Strain of hybrid cultured animal cells-producer of rat monoclonal antibodies to hypoglycosylated and deglycosylated isoforms of tumours associated with human muci antigen

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry, specifically to obtaining a hybrid, and can be used for obtaining a strain-producer of monoclonal antibodies to the MUCI human antigen. Using monoclonal antibody technology, a strain of hybrid cultured animal cells "ВКПМ Н-105" - producer of clinical rat monoclonal antibodies, specific for hypoglycosylated and deglycosylated isoforms of tumours associated with the human MUCI antigen can be obtained.

EFFECT: possibility of identifying clinical isoforms of MUCI antigen using antibodies, produced by an obtained strain, the antibodies of which can be used determining concentration of MUCI in blood plasma of a person previously diagnosed with tumours.

1 dwg, 3 ex

 

The invention relates to biotechnology and immunology and is concerned with the obtaining of monoclonal antibodies rat to the tumor-associated antigen human MUCI (marker MUCI) using cultivated hybrid cells (hybridomas) animals. The panel of monoclonal antibodies to tumor marker MUCI promising for creation on their basis based assays used in clinical Oncology, in particular for immunohistochemical studies and for immunopathogenicity (ELISA) methods for determining the concentration of MUCI in the serum of human blood (EGTM // Tumor markers in breast cancer - EGTM recommendation, 1999).

MUCI - glycoprotein, expressed on the surface of epithelial cells. The overexpression of this protein observed in cancer. Molecule marker MUCI is a strong polymorphism. The greatest interest from the point of view of early diagnosis of cancer are hypoglycosylated and deglycosylation isoforms antigen human MUCI characteristic of tumors of epithelial cell origin (Br.Cancer Res. And-Treat. 81: 195-207, 2003).

Currently, almost all known antibodies to marker human MUCI obtained in mice (Tumor. Biol. 19 (suppl.1): 1-152, 1998). At the same time, the use of monoclonal antibodies such animal, like a rat, allows you to:

1) to induce a stronger immune response is and antigen MUCI rats than in mice;

2) to obtain monoclonal antibodies rat to the marker human MUCI, differing in specificity of antibodies mouse;

3) during the development of ELISA methods aimed at identifying the antigen MUCI, use species (antiaritmicheskie) antibodies, which are well characterized in rats and little studied for mice (RDI, Inc // Fitzgerald Industries International, USA, 2007).

Information about the equivalents of the claimed invention in available open sources of information are missing. In electronic information sources monoclonal antibodies rat MFGM-5-11 (ICR-2), specific to the antigen of human MUCI, offered as a commercial product by the Russian company "Alabin" for use in immunohistochemistry.

The task of the invention is to expand the Arsenal of hybrid strains of cultured animal cells producing monoclonal antibodies to tumor-associated antigen human MUCI.

The problem is solved by obtaining strain 5F8F3 hybrid of cultured animal cells producing monoclonal antibodies of rats to hypoglycosylated and deglycosylation isoforms marker of human MUCI.

The strain is deposited in Russian national collection of industrial microorganisms and has a number VKPM N-105.

The strain obtained by hybridization of lymphocytes immunized rats August with myeloma cells m the Chi (Sp2/0/Ag14) and the use of subcutaneous immunization scheme, followed by separation limosella In lymphocytes of the rat. As immunogen used conjugate of streptavidin with VNR22-peptide (Sav-TR).

To assess the obtained hybrid and specificity of the produced antibodies was performed by the method of enzyme-linked immunosorbent assay (ELISA) (ELISA) (In Russian). "Antibodies", Vol.2. M.: Mir, edited Dcache, 223-230, 1991) using multiple markers of specificity, namely:

natural purified antigen MUCI isolated from the milk man (J.Immunol.: 135, 3610-3616, 1985);

- VNTR22-polypeptide (J.Biochem.: 263, 12820-12823, 1988);

- synthetic Monomeric polypeptide (TR1) (Encyclopedia of Polimer Science and Technology // by J.Wihey and S.Iuc, 3-49, 2004);

- hypoglycosylated MUCI (MUCI), the resulting controllable periodic destruction of the natural oxidation antigen (J. Immunol. Methods: 78, 143-153, 1985).

Natural purified antigen MUCI, VNTR22-polypeptide and hypoglycosylated antigen (MUCI) represent isoforms of the tumor marker MUCI with varying degrees of glycosylation. Isolated from milk antigen MUCI contains a complex of glycoproteins consisting of a polypeptide of the skeleton with variable number of tandem repeats of 20 amino acids (VNTR) and oligosaccharide chains. This antigen was used as a control in evaluating the interaction of monoclonal antibodies with less glycosylated antigens. Deglycosylation antigen (VNTR22does not contain carbohydrate chains, and on-MUCI, gender is received in the softer controllable periodic destruction of oxidation, characterized abberant glycosylation (hypoglycosylated antigen). Thus, the above markers specificity allow you to set individual specificity of monoclonal antibodies against clinically significant hypoglycosylated and deglycosylation isoforms marker of human MUCI.

The end product.

Monoclonal antibodies of the IgG class 1 (test system to ittipiboon - ISO 2-CT, Sigma), having specificity for the antigen MUCI in hypoglycosylated and deglycosylated form.

Cultivation of the strain in a nutrient medium.

For cultivation use environment DMEM (firm Sigma)containing 10% fetal serum, 1% glutamine and pyruvate, 50 U/ml penicillin and 25 μg/ml of streptomycin. Cells were cultured at 37°C in an atmosphere containing 5-7% CO2. For cultivation use plastic or glass tablets or vials of 50 ml volume (firm Lux). Character growth is stationary suspension. The frequency of passage 2-3 days. The multiplicity of sieving 1:3-1:4. The titer of antibody in culture liquid of 1:128.

Cultivation of the strain in the animal body.

Received hybridoma cultivated in mice Balb/c-nude (H-2d, absent thymus). To obtain ascites mice injected vnutriarterialno 0.5 ml of piers (In kN. "Monoclonal antibodies". M.: Medicine, Ed. by Rtnet is, 396-397, 1983). Prepared animals rest for 3 weeks. Hybridoma cells were washed off contained in the environment serum and injected 4·106cells in Hanks solution (producer - Moscow plant of bacterial preparations). The growth of ascitic tumors noted in 7-10 days. The antibody titer in the culture fluid is 1:128, and in ascitic - 1:10000.

The productivity.

The concentration of antibodies produced by the strain is in the culture liquid of 15 μg/ml, in ascitic fluid 8 mg/ml Stability producing antibody persists for over 20 passages in culture and 7 passages on animals, if hybridoma perebivaetsya.

The contamination.

Bacteria and fungi in cultures not detected during long-term observation and fields on standard nutrient medium (MPA was used to identify bacteria and agar Saburo - for fungi). Infection by Mycoplasma is not detected (test system Mycoplasma Stain Kit, company Flow Lab.).

Preservation of cells.

Cells frozen after the 2nd and 3rd cloning for 10 and 15 passages in culture and after growth in the form of ascitic tumors in Nude mice. Cells in createsite medium of the following composition (wt.%): DMEM - 50, fetal serum - 40 and dimethyl sulfoxide - 10, placed in the fridge for at - 70°S, and then transferred into liquid nitrogen. The survival rate after thawing is 70%.

Izobreteny is illustrated by the following graphic image, which presents immunospecificity monoclonal antibodies of the claimed strain to hypoglycosylated and deglycosylation isoforms tumor-associated antigen human MUCI.

Example 1. Obtaining the claimed strain

Rats August subjected to immunization Sav-TR in complete Freund's adjuvant in pads rear pads (75 μg per animal). On the 13th day of mice subjected to immunization in the pads of the hind legs the same amount of Sav-TR in incomplete Freund's adjuvant (In kN. "Monoclonal antibodies". M: Medicine under. edit Rkennedy, 371-382, 1983). On the 16th day rats hammer, isolated popliteal lymph nodes and lymph node cells merge with myeloma cells Sp2/0/Ag14 using polyethylene glycol (PEG). The ratio of lymph node cells and myeloma cells is 5:1. After merging cells scatter in the wells of 96-hole tablet (firm Nunc) in an amount of 200 thousand cells per well, in which pre-seeded peritoneal macrophages mouse 10 thousand cells per well. Breeding hybrid lead in the environment DMEM containing gipoksantin, aminopterin and thymidine (GAT) (IN Russian). "Monoclonal antibodies". M: Medicine under. edit Rkennedy, 371-382, 1983). The growth of hybrids in the cultural medium containing 15% fetal serum, see 3-7 day.

The growing selection of cell clones secreting antibodies of defined specificity, spend 10-12 day with use the of the ELISA test and the previously mentioned markers specificity.

The result is a hybrid strain of cultured animal cells PMBC N-105 producing monoclonal antibodies rat Hypo - and deglycosylation isoforms tumor-associated antigen human MUCI.

Held twice cloning the obtained hybrid method of limiting dilutions by sieving into the wells of 96-hole tablet at the rate of 1 cell per well in medium DMEM and subsequent tests on the secretion of antibodies showed that the proportion of clones that retain the production of antibodies of defined specificity is 100%.

Example 2. Production, isolation and purification of monoclonal antibodies produced by the strain VKPM N-105

To obtain monoclonal antibodies, the cells of the inventive strain grown in 24-hole plates (firm Nunc) in the cultivation environment (In kN. "Monoclonal antibodies". M: Medicine under. edit Rkennedy, 371-382, 1983). After reaching the concentration of cells in the well 106cells/ml of culture fluid is removed by centrifugation at 2000g for 10 minutes the Precipitated cells are transferred to serum-free medium (DMEM) and injected Nude mice intraperitoneally 4·106cells/mouse. 7-10 days see the visual growth of ascitic tumors, mice hammer and syringe collect ascites in the abdominal cavity. Ascitic fluid is centrifuged 10 min at 2000g to Osvoboditel is from the cells, and the supernatant used for the selection of monoclonal antibodies.

With this objective, the ascitic fluid of mice containing monoclonal antibodies, add an equal volume of saturated (4 M) solution of ammonium sulfate and centrifuged at 5000g for 20 min the Precipitate is dissolved, carefully cialiswhat against 0.5 M phosphate buffer pH 7.2 and purified on a column of DEAE cellulose (DE-52). (Immunology: 4, 40-44, 1980). From 1 ml of ascitic fluid receiving 8 mg of monoclonal antibodies. The purity of the selected thus monoclonal antibodies confirmed electrophoretically and is 90-95%.

Example 3. Determination of specificity and concentration of monoclonal antibodies in the culture and ascitic fluids by ELISA

To the wells of the 96-hole tablet make 100 μl of antigen solution (MUCI, VNTR22-polypeptide, MUCI) with a concentration of 10 μg/ml and incubated for 2 h at 37°C. After 4 times washing tablet from an excess of antigen in the wells contribute a work buffer (phosphate buffer with a pH of 7.2, containing 0.05% bovine serum albumin and tween-20) (In Russian). "Antibodies", Vol.2. Ed. Dcache, 153-183, 1991). Incubated for 30 minutes to Prepare serial 10-fold dilutions of the culture or ascitic fluid in the working buffer. The analyzed sample volume of 50 ál with 2-fold repetition contribute in wells with antigen and incubated for 2 h at room temperature. After the Incubus the AI remove the contents of the wells, wash out the tablet and make 50 ál per well of a solution of the conjugate rabbit antibodies to mouse immunoglobulin horseradish peroxidase at a dilution of 1:3000. Tablets incubated for another 2 h, washed and contribute to the wells in 100 μl of substrate buffer (phosphate-citrate buffer with pH 4.5) (In Russian). "Antibodies", Vol.2. Ed. Dcache, 153-183, 1991)containing 4 mg/10 ml O-phenylenediamine and 0.03% of hydrogen peroxide. Tablets incubated for 10-15 min and stop the reaction by adding 10% sulfuric acid at 100 μl per well. Tablets scan on the reader for microplates (firm Labsystems) at a wavelength of 492 nm. The antibody concentration is determined by a calibration curve which is obtained simultaneously with the test. To construct the calibration curve instead of the samples in the wells contribute breeding standard preparations of purified antibodies of the claimed strain in a known concentration.

The antibody concentration in the culture fluid is 15 µg/ml, and in ascitic 8 mg/ml

The drawing shows data glycopeptides specificity of the obtained monoclonal antibodies. On X axis is the concentration of monoclonal antibodies, on the Y-axis is the optical density at a wavelength of 492 nm. Activity interaction of monoclonal antibodies with antigen increases with increasing antibody concentrations from 1 ng to 1 μg.

In the drawing, Yes the data should that monoclonal antibodies of the claimed strain weakly interact with the natural antigen MUCI, but have sharply increased level of binding to its tumor-specific forms: hypoglycosylated (o-MUCI) and deglycosylated (VNTR) antigen human MUCI, which allows to make a conclusion about the clinical significance of the obtained antibodies.

Thus, the obtained hybrid strain of cultured animal cells PMBC N-105 producing monoclonal antibodies rat, with individual specificity to Hypo - and deglycosylation isoforms marker of human MUCI.

Produced by the claimed strain monoclonal antibodies have the ability to detect clinically significant antigen isoforms MUCI and suitable for determination of its concentration in the serum and tissues at early diagnosis of tumors.

The hybrid strain of cultured animal cells PMBC N-105 producing monoclonal antibodies rat to hypoglycosylated and deglycosylation isoforms tumor-associated antigen human MUCI.



 

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