RU2607029C1 - Cultivated hybrid animal cells of mus musculus l.-en-4c9 - producer of monoclonal antibodies against endoglin (cd105) - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry. Strain of cultivated hybrid animal cells is disclosed, Mus musculus L. - EN-4C9 – which is producer of monoclonal antibodies against human endoglin (CD105). Strain is created as result of merging of cultivated cells of mouse myeloma SP-2/0 and splenocytes of mice-hybrids F1 (SJL/J x BALB/c), immunized with recombinant human endoglin. Obtained strain EN-4C9 is deposited in Russian collection of cell cultures of vertebrates (Institute of Cytology of RAS) under number RKKK (P) 771D.

EFFECT: invention enables to obtain monoclonal antibodies against human endoglin with wide spectrum of application, in particular for detecting native endoglin on membranes of living and dead cells, as well as for determining concentration of endoglin in human blood serum by ELISA.

1 cl, 5 dwg, 6 tbl, 6 ex

Description

The invention relates to the field of biotechnology and is intended for the production of monoclonal antibodies (MCAs) against human endogline (CD105) using genetic engineering and is intended for use in medicine and biotechnology, in particular in flow cytometry to detect endogenous expressing cells such as endothelial or mesenchymal cells stem cells (MSCs), as well as in an enzyme-linked immunosorbent assay (ELISA) to determine the concentration of endoglin in blood serum in the diagnosis of a syndrome that complicates pregnancy ( preeclampsia) and in the detection of progressive forms of some malignant tumors.

Endoglyn is a homodimeric membrane glycoprotein with a molecular mass of 180 kDa, most represented on endothelial cells lining the inner surface of blood vessels. The participation of endoglin is critical to the development of a healthy body. Turning off the gene encoding this protein leads to fetal death of embryos due to defects in the circulatory system. Mutations in the endogline gene in humans are associated with congenital defects in the structure of blood vessels, which manifest themselves in the form of a clinical syndrome of hemorrhagic telangiectasia.

According to modern concepts, endoglin located on the membrane of endothelial cells provides the reception of cytokines that determine the activation of endothelial cells, i.e. their transition from a state of rest to a state of reproduction and migration. Endothelial activation is associated with the formation of new blood vessels (angiogenesis), which accompanies the formation of the placenta, wound healing and the growth of malignant tumors. Identification of the membrane form of endoglin is used to identify vascular endothelial cells and MSCs by flow cytometry or immunohistochemistry. Along with the membrane form, endoglin is detected in the body as a soluble molecule released from the surface of cells as a result of proteolysis. Soluble endoglin in the circulating blood is in the form of complexes with its natural ligands. The content of soluble endoglin in serum according to the literature varies from units to hundreds of nanograms per 1 ml. Determining the concentration of endoglin in serum is used to diagnose pregnancy pathology (preeclampsia), as well as to identify cases of progressive development of some neoplasms.

The specificity and sensitivity of modern methods for detecting nanogram concentrations of soluble endoglin are ensured by the implementation of the principle of two-determinant ELISA, according to which the endoglin contained in the test sample binds to adsorbed on the solid phase of MCA that recognizes one antigenic determinant of endoglin. After this, the formed immune complex is detected using the second enzyme-labeled MCA directed to another endogen antigenic determinant. The use of two MCAs directed to different antigenic determinants of the studied protein guarantees a high specificity of the analysis.

In this regard, reagents suitable for detecting soluble endoglin by the method of two-determinant ELISA are selected among the obtained MCAs according to the criteria for maintaining functional activity during immobilization on the solid phase or by covalent binding to peroxidase enzyme molecules. In addition, MABs specific for antigenic sites of endoglin are selected, which, firstly, are not masked during the formation of the immune complex, and, secondly, are spatially removed from each other, which excludes the competition of MABs for binding to endoglin molecules. Promising for practical use are MCAs with one or more of the listed properties.

Known hybridoma strains (Table 1) synthesizing MCAs against human endoglin [Haruta Y., Seon B.K. Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6. Proc Natl Acad Sci USA. 1986; 83: 7898-7902., Gougos A., Letarte M. .. Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line. J. Immunol. 1988. 141: 1925-1933].

The disadvantage of these developments is that they do not allow to achieve high accuracy analysis in complex cases.

The closest in technical essence to the claimed invention is a strain of mouse myeloma P3X63.Ag8.653, containing a hybridoma - producer of MCA that recognize different parts of the endoglin molecule [Seon B.K. Anti-endoglin monoclonal antibodies and their use in antiangiogenic therapy. US 6,200,566 B1, 2001]. Hybridomas of the SN6 series were obtained on the basis of the strain of mouse myeloma P3X63.Ag8.653 and lymphocytes of BALB / c mice immunized with endogline isolated from cells of the cultured human acute lymphoblastic leukemia line. The study of the properties of the obtained MCA was aimed at assessing their ability to inhibit the growth of transplanted tumors, suppressing angiogenesis in them, or to detect endoglin on histological sections.

The disadvantage of the strain is that the hybridomas obtained on its basis do not allow obtaining sufficient analysis accuracy, since they recognize only the same antigenic determinants (epitopes) of the studied antigen.

The problem solved by the authors was the creation of a technology that allows one to obtain MCAs against endoglin (CD105), which have a wider range of applications, namely, they are suitable for detecting endoglin on human cell membranes and for determining the concentration of soluble endoglin in human serum by the method of solid-phase ELISA.

The technical result was achieved by creating a strain of hybrid cultured animal cells of animals Mus musculus L., En-4C9, a producer of human anti-endoglin (CD105) MCA, obtained by fusion of cultured mouse myeloma cells SP-2/0 and splenocytes of F1 hybrid mice (SJL / JxBALB / c) immunized with recombinant human endoglin synthesized in NS0 cells (R&D).

The strain is assigned the copyright name En-4C9 (En-4C9).

The resulting strain En-4C9 was deposited in the Russian Collection of vertebrate cell cultures (Institute of Cytology RAS) under the number RKKK (P) 771D from 01/10/2015

Unlike the analog, the synthesized by the MCA strain synthesized have properties that the analog is not known to have: they recognize native endoglin on the membranes of living and fixed human cells, and in the form of antibodies immobilized on the solid phase (in combination with another labeled peroxidase MCA against endoglin, designated En -4E4) allow you to determine the concentration of endoglin using the method of two-determinant ELISA.

The strain also differs from the analogue in the origin of the line of malignant cells (hybrid line SP-2/0) and the genotype of donor mice of immune lymphocytes, which were used interline hybrids F1 (SJL / J x BALB / c). The combination of the described properties allows us to consider the proposed hybridoma strain En-4E4 as an object that has significant differences from the analogue.

Strain En-4C9 is characterized by the following cultural-morphological and physico-biochemical properties.

Cultural and morphological features of the strain: suspension culture, inoculum when passivated - 100 thousand cells per ml, sieving coefficient 1: 2, subculture time - 48 hours.

Hybridoma strain pedigree: obtained by fusion of cultured murine myeloma cells SP-2/0 and splenocytes of F1 hybrid mice (SJL / J x BALB / c) immunized with recombinant human endogline synthesized in NS0 (R&D) cells. Merging agent - 50% PEG (Sigma), selection of hybrids in HAT medium (Sigma). The cells were cloned by the method of limiting dilutions 4 times, the percentage of producer clones was 100%.

Characteristics of the useful substance synthesized by the strain: Hybridoma produces MCAs specific for the conformational epitope of the extracellular region of human endogline. MCAs recognize endoglin on the membrane of living endothelial cells, HEP G2 cells, human MSCs (flow cytometry, immunofluorescence). Antibodies recognize endoglin on the same cells fixed with 0.025% glutaraldehyde (Cell ELISA method).

Peroxidase-labeled MABs can be used to detect soluble endoglin by a two-center ELISA in combination with another MAB (En-4E4) adsorbed on plastic.

Hybridoma cells produce and secrete monoclonal murine IgG into the medium. To control the productivity level, the method of titration of culture or ascitic fluid in tablets sensitized with recombinant human endoglin obtained in mammalian cells (Recombinant human endoglin / CD105, 1097-EN-025 / CF, R&D, USA) followed by identification of the immune complex using labeled anti-mouse antibody enzyme. The titer in the culture medium is 1 / 10000-1 / 15000, in the serum or ascitic fluid of hybridoma-bearing mice - 1 / 100000-1 / 200000. Stable antibody production is maintained for 20 passages (observation period) in culture and 6 passages when transplanted on animals (Table 5).

Method of cryopreservation: the cryomedium contains 45% of DMEM medium, 45% of blood serum of cow embryos and 10% of DMSO. Freezing and storage in polypropylene cryovials. Freezing mode - the rate of temperature decrease is 1 degree per minute until it reaches minus 70 ° C, then transfer to liquid nitrogen for storage. Decryoconservation: by immersing the cryovial in a water bath with a temperature of + 42 ° C until thawing. Then the contents of the ampoule are transferred to 5 ml of growth medium and centrifuged at 1000 rpm for 7 minutes. Then the medium is replaced with growth medium at the rate of 1 ml of medium per 500 thousand cells, carefully suspended, seeded into culture plates and further cultured under the conditions described above. Cell viability, estimated using trypan blue dye, is 72-76%.

Strain cultivation: DMEM medium with 10% serum of cow embryos. The temperature is 37 ° C, the gaseous medium contains 7% CO ₂ in air at 100% humidity. From the moment of receipt, the cells were cultured in a medium without antibiotics.

Contamination control by bacteria, fungi, mycoplasmas, viruses. The cells were checked for the absence of mycoplasma by staining with Hoechst 33258 dye (Serva).

Materials explaining the essence of the invention

Tab. 1. Hybridoma strains producing ICA against endoglin (prototype and analogues).

Tab. 2. Scheme for the detection of antibodies against endoglin using indirect ELISA on adsorbed antigen or on the membranes of fixed cells by the method of cellular ELISA.

Tab. 3. Interaction of MCA with recombinant molecules of endoglin and endoglin on the membranes of fixed cells of the HEP G2 line and human MSC.

Tab. 4. Scheme for identifying the soluble form of endoglin using two-determinant ELISA

Tab. 5. Average values of reverse titers of MCA synthesized by cells of strain En-4C9 during cultivation and in ascitic fluids when passaged in mice.

Tab. 6. Concentrations of soluble endoglin in the blood plasma samples of healthy volunteers and women suffering from preeclampsia, determined using the two-determinant ELISA method based on adsorbed on the solid phase of MKA En-4E4 and labeled with peroxidase MKA En-4C9.

In FIG. 1. presents the determination of the titer of antibodies in the blood serum of F1 mice (SJL / J x BALB / c) immunized with recombinant glycosylated endoglin En-NS0, the method of solid-phase ELISA on the immunogen (A) and the method of cellular ELISA on fixed cells of the HEP G2 line (B) .

In FIG. 2. presents a selection of combinations of immobilized on a solid phase and labeled with peroxidase MCA to determine the concentration of soluble endoglin in human serum by the method of two-determinant ELISA. The abscissa shows the tried and tested combinations of MCAs. The ciphers of immobilized reagents are listed first, the ciphers labeled with peroxidase MCA are indicated second.

FIG. 3. shows a calibration graph for determining the concentration of soluble endoglin in a two-determinant ELISA based on En-4E4 solid-state MCA immobilized and En-4C9 peroxidase-labeled MCA. The straight line is the regression line, the gray zone is the 95% confidence interval.

Figure 4 shows the detection by flow cytofluorimetry of the expression of endoglin on the membranes of human MSCs using commercial MCAs (CD105, Beckman Coulter) against endoglin (A) and using MCA En-4C9 (B).

FIG. 5 shows a comparison of the levels of expression of endoglin detected by the method of cellular ELISA on the membranes of human cells of several lines. Above the graphs are the names of the cell lines. The absorbance values obtained on HEP G2 hepatocarcinoma cells are taken as 1 and are shown in horizontal lines on the graphs. The abscissa shows the codes of the studied MCAs.

Strain EN-4C9 receive as follows. Immune lymphocyte donors are male mice F1 (SJL / J x BALB / c), which are injected with 2-3 μg of recombinant glycosylated endoglin (a commercially available drug from R&D Systems, USA) in complete Freund's adjuvant. After 4 weeks, 1.5-3.0 μg of antigen is administered in incomplete Freund's adjuvant. After 5-7 days receive blood samples to detect antibodies. As donors of splenocytes use animals with the highest titer (more than 1: 100000) of circulating antibodies (Fig. 1). Mice donating splenocytes 72 and 48 hours before hybridization were injected intraperitoneally with 1-3 μg of antigen dissolved in phosphate-saline solution.

Hybridization partners are hybrid cells of the SP-2/0 hybrid line grown on Eagle's medium in the Dulbecco modification (DMEM) with 5% serum of cow embryos. For fusion, 100 million spleen cells and 50 million SP-2/0 cells are used. The mixture of cells is treated with a solution of polyethylene glycol (50%) with a molecular weight of 1000. For the cultivation of hybrids, a nutrient layer from macrophages of mice and a nutrient medium of the above composition containing 100 ml (μg) aminopteriin (10), hypoxanthine (500) and thymidine (500) are used )

The selection and testing of culture supernatants is carried out three times from 11 to 28 days after the merger. To identify producers of MCA specific for endogline, two ELISA modifications were used.

MCAs reacting with isolated endoglin molecules are detected by the method of solid-phase ELISA, according to which either recombinant glycosylated endoglin synthesized in mouse NS0 mouse myeloma cells (immunogen) or recombinant non-glycosylated endoglin molecules synthesized in E. coli bacteria cells are adsorbed in the wells of the plates (Table 2 )

MCAs that recognize the endoglyn membrane form are detected by ELISA on cells (Cell-ELISA), according to which (Table 2), cultured human HEP G2 hepatocarcinoma cells or cultured human MSCs are fixed in tablets with a glutaraldehyde solution (0.025%) and after washing add supernatants of hybridoma cultures. Antibodies bound to cell membranes are detected using peroxidase-labeled goat antibodies against mouse immunoglobulins.

Of the 480 primary cultures, ten synthesize antibodies that recognize the glycosylated molecules of the recombinant endoglin (immunogen). Interaction with the non-glycosylated analogue synthesized in E. coli cells is less pronounced, but significant. Eight out of 10 supernatant samples recognize endoglin on the cell membranes of the HEP G2 hepatocarcinoma cell line and human MSCs (Table 3).

Hybridoma cells are cloned 4 times by the method of limiting dilutions on the feeding layer of mouse peritoneal macrophages. Clones that, when two consecutive clonings with a seeding density equal to 1 cell per 1 or 2 wells, gave more than 95% of the subclones synthesizing antibodies against endoglin, are transferred to mass culture. Cells grown in the culture are inoculated with intraperitoneal histocompatible mice (5 million cells per 1 specimen), which were injected with pristan (0.5-1.0 ml) in the abdominal cavity 7-10 days before. On day 7-14, ascitic fluid was collected, the globulin fraction of which was precipitated with ammonium sulfate at 50% saturation and tested using one of the methods given in the examples. MCA was purified on a protein G-Sepharose column (Pharmacia, Sweden).

The resulting preparations of purified MCAs are screened for their ability to maintain antigen-binding activity when an enzyme label is attached to them or immobilized on a solid phase. For this, conjugates with peroxidase are prepared on the basis of each of the obtained MCAs, and their activity was tested using the direct ELISA method. At the next stage, combinations of adsorbed on the solid phase and labeled with peroxidase MCA are tested and selected, which most sensitively detect soluble endoglin in the two-determinant ELISA (Table 4). From the data obtained (Fig. 5), it follows that the combination of En-4E4 immobilized on the solid phase of MCA and En-4C9 peroxidase-labeled MCA is optimal.

Thus, as a result of multistage selection, the strain En-4C9 (En-4C9) is produced that produces MCAs that possess the necessary set of properties: they recognize the membrane form of endogline on living and fixed human cells, and in the form of a peroxidase-labeled reagent (in combination with adsorbed on solid phase MCA En-4E4) provide a determination of the concentration of the soluble form of endoglin in the two-center ELISA.

Examples of the preparation and use of MCA synthesized by the strain En-4C9

Example 1. Cultivation of cell culture hybridoma En-4C9 for subsequent vaccination with mice

The seeds are hybridoma cells stored in liquid nitrogen. For defrosting, a test tube containing 5 million cells is removed from liquid nitrogen and immersed for 3 min in a water bath with a temperature of 42 ° C. After complete thawing, the tube is centrifuged for 3 min at 1000 rpm, the supernatant is removed, the cells are resuspended in 10 ml of growth medium (DMEM 95%, cow embryo serum 5%) and placed in a culture bottle. The bottle is placed in an incubator with a temperature of 37 ° C, a humidity of 100% and 7% carbon dioxide in the air. After 1 day, a visual control of the state of the cells is carried out using an inverted microscope and sifted in 2 bottles. On the 4th day after thawing, the cells are subcultured at a dilution of 1: 5. After 2 days, a sample is taken to count the cells - 800 ml of hybridoma cells are contained in 1 ml of suspension. The total culture contains 120 million cells, which is 24 vaccination doses. Cells are pelleted by centrifugation (1000 rpm, 20 min), suspended in a Hanks solution, and 5 ml each are injected 1 ml intraperitoneally to F1 inter-mice hybrids (SJL / J x BALB / c) 7-10 days before vaccination injection of pristan intraperitoneally.

Example 2. Obtaining hybridoma ascitic liquids and the allocation of them globulin fraction containing MCA

The accumulation of ascites in the abdominal cavity of mice begins on the 7th-9th day after inoculation of the cells. On the 10-11th day, the first puncture is performed. Moreover, from every 10 mice receive 45-50 ml of ascites. On the 17th day, the surviving mice are re-punctured and another 40-45 ml of ascites are obtained from them. By day 20, 50% of vaccinated animals remain alive. As a result of the third puncture, 10-15 ml of ascites are obtained from them. The total amount of ascites obtained from 10 mice is 105-110 ml. After the formation of the fibrin clot and the separation of it and the cell mass by centrifugation from 110 ml of ascites, 100 ml of the supernatant are obtained. To isolate the globulin fraction, an equal volume of a saturated solution of ammonium sulfate with a pH of 7.0 is poured dropwise to the obtained liquid under constant stirring on a magnetic stirrer. The precipitate was separated by centrifugation (4000 rpm, 20 min), the supernatant was discarded, and the precipitate was dissolved in distilled water, bringing the solution to its original volume. The salting out procedure is repeated, the resulting precipitate is suspended in half the volume of a semi-saturated solution of ammonium sulfate and stored in this form at 4 ° C.

Example 3. Determination of the titer of murine antibodies against human endoglin

The source of murine antibodies against human endoglin are: blood serum of mice immunized with endogline, culture fluid from growing hybridoma cells in culture, ascitic fluid of mice with growing hybridoma tumors, globulin fraction containing MCA isolated from ascitic fluids. The titer of antibodies against human endoglin is determined using the method of solid-phase ELISA.

The reaction is carried out in 4 stages. At all stages, a tablet containing sodium chloride (0.85%), 0.01 M phosphate buffer with a pH of 7.0 and tween-20 (0.05%) are used to wash the tablets and dilute the reagents.

At the first stage, antigen is adsorbed on the solid phase. For this, 100 μl of a solution containing 10 μg of endoglin in 1 ml of carbonate-bicarbonate buffer (0.01 M, pH 9.5) is added to the cells of the immunoassay plates. Incubate for at least 10 hours at 4 ° C. Remove the antigen from the cells and rinse once.

At the second stage, serial dilutions of the test samples with a volume of 100 μl are prepared in the wells of the plates, incubated for 1 h at 37 ° C. At the end of the incubation, the wells are freed from the samples and washed three times.

In a third step, peroxidase-labeled rabbit antibodies against mouse immunoglobulins are attached to solid phase-bound murine antibodies. 100 μl of working dilution of the conjugate are added to the wells, incubated for 45 min at 37 ° C, the conjugate is removed and washed 3 times.

At the last stage, using the color reaction, the activity of peroxidase bound to the solid phase is detected. 100 μl of a substrate mixture containing tetramethylbenzidine (R055, Hema-Medica, Moscow) was added to the wells, incubated for 5-7 minutes at room temperature in the dark, and then the reaction was stopped by adding 50 μl of 1 n sulfuric acid. The optical density of the samples recorded on a tablet photometer "Multiscan-MS" at a wavelength of 450 nm. The results of determining the titers in the sera of mice are presented in figure 1, the results of determining the titers in culture and ascitic fluids are shown in table. 5.

Example 4. The use of peroxidase-labeled MCA En-4C9 to determine the concentration of endoglin in human serum

The studied material is the blood serum of healthy donors and patients with preeclampsia. To determine the concentration of endoglin using the method of two-determinant ELISA. To capture endoglin from the studied samples, MCAs immobilized on the solid phase, synthesized by the hybridoma strain En-4E4, are used. To identify endoglin, which is included in the immune complex on the solid phase, peroxidase-labeled MCA En-4C9, directed to another antigenic determinant of endoglin, is used.

The reaction is carried out in polystyrene ELISA plates. To dilute the reagents and launder the tablets using a solution of the same composition as in example 3.

At the first stage, a solution containing MCA En-4E4 (5 μg protein in 1 ml of 0.01 M bicarbonate buffer with a pH of 9.5) is added to the wells of the plate. Incubated for 10-18 hours at 4 ° C, then the solution is removed and the wells are washed once.

At the second stage, samples of the studied sera and samples with a known endoglin content are introduced into the wells, intended for constructing a calibration graph. Incubated at 37 ° C for 1 hour, then the samples are removed and the wells are washed three times.

At the third stage, the conjugate of MCA En-4C9 with peroxidase in working dilution is added to the wells, incubated for 1 hour at 37 ° C, the solution is removed, the plate is washed three times.

At the 4th stage, a substrate mixture is added to the wells (see Example 3) to detect peroxidase activity. Incubated for 5-7 minutes in the dark at room temperature, the reaction was stopped with sulfuric acid and the optical density of the solutions in the wells was measured at a wavelength of 450 nm.

Based on the data obtained, a calibration graph is constructed. On the abscissa, the concentration of endoglin in ng / ml is plotted, on the ordinate, the optical density of the samples (Fig. 3). Using the calibration graph and taking into account the dilution coefficient of the studied samples, determine the content of endoglin in them (table. 6).

Example 5. The detection of endoglin on the membranes of MSCs by flow cytometry using MCA En-4C9

MSCs grown as a monolayer in a plastic bottle are removed from the surface, first replacing the growth medium with phosphate-buffered saline (6 ml), then with 2 ml of trypsin-versene solution (Biolot, St. Petersburg). Incubate at room temperature for 10 minutes. Cells separated from the surface are transferred to a test tube containing 6 ml of DMEM medium with serum of cow embryos (10%), and a sample is taken to calculate the concentration of cells in suspension.

The number of cells is counted using a particle counter Z1 (Beckman Coulter). For the analysis of one sample on a flow cytometer, from 100 to 300 thousand cells are required. The suspension is washed from trypsin-versene by centrifugation at 1000 rpm for 7 minutes, after which the precipitate is suspended in 1 ml of washing phosphate-buffered saline containing serum of cow embryos (3%) and sodium azide (1 mg / ml). After repeated washing, the precipitate is suspended, bringing the concentration to 100-300 thousand cells in 1 ml and 1 ml is transferred into plastic tubes.

In one test tube with cells (experiment) add 1 μl of MKAT 4E4 solution with a concentration of 1 mg / ml. In another tube with cells (isotypic control) add 1 μl of a solution of MCA that does not recognize endoglin. The contents of both tubes are thoroughly mixed and incubated in the dark at + 4 ° C for 40 minutes.

Wash cells from unbound MCA. To do this, add 1 ml of a washing solution of the above composition, centrifuged at 2000 rpm for 5 minutes and repeat the washing procedure. The cells in the experiment and in the control are suspended in 200 μl of a solution of goat antibodies against immunoglobulins of mice labeled with fluorescein (1: 1000 dilution with the buffer of the above composition). Cells are incubated at 4 ° C for 30 min in the dark. At the end of the incubation, 1 ml of washing solution is added to each tube and the cells are centrifuged at 2000 rpm for 5 minutes.

The supernatant was removed and the cells were suspended in 600 μl of a cytometric solution (BD Cell Wash).

Following the instructions of the manufacturer, the analysis is carried out on a BD FACSAria III flow cytometer, using the settings for registering fluorescin fluorescence.

For comparison, endoglin was detected on the MSC membrane using commercial MCAs (CD105-PE, Beckman Coulter). The first stages of cell preparation are as described above. Then, 5 μl of phycoerythrin-labeled MABs against endoglin are added to one tube with cells (experiment), 5 μl of labeled MABs not binding to endoglin are added to another tube (isotypic control, Beckman Coulter). Cells are incubated with antibodies for 40 min at 4 ° C, then washed, suspended in cytometry liquid, and the samples are analyzed on a cytometer as described above.

Comparison of the results obtained (Fig. 4) leads to the conclusion about the identity of the expression of endoglin on MSCs detected using commercial MCA and MCA En-4C9.

Example 6. The detection of endoglin on the membranes of fixed cells by ELISA (Cell-ELISA) using MCA series En

MSCs and umbilical vein endothelial primary culture cells (HUVEC), transplantable lines of hepatocarcinoma (HEP G2), glioblastoma (T98, A172), endothelium (ECV304) and human embryonic kidney (HEK 293) are grown in cells of a 96-well culture plate in growth medium alpha-MEM with serum of cow embryos (5%) to the state of the confluent monolayer.

The medium is removed from the cells, the cells are washed from the serum, filling the cells with phosphate-buffered saline (FSF) containing sodium chloride (0.85%) and phosphate buffer (0.01 M, pH 7.0). Remove the solution from the cells, repeat 2 times.

Cells are fixed by adding 60 μl of freshly prepared glutaraldehyde solution (0.025%) to the cells in the cells and incubated for 10 min at room temperature. The cells are washed twice from the fixative, introducing 100 μl of SDF and incubating for 1 min. To block nonspecific binding, add 60 μl of casein solution to the cells and incubate for 1 h at 37 ° C.

Dilutions of MCA test preparations are prepared in 1.5 ml plastic test tubes using TCR-Tween solution containing sodium chloride (0.85%), Tris buffer with a pH of 7.4 (0.01 M) and Tween-20 for dilution (0.05%).

Casein blocking solution is removed from the cells and 60 μl of diluted antibody solutions are added. Instead of antibodies, 60 μl of TCP-Tween are added to the control wells. Incubated for 1 h at 37 ° C. Introduce 60 μl peroxidase-labeled goat antibodies against immunoglobulins of mice diluted on TCR-Tween (1: 2000) and incubated for 45 min at 37 ° C. The contents of the cells are removed, washed three times from components not bound to the immune complex, introducing 100 μl of TCR-tween. 60 μl of a substrate-chromogenic solution of tetramethylbenzidine (R055, Hema-Medica, Moscow) are introduced into the cells. Incubate the plate for 7 minutes with visual inspection. To stop the enzymatic reaction, 50 μl of 1 N sulfuric acid are added. The optical density of the samples recorded on a tablet photometer "Multiscan-MS" at a wavelength of 450 nm. The results are shown in FIG. 5. From the presented data, it follows that MCA 4E4 detect 2-3 times higher levels of endoglin expression on MSCs and HUVEC cells than on ECV304 cells, glioblastomas, and human embryonic kidney cells.

Claims (1)

The strain of hybrid cultured animal cells Mus musculus L., EN-4C9, is a producer of monoclonal antibodies against human endogline (CD105), deposited in the Russian Collection of Vertebrate Cell Cultures under number PKKK (П) 771Д.

Images