SU1759872A1 - Strain of hydride cultured cells mus musculus l, used for preparation of monoclonal antibodies to the human transferring - Google Patents

Abstract

Uses: biotechnology and medicine, the isolation and purification of human trisvirrin. The essence of the invention: obtaining a strain of hybrid cultured mouse cells producing monoclonal antibodies to human transferrin, which do not cross-react with closely related proteins. The strain is obtained by hybridization of Balb / c mouse splenocytes with Sp EBR-5 mouse myeloma cells. MCAs are related to lgG1, their titer in the culture fluid is 1:10, in ascites - 1: 107. MCA does not bind to hemoglobin, albumin and human C-reactive protein.

Description

The invention relates to biotechnology and can be used to make an immunosorbent for the purpose of isolating and purifying human transferrin.

Known strains of hybrid cells that produce monoclonal antibodies (mAbs) to human transferrin 1,2. However, in these papers, only the properties of the ICA themselves are considered and no description of any signs of hybridomas producing these ICA is given. In addition, there is no information on the interaction of ICA with closely related and related proteins (albumin, hemoglobin and C-reactive protein) in tissues and biological fluids,

The aim of the invention is to obtain a new strain of cultured hybrid mouse cells producing ICA that are detected by transferrin in biological fluids and human tissues.

The strain was prepared as follows. Balb / c mice were immunized subcutaneously with 50 μg of human transferrin dissolved in 0.01 M Na-phosphate buffer with 0.14 M NaCI, pH 7.2 (FBS) muli-fused in complete adjuvant Freund in a volume of 1 : 1 (0.5 ml per mouse). On days 21 and 42, the immunization is repeated, and 21 days after the last immunization, the test is carried out, injecting (in the tail vein) 100 µg of human transferrin dissolved in PBS (0.5 ml of solution per mouse) intravenously. The boosting is repeated over the next two days and one day after the last, the cells are fused as follows.

A polyethylene glycol-1000 solution (50% of Dulbeco's Modified Eagle (DMEM) solution with 10% dimethylsulfo-PdG) (DMSO) was added dropwise to the cell pellet with constant shaking of the tube.

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1 ml for 1.5 minutes). Then, using a Pasteur pipette, dropwise 10 ml of serum-free serum DMEM without serum for 5 minutes and with constant shaking. The mixture of cells is centrifuged (1500 rpm, 5 min), the precipitate is resuspended in DM EM medium with serum. The cell suspension is digged into the wells of 96-well plates with macrophages (104), placed there the day before. The concentration of myeloma cells in the microplates is 104 cells per well, splenocytes - 10 cells per 100 µl of medium. The boards are placed in a C02 incubator (6% C02, 37 ° C). After 24 hours of incubation, microplates with cells (total volume of medium in the well 150 µl: 50 µl of medium with macrophages and 100 µl of medium with myeloma cells and splenocytes) are added to each well of the microplates with 50 µl of 4-fold GAT medium with ethidium bromide at a concentration of 1 μg / ml, GAT medium includes, μg / ml: hypoxanthine 136; aminopterin 0.18 and thymidine 3.78. Feeding the cells is carried out by replacing half of the medium in the wells.

As a result of screening the obtained hybrids for the sign of the production of antibodies to transferrin, their subsequent cloning and recloning, they select a strain that stably produces mAb to transferrin. The strain is stored in the Specialized Cell Culture Collection of the Institute of Cytology of the Academy of Sciences of the USSR under the number 452D and is characterized by the following features.

The strain is cultivated on Eagle's growth medium in the Dulbeko modification (DMEM) with the addition of 10% bovine fetal serum, as well as 1 mM of essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 5 x mM 2-mercaptoethanol, and 40 units / ml gentamicin.

Cells are cultured at 37 ° C in an incubator in an atmosphere of 5-7% C02 or in sealed bottles in a thermostat without C02 supply. The cultivation method is suspension. With growth on this medium, the strain forms rounded cells. The population doubling time is 36 hours. The multiplicity of sowing is 1: 2 when reseeding after 48 hours. A seeding dose of 150 thousand cells in 1 ml, the maximum population density of 800 thousand cells in 1 ml. Cell cloning is carried out in 96-well plates without the addition of feeder cells. When seeding 3-4 cells per well, the efficiency is 70%. The ability to produce ICA is maintained for 20 passages in cell culture.

Cryopreservation is carried out in fetal bovine serum with the addition of 10% dimethyl sulfoxide (DMSO) at a concentration of 1 x 106 cells in 1 ml. Cell immunity after thawing is 70%.

Vaccination of cultivated hybrid

cells of strain 452D to syngeneic mice (Balb / c line, 106 cells per mouse) causes

them the formation of ascites tumor cells

grafted with 100% efficiency

and explanted onto tumors into culture. AT

ascites fluid contains ICA to

0 transferrin.

The productivity of the strain. Under standard culture conditions of the 452D strain in vitro, the antibody titer in a medium conditioned by hybridoma cells (supernatite) is 1: 104. The titer of ICA in ascites fluid, determined by E, is 10.

MABs produced by the strain 52D cells into the culture medium are specifically bound to human transferrin, as indicated by standard immunoassay, as well as by immunoblotting. The produced mAbs do not bind to hemoglobin, albumin and human 5 C-reactive protein. When conducting radial immunodiffusion in agarose, ICA does not form transferrin lines.

Using standard specific antisera, it has been established that MCAs produced by the 452D strain belong to the IgG1 isotype.

Example 1. To obtain ICA, strain cells are cultured in Correll 5 vials with a capacity of 100 ml in DMEM medium supplemented with 10% fetal bovine serum, 1 mM replaceable amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 5 x M mercaptoethanol, and 40 units / ml of gentamicin 0 at 37 ° C5-7% C02.

Seed dose of 1.5 x 105 cells / ml. Reseeding is carried out every 2 days by pipetting. When reseeding, the cells of the strain are sterilely separated by centrifugation (3000 rpm, 5 minutes) and used for further cultivation, and the medium conditioned by the cells of the strain (supernatant) is sterilely drained. Thus, 20 passengers were conducted.

Using standard immunoassay (ELISA), it is established that the supernatant contains ICA to human transferrin. To do this, transferrin (3 5 µg / ml in 0.2 M bicarbonate buffer, pH 9.5), sorbed in the wells for immunological studies (50 µl each), are added washed with distilled water and phosphate buffer (0.01 M phosphate buffer with 0.14 M NaCI and 0.05%

Tween-20, pH 7.2) (PBS) the supernatant is diluted and incubated for 2 hours at room temperature. Then it is washed with water and PBS and 50 µl of rabbit anti-mouse conjugate anti-horseradish peroxidase is added to each well at a dilution of 1: 500. After 1 h, the plates are washed with water and PBS and 50 µl of substrate (0.1% orthophenyldiamine, 0.03% H200, 0.2 M citrate buffer, pH 4.5) is added to each well. At the end of the enzymatic cleavage reaction of NaOO in the presence of ortho-phenylenediamine in the presence of MCA, a yellow color develops, which is recorded on a Multiscan spectrophotometer at a wavelength of 450 nm. Binding of ICA to human transferrin is detected by dilution of the supernatant before.

The lack of interaction of the obtained µa with hemoglobin, albumin and human C-reactive protein is determined as follows. 50 µl of each antigen used (transferrin, albumin, hemoglobin, and human C-reactive protein) at a concentration of 3 µg / ml carbonate buffer (0.2 M NaaCOa, 0.2 M №) are applied to a portion of the wells of a 96-well plate. HCO3 (pH 9.5) and incubated for 1 hour at 37 ° C. Then the boards are washed as follows; rinse with distilled water, add 200 µl of PBS (0.1 M Na-phosphate buffer, 0.14 M NaCI, pH 7.2) with the addition of 0.05% Tween-20 (FBI), incubate for 3 minutes and once rinsed with distilled water. Subsequently, all washes are carried out in a similar manner. After washing, the media conditioned with hybrid cells, 50 µl per well, are applied to all wells and incubated for 2 hours at room temperature. Next, the plates were washed as described above and rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase and diluted 1: 100 in PBS were applied, incubated for 1 hour at room temperature, washed and added to the wells with 50 µl of substrate solution (10 µg 0-phenylene - diamine, 10 ml of 0.1 M citrate buffer, pH 4.5, 4 μl of 30% NaOo) and incubated for 30 minutes at room temperature. If the reaction is positive, yellow staining of the wells is observed. The extinction of individual cells was measured at 450 nm using an automatic instrument.

The measurement results are shown in the table.

The results in the table indicate that the obtained ICA interact specifically with transferrin and do not bind hemoglobin,

albumin and human C-reactive protein.

Example 2. Preparing samples for immunoblotting. Proteins (transferrin, albumin, hemoglobin, and human C-reactive protein) are placed in sample buffer (0.125 M Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol) and heated for 30 minutes at 56 ° C. Then it is subjected to electrophoresis in a 12% polyacrylamide gel according to the method of Laemmley (voltage 40 V, 4 h). Then one part of the gel is fixed in a mixture of isopropanol (25%), acetic acid (10%). water the rest for 3 h, stained in the same solution with the addition of 0.1% amido black 10 V and clarified in fixing solution.

Another part of the gel is used to conduct electrophoretic transfer of proteins from the gel to a nitrocellulose filter with a pore diameter of 0.45 µm. Protein transfer to nitrocellulose is carried out at a current of 500 mA in an electroblotting device in electrode buffer: 25 M Tris. 192 M glycerol, 20% (1: 1) methanol. pH 8.3 for 1.5 hours. Transfer quality is assessed by staining the gel used to transfer proteins to proteins. Under these conditions, almost complete protein transfer is observed. After electrotransfer, immunological detection of proteins on nitrocellulose is carried out. For this, the electrophoretic blots are rinsed with PBS solution and soaked in PBS solution with the addition of 0.3% Tween-20 (PBSL) for 1 hour at 37 ° C. The blots are cut in half, One part is used to control non-specific binding of rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase, and incubated with DM EM culture medium with 10% fetal serum added. The other part of the blot is incubated with the medium conditioned by the hybrid cells without dilution. Incubation is carried out for 18 h at 4 ° C. After incubation, both parts of the blot are washed with PBST solution four times for 5 minutes and incubated with rabbit anti-mouse immunoglobulins conjugated with horseradish peroxidase at a dilution of 1: 1000 in PBST for 1 hour at room temperature, washed with PBBS 4 times for 5 minutes, 1 hour for PBS and substrate was added (20 mg diaminobenzidine, 0.6 ml 0.1 M imidazole, 10 µg 30% H 200. 19.4 ml PBS, pH 7.2). Incubated for 20 minutes

The reaction is stopped by washing the blots with distilled water. Gels and blots are photographed on the film Mikrat-300. Only one band is observed on the film.

binding ICA corresponding to human transferrin. At the same time, there is no specific adhesion of the second antibodies in the control. Thus, the obtained ICA specifically recognize human transferrin.

Example 3. Synthesis of grasferrin is one of the many tissue-specific functions of the liver. For various diseases of the liver for diagnostic purposes, it is of interest to characterize the temporal and spatial organization of this function in the liver tissue, to estimate the proportion of cells synthesizing transferrin, which can vary during the development of organ damage. Such an approach makes it possible to reveal dysfunction and to trace the dynamics of the functional state of hepatocytes and the liver as a whole. ICA, in comparison with traditional cytochemical methods, allows to detect individual antigens and, in particular, transferrin in liver tissue with high sensitivity and accuracy, and thus opens up possibilities in the differential diagnosis of liver diseases. Transferrin is detected in liver tissue in the following way.

Liver pieces from two people (relative norm and chronic hepatitis}, obtained by intravital puncture biopsy, are fixed in 4% formalin, in 0.1 M phosphate buffer, pH 7.4 for 2 hours, washed in the same buffer, dehydrated in alcohols of upward concentration, carried out through chloroform and embedded in paraffin.Cut slices 5 microns thick. Before immunohistochemical analysis, dewaxing is carried out in 2 xylene shifts (3 min each) and rehydrated in alcohols descending in strength (3 min in each.) Washed in PBS. For submitting tim own peroxidase activity, sections after 100 ° alcohol (to 96 ° alcohol) are incubated in methanol with 0.3% NaOo for 30 min.

After washing in FGTS, sections were incubated in FFM with O, I% CTI onin added for 1 h, rinsed in PBS and incubated with ICA without dilution of 30 pins. As a control for non-specific binding of vyrchantitel, sections were simultaneously incubated without first antibodies with PBS.

After priming in FBL (3 times for 5 min), the sections are incubated with va sec (in mice - anti-mouse immunogloins, with rabbit peroxide, with horseradish peroxide at a dilution of 1 100 o FBS for 30 min. After washing with cDBC ( 4 times for 5 minutes) incubated with subtrlt {1 mg diaminobeninide (DAA) gl Trio-ChS pH 7.5 4 μl 30%) 10 min dark Then rinse or in FPG (3 times for 5 min) and amplify the reaction of r about i% C "-SM for min, satacer s pv nv a t in PBS (3 times in g, -, rfj, ST - alcohols in descent qPCR, |" yr m (J times each), the OS 5 5 changed the silol (POS min) and zak yuchayut in Sections balm Photog raFiru or m grmbsoe f / o r fore- shrouds object x and 2nd flow (Likrag-ZOO.

The results of the study r show that there is no evidence that the meshing KiMTf j} J ix gtit tsgda kek when processing sections almost all hepatocytes with different severity of severity, especially in the lesion, contain transferrin, i c there is a heck of heterogeneity of hepato- sis to support transferrite compared with the relative norm

Thus, the obtained mAbs specifically expose human grlisferrin in biological fluids and human tissues and can be used for diagnostic purposes.

Claims (1)

The claims of the strain of cultured cultured cells of Mus musculus L, h 452D, used to obtain monoclonal antibodies to human transferrin