterocolitica of other serovars (from O1, 2a, 3 to O34) 34 - Other microorganisms 42 - (Y. pseudotuberculosis, Y. pestis, E. coli, Shigella sp., Salmonella sp., Br.abortus, Fr.tularensis) Note:
+ - the presence of a reaction
- - lack of reaction

Claims (1)

The strain of hybrid cultured animal cells Mus musculus L. is a producer of monoclonal antibodies specific for the protein-polysaccharide antigen of Yersinia enterocolitica O3 and O9 of serovars, deposited in the Specialized Collection of Vertebrate Cell Cultures of the Russian Collection of Cell Cultures under the number RKKK (P) 679D.