SU1604849A1 - Strain of hybride cultivable mus musculus l. cells producing monoclonal antibodies to bacteria of brucella variety - Google Patents

Abstract

The invention relates to hybridoma technology and can be used in veterinary medicine and medicine in the diagnosis of brucellosis. The purpose of the invention is to obtain a hybridoma-producer of monAT, suitable for differentiating bacteria of the genus BRUCELLA and not giving cross-reactions with YERSINIA ENTEROCOLITICA serovar 0: 9 bacteria. The BAMA strain is deposited under a VSKK (P) number 302D. The strain retains production capacity for 5 passages on syngeneic mice. The yield of monat on 3-4 days of cultivation in the medium is 35-45 µg / ml, IN VIVO - 8 mg / ml of ascites fluid. Produced by the monAT strain belong to the JGG class, subclass 2a

epitope-specific parts of the polysaccharide chain of LPS Brucella are epitope-specific. 1 tab.

Description

The invention relates to hybridoma technology and can be used in veterinary medicine and medicine in the diagnosis of bronze.

The aim of the invention is to obtain a monobat hybridoma-producer suitable for differentiating bacteria of the Brucella genus and not cross-reacting with Yersinia eoterocolitica bacteria of the serovar 0: 9.

Strain hybridoma obtained as follows.

On the first day of immunization, a polysaccharide antigen was injected intraperitoneally to mice of the BALB / c line. - poly-B, obtained from R-forms of Brucella melitensis VP5 at a dose of 30 µg in O, 1 ml of Freund's incomplete adjuvant. Poly-B of the R-forms of Brucella does not contain the 0-antigen of LPS and consists of lipid A and the core) (core) part of the bacterium. polysaccharide. On days 7, 11, 12, and 13 of immunization, animals are injected with 15 μg of antigen into the OO5.

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rhein physiological solution (SFR). 4 days after the last injection, the spleen of those mice that produce higher

antibody titers To poly-B antigen by the solid-phase and enzyme immunoassay. Splenogosts of 7.5 x 10 immune mice are hybridized with 1.5 X 10 X 63 Ag JQ 8-6.5.3 myeloma cells. in the presence of 1 ml of a solution containing 45% polyethylene glycol with a molecular weight of 4000 and 10% of dimethyl sulfoxide for 1 minute. After hybridization and washing of the tails 15 from polyethylene glycol, they are seeded in the wells of a 96-well panel on oly peritoneal macrophages.

Hybrid cell selection was carried out on medium containing HAT-hypoxan-20 tin-aminopterin-thymidine. Producer hybrids were cloned twice by limiting dilution method, sowing into cells of a 96-well panel with a feeding layer of peritoneal macrophages. 25 After the second cloning, almost 100% of the subclones produce antibodies to the antigen under test. The productive hybrid cell clone (hybridomas) is designated as BAMA-Brucella Q

Abortus Mono-clal Antibody.

The BAMA hybridoma strain is stored in the specialized collection of transplantable somatic cells of the vertebrates of the Institute of Cytology, Academy of Sciences of the USSR, under the number VSKK / P / 302 D and is characterized by the following properties.

Morphological characteristic. The culture consists of rounded cells weakly attached to the substrate;

the size of the original myeloma cell. The nucleus occupies most of the cell. Cytoplasm has the appearance of a thin rim.

Cultural properties. Sredad

Cultivation - RPMI-1640 medium with 10% 5, mbrional calf serum, 2 x M glutamine, 1 x m sodium pyruvate, 5 x 10 m 2-mercaptoethanol, 1 x y. mnEreb, 50 µg / ml gentamicin. Cultivation of the strain is carried out at 37.5, C in an atmosphere with 5% CO. The growth pattern is a stationary suspension. Passing frequency - after 2-3 days, short.

sowing 1: 3-1: 4. The seed concentration of cells is 1-2 x 10 per 1 ml. Maximum cell density is 0.5 ppm.

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With intraperitoneal inoculation of a mouse sperm gene, the hybridoma strain forms a tumor in the ascitic form on day 10. Cell dose during inoculation of 2 X 10 cells. 7 days prior to the introduction of the hybridoma, it is necessary to inject animals with pristane - 2, 6, 10, 14 - tetramethylpentadecane in a dose of 0.5 mi per mouse. The strain retains the ability to produce specific monoclonal antibodies for 5 passages on syngeneic mice (observation time).

The productivity of the strain. Hybrid cells are placed in a plastic vial with an area of 25 cm 5 × 10–5 ml of cultivation medium, incubated for 3-4 days at 37.5 ° C in an atmosphere containing 5% C02. The culture medium is harvested, centrifuged for 10 minutes at 800 g and 4 s in order to separate the catches from the supernatant — a supernatant containing antibodies. On the 3–4th day of hybridoma cultivation, the monAt concentration reaches 35-45 µg / ml i culture medium.

When an in vivo hybridoma is cultivated, the immunoglobulin fraction from ascites fluid is obtained by salining out the proteins with ammonium sulfate to 45% saturation followed by dialysis against PBS (pH 7.2-7.3) at 4 s overnight. The resulting antibody-containing materials: the supernatant and purified ascitic fluid with a final concentration of immunoglobulins of 8 mg / mp: are used as reagents for emitting the specificity of the interaction of monAt with the tested antigens using PMA and TYPE.

The characteristics of a useful product, MonAT, belong to the IgG class, subclass 2a. They are specific to the zitope core of the polysaccharide of the LPS of Brucella. The coupling constant is 0.6 x X 10 M.

Methods for assessing the specificity of ICA: microagglutination reaction (PMA) and solid phase immunoferm assay (TYPE).

Contamination strain. Contaminants. no lines, including bacteria, fungi, yeast and mycoplasma, were detected.

Cryopreservation Hybrid cells are added to the pellet. fetal calf serum, and for those dimethyl sulfoxide to a final concentration of -, 10%. Cell suspension is poured into

Sterile plastic ampoules with screw caps of 1-5 mln. cells in a volume of 1 ml are placed in a foam box and left for 1 day at 70 ° C, after which the ampoules are transferred to liquid nitrogen. Thawing: a frozen ampoule is quickly placed in a 37 ° C water bath. Once the last pieces of ice have melted, the hybridoma suspension is transferred with a sterile pipette into a centrifuge tube containing 10 MP of cold-free medium, washed once and seeded into 24-well cells with a feeding layer of peritoneal macrophages. The number of viable cells after thawing at least 73%

EXAMPLE 1. Investigation of the interaction of monobAT BAMA in the microagglutination reaction (RIA) with various species and strains of bacteria of the genus Brucel-1a, as well as with bacteria Yersinia enterocolitica 0: 9, Escherichia coli, Campylobacter fetus veneralis.

PMA is conducted in cells of a 96-well microtiter plate. 50 µl of the undiluted supernatant is added to the first cell, and from the second to the eighth cells, its double dilutions. The purified ascitic fluid is diluted in 16 wells, starting at 1: 100. PBS, pH 7.2-7, is used as a dilution buffer. Then 5 μl of the suspension of bacterial cells is placed in each well centration 1 x cells / ml. The results of the reaction take into account under a light microscope or visually after 2 hours of incubation at 37 ° C.

The data obtained indicate a high specificity of BABA monAt for bacteria of the genus Brucella and the absence of cross-reactivity with the bacteria Y. enterocolitica, E. coli, Campylobacter fetus veneralis.

P p and M e p.2. Investigation of the interaction of monAt with purified polysaccharide antigens and (LPS and) from various species and strains of bacteria of the genus Brucella and Yersinia enterocolitica 0: 9, in an immunoassay.

Olysaccharide antigens (lipopolysaccharides and poly-B) of Brucella and Yerinia enterocolitica 0: 9 at a concentration of 1 μg / ml in 100 μl of PBS () and 7.2, 3) are adsorbed on a polystyrene 96-well Microtitration anel; during

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1604849

nights The panels were washed three times with PBS and three times with PBS with 0.05% Tween-20 (PBS-TV), and then doubled dilutions of the culture medium or purified ascites fluid in an amount of 100 µl were added to the panel cells and incubated for 1 , 5 hours at 3 s. The panel is washed in the manner described in Q and rabbit antibodies against mouse immunoglobulins covalently bound to horseradish peroxidase are added to the cells. After the end of incubation. (1 h, 37 C), the washing procedure is repeated to remove unbound reaction products, and the bound conjugate is quantitatively determined by the cleavage of the substrate, in the presence of the chromogen-orthophenylene diamine. The life of the reaction is characterized by brown staining and is taken into account spectrophotometrically at a wavelength of 492 nm.

The results of an experiment to determine the 25 specificity of immunoglobulin binding from cultured and purified ascites fluid in TIFA show that monAT at the highest dilution (1: 12800) reacts with antigen Q poly-B from Brucella melitensis cells

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B-115, which is devoid of the LPS 0-chain and consists mostly of a polysaccharide site. Thus, it is precisely to the latter that BAMA monats are specific.

Similar epitopes were detected by BAMA antibodies in Brucella abortus LPS.

Example 3. The use of MonAT BAMA as the 1st: lo on a solid phase in an ELISA for the detection of Brucella antigens in the test material.

MonAT (20 µg in 1 MP), purified by salting out ammonium sulfate, in 100 µL of bicarbonate buffer (pH 9.5) was adsorbed onto a polystyrene 96-well microtitre panel with ammonium sulfate in 100 µl overnight. Then the panel is washed three times with PBS and PBS-TV, 100 µl of polysaccharide antigen at a concentration of 1 µg / MP in PBS-TV is added to the cells and incubated for 1.5 hours at 37 C. The panel is washed in the described manner, prepared in cells two-fold dilutions of polyclonal 5 bovine serum in ZFR-Tv (1: 1000 and above), having equal antibody titers against the used species of brucella and yershim for PMA and TYPH. After giving pa /

716048498

At 37 ° С for 1 h, the repeats of the Brucella genus from Yersinia entailed the washing procedure and in the cells of the serovar 0: 9, and

Anti-immunoglob antibodies are introduced in the development of bovine-labeled peroxidase-specific specimens. Physical methods for diagnosing brucelconcentration of binding antibodies of op-leuza. determined according to example 3.

Claims (1)

The results of the study-specific formula of the invention te monat in sandwich variant TIFA consistent with the data of the preceding Strain hybrid cultured examples of animal cells Mus musculus L. The strain of hybrid cells BAMA proSCAS (P) No. 302D, producing monoduciate monoclonal antibodies, clonal antibodies to bacteria of the genus allowing differentiation of bakte-Brucella.