RU2439160C1 - Method of obtaining monoclonal antibodies to abluminal membrane antigen of cerebral endotheliocytes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: mouse of line Balb/C is immunised with preparation of membrane proteins of cerebral endotheliocytes. B-lymphocytes of said mouse spleen are isolated and fused with cells of myeloma of line Sp2/0-Ag14 mouse, hybridomas are obtained, supernatants of obtained hybridomas are tested in immune-chemical way and hybridomas, producing monoclonal antibodies to abluminal membrane antigen of cerebral endotheliocytes, are separated. From the selected is hybridoma, characterised by the largest number of antibody-producing hybrid cells, synthesising antibodies belonging to class of G immunoglobulins preserving biological activity in blood serum for not less than 24 hours, monoclonal antibodies are separated from supernatant of selected hybridoma, in particular, by method of affine chromatography on protein-G-agarose.

EFFECT: method by invention ensures obtaining monoclonal antibodies to antigen of cerebral endotheliocytes with high immune-chemical activity, making it possible to visualise cerebral endotheliocytes both ex vivo, and in conditions in vitro and in vivo for characteristics of vascular system of gliomas.

2 cl, 9 dwg, 1 ex

Description

The invention relates to the field of molecular biology, in particular to the problem of producing biologically active antibodies that bind to a living cell.

Known work on the production of polyclonal antibodies and monoclonal antibodies that visualize the capillaries of the brain [Lin J.H. et al. 2002]. The most selective marker of cerebral endotheliocytes, EBA, is visualized by SMI-71 antibodies only on sections of the rat brain (Sternberger L.A. et al. 1992). Similarly, OX-26 antibodies stain the CD-71 transferrin receptor only in rat microvessels (Pardridge at al. 2002). Monoclonal antibodies specific for the insulin receptor of cerebral microvessels of humans and various animal species were obtained. Also known are commercial preparations of monoclonal antibodies to VEGF, FV for visualization of cerebral vessels [Zymed, USA]. It is known that not all monoclonal antibodies obtained to visualize cerebral endotheliocytes can be used to visualize living cells. In addition, the open press provides conflicting data on the kinetics of allogeneic antibodies introduced into the bloodstream of healthy animals. Some researchers suggest that allogeneic antibodies, when they enter the systemic circulation, very quickly lose their immunochemical activity due to interaction with blood plasma proteins and are eliminated as a result of uptake by liver and spleen cells. Moreover, an objective assessment of the immunochemical activity of allogeneic antibodies in the vascular bed is sometimes significantly limited due to methodological difficulties.

The objective of the invention is to obtain biologically active monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes with high immunochemical activity, allowing visualization of cerebral endotheliocytes both ex vivo and in vitro and in vivo.

An object of the present invention is to develop a method for producing biologically active monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes that bind to a living cell.

The authors found that the abluminal antigen of cerebral endotheliocytes is available for circulating in the blood nanosystem based on specific monoclonal antibodies in the development of a tumor process in the brain of rats (C6 glioma). The resulting nanosystem is able to penetrate the pathological blood-brain barrier from the bloodstream into the glioma region, which allows visualization of the vascular system of C6 glioma.

The invention consists in the following.

A cerebral endotheliocyte membrane protein preparation immunizes a Balb / C mouse. The spleen B lymphocytes of this mouse are isolated and fused with Sp2 / 0-Ag14 mouse mouse myeloma cells, hybridomas are obtained, the supernatants of the obtained hybridomas are immunochemically tested, and hybridomas producing monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes are selected. A hybridoma is selected from them, characterized by the largest number of antibody-producing hybrid cells synthesizing antibodies belonging to the class of immunoglobulins G, preserving biological activity in blood serum for at least 24 hours, and monoclonal antibodies are isolated from the supernatant of the selected hybridoma.

In particular, the isolation of monoclonal antibodies from the supernatant of the selected hybridoma is carried out by protein G-agarose affinity chromatography.

The invention is illustrated by the following figures.

The Figure 1 shows the disk electrophoresis of a solubilized preparation of membrane proteins of rat cerebral endotheliocytes. A. - Markers of molecular weight. B. - The preparation of membrane proteins (Coomassie staining G-250).

Figure 2. Confocal laser microscopy of cerebral endotheliocytes on sections of a rat brain using anti-BEMP-1 antibodies.

Figure 3. The accumulation of Anti-BEMP-1 in the microvasculature of the brain and liver (%) in vitro, depending on the amount of labeled antibodies introduced.

Figure 4. Immunohistochemical staining of pial microvessels Anti-BEMP-1. Magnification 1 × 100.

Figure 5. Immunoperoxidase staining of cerebellar microvessels using exvivo anti-BEMP antibodies, magnification 1 × 100.

Figure 6. Distribution of I ¹²⁵ -labeled Anti-BEMP-1 in the microvasculature of the brain, liver and avascular fraction of the brain (in vitro).

Figure 7. Distribution of IgG in the microvasculature of the brain and liver (%) in vitro with different amounts of labeled antibodies.

Figure 8. The results of laser confocal microscopy imaging of cerebral endotheliocytes using anti-BEMP-1 antibodies. According to the location of the nucleus stained with DAPI (blue color), the antigen to which antibodies are obtained is localized on the abluminal surface of cerebral endotheliocytes.

Figure 9. Immunohistochemical analysis of sections of the liver (A), lung (B, C) and kidneys (G) using anti-BEMP-1.

The method is as follows.

The modifications of the preparation of the preparations of the abluminal antigen of cerebral endotheliocytes used in this work are based on the methodology of Lidinsky et al. for membrane proteins of the microvasculature of the brain. The main requirement for the modified production methods is the maximum preservation of the antigenic spectrum of the preparation of membrane proteins of cerebral endotheliocytes with the greatest extraction of integral membrane proteins.

The technique used by the authors to obtain a preparation of membrane proteins of cerebral endotheliocytes includes the following steps.

1. Homogenization. To 50 g of rat brain tissue was added 500 ml of PBS containing 5.5 mM glucose, 2 mM EDTA, 4 mM phenylmethylsulfonyl fluoride and aprotinin at a dose of 0.5 U / ml and ground in an ETA 0010 homogenizer for 30 seconds at maximum speed.

2. Filtering. The resulting homogenate is passed once through a sieve with a pore diameter of 360 μm, and then the resulting filtrate is doubled through a sieve with a pore diameter of 160 μm. The resulting filtrate was centrifuged at 1000 g for 10 minutes.

3. Gradient centrifugation in dextran. The precipitate was suspended in a solution of T-70 dextran to a final concentration of 15% (mass / vol), layered in 10 ml each in a 25% solution of dextran in 50 ml tubes and centrifuged at 25,000 g for 10 min in a bucket rotor. The fraction of microvessels that formed a line at the interphase of dextran solutions of various densities was collected and centrifuged at 25,000 g for 10 minutes.

4. Osmotic lysis of cells. The microvessel preparation is suspended in 50 ml of distilled water, stirred for 2 hours at 4 ° C and centrifuged at 15,000 g for 10 minutes. The procedure is repeated twice, taking the precipitate.

5. Sonification. The lysed cell pellet was dissolved in a minimal volume of H ₂ O and sonicated on a G112SP1T ultrasonic disintegrator (Laboratory Supplies Co. Inc.) at a frequency of 80 kHz for 20 min (3 times) in chilled distilled water with Triton-X100 detergent. The drug is centrifuged at 25,000 g for 10 min and the supernatant is taken.

The resulting gradient centrifugation in dextran and subsequent osmotic lysis with a purified fraction of endotheliocyte membranes immunize a Balb / c mouse in an amount of 20 μg for the total protein. A preparation of membrane proteins of cerebral endotheliocytes (20 μg) is injected subcutaneously at three points with an equal volume of Freund's complete adjuvant with an interval of 1 time in 10 days, followed by a 2-month break. Then the immunization cycle is repeated, and on the 5-6th day after the second immunization cycle, 50 μg of the preparation of membrane proteins of cerebral endotheliocytes mixed with incomplete Freund's adjuvant are administered intraperitoneally. The effectiveness of immunization is evaluated by immunodiffusion with a test system (brain extract).

Hybridization is carried out on the 3-4th day after the last injection of the preparation of membrane proteins of cerebral endotheliocytes in the ratio of myeloma cells and B-lymphocytes 1:10. Hybrid clones are selected on a selective medium containing hypoxanthine, thymidine and aminopterin. Hybridomas are screened for the production of specific antibodies by in vitro and in vivo immunohistochemistry and immunocytochemistry methods. Namely, hybridoma cells are carried out through a series of reclations under the control of the highly sensitive immunohistochemical method based on the ABC-Vectastain avidin-biotin peroxidase whale (Vector Lab., USA) with enhanced DAB staining using CoCl ₂ and laser confocal microscopy using secondary fluorescence antibodies (Invitrogen, USA).

From a large number of positively reacting hybridomas, those whose supernatants contained affinity monoclonal antibodies to cerebral microvessel endotheliocyte membrane antigens are selected, after which consecutive series of reclamation and analysis of supernatants for each of these hybridomas are carried out.

One hybridoma is selected that produces monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes, characterized by the highest percentage of antibody-producing hybrid cells with the production of class G monoclonal antibodies that retain biological activity in the blood serum of a laboratory animal for at least 24 hours.

The selection of a hybridoma producing monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes is carried out according to the results of confocal microscopy. The determination of the class of immunoglobulins is carried out according to the results of isotyping using commercial sera (Sigma, USA). The study of the biological activity of antibodies in the blood serum of a laboratory animal is carried out according to the results of immunochemical testing on sections of the rat brain after 24-hour circulation of monoclonal antibodies introduced into the bloodstream of the rat.

To perform subsequent experiments, hybrid cells of the selected hybridoma are intraperitoneally administered to Balb / c mice to obtain ascites. Ascites is also checked by immunohistochemistry and immunofluorescence methods, positive samples are combined and stored in a deep cold refrigerator (-80 °). Hybrid cells from ascites are separated by centrifugation and either reintroduced into the mouse or sterile frozen in liquid nitrogen in a medium with DMSO and normal horse serum.

This technology allows defrosting hybrid cells at any time and, after the reclone procedure, reintroducing them into the mouse and receiving ascites in unlimited quantities (at least 300 ml ascites with an average MAb concentration of 4 mg / ml). The obtained antibodies to the abluminal membrane antigen of cerebral endotheliocytes are named by the authors as anti-BEMP-1 antibodies.

To carry out immunohistochemical analysis for specificity and / or the subsequent creation of vector immunoliposomes, anti-BEMP-1 antibodies are isolated from the obtained ascites using affinity chromatography on protein-G agarose. The IgG fraction is taken under the control of a photometer, concentrated by ultrafiltration and stored at -20 ° in a solution of 50% glycerol. The immunochemical integrity of antibodies is monitored immunohistochemically at each stage of purification and concentration. The purity of the obtained anti-BEMP-1 antibodies is evaluated by disc electrophoresis in SDS page.

For tissue and species specificity, anti-BEMP-1 antibodies are tested by immunohistochemical analysis on sections of other rat organs and brain sections of other animals.

To assess the specificity of the interaction of anti-BEMP-1 antibodies with endothelial antigens of cerebral microvessels, a radioimmunoassay of the accumulation of labeled anti-BEMP-1 antibodies in liver and brain microvessels is carried out in vitro. Anti-BEMP-1 antibodies and non-specific mouse IgG (control) are conjugated to the radioactive I ¹²⁵ T-chloramine method (Fraker PJ and Speck JCJr). Formulations ^{of 125} I-labeled antibody was separated from free iodine and incubated with a preparation of native microvessels of freshly prepared rat brain homogenate. As a control, labeled non-specific mouse immunoglobulins G (IgG-I ¹²⁵ ) are used. Calculation of the administered dose is carried out by the specific radioactivity of protein preparations (CPM / μg), the accuracy of the calculations is checked by determining the radioactivity in the samples (CPM / ml) with a known protein concentration. After incubation with labeled antibodies, the preparations are washed three times with a 50-fold volume of PBS by centrifugation for 10 min at 50,000 g. After washing, the preparations are placed in a γ-counter and the total and specific radioactivity of the crude substance of microvessels is determined.

To prove the feasibility of implementing the proposed method with the achievement of the stated purpose and the specified technical result, the following data.

EXAMPLE 1

To obtain a preparation of membrane membrane proteins of the brain, 500 ml of PBS containing 5.5 mM glucose, 2 mM EDTA, 4 mM phenylmethylsulfonyl fluoride and aprotinin at a dose of 0.5 U / ml were added to 50 g of rat brain tissue and ground in an ETA 0010 homogenizer 30 seconds at maximum speed. The resulting homogenate was passed once through a sieve with a pore diameter of 360 μm, and then the resulting filtrate was doubled through a sieve with a pore diameter of 160 μm. The filtrate was centrifuged at 1000 g for 10 minutes. The precipitate was suspended in a T-70 dextran solution to a final concentration of 15% (w / v), 10 ml each was layered in a 25% 50 ml dextran solution and centrifuged at 25,000 g for 10 min in a bucket rotor. The fraction of microvessels that formed a line at the interphase of dextran solutions of various densities was collected and centrifuged at 25000 g for 10 min. The microvessel preparation was suspended in 50 ml of distilled water, stirred for 2 hours at 4 ° C and centrifuged at 15,000 g for 10 minutes. The procedure was repeated twice, taking the precipitate. The lysed cell pellet was dissolved in a minimal volume of H ₂ O and voiced on a G112SP1T ultrasonic disintegrator (Laboratory Supplies Co. Inc.) at a frequency of 80 kHz for 20 min (3 times) in chilled distilled water with Triton-X100 detergent. The drug was centrifuged at 25,000 g for 10 min, selecting a supernatant. The obtained heterogeneous preparation of membrane proteins of the microvasculature of the brain was characterized by the method of disk electrophoresis (Figure 1).

The purified fraction of endotheliocyte membranes obtained as a result of gradient centrifugation in dextran and subsequent osmotic lysis was used for immunization and subsequent fusion, as a result of which several hybridomas were produced that produce antibodies visualizing on sections of the brain of a rat microvasculature. Under the control of immunofluorescence analysis using confocal microscopy, a hybridoma was selected that produced monoclonal antibodies (anti-BEMP-1 antibodies) specific for the abluminal membrane antigen of cerebral microvessel endothelial cells.

Initially, the cells of the selected hybridoma were subjected to a series of reclocations under the control of a highly sensitive immunohistochemical method in the author's modification based on the ABC-Vectastain avidin-biotin peroxidase whale (Vector Lab., USA) with enhanced DAB staining using CoCl ₂ . After the seventh reclamation procedure, supernatants from 97% of the wells stained endothelial cells of the microvasculature of the brain with the same intensity, indicating stable secretion of monoclonal antibodies by hybrid cells. Thus, more than 50 positively reacting subclones were obtained, of which one producing the maximum amount of high-quality anti-BEMP-1 antibodies to the abluminal membrane antigen of microvasculature of the brain was monitored by immunofluorescence analysis using laser confocal microscopy (Figure 8, D).

To obtain ascites, hybrid cells of the selected hybridoma in an amount of 2 × 10 ^{6 were} intraperitoneally administered to Balb / c mice. He less than 7 days after intraperitoneal injection of hybrid cells in mice ascites were collected. During the collection, ascites was also checked by immunohistochemistry and immunofluorescence methods, positive samples were combined and stored in a deep cold refrigerator (-80 °).

In order to carry out immunohistochemical analysis for specificity and subsequently to create vector immunoliposomes for in vivo work, anti-BEMP-1 antibodies were isolated from ascites of mice immunized with a membrane protein preparation of cerebral endotheliocytes by affinity chromatography on protein G agarose. The output of anti-BEMP-1 antibodies in affinity chromatography was at least 50%.

The IgG fraction (anti-BEMP-1 antibody) was taken under the control of a photometer, concentrated by ultrafiltration and stored at -20 ° in a solution of 50% glycerol. The immunochemical functionality of antibodies was monitored immunohistochemically at each stage of purification and concentration. The purity of the obtained anti-BEMP-1 antibodies was evaluated by disc electrophoresis in SDS page with SDS and was not less than 90%.

Subsequently, having obtained a purified preparation of anti-BEMP-1 antibodies, the authors showed the presence of a large amount of antigen in the preparation of membrane proteins of the microvasculature of the brain using the Western immunoblot method.

Immunohistochemical and immunofluorescence analysis of the purified anti-BEMP-1 preparation showed that they perfectly bind to the abluminal membrane antigen of cerebral microvascular endotheliocytes on sections of the rat brain. Microvessel staining was well visualized on rat brain sections using dilutions from 1: 1000 to 1: 100000. At large dilutions (1: 200000, 1: 300000), the microvessels were also visualized by immunohistochemistry on sections of the rat brain, but the intensity of staining of the microvessels significantly decreased. At dilutions of less than 1: 1000, background tinting increased dramatically due to non-specific sorption of antibodies. The recommended working range of dilutions of the obtained anti-BEMP-1 antibody preparation for immunohistochemistry and immunofluorescence on brain sections is 1: 5000-1: 10000.

The most pronounced immunohistochemical reaction was observed in the rostral sections of the neocortex, in the hypothalamus, in the cerebellar cortex and in the medulla oblongata. Both small microvessels (8-10 microns) and rather large elements of the vascular bed (100 and more microns) were visualized.

The absorption of primary antibodies by dry plasma and extracts of organs and tissues did not affect the staining intensity. At the same time, the absorption of brain tissue by a homogenate led to a sharp decrease in the intensity of staining, up to its disappearance. Along with staining of the microvasculature of the brain parenchyma, intense staining of the vascular system of the meninges (pial microvessels) and microvessels of the choroid plexus was observed (Figure 4).

Subsequently, anti-BEMP-1 antibodies were tested for tissue specificity using immunohistochemical analysis on sections of other organs. According to the selected scheme, all supernatants and ascites from subclones of the selected hybridoma, and then purified anti-BEMP-1 antibodies from ascites, were tested on sections of the liver, heart, lungs, kidney and spleen (Figure 5).

Thus, the study showed that anti-BEMP-1 visualize the abluminal membrane antigen expressed on the abluminal surface of the endothelial cells of the microvasculature of the brain and, to a much lesser extent, in the microvessels localized in the mucous and muscle membranes of large bronchi. This suggests a high tissue specificity of anti-BEMP-1 antibodies.

The species specificity of anti-BEMP-1 antibodies was characterized by immunoperoxidase and immunofluorescence analysis on sections of the brain of rat, mouse, rabbit, bovine and human. Microvascular staining was determined on sections of the mouse brain using both immunoperoxidase and immunofluorescence imaging techniques. At the same time, the staining intensity was slightly lower than that on rat brain slices. This indicated a cross-immunochemical reaction of anti-BEMP-1 antibodies with homologous mouse and rat epitopes. On sections of the brain of a rabbit, a bull, and a human, a positive immunohistochemical reaction to any structures was not observed. Thus, a conclusion was drawn on the limited species specificity of anti-BEMP-1 antibodies.

To assess the specificity of the interaction of anti-BEMP-1 antibodies with endothelial antigens of cerebral microvessels, a radioimmunoassay of the accumulation of labeled Anti-BEMP-1 in liver and brain microvessels was carried out in vitro. For this purpose, anti-BEMP-1 antibodies isolated from ascites and non-specific mouse IgG (control) were conjugated to radioactive I ¹²⁵ . Iodination was carried out by the T-chloramine method in accordance with the procedure described by Fraker PJ and Speck JCJr. Formulations ^{of 125} I-labeled antibody was separated from free iodine on microcolumns with Sephadex G-50. The total protein content in the preparations was determined using bicinchoninic acid (BCA Protein Assay reagent, Pierce, USA).

The preparation of native microvessels was obtained from freshly prepared rat brain homogenate by ultracentrifugation in a stepwise gradient of T-70 dextran.

A suspension of microvessels was divided into equal aliquots and incubated for 12 hours with anti-BEMP-1 labeled antibodies (anti-BEMP-1-I ¹²⁵ ). As a control, labeled non-specific mouse immunoglobulins G (IgG-I ¹²⁵ ) were used. Calculation of the administered dose was carried out by the specific radioactivity of protein preparations (CPM / μg), the accuracy of the calculation was checked by directly determining the radioactivity in the samples (CPM / ml) with a known protein concentration. The specific radioactivity of the protein preparations did not differ significantly: in different series of the experiment, the fluctuations in the specific radioactivity for anti-BEMP-1-I ¹²⁵ and for IgG-I ¹²⁵ did not go beyond 3-5 · 10 ⁵ CPM / μg. In this regard, the standardization of the administered dose in the experiment and in the control was carried out according to the total protein. The following concentrations of labeled antibodies were evaluated: 50, 10, 5, and 1 μg per aliquot of microvessels. All samples were examined in duplicates.

After incubation with labeled antibodies, the preparations were washed three times with a no less than 50-fold volume of PBS with centrifugation for 10 min at 50,000 g at each washing step. The resulting precipitate was placed in a γ-counter and the total and specific radioactivity of the crude substance of microvessels was determined. The results of the study are presented in histograms (Figure 6, Figure 7).

A significant accumulation of labeled anti-BEPP-1 antibodies in rat brain microvessels was found. In this case, an inverse dose-dependent effect was observed. At an initial anti-BEMP-1 antibody concentration of 50 μg / sample, the brain / liver radioactivity ratio was approximately 3: 1. With a decrease in the number of introduced antibodies, the degree of specific interaction increased significantly and at a concentration of 1 μg / sample in the initial preparation, the brain / liver ratio was about 20: 1.

In the control with non-specific mouse IgG labeled, the effect of antibody capture by microvasculature of the brain was not observed. Regardless of the amount of protein administered, label accumulation in the brain ranged from 2.12 to 4.1% of the administered dose (Figure 3). The accumulation of IgG-I ¹²⁵ in the liver and cerebral microvessels did not differ significantly.

When comparing the capture of anti-BEMP-1-I ¹²⁵ antibodies with IgG-I ^{125 by the} microvasculature of the brain, the effect of significantly greater accumulation of the former, most pronounced at the initial antibody concentration of 1 μg / sample, was also revealed. With a further increase in the concentration of anti-BEMP-1-I ¹²⁵ , the microvessels were saturated with antibodies and the fraction of captured antibodies from the administered dose decreased.

The data obtained were reproduced in vitro experiments on preparations of isolated cerebral microvessels several times. As a second control, in addition to the liver microvasculature, a preparation of the avascular fraction of the rat brain was used. The total data of all experiments are presented on the histogram (Figure 6).

The accumulation of labeled anti-BEMP-1 antibodies, calculated on the basis of the weight of the crude substance of cerebral microvessels, was 19.79% / 100 mg, which is 27 times higher than their accumulation in liver microvessels and 11.5 times in the non-vascular fraction of the brain. The accumulation of non-specific IgG in cerebral microvessels was 1.03% / 100 mg.

The following experiment, using the obtained and purified anti-BEMP-1 antibodies, was carried out with an assessment of the immunochemical activity and kinetics of antibodies after the introduction of a laboratory animal into the bloodstream. Healthy rats (8) under ketamine anesthesia injected 1 mg of anti-BEMP-1 antibodies (3 rats), anti-GFAP antibodies (3 rats) and non-specific mouse IgG (2 rats) into the femoral vein. After 10 and 30 minutes, 1, 2, 3 and 12 hours, 0.3 ml of blood was taken from the opposite femoral vein, the obtained blood serum was centrifuged and incubated with rat brain sections (as primary antibodies) in 1: 100, 1 dilutions: 500 and 1: 1000. The results of the study are presented in Figure 5.

Incubation with rat serum containing non-specific rat IgG did not lead to staining of rat brain sections in any of the 3 cases. Unlike IgG, incubation with serum samples containing specific monoclonal antibodies led to staining of rat brain sections. The latter indicates the ability to circulate in the blood for a long time without loss of immunochemical activity.

The following experiment was conducted to evaluate the immunochemical activity of anti-BEMP-1 antibodies after the introduction of rats with experimental C6 glioma into the bloodstream. Three rats were injected with 1 mg of anti-BEMP-1 antibodies covalently linked to liposomal nanocontainers containing DTPA gadolinium in the femoral vein, after which an MRI scan of the brain was performed after 24 hours of immunolipos circulation. The results of an MRI scan on the accumulation of a diagnostic label at the VEMP-1 expression sites indicate the ability of anti-VEMP-1 antibodies to circulate in the blood for a long time (at least 24 hours) without loss of immunochemical activity and reach the target cell antigen (abluminal surface of cerebral endotheliocytes).

Thus, the monoclonal anti-BEMP-1 antibodies obtained by the authors, introduced into the bloodstream of a living rat, retain immunochemical activity in blood plasma for at least 24 hours after injection and are able to reach the target antigen.

The proposed method can be used to obtain analogues in humans. Immunohistochemical analysis of endothelial antigens using monoclonal antibodies obtained by the method proposed by the authors can be used in experimental in vitro and in vivo studies to characterize the vascular system of gliomas.

Claims (2)

1. A method for producing monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes, in which a preparation of membrane proteins of cerebral endotheliocytes is obtained, a Balb / C mouse is immunized with it, spleen B lymphocytes of this mouse are isolated and fused with Sp2 / 0-Ag14 mouse mouse myeloma cells, hybridomas are obtained, supernatants of the obtained hybridomas are tested immunochemically, and hybridomas producing monoclonal antibodies to the abluminal membrane antigen of cerebral endotheliocytes are selected, hybrids are selected CB, characterized by the higher number of antibody-producing hybrid cells synthesize antibodies related to the immunoglobulin class G, retain biological activity in the serum for at least 24 hours, the monoclonal antibodies isolated from the supernatant of the selected hybridomas. 2. The method according to claim 1, characterized in that the selection of monoclonal antibodies from the supernatant of the selected hybridoma is carried out by affinity chromatography on protein-G-agarose.

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