SU1514768A1 - Strain of hybrid cultivable mus musculus cells as producer of monoclonal antibody to recombinant necrosis factor of human beta-tumours - Google Patents

Description

LOVEK

(57) The invention relates to hybridoma technology. The purpose of the invention is the production of a hybrid strain producing monoclonal antibody (monAT) to human tumor necrosis factor neurosis factor (FIO- / 3), which has biochemical uniformity and high specificity. The strain deposited under the number VSKK (I) No. 17YU.

MonAT belongs to the Ιβ С2Ь isotype, binds to human TNF-β. The content of monat in 1 ml of culture medium and ascitic fluid is, respectively, 55-60 μg and 6-7 mg. About Production of antibodies persists for 20 passages in culture. MonAT A can be used to determine human P TNF- / 5 by the method of im- * monobdoting. 1 tab.

The invention relates to hybridoma technology.

The purpose of the invention is to obtain a hybrid strain that produces monoclonal antibody (monAT) to human tumor necrosis factor-beta, which has biochemical homogeneity and high specificity.

Strain was prepared as follows.

Mice of the Ba1b / c line are immunized by intraperitoneal administration of 50 μg P of TNF-α (3 in 0.15 ml of 0.15 M MaCl in a mixture with an equal volume of Freund's complete adjuvant. Immunization is repeated after 14 days with the same amount of antigen in a mixture with Freund's incomplete adjuvant.

After another 14 days, 50 μg of P TNF-β is given intraperitoneally in 0.5 ml of 0.15 M NaCl. After 3 days, 6x10 ⁷ cells of spleen of immune mice and 1.2x10 ⁷ myeloma cells Χ63-Αβ8-653 were hybridized with 50% polyethylene glycol solution, mass 4000, containing 5% dimethyl sulfoxide. After hybridization and washing of cells from polyethylene glycol, the latter are seeded in 96-well panels of 1x10 ⁵ splenocytes per well. For the cultivation and breeding of hybridomas use 1MDM medium (modified 1 in 'on Dulbecco's medium) with the addition of 10% fetal calf serum

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(OTS), 1 (G ⁴ M hypoxanthine, 4'10 ⁷ M aminonterin, and 1.6 · 10 M thymidine. Producer hybrids are cloned 2 times by the final dilution method, sowing 1 cell per well, containing 4'10 spleen cells. After two cloning, almost 100% of the subclones produce MonAT to P. FIO-. The antibody production is preserved for at least 20 passages in culture. The hybridoma has received the conditional name LTD9.

The strain is characterized by the following features.

Cultural features. Standard cultivation conditions. Wednesday 1MDM with 10% donor calf serum, 2 x 10 ' ⁴ 1 - glutamine,

100 μg / ml penicillin, 100 μg / ml streptomycin, 0.05x10 ”^ M mercaptoethanol, at 37 ° C and with an atmosphere of 5% C0 ₂ . For growing strain using glassware. Sowing dose of 100-200 thousand cells per 1 ml. Cells are passaged once every 2-3 days. The multiplicity of sieving - 1: 6-1: 8. Suspension culture.

Cultivation in the animal. VABB / C mice are suitable for growing ascites. For 7–30 days before the injection of strain cells, intraperitoneal (ip) in 0.5 ml of pristane is injected. Cells are injected ip at 2-5x10 cells / ml of IMMD medium. Ascites is formed in 12-14 days.

The productivity of the strain. MonAT secretion for 2-3 days of cultivation is 55-60 µg / ml of culture medium and 6-7 mg in 1 ml of ascites fluid when determined by an enzyme immunoassay.

Characteristics of the resulting product. The strain produces monat Ιβ C2B. Specificity - name — β.

Methods for assessing the preservation of specificity: a test for the binding of monAT to an immobilized FULL NAME-β (enzyme immunoassay). The antigen is sorbed on plastic: 500 ng in 50 μl of 0.1 M bicarbonate buffer, pH 9.6, overnight at 4 ° C. Then, after washing, the test culture medium is applied after incubation, and the amount of sorbed antibodies with peroxidase-labeled rabbit anti-mouse antibodies is determined by washing. All washes

lead bidistilled water. The control is bovine serum albumin as an antigen.

Cryoconservation. For long term storage, strain cells are frozen in fetal calf serum with the addition of 10% dimethyl sulfoxide. Freezing mode: 1 C in 1 min to -70 ° C. After freezing, cells are transferred to liquid nitrogen. Razo

freezing in a water bath at 37 C. Cell viability after thawing 50-55% by trypan blue staining.

Contamination. Bacteria and fungi were not detected in culture during long-term observation and culture on nutrient media. Infection with mycoplasma was not detected by staining with dyes on DNA and the nature of the inclusion of thymidine label.

Example 1. Test for determining the specificity of the interaction of monat with antigens immobilized on plastic.

P FIO- O4, P TNF-β and bovine serum albumin (BSA) in the amount of 0.5 μg in 50 μl of 0.1 M carbonate buffer, pH 9.6 is added to the wells of 96-well microplates and sorb overnight at 4 ° C . After washing, 50 μl of culture medium of strain LTD9 or diluted 1: 5000 in 0.01 M phosphate buffer, pH 7.4, 0.15 M LaCa, 1% BSA (PBS-BSA) polyclonal rabbit antibodies specific to P are introduced into the cells. TNF ~ (5. The panel is incubated for 2 hours at 20 ° C, then washed 5 times with double-distilled water and 50 μl per well of rabbit anti-mouse antibodies, labeled with peresidase, diluted 1/2500 in PBS-BSA and labeled with peroxidase anti-rabbit antibodies 1/2500 SFR-BSA. After incubation for 1 h at 20 ° C, the plate is washed and the substrate, orthophenylene-dia, is added to the wells. yn hydrochloride, 0.6 mg / ml in citrate-phosphate buffer, pH 4.7, containing 0.03% H _r ^ 0. The reaction was stopped after 10 min with 1 M sulfuric acid and allow for a vertical beam spectrophotometer at a wavelength of 492 nm.

The results of the experiment on the study of the specificity of binding of monat produced by cells of the strain LTD, with

-antigens immobilized on plastic are presented in the table.

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The results of the experiment on the study of specificity testify to the high specificity of monAT produced by the cells of the LTD9 strain, and show the possibility of using this monAT to determine the P FIO-β using an enzyme immunoassay method.

Example 2. The interaction of monat LTD with R FIO-β person, transferred to nitrocellulose 'after the disc electrophoresis in polyacrylamide gel.

Electrophoresis in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE) is carried out using a vertical electrophoresis apparatus, P ΦΗΟ-β 10 μg, in a sample buffer with mercaptoethanol and an equivalent amount of E. coli lysate in the same buffer is heated 2 min at 100 ° C and applied to the cells of the gel. Electrophoresis is carried out at a constant current of 50 mA.

The electrical transfer of the separated components from the gel to nitrocellulose (NC) with a pore diameter of 0.45 μm is performed in the apparatus for 1 hour. After the transfer, the NC is washed with distilled water and further processed using an immunoblot kit.

Nonspecific protein sorption on NC is blocked in 0.02 M tris-HC1 buffer, pH 7.4 with 0.5 M LaCl (TBR) containing 3% gelatin. After that, the SC is washed 2 times with TBR with 0.05% tween20 (TTBR) and cut into strips.

Binding specificity

The strips corresponding to the 'LTO-PAGE P FIO-β and E. coli lysate are placed in 15 ml glass tubes and 10 ml of the supernatant MonAT LTD9 or rabbit antisera at a dilution of 1/5000 in TTBR with 1% BSA are added. The incubation is continued for 2 hours at 20 ° С on a device with horizontal stirring. After that, the NC wash

3 times 5 min each of TTBR, goat anti-mouse or anti-rabbit anti bodies added, diluted with 1/2000 in TTBR, 1% BSA and incubated for 1 h, at 20 ° C with stirring. After 2 washes with TTBR and one washed with TBR, the reaction is shown in a substrate solution containing 4-chloro-1-naphthol and 0.015% H ^ O ^, for 20 minutes. For storage and foto29 graphing NTS washed in distilled water and dried in air.

The results of this example indicate the binding of monAt, produced by the cells of the LTD9 strain, with P FIO-p after DSP-PAGE and electrotransfer to nitrocellulose. MonAT strain LTD9 can be used to determine the P FIO- / 3 method im30 munoblotting.

Claims (1)

<claim-text> Formula of Invention </ claim-text> <claim-text> Strain of hybrid cultured animal cells Mizzizi5 SCC (II) £ 171P - producing monoclonal antibodies to recombinant human tumor necrosis factor-beta. </ claim-text> <claim-text> monat with antigens </ claim-text> <table border = "1"> <tr> <td> <claim-text> Antigens </ claim-text> </ td> <td> <claim-text> Culture - </ claim-text> </ td> <td> <claim-text> Rabbit anti- </ claim-text> </ td> </ tr> <tr> <td> <claim-text> </ claim-text> </ td> <td> <claim-text> Wednesday </ claim-text> </ td> <td> <claim-text> serum </ claim-text> </ td> </ tr> <tr> <td> <claim-text> </ claim-text> </ td> <td> <claim-text> LTD9 strain </ claim-text> </ td> <td> <claim-text> 1: 5000 </ claim-text> </ td> </ tr> <tr> <td> <claim-text> </ claim-text> </ td> <td> <claim-text> A492x10 <sup> 3 </ sup> </ claim-text> </ td> <td> <claim-text> A492x10 <sup> 5 </ sup> </ claim-text> </ td> </ tr> <tr> <td> <claim-text> _ ______ </ claim-text> </ td> <td> <claim-text> </ claim-text> </ td> <td> <claim-text> </ claim-text> </ td> </ tr> <tr> <td> <claim-text> BSA </ claim-text> </ td> <td> <claim-text> 659 </ claim-text> </ td> <td> <claim-text> 261 </ claim-text> </ td> </ tr> <tr> <td> <claim-text> FIO-p </ claim-text> </ td> <td> <claim-text> 2444 </ claim-text> </ td> <td> <claim-text> 2656 </ claim-text> </ td> </ tr> <tr> <td> <claim-text> full name </ claim-text> </ td> <td> <claim-text> 667 </ claim-text> </ td> <td> <claim-text> 342 </ claim-text> </ td> </ tr> <tr> <td>