SU1747484A1 - Strain of hybrid cultured cells mus musculus l, used for preparation of monoclonal antibodies to recombinant l-factor necrosis of human tumors - Google Patents

Description

Yes, for 1.5 minutes After hybridization and washing of cells from polyethylene glycol, the cells are seeded into 96 well panels of 1 105 splenocytes per well. For the cultivation and breeding of hybridomas, IMDM medium (modified Iskove s Dulbecco's medium) is used with the addition of 10% fetal calf serum (ETS), 10 M hypoxanthine, 4 M aminopterin and 1.6 M thymidine. Producer hybrids are cloned once by the final dilution method, seeding 1 cell per well containing spleen cells. After cloning, almost 100% of the subclones produce µA to ra-TNF. Antibody production is maintained for at least 15 passages in culture. The strain was given the designation YURM.On it is stored in the ETS with the addition of 10% dimethylsulfoxide in liquid nitrogen. The strain produces isotype µa G2a having specificity for human α-TNF. The secretion of monoclonal antibodies for 3-4 days of culture is 100-125 µg / ml of culture medium.

The use of the strain is illustrated by the following examples.

Example 1 Hybrid cells are placed in a glass vial with a volume of 50 ml per cell in 5 ml of IMDM medium with 10% ETS, 2 mM L-glutamine, 100 µg / ml penicillin and streptomycin, 0.05 mM mercaptoethanol. Cells are cultured for 2-3 days at 37 ° C in an atmosphere of 5% C02. ICA was isolated from the resulting supernatant by affinity chromatography on an immobilized recombinant protein A St. aureus Cowan I and used in immunofermental assay (ELISA). pa-TNF, p / Ј-TNF and bovine serum albumin (BSA) in an amount of 0.3 µg in 100 µl of 0.1 M carbonate buffer (pH 9.5) are added to 96-well microplates and left overnight at 4 ° C. Wash with tap water and add 1 µg of µa in 100 µl of 0.1 M phosphate buffer (pH 7.4) containing 0.15 M NaCI, 1% BSA (FSB-BSA). Incubate for 1 hr on a shaker. After washing, they are incubated with peroxidase-labeled antibodies to mouse immunoglobulins at a dilution of 1: 2000 on PBS-BSA for 1 hour. The enzyme reaction is carried out for 10 minutes. The substrate solution is 0.1 M citrate buffer (pH 4.7) with 0.6 mg / ml orthophenylenediamine hydrochloride and 0.01% hydrogen peroxide. The reaction is stopped after 10 minutes with 1M sulfuric acid and taken into account on a spectrophotometer with

vertical beam at a wavelength of 492 nm.

The results of an experiment to study the binding specificity of ICA produced by cells of strain 10FN with antigens immobilized on plastic are presented in Table 1.

Research results indicate a higher specificity of ICA,

produced by cells of the 10FN strain in comparison with the prototype, and confirm the possibility of using ICA data for the determination of human Pa-TNF using the ELISA assay.

PRI mme R 2. Biotin-labeled MCAs are used in competitive ELISA with human p α-TNF adsorbed on the solid phase in the presence of two concentrations of unlabeled antibodies.

On a 96-well polystyrene ELISA plate, ra-TNF (2 μg / ml) is adsorbed in 100 μl of 0.1 M bicarbonate buffer (pH 9.5) overnight at + 4 ° C. Non-specific binding is blocked with a solution

FSB-BSA. After each incubation, the plate is washed several times with PBS containing 0.01% Tween 20. Diluted solutions of PBS-BSA biotinylated MAb are mixed with different concentrations of unlabeled antibodies. The mixture was added to the wells and incubated for 1 hour at room temperature on a stirrer. After washing, Avidin-conjugated peroxidase is added (dilution 1: 2000 per

PBS-BSA) and incubated for 1 hour. The board is then washed and an enzyme reaction is carried out for 10 minutes as indicated. The research results are presented in table 2.

The results presented in Table 2 indicate that MCA 10FN to a certain extent suppresses the binding of 3A7N ICA to human p-TNF adsorbed on plastic and does not interfere with the interaction with the antigen MCA AO. Thus, mAb of the tOFN and 3C7N strain recognize non-identical, partially overlapping epitopes. The epitopes recognized by MCA 10FN and AO are different,

PRI me R 3. The biological activity of various TNF samples was determined by lysis of cells of murine fibroblastoid line L929 in the presence of actinomycin D. On the eve of the test, L929 cells were seeded

50 l 10-well per flat-bottomed 96-well microplates in minimal medium modified by Dulbecco (DMEM) with 5% ETS, glutamine, penicillin and streptomycin. In a separate board, dilutions of the full name samples in a volume of 50 µl are prepared, an equal volume of the corresponding antibody dilution is added and incubated for 30 minutes at room temperature. After incubation, the samples are transferred to a plate with L929 cells, added actinomycin Up to a concentration of 1 µg / ml and placed in a thermostat with a temperature of 37 ° C for 20 hours. The results are taken into account after staining the cells with a 0.2% gentian violet solution in 20% methanol within 15 min The optical density of the wells was measured at a wavelength of 540 nm on a vertical beam spectrophotometer. The decrease in the optical density of the wells corresponded to the lysis of L929 cells. Per unit of the biological activity of the samples, the full names are taken by their dilution, which causes a 50% lysis of the cells in relation to the intact ones.

The results of the experiment to neutralize the biological activity of p «-FNO human ICA strains 10FN, 3C7N and AO are presented in Table 3.

The data of table 3 indicate that the ICA of the resulting strain 10FN have the highest neutralizing activity.

In addition, MCA 10FN neutralizes the biological activity of both recombinant and natural human TNFN (Table 4). Macrophage a-FIO received

us when stimulating human monocytes aureus to a final concentration in the culture medium of 0.001% (by volume) for 18 hours at 37 ° C.

The data of this example testifies to an equal degree of efficiency of neutralization of biological activity as a recombinant one. Such a natural human s-name.

Thus, the proposed strain

5 produces human lactic acidophilic arrhythmia ka-TNF in large quantities compared with the known. The resulting mAbs have a high neutralizing activity and can be used to study antigenic

0 structure and biological effects of α-TNF in humans in vitro and fn vivo, as well as in the process of genetic engineering production of α-TNF.

Claims (1)

Invention Formula 5 Strain of hybrid cultured cells Mus musculus L SCC (P) N 441D used to produce monoclonal antibodies to its recombinant α-factor of human tumor necrosis. Table 1 table 2 ) -expressed in the ME p a-TNF of a person neutralized by 1 μg Jq G -activity of human macrophage a-TNF is 235 IU / ml ) The human p-a-TNF concentration is Young / ml, which corresponds to 750 IU / ml. Table 3 Table 4