Strain of mammalian hybrid cell c3/s-3e5 of mus musculus l. producing monoclonal antibody to bernet's coxiellas

FIELD: biology, hybridoma technology.

SUBSTANCE: invention represents a new strain of mammalian hybrid cells C3/S-3E5 of Mus musculus L. producing monoclonal antibodies (MCAb) to Bernet's coxiellas (strain "Grita") in cell cultures and abdominal cavity of syngenic animals. Hybridoma C3/S-3E5 producing MCAb to this pathogen is obtained by fusion of murine myeloma of strain Sp-2/0 and murine splenocytes of strain BALB/c immunized with the concentrated and purified Bernet's coxiella preparation (strain "Grita) inactivated with formalin using polyethylene glycol of molecular mass 1000 Da as a fusing agent and the following cloning by method of maximal dilutions. Specificity of prepared MCAb: absence of cross-reactions in IFA with Provacheck's rickettsia antigen and with the non-infected accumulation substrate. Using prepared MCAb it is possible to carry out specific detection of Bernet's coxiellas by method IFA (direct and indirect variants). IFA sensitivity based on these MCAb is 2.0 x 10³ ID₅₀ x cm^-3 for white rats. Applying the present invention allows detecting and identifying pathogens of rickettsial etiology.

EFFECT: improved method preparing, valuable properties of strain.

3 tbl, 1 dwg, 1 ex

The invention relates to the field of Microbiology and biotechnology, namely, hybridoma technology, and represents a new strain of hybrid cells C3/S-3E5 animals Mus musculus L. producing cell cultures and peritoneal cavity of syngeneic animals monoclonal antibodies (hereinafter MCAT) to Coxiella of Berneta (strain “Grita”), which can be used in scientific research and in the manufacture of medical immunobiological preparations.

The invention can be used in microbiological and immunological research in creating a diagnostic to detect in samples from humans and vectors of Coxiella of Berneta agents fever Ku.

Q fever refers to the zooantroponozes with natural focality and distributed in many countries of the world [1-7]. Features of the pathogenesis of infection in humans and originality of clinical manifestations of Coxiella creates great difficulties in the recognition of the disease [1,8-10].

For detection of specific antigens of the causative agents of various infectious diseases of viral and rickettsioses etiology recently widely used immunochemical methods, in particular, enzyme-linked immunosorbent assay (hereinafter ELISA). One of the most promising ways of increasing the efficiency of ELISA is the use of MCAT as anticaste the containing substrate, used as diagnosticum. However, the question about the possible use of MCAT for detection and identification of pathogens rickettsioses etiology remains up to date unresolved.

The use of MCAT to Coxiella of Berneta for the diagnosis of fever Ku in the early stage of the disease may exclude false-positive reactions often occur when you use the test-systems based on polyclonal immune sera. It should also be noted that the use of MCAT will allow you to create a standardized test systems due to the possibility of using antibodies permanent avidity and specificity.

If you are developing on the basis of MCAT-based assays designed to identify agents of dangerous and especially dangerous infectious diseases of viral and rickettsioses etiology, it is important for us to obtain sufficient biomass of the latter. For this purpose, conduct the cultivation of hybrid cells by various methods in vitro or in vivo.

A promising direction of research is the selection and justification of the conditions of the method of culturing the hybridomas in vitro for obtaining biomass of MCAT, quantitatively comparable with those of the cultivation of hybrid cells in vivo.

The aim of the present invention is to obtain hybridoma producing MCAT to Coxiella of Berneta, is the quiet use as antitelomerase substrate in serological tests in the detection and identification of the causative agent.

The essence of the invention lies in the fact that as a result of the fusion of mouse myeloma line SP2/O and splenocytes of BALB/c mice immunized inaktivirovannye purified concentrated antigen of Coxiella of Berneta, when used as a melting agent, polyethylene glycol (hereinafter PEG) with a molecular weight of 1000 and subsequent cloning method of limiting dilutions obtained hybridoma C3/S-3E5, producing MCAT to Coxiella of Berneta. Data MCAT allow specific identification of Coxiella of Berneta by ELISA (direct and indirect ways). The sensitivity of ELISA-based MCAT is 2.0· 10³ID₅₀·cm^-3.

An example of executing

The resulting strain hybrid cells C3/S-3E5 animals Mus musculus L. producing monoclonal antibodies to Coxiella of Berneta, has the following characteristics:

1. Pedigree of hybridoma shown in the drawing.

2. The number of passages by the time certification is 14 times.

3. Standard growing conditions in vitro:

- seeding concentration when grown in vitro 2,0· 10⁵CL· cm^-3;

the environment of the cultivation medium Needle in the modification Dulbecco (DMEM) supplemented with 10% fetal calf serum (FTS);

the temperature of cultivation (37±0,5)° C;

- the CO₂in the atmosphere of cultiva the Finance 5%.

4. Cultural properties strain

The strain is monocline-suspension: about 30% of the cells are in suspension, being attached to the surface of the culture vessel. The frequency of passage when planting a dose of 2.0· 10⁵CL· cm^-3is 3 to 4 days. The index of proliferation when grown in vitro is equal to 7.2.

5. Growth (kinetic) characteristics hybridoma presented in table 1.

6. Characteristics of cultivation in the animal organism

Intraperitoneal administration to mice of BALB/c mice from 2.0· 10⁶to 5.0· 10⁶cells hybridoma on 9-21 day formed serous tumors. From one mouse get from 2.0 to 3.0 cm³immune ascitic fluid (hereinafter IAG). The concentration of cells hybridoma in IAG is from 30· 10⁶up to 50· 10⁶CL· cm^-3.

7. Cytogenetics (karyotype) feature:

modal class from 88 to 96 chromosomes;

- the proportion of cells in the modal class - 54%;

modal class for the parent myeloma line SP2/O - 57 to 59 of chromosomes.

8. Cytomorphological characteristics

Clone hybridoma presents a large, round cells with sizes ranging from 20 to 25 μm, similar in morphology to cells of the initial myeloma line SP2/O.

9. Species Mus musculus.

10. Carcinogenesis

Hybridoma intraperitoneal injection causes serous (in 70% of cases) and solid tumors.

11. Marker features

The clone producing MCAT to Coxiella of Berneta.

12. Control of contamination

Fungal and bacterial microflora in the culture of strain hybridoma missing.

13. Biotechnological characteristics

The strain produces MCAT to Coxiella of Berneta:

- the title of MCAT when grown in vitro to 1:2560 (ELISA);

- the title of MCAT when grown in vivo - 1:163840 (ELISA).

Feature produced MCAT to Coxiella of Berneta:

protein is the target antigen of the first phase of Coxiella of Berneta, structural protein with a molecular mass (MM) 62 kDa;

- isotype produced immunoglobulins - IgG;

- the number of explored passages: in vitro - 14; in vivo - 5.

Secretory characteristics hybridoma C3/S-3E5 presented in table 2.

14. Method of cryopreservation

Sediment cells obtained after low-speed centrifugation, resuspended in medium containing 90% FCS and 10% dimethyl sulfoxide (DMSO), adjusted to a final concentration of 3.0· 10⁶CL· cm^-3and poured into plastic ampoules of 0.5 cm³. The ampoule is cooled to a temperature of minus 70° in the software installation for freezing during freezing rate of 1 to 2° With a minute and then placed in a Dewar flask with liquid nitrogen.

Cell hybridoma C3/S-3E5 grown in vitro in 24-hole tablets to a final concentration of 1.0· 10 1.5· 10⁶CL· cm^-3, precipitated using low-speed centrifugation and injected intraperitoneally to mice of BALB/c, pre (7 days) treated with adjuvant of the Wharf, in the amount of from 2.0· 10⁶to 5.0· 10 cells in a volume of 0.5 cm³. For 9-21 days after the injection of hybridoma approximately 70% of animals are formed ascitic tumor. Cell hybridoma precipitated from IAG using low-speed centrifugation (1500 rpm· min^-1within 10 minutes) and used for subsequent passage in vivo. The supernatant containing MCAT in high titers, is used as a material for the selection of immunoglobulin, which is used for diagnosticum. Data on the sensitivity and specificity of ELISA-based MCAT C3/S-3E5 presented in table 3.

As shows the analysis of the data presented in table 3, the sensitivity of the indirect variant of ELISA-based MCAT in determining coxall of Berneta (C. Bumetii) in the composition of native products is 1.2· 10³ID₅₀·g^-1. The specificity of the indirect variant of ELISA-based MCAT confirmed by the lack of positive reaction to the antigen of Rickettsia Prowazeki (R. Prowazekii) [12].

Sources of information

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The strain of hybrid cells C3/S-3E5 animals Mus musculus L., producing within 14 studied passages in vitro of monoclonal antibodies to Coxiella of Berneta (strain “Grita”)relating to the IgG isotype, suitable for cooking on their basis based assays for specific detection of Coxiella of Berneta (1st phase) using enzyme immunoassay.