LT3879B - Hybridoma producing murine monoclonal antibodies against human growth hormone - Google Patents

Abstract

This invention applies to biotechnology. The main point is the creation of hybridomas and the production of monoclonal antibodies against human growth hormone. Hybridomas differ from earlier described analogues according to the origin of mother-cells, method of hybridisation and specifics of the produced monoclonal antibodies. The monoclonal antibodies may be applied for the determination of human growth hormone.

Description

The invention is in the field of immune biotechnology. Its essence is the development of a novel hybridoma and the generation of monoclonal antibodies (MA) against human growth hormone.

Hybrid cell lines (hybridomas GH-1 and GH-2) are MA anti-human growth hormone producers. Both of these cell lines were obtained by the same method, characterized by the same characteristics. They differ in one property - MA produced by GH-1 and GH-2 recognize different epitopes on human growth hormone moiety.

Hybridoma analogs GH-1 and GH-2 may be considered hybrid cell lines producing MA against human growth hormone A64-D / E3, A64-E / C8, A64-E / B12 and A64-D / F2 [DD 263537, A61K39 / 395, 1987] and against bovine growth hormone, MMB 2/3 [SU 1664184, A61K39 / 395, 1988]. Hybridomas GH-1 and GH-2 differ from hybridoma HMB 2/3 in the specificity of MA produced (MME 2/3 produces MA against bovine growth hormone) and from hybridoma A64-D / E3, E / 08, E / B12, D / E2 - according to the structure of the antigen used for immunization, the method of hybridization and the origin of the parental cells (myeloma).

The object of the present invention is to create hybridomas which produce specific antibodies against human growth hormone. For this purpose, mice were immunized with recombinant human growth hormone, followed by crossing of spleen lymphocytes of immunized mice with murine myeloma UFO / 1 according to the Koller and Milstein method [Kohler,

Milstein, Eur. J. Immunol. 6: 511-919 (1976)]. After screening and cloning the hybrid cells, hybridomas GH-1 and GH-2 were obtained.

Hybridomas deposited at Institute of Biotechnology Enzyme: GH-1 deposit number H 0304, GH-2 deposit number H 0305.

Hybridoma GH-1 and GH-2 are characterized by the following traits.

Morphological features. Hybrid cells are spherical in shape with smooth edges and a large nucleus. Multiplies in suspension and exhibits weak adhesion to glass or plastic surfaces.

Signs of cell culture. Culture conditions are standard: DMEM medium with 10% embryonic cow serum, 2 mM L-glutamine, 20 mM HEPES buffer, 200 ug / ml gentamicin, grown at 37 ° C, 5% CO ₂ . Glass or plastic containers are used for growing the cells, transplanted every 3-4 days, the grafting dose is 100 thousand. cells / ml.

Animal production in the body. In BALB / c singletal mice, hybridomas GH-1 and GH-2 cause ascites in 100% of cases by intracellular injection of 1-2 million. cells. Mice are sensitized with 0.5 ml pristan 7-10 days before cell injection. Ascites develops 8 to 12 days after injection of hybrid cells.

Cryopreservation. For long-term storage, cells are frozen in embryonic cow serum containing 10% dimethylsulfoxide. Freezing mode - 1 ° C / min. to -70 ° C, after which the cells are transferred to liquid nitrogen. Thawing - water bath 37 ° C. Cell viability after thawing - 70-80%.

Contamination. Not found in bacterial and fungal cultures, negative for mycoplasma test.

Cell productivity. At 3-4 culture days, MA secretion in the medium is 10-15 ug / ml, in the ascitic fluid 5-10 mg / ml, and the MA titer in the ascitic fluid is 1: 30,000. MA production remains up to 15 passages in culture and up to 5 passes in ascites.

Product Characteristics. Hybridomas GH-1 and GH-2 produce an IgG class, an IgG1 subclass MA, specific for human growth hormone. These MAs have been shown to recognize different epitopes (amino acid sequences) in the human growth hormone molecule by two-way analysis.

The following examples illustrate the invention.

example. Obtaining hybridomas. BALB / c mice are immunized with recombinant human growth hormone with a deletion of the first N-terminal amino acid (des-Phe-1-STH). This hormone is made by the Enzyme Institute of Biotechnology. Mice are immunized by injection of antigen into the abdominal cavity. The first 3 immunizations are given every 10 days with 100 μg of growth hormone suspended in complete Proindo adjuvant per mouse. The final immunization is given 5 weeks later - 100 μg of growth hormone in saline 5, 3 and 2 days before hybridization.

The murine myeloma line NSO / 1 in logarithmic growth phase and immunized mouse spleen lymphocytes were used for hybridization. Hybridization Agent - 50% Polyethylene Glycol (PEG-4000) in DMEM with 15% Dimethylsulfoxide, Myeloma to Spleen Cells in Hybridization 1: 7. Hybridization is performed for 1 min, then cells are washed from PEG in serum-DMEM, centrifuged, suspended in HAT medium and plated in 10 cells / well. Prior to this, the plates were seeded with macrophages from the mouse abdominal cavity. Hybrid cells are selected on HAT medium, which is gradually replaced with HT medium (hypoxanthine, thymidine) on culture day 7, followed by normal culture medium with double serum concentration (20%). The hybrid producers are cloned by the high dilution method, plating 1 cell per well. Selected clones were propagated and further cultured in the peritoneal cavity of BALB / c mice, where ascites formed.

example. Verification of MA specificity by indirect IFA. The indirect immunoenzyme assay (IPA) method is used to select positive hybrids. The plates are sorbed with human growth hormone: 0.5 μg of antigen in 100 μulin of 0.05 M Na-carbonate buffer (pH 9.5) is added to the wells and incubated for 16 hours. At 4 ^U G. Plate wells are then blocked with 1% albumin solution, rinsed with water, and filled with 100 μΐ of hybrid medium. After incubation (1 h at room temperature) and wells, 100 μΐ of rabbit anti-mouse immunoglobulin conjugated with horseradish peroxidase is added. After incubation (30 min at room temperature) and rinsing, develop the enzymatic reaction by adding 100 μΐ o-phenylenediamine solution (0.5 mg / ml) in 0.05 M Na-acetate buffer, pH 5.5, to the wells, 03% hydrogen peroxide. The reaction is stopped after 10-15 min by the addition of 50 μΐ of 10% sulfuric acid solution. The optical density was measured with a Tltertek MultilSCCLT1 (Flow) photometer at 492 nm. Wells containing growth media of hybridomas GH-1 and GH-2 had an optical density in excess of 2 opt. units, and less than 0.3 opt. in wells containing MA medium non-producing hybrid hybrids. pcs.

example. Verification of MA specificity by two-epitope IFA. The plates are sorbed with MA GH-1 (1 μβ per well). After blocking and rinsing (see Example 2), 100 μΐ of human growth hormone solution (20-200 ng / ml) in phosphate buffer containing 0.05% Tween-20 detergent is added to the wells. After incubation (1 h at room temperature) and rinsing, 100 μΐ of conjugate MA GH-2 conjugated to horseradish peroxidase was added. Incubate for 1 hour. at room temperature. The plate is then washed and the enzymatic reaction developed as described above (see Example 2). The results of this analysis showed that MA GH-1 and GH-2 react with different epitopes of human growth hormone, i.e. a direct dependence of optical density on antigen concentration was observed. When used in such an assay system, antibodies of the same specificity or competing antibodies (e.g., GH-1 with GH-1 conjugate) do not react with the conjugate and no increase in optical density is observed.

pavy z d y s. Determination of Human Growth Hormone by Two-Epitopic IFA. The assay is performed as described in Example 3. The concentration of growth hormone in the test solutions is determined from a calibration curve which represents the dependence of the optical density on the hormone concentration. The linear dependence is observed at a concentration of 5-100 ng / ml. A standard growth hormone solution (1 pg / ml) is used for the calibration curve and diluted accordingly. The sensitivity of the method is 10 ng / ml growth hormone. By this method, MA GH-1 and GH-2 were found to respond equally well to recombinant human and natural (pituitary) human growth hormone, but not to bovine growth hormone. The method can be used to control the technological process by producing both recombinant ini and natural human growth hormone as well as to analyze the end product. The method can also be used in medical diagnostics - in cases where serum growth hormone levels are elevated (above 10 ng / ml).

Hybridomas GH-1 and GH-2 differ from the analogue described above, hybridoma MMB 2/3, in the following features: 1) Hybridoma MMB 2/3 produces antibodies against bovine growth hormone; 2) Hybridoma MMB 2 / ?; derived from another myeloma cell line, Χ-63 Ag8653.

Hybridomas GH-1 and GH-2 differ from the analogs described above - Hybridomas A64-D / E3, A64-E / C8, A64-D / E2, A64-E / B12 in the following features: 1) said hybridomas obtained by immunization natural human growth hormone, 2) sophisticated electrofusion method for cell hybridization, 3) radioimmune method for selection of positive hybrids requiring special precautions, 4) myeloma cell line used for hybridization Ag-63 Ag8653Hybridomas GH-1 and GH-2 In Table 1.

table.

Characterization of hybridomas producing MA against growth hormone

Hybridoma ί name ί I i Specificity Aug hormone Immunogen origin i Hybridization- ! her way 1 { I Myeloma I I I l MMB 2/3 bull bull PEG 1500 X-63Ag8653

A64-D / E3, A64-E / C8, A64-E / B12, A64-D / F2 human human, natural electrofusion X-63Ag8653 GH-1, human human, PEG 4000 NSO / 1 GH-2 recombinant,

des-Phe-1

Claims (1)

DEFINITION OF INVENTION Hybridomas producing murine monoclonal antibodies to human growth hormone (deposited at the Institute of Biotechnology, accession numbers H 0304 and H 0305).