SU1507791A1 - Strain of hybrid cultivable mus musculus cells as producer of monoclonal antibody to factor of alpha-tumor necroses of man - Google Patents

Abstract

This invention relates to the field of hybridoma technology. The purpose of the invention is to obtain a hybrid strain producing monoclonal antibody (mon AT) to human tumor necrosis factor-alpha (TNF-α), which has biochemical uniformity and high specificity. The strain deposited under the number VSKK / P / N173D. The strain produces mon AT JGG1 isotype that binds to human TNF-α. The content of mon AT in 1 ml of culture medium and 1 ml of ascites fluid is 14-20 µg and 5-7 mg, respectively. Antibody production is maintained for at least 15 passages in culture. The mon Abs of the hybridoma strain can be used for the immunosorption of P TNF-α as well as for the purification of the latter. 2 tab.

Description

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31507791

; This invention relates to hybridoma technology.

i The purpose of the invention is to obtain a hybrid strain that produces monoclonal antibody (monat AT) to tumor HiiKposa alpha factor (TNF-E human Bi Ka, which has biochemical homogeneity and high specificity.

Strain was prepared as follows. ten

The lower line BA1B / C is immunized according to a 2-week schedule. 20 μg of P in 0.15 m of NaCl in a mixture with an equal volume of Freund's adjuvant adjuvant twice injected intraperitoneally with an interval of 7 days. 3, 2 and 1 day before the merger, 1} µg of P TNF-o was administered intraperitoneally in 0.5 ml of 0.15 M NaCl. Hybridization of 1.2 X 10 cells of spleen immune cells and 4 X 10 mouse myeloma cells} i.63-Ag8-653 is carried out with a 50% solution of IOM polyethylene glygol mol. Mass 000 containing 5% dimethylsulfoxy ;; a, in for 1.5 minutes After hybridization and removal of polyethylene glycol cells, cells are seeded in 96-well 1 aneli of 1 X 10 splenocytes per well. For the cultivation and selection of hybridomas, 1ChCH4 medium (modified Iskove S Dulbecco's medium) is added by adding 10% fetal serum 5 serum

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4 X 10 M aminopterin and 1.6 x 10 | himidine. Producer hybrids are cloned 2 times by the finite-dilution method of a syphilus per 1 cell per well, containing 4 x 10 spleen cells. After Z cloning, almost 100% of the palonons produce mon AT to P TNF-c /. Antibody production is stored as (for at least 15 passages in culture.

The hybridoma received the conditional name 13D10, the strain was deposited under the VSKK (P) number 173D.

Cultural features.

Standard cultivation conditions. IMDM medium with 10% donor; tel short; 2x10 M glutamine; 100 µg / ml penicillin, 100 µg / ml: Streptomycin, 0.05x10 M mercapto-ethanol, 5% COj atmosphere

For the cultivation of the strain, the following was used: glassware, a sowing dose of 100-: 200 thousand cells / ml, cells were passaged once every 2-3 days, the multiplicity of sowing was 1: 6-1: 8, suspension culture

Cultivation in animals. Suitable for growing ascites

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50

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BALB / C mice. Mice 7-30 days before the injection of strain cells were injected intraperitoneally with 0.5 ml of prisant. Cells are injected intraperitoneally with 2-5x10 cells / ml IMDM medium. Ascites forms after 12-14 days. Productivity shtamma. The secretion of monoclonal antibodies on the 2-3rd day of cultivation is 14-20 µg / ml of culture medium and 5-7 mg / ml. ascitic fluid when determining the enzyme immunoassay.

Characteristics of the resulting product. The strain produces monoclonal IgGI antibody. Specificity - NFOch.

Methods for assessing the preservation of specificity: a test for binding AT-mon with immobilized TNF-c (immunoassay).

The antigen is sorbed on a plate: 500 ng in 50 μl of 0.1 M bicarbonate buffer, pH 9.6, overnight at 4 ° C. After washing, the test culture medium is applied, after incubation and washing, the amount of sorbed antibodies is labeled with peroxidase-labeled rabbit anti-mouse antibodies. All washes were carried out with bi-distilled water. The control is bovine short-lived albumin (BSA) as an antigen.

Cryoconservation For long term storage, the strain cells are frozen in fetal calf short with the addition of 10% dimethyl sulfoxide. Freezing mode: 1 ° C / min. After freezing, the cells are transferred to liquid nitrogen. Thawing in a water bath at 37 C. Cell viability after thawing 55-60% by trypan blue staining

Contamination Bacteria and culture, not detected with prolonged observation and culture. On nutrient media. Infection with mycoplasma was not detected by staining with DNA stains. and the nature of the inclusion of the tmidine label.

P R and M e R 1, Test for determining the specificity of the interaction of mon AT with antigens immobilized on the plate R TNF-α, P TNF-α and VSA in the amount of 0.5 μg in 50 μl of 0.1 M carbonate buffer PJ 9.6 t into the wells of 96-well microplates and sorb overnight at 4 ° C. After the cell, 50 µl is poured into the wells.

culture medium of strain 13D10 or diluted 1: 5000 in 0.01 M phosphate buffer, pH 7.4, 0.15 M NaCl, 1% BSA (FDA-BSA) polyclonal rabbit antibodies specific for P TNF-o. The panel is incubated for 2 hours at 20 ° C, then washed 5 times with bidistilled water and 50 µl / well of rabbit anti-mouse antibodies labeled with 10 peroxidase, diluted 1: 2500 in PBS-BSA, and labeled with a progugi peroxidase are added. 5 mg of the hybridoma 13D10 sulphate fraction of ascites is mixed with 0.3 ml of the obtained sorbent, incubated on a horizontal shaker for 1 hour at room temperature, filled in a gel with a microcolumn from a Pasteur pipette and washed with TBR. Samples of P TNF-o and P TNF-α / E are prepared on minimal medium modified by Dulbecco (DMEM) with 5% ETS, glutamine, mercaptoethalone, penicillin, and 1: 2500 strepto-rabbit antibodies in PBS-BSA. tomycin (full medium (DMEM) .0 ml

fugirovaniya. 5 mg of the hybridoma 13D10 sulphate fraction of ascites is mixed with 0.3 ml of the obtained sorbent, incubated on a horizontal shaker for 1 hour at room temperature, filled in a gel with a microcolumn from a Pasteur pipette and washed with TBR. Samples of P TNF-o and P TNF-α / E are prepared on a minimal medium modified by Dulbecco (DMEM) with 5% ETS, glutamine, mercaptoethal, penicillin and strep

After incubation for 1 h at

20 C, the plate is washed and 15 minutes is added to the wells, then the substrate is collected and sterilized — orthophenylene diamine is hydrated by filtration through a filter with 0.6 μg / ml diarochloride in a citrate-phosphorus pore of 0.22 μm. . fat buffer, pH 4.7, containing the biological activity of factors 0.03% HjOg. The reaction is stopped by cell lysis by murine 10 min cut with 1 M sulfuric acid and 20 fibroblastoid line b) 29 V is present on a spectrophotometer with a vertical beam at a wavelength of 492 nm. The results of an experiment to study the specificity of binding my ATs produced by the cells of strain 13D10 to the antigens immobilized on the plate are presented in Table 1.

Table 1

1 PP,

The results indicate a high specificity of mont AT produced by the cells of strain 13D10, and shows the possibility of using this mon AT to determine P FULL NAME-c using the immuno-enzyme method. The high affinity constant of monat AT strain 13D10 creates additional advantages when used in immunofermental analysis.

Example 2. Sorbtsi P FULL NAME-OI MON AT strain 13D10. To 1 ml of 5Pa-Sephrase-4B, add 1 ml of rabbit

clutches to mouse immunoglobulins and., speculate about the specific binding of the bir- incubate for 1 h with room temperature activity of the recombinant TNF-mont AT strain 13D10. Thus, mont AT strain 13D10 may

The procedure of Nesv ’s antibodies is RTM-0.05 M Tris-HC1.0.15 M NaCl, pH 7.4 (TBR), by multiple centrifuges used for immunosorption

the sample is passed through the column in teS

actinomycin, on the eve of the test, L929 cells disperse 50 thousand / well into flat-bottomed 6-cell microplates in complete DMEM medium. Samples of the first name are patched in a separate plate in a volume of 100 µl, then transferred to the plate with L929 cells, actinomycin is added to a concentration of 1

µg / ml and place the B thermostat with a temperature of 37 ° C for 16-20 hours. The results are taken into account after staining the cells with a 0.2% solution of a gentian violet in 2% methanol for 10-12 minutes. The optical density of the wells was measured at 540 nm using a vertical beam spectrophotometer. The results of the experiment on the sorption of P TNF-i / mon AT strain 13D10 are presented in table 2.

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table 2

Recombinant FULL NAME-0 445

Recombinant TNF-316

1308

413

The data of this example is evidence to be used for immunosorption.

715077918

Claims (1)

with the whole study of the biological effects of the formula of the inventions of the P R TNF-o (and also for its purification - the strain of hybrid cultured ki. cells of animals Mus Musculus VSC 5 (P) No. 173D - producing monoclonal. The optical density of stained antibodies to the necrosis factor of intact cells is 1290. human cholea-alpha.