SU1530638A1 - Strain of hybrid cultivable mus musculus cells as producer of monoclonal antibody to necrosis factor of alpha-tumors of man - Google Patents

Abstract

The invention relates to hybridoma technology and can be used for the determination and immunosorption of human tumor necrosis factor-α (TNF-α). The purpose of the invention is to obtain a strain producing monoclonal antibody to TNF-α of G2B isotype with an increased titer in ascites. Strain 7G 1 obtained by immunization of mice of BALB / C mice with recombinant TNF-α. Next, spleen cells of immune mice and X63-AG8-653 mouse myeloma cells are hybridized and then selected and cloned. The resulting strain deposited under the number VSKK (P) N 172D. The strain produces monat JGG2B, specificity - TNF-α. Ascites titer exceeds 1: 1,000,000. 2 tab.

Description

The invention relates to hybridoma technology and can be used to determine and immunize the necrosis factor of human oleu-human necrosis (TNF-oi).

The purpose of the invention is to obtain a strain that produces monoclonal antibody to the full name of the G2b isotype with an increased titer in ascites.

Strain get the following way.

BaLb / c mice are immunized on a two-week schedule. 20 μg of recombinant TNF-oC (p full name) in 0.15 M NaCl in a mixture with an equal volume of ps; Freund's adjuvant two centers APP intraperitoneally with an interval of 7 days, 3, 2 and 1 day before fusion 10 mcg

TNF-ii is administered intraperitoneally. About 0.5 ml of 0.15 M NaCK bryd laniDO 1,2x10 cells of the Spleen immune Mitiueff and

 M1 cell cellulose bhp HbZ-AaYa-653

carried out with 50% -ui.jM solution of polyethylene glycol mol. mass 4000, containing 5% dimethyl sulfoxide, for 1.5 KtiH. After hybridization and washing of polyethylene cells, cells are seeded in a 9 in-1 panepea with 1 x 10 splenoitis per well. For the cultivation and breeding of hybridomas, use is made of 1M1) M medium (modified by Iskove s S. da Dulbeks; with the addition of 10% fetal calf serum (IT),

ate

Od

ABOUT

but

with

00

10 M hypoxanthin, 4 x

aminopterin and

, 6 X timidnna. Hybrid

producers of CLS are caus of final dilutions, seeding 1 cell per well containing 4 x 10 spleen cells. After two cloning, almost 100% of the subclones produce AT to P FIO-. Antibody production is maintained for at least 15 passages in culture.

The resulting 7G1 strain is deposited under the FCC (P) number 172D and is characterized by the following: w; hrmi signs.

Cultural features. Standard culture conditions, IMDM medium with 10% of donor bovine serum, 2 X 10 M LH. 1 utamine, 100 μg / m penicillin, 100 μg / ml streptomycin, 0.05 X 10 M mercaptoethanol, at 37 ° C and 5% CO atmosphere. To grow the strain, glassware was used, a sowing dose of 100-200 thousand cells per 1 ml, the cells were passaged i every 2-3 days, the multiplicity of sowing was 1: 6-1; 8. Suspension culture,

Cultivation of animal rganism. BALB / C mice are suitable for growing cites. For 7-30 days before the injection of cells pc, -,. intraperitoneally injected in 0.5 ml of npucTai a, cells of H1 are injected intraperitoneally in 2-5 x 10 cells of I ml of If-ffiM medium. Lscyte forms after 12-14 days

Biosynthesis of a useful product. The secretion of monoclonal antibodies (MMAT) for 2-3 days of cultivation is 25-30 µg / ml of culture, culture medium and 7-8 mg in 1 ml of aging fluid in the determination of immunity;. rmsntn1 1m m t odom,

Characteristics of the product received. The strain produces monoclonal antibody IgG2b. Specificity - TNF-oi

Methods for assessing the retention of specificity; a test for coupling with immobilized TNF - C (s) (ucoferms (nt analysis)).

The antigen is sorbed onto PLO static: 500 ng in 50 µl of 0.1 M bicarbonate buffer, pH 9.6, overnight at ° C. Then, after washing, the test culture medium is applied and after incubation of TSH and washing, the following are determined: n-quality sorbed antibodies labeled with peroxidase, rabbit anti-mice and antibodies. All washes were carried out with Oidyl distilled water. The control is bovine serum al bumin n quality antigen.

Product stability MonAT production is maintained at least up to 15

P

-

Q

five

0

five

na (-: c fiA; and ill vitco. B; Sci ji ii,;.: it strain didn’t psize off )— times.

Contamination Bacteria and fungi in culture were not detected during long-term observation and culture on nutrient media. Infection with 1plasma was not detected by staining with dyes on DNA and by the nature of the release of the thymidine label.

Cryoconservation For long-term storage, strain cells are frozen in fetal bovine serum supplemented with 10% dimethyl sulfoxide. Freezing mode: 1 ° C / min to -70 ° C. After freezing, the cells are transferred to liquid nitrogen. Defrosting in the bath at 37 ° C. Cell viability after thawing is 65-70% with trypan blue staining.

Example 1. Hybridoma cells are placed in a glass vial with a volume of 50 ml of 1 X 10 cells in 5 ml of medium with 10% ETS, 2 mM of glutamine, 100 μg / ml of penicillin and streptomycin. 0.05 m11 mercaptoethanol. Clet-1S cultured 2-3 days at 37 ° C in an atmosphere of 5 CO. The resulting supernatant is used as a reagent in chemical chemical rodents (as a monAT preparation).

The test for the determination of the specificity of monoAT anti-genes with antigens, which are mobilized on plastic.

Method used: and; : ykofferment analysis.

P TNF-cC, P and bovine serum albumin. (BSL) in the amount of 0.5 µg in 50 µl of 0.1 M carbonate buffer, pH 9.6, are added to the wells of 96-cell sprinkles and sorbed overnight at, After washing, 50 µl of culture medium are added to the wells strain 7G1 or diluted 1: 5000 in 0.01 M phosphate buffer, pH 7.4, 0.15 M NaCl, 1% BSA (SFR-BSA) polyclonal rabbit antibodies specific for P FIO-vol. The panel is incubated for 2 hours at, then washed 5 times with double-distilled water and 50 µl / well of rabbit anti-mouse antibodies labeled with peroxidase, diluted 1/2500 in PBS-BSA, and peroxidase labeled anti-rabbit antibodies 1/2500 in PBS are applied. -BSA, after incubation for 1 hour at 20 ° C, the fee is washed and into the wells

the substrate is orthophenyleidiamine hydrochloride, 0.6 mg / ml in citrate-phosphate buffer, pH 4.7, containing 0.03%. The reaction is stopped after 10 mines of M with sulfuric acid and taken into account on a spectrophotometer with a vertical beam at a wavelength of 492 nm.

The results of the experiment to study the binding specificity of monat produced by cells of the 7G1 strain with antigens immobilized on plastic are presented in Table 1.

The results of this example indicate the high specificity of monAT produced by cells of the 7G1 strain, and show the possibility of using this monAT to determine the P FULLY-in using an enzyme immunoassay method.

The data of Table 1 show that monat 7G1 from the culture medium of the hybridoma interacts with TNF-about-the same with the rabbit antiserum at a dilution of 1: 5000 (2563 and 2584, respectively). MonAT binding specificity c. moreover, higher than that of papiclonal antibodies (with respect to binding to BSA and FIO-ji),

PRI mme R 2. Sorbtsi R TNF-os monAT strain 7G1.

To 1 ml of Spa-sepharose 4B, 1 ml of rabbit antisera was added to the immunoglobulin mass and incubated for 1 hour at room temperature. Nesv zavshaysh antibodies washed with 0.05 M Tris-HCl, 0.15 MNaCl., PH 7.4 (TBR) by multiple centrifuging rutem, 0.3 ml of the resulting sorbent is mixed with 5 mg of the ascite sulfate fraction of hybridoma 7G1, incubated on a horizontal shaker 1 at room temperature, the microcolumn of the Pasteur pipette is filled with gel and washed with TBR. Samples F of FIO-about and P FIO-p are prepared on a minimal medium modulated by Dulbecco (D- NEM) with 5% ETS, glutamine, mercaptoethanol, penicillin and streptomycin (complete medium DMEM). 0.5 ml of the sample is passed through the column for 15 minutes, then collected and sterilized by filtration.

The biological activity of the factors is determined by the lysis of cells of the 1929 fibroblastoid line in the presence of actnomycin D.

in flat-bottomed UJ 96-chewed myrghtlats in complete medium, Q ;; m. Samples of the full name are diluted in a separate 1-7 tablet in a volume of 100 µl, then transferred in 1929 cells, actinomycin D is added to a concentration of 1 µg / ml and placed in a thermostat with a temperature of 16-20 hours. staining cells with a 0.2% solution of gentian violet in 2% methanol for 10–12 min. The optical density of the wells was measured at a wavelength of 540 nm on a vertical beam spectrophotometer. The lysis of 1929 cells corresponds to a decrease in the optical density of the wells.

The results of the experiment on the sorption of P FIO- oL monAT strain 7G1 are presented in Table 2.

Optical density of stained intact cells 1290.

25

0

five

0

0

five

From the data of Table 2, it follows that passing a FIO-about sample through a column with monAT 7G1 leads to a loss of its cytotoxic activity, which confirms the specificity of monAT and the possibility of their use for immunosorption.

Example 3: The determination was carried out in accordance with Example 1, but instead of the culture medium of strain 7G1, the appropriate dilutions of 7G1 ascites were made in PBS-BSA in the wells with sorbed FIR.

The results show that at a dilution of ascites of 1: 1,000,000, the optical density in ELISA (A492x10) is 380 and, thus, the titer of ascites exceeds 1: .1000000.

Using hybrido-1s 7G1 allows you to get monat isotype G2b, 4To creates advantages over monat isotype G1 when cleaning on affinity columns with protein. A Staphylococcus aureus, and also creates the possibility of saving ascites with

definition of name.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Musmusculus SCC (N) No. 172D - producing monoclonal antibodies to tumor necrosis factor - human alpha. Table 1 A492 is the optical density of the wells at 492 nm. 15 table 2