RU2360707C1 - Sorbent for antibody removal from whole blood and method for making thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: sorbent for antibody removal from whole blood includes microspheres sized 0.2-1.2 mm consisting of antigen and alginic acid or calcium alginate enclosed in a semipermeable membrane made of polycationic polymer passing proteins of molecular weight 1000 kDa and less. As a polycationic polymer, the sorbent can contain either chitosan, or poly-L-lysine, or poly-L-arginine. Herewith the antigen represents a high-molecular water-soluble polymer conjugate with saccharide. Polymer represents either polyacrylamide, or polyacrylic acid, or their copolymer, or chitosan. Besides, saccharide can represent oligosaccharide of blood group A or B, or oligosaccharide component of ganglioside or sulphated glycolipid, or tumour-associated oligosaccharide. The method for making the sorbent involves injection of sodium alginate solution drops and antigen into calcium salt solution. The prepared material is kept in polycationic polymer solution to form microspheres then to be processed with complexon, e.g. ethylenediamine tetraacetate (EDTA), to remove calcium ions.

EFFECT: improved effectiveness.

6 dwg, 5 tbl, 17 ex, 10 cl

Description

The invention relates to medicine and can be used in transplantology to remove pathological antibodies from the blood.

Removing antibodies from the patient’s whole blood is a promising method for solving a number of medical problems in transplantology, in the treatment of autoimmune diseases, as well as complications caused by errors in blood transfusion or red blood cells. In particular, an allograft transplant requires careful selection of a donor-recipient pair not only for histocompatibility antigens, but also for blood antigens of the AB0 system, and in the treatment of Guillain-Barré syndrome, the removal of autoantibodies can prevent the destruction of nerve endings.

There are various ways to solve the problem of removing pathological antibodies from the blood, specific and non-specific. The latter include hemodialysis, when, together with pathological antibodies, all proteins are totally removed from the blood. An intermediate solution is the removal of all or most of the antibodies, including pathological ones, using an affinity sorbent with protein A or G. An obvious drawback of these approaches, in addition to the high cost, is the patient’s loss of proteins that are most important for his health, the need for their replacement and, as a consequence, the risk infection with frequent use of blood products.

A fundamentally different solution is the specific sorption of antibodies on the corresponding sorbent, which is an antigen (hapten) attached to the solid phase. The use of this sorbent allows you to remove exclusively pathological antibodies, leaving all other proteins in the blood, including normal antibodies. Usually, the methods currently used require preliminary separation of blood into plasma and blood cells (plasmapheresis), then a sorption procedure on a column with a sorbent and subsequent reunion of cells with purified plasma before returning them to the patient; such techniques require expensive equipment and qualified personnel at the plasmapheresis stage. If, when using ordinary sorbents, plasmapheresis is not carried out, but undivided blood is passed through the column, then due to the small size of the particles and channels between the particles, platelets and other blood cells are damaged and result from this complication. In addition, the oxygen capacity of the blood, the overall level of immunoglobulins and blood pressure are reduced. Therefore, modified carriers are used for hemosorption, for example, ordinary particles of a sorbent are coated with a semipermeable membrane [Chang T.M.S. // Trans Am Soc Artif Intern Organs. 1980. V. XXVI, P.546-549]. The particle size of traditional carriers, such as Sepharose, agarose granules, silica gel, about 5-20 microns provides high sorption capacity and efficiency of the sorption column, but leads to the creation of significant hydraulic resistance and, as a result, to cell damage. To overcome this obstacle, particles of a significantly larger size are needed, not traumatic blood cells. However, an increase in the particle size of the sorbent leads to a decrease in its surface area and, as a result, to a sharp drop in the specific capacity (ability to absorb antibodies).

The objective of the invention is the creation of new materials that, with millimeter particle sizes, would retain the capacity of traditional sorbents, that is, were suitable for practical hemosorption, when the column volume remains small, and the sorption time is comparable to the time of plasma absorption procedures. In addition, the material of the carrier matrix must be non-toxic and biocompatible, the antigen (hapten) must not enter the bloodstream, and both IgG and IgM antibodies must be adsorbed.

The problem is solved by a sorbent for removing antibodies from whole blood, which is a hydrogel microspheres consisting of salts of alginic acid - insoluble in water (calcium alginate) or soluble (sodium alginate), and from glycoconjugate (antigen). All of these components are enclosed in a polyelectrolyte alginate-polycationic membrane.

As the polycationic polymer, the sorbent may include chitosan, or poly-L-lysine, or poly-L-arginine, or poly-L-ornithine, or DEAE-dextran.

The antigen is a high molecular weight water soluble polymer conjugate with a saccharide. Typically, the molar% of the saccharide-substituted monomer units of the polymer in the conjugate is 1-30% (preferably 1-20%).

The polymer is polyacrylamide, or polyacrylic acid, or a copolymer thereof, or chitosan.

As a saccharide, an oligosaccharide of blood group A or B, or an oligosaccharide part of a ganglioside or sulfated glycolipid, or a tumor-associated oligosaccharide can be used.

Using a special device, a solution containing sodium alginate and antigen is added dropwise to a solution of calcium salt, and the obtained microspheres based on calcium alginate are kept in a solution of a polycationic polymer in order to form an additional polyelectrolyte membrane (consisting of alginate and polycation) on the surface of these microspheres. The size of the microspheres lies in the range of 0.2-1.2 mm.

Then, the obtained microspheres can be treated with a complexon for calcium ions, for example ethylene diamine tetraacetate (EDTA), resulting in microspheres filled with a more liquid gel.

The idea of a sorbent that allows you to work with whole blood is to replace small particles (ordinary sorbent) with significantly larger microspheres. The microspheres have a semi-permeable membrane, transmitting proteins of molecular weight less than 1000 kDa, including immunoglobulins G and M; inside the microsphere is a soluble conjugated antigen with a molecular weight of more than 1000 kDa. Microspheres are formed in such a way that antibodies pass through the membrane and bind internally to the antigen; at the same time, the antigen cannot exit the microsphere. Thus, all antibodies can enter and exit the microspheres, but only pathological antibodies that bind to their own antigen inside the capsule are eliminated from the bloodstream. Traditional sorbents have a highly developed surface on which antigens are immobilized, which leads to their high efficiency. In the proposed solution, the surface area is orders of magnitude lower, and the efficiency of antibody binding is achieved due to volumetric placement of antigen inside the microsphere in dissolved form. The microspheres are filled with either Ca-alginate hydrogel or alginic acid (when calcium ions are removed by treating the microspheres with a complexon). In the first case, the hydrogel becomes more rigid.

Such a general solution to the problem required solving a number of particular problems:

- Ensure the semi-permeability of the membrane of the microspheres.

- Synthesize such an antigen (in general form) that would have a significantly larger mass (hydrodynamic size of the molecule) than immunoglobulin M, but at the same time remain soluble.

- To form a membrane of microspheres having a permeability sufficient to ensure diffusion of antibodies inward, but at the same time sufficiently mechanically strong.

- The material of the microspheres should be resistant to the action of blood components, non-toxic, not sorb the blood components non-specifically.

- The speed of the sorption process should be high.

- The sorption capacity of the microspheres should be high to minimize the volume of the column packed with microspheres.

These particular problems were solved as follows.

To obtain the microspheres, sodium alginate and a polycationic polymer were used. Alginate is the most widely used polymer for incorporating various biological objects. Alginate is a natural polysaccharide consisting of irregularly alternating residues of β-D-mannuronic and α-L-guluronic acids linked by 1-4 glycosidic bonds. Alginate-based carriers are usually prepared by crosslinking carboxyl groups with Ca ²⁺ cations. In this invention, to stabilize Ca-alginate microspheres under physiological conditions and to prevent the release of glycoconjugates (antigens) included therein, the particles were coated with a polycationic polymer membrane. As such a polymer, for example, polylysine or polyarginine, or others, may be used, but chitosan is most preferred. Chitosan is a deacetylated chitin consisting of beta-1-4-linked residues of 2-amino-2-deoxy-D-glucopyranose. An additional role of the polycationic layer is to control the permeability of the membrane of microspheres.

The size of the microspheres (0.2-1.2 mm) can be varied by varying various parameters, such as the viscosity of the sodium alginate solution, its feed rate, the diameter of the needle through which the alginate solution is added dropwise to the calcium chloride solution, etc.

The thickness of the membrane is regulated by varying several parameters, namely, the molecular weight of chitosan, the concentration of chitosan and the formation time of the alginate-chitosan membrane, the pH of the chitosan solution.

Figure 1 presents the scheme for producing the sorbent in the form of microspheres.

Figure 2 shows a photograph of the obtained microspheres.

Figure 3 shows the size distribution diagram of alginate-chitosan microspheres.

Figure 4 shows a graph characterizing the kinetics of the release of fluorescently-labeled glycoconjugate from alginate-chitosan microspheres.

5 (A, B) are diagrams illustrating the sorption of monoclonal IgM antibodies by saccharide-containing microspheres and Sepharose (to which the same carbohydrate is attached, for comparison) after 1 hour (A) and 24 hours (B) of incubation.

6 shows an experimental setup for producing microspheres.

A method of manufacturing a sorbent is described below on the example of the use of chitosan. Microspheres were similarly made using other polycationic polymers, including poly-L-lysine, or poly-L-arginine, or poly-L-ornithine, or DEAE-dextran.

The experimental setup for the preparation of Ca-alginate microspheres is presented in Fig.6. The solution containing alginate and glycoconjugate was supplied using a peristaltic pump (0.28-3 ml / min) to a device 1 equipped with a needle 2 (needle fastening elements 3, 4), where this solution was sprayed with a stream of compressed air and in the form of small drops got into a solution of CaCl ₂ .

The formed Ca-alginate microspheres were washed with 0.9% NaCl, transferred to a polycation solution (pH 4.5), incubated for 5-30 minutes at room temperature, and again washed with 0.9% NaCl. Thus, alginate-polycationic microspheres were obtained (see the scheme in FIG. 1). Next, the microspheres were treated with 0.1 M EDTA solution (room temperature, 10-30 min). The prepared alginate-chitosan microspheres were stored in 0.9% NaCl at 4 ° C.

The size of the microspheres was controlled by the concentration of the sodium alginate solution, its feed rate using a peristaltic pump, compressed air pressure, needle diameter 2, the distance between the surface of the CaCl ₂ solution and needle 2. The optimal conditions were found for obtaining Ca-alginate microspheres of the correct spherical shape: pressure - 5 bar; CaCl ₂ solution volume 25 ml; the flow rate of the polymer solution of alginate 0.28 ml / min; needle diameter 0.8 mm. The mass ratio of antigen and alginate in the microspheres was in the range from 1:40 to 1: 2.

An increase in the feed rate of the polymer solution (above 3 ml / min) or a decrease (below 0.28 ml / min) leads to deformation of the spherical shape of the microparticles and, as a result, to a loss of antigen, which, in turn, leads to a decrease in the antibody sorption capacity. Therefore, the optimum rate of 0.28 ml / min was chosen, and the resulting size was ~ 900 μm. Under these conditions, microspheres of the correct spherical shape were obtained.

The thickness of the membrane (from 40 to 100 μm) is controlled by varying several parameters, namely, the molecular weight of chitosan, the concentration of chitosan and the formation time of the alginate-chitosan membrane, the pH of the chitosan solution. To form a stable membrane, Ca-alginate microspheres are incubated in a polycation solution for at least 5 minutes at pH 4.5.

As can be seen from figure 2, 3, the obtained alginate-chitosan microspheres have the correct spherical shape and a narrow size distribution.

The antigen is introduced into the system at the stage of preparation of the mixture to form particles, technically this is realized by adding it to the alginate solution.

The efficiency of incorporating a glycoconjugate (antigen) into the microspheres was determined using its labeled variant, namely A _tri- PAA-fluo, where A _tri is a trisaccharide antigen of blood group A, PAA is a polyacrylamide with a molecular weight of about 2000 kDa, and fluo is a fluorescein residue. The degree of incorporation of A _tri- PAA-fluo into alginate-chitosan microspheres was ~ 40% of the initial amount. After treatment of alginate-chitosan microspheres with an EDTA solution, the content of A _tri- PAA-fluo in them decreased almost twofold. It can be assumed that the removal of calcium ions from the polymer membrane led to an increase in its permeability and, as a result, to an additional exit of low molecular weight fractions of the glycoconjugate from the microspheres. The methodology used allows you to remove the low molecular weight fraction of the glycoconjugate (which is always present even in the composition of the highest molecular weight polymers) during the manufacturing (washing) of the microspheres, therefore, special preliminary fractionation of the glycoconjugate is not required.

Figure 4 shows the time dependence of the yield of A _tri- PAA-fluo in the supernatant (saline solution) from alginate-chitosan microspheres. It can be seen that even after 16 days of storage, the loss of glycoconjugate did not exceed 3.5%.

At the same time, sorption from a solution of anti-B _di and anti-A _tri antibodies was studied with microspheres not containing antigen (control); sorption in the control (i.e., non-specific binding of antibodies to microspheres) was less than 5%.

The sorption capacity of alginate-polycationic microspheres was compared with the most frequently used today sorbent-carrier Sepharose FF, to which the same antigen is covalently attached. The load of A _tri in both cases was the same, namely 0.6 mmol / ml. The results are presented in figure 5. It is seen that the specific sorption of antibodies by microspheres at low antibody dilutions proceeds more slowly than in the case of sepharose. However, after 24 hours, in both cases, the process reaches a plateau.

Thus, the obtained data confirm the possibility of using the proposed material for sorption of anti-carbohydrate antibodies. The resulting microspheres in sorption capacity are not inferior to modified sepharose. The compatibility of the developed sorbent with whole blood was tested on rabbit blood, that is, blood was repeatedly passed through a layer of microspheres. At the same time, neither damage to blood cells (hemolysis, thrombosis), nor capsule destruction by blood components was observed. The optimal average size of the microspheres lies in the range of 0.2-1.2 mm; larger microspheres slowly adsorb antibodies; smaller microspheres can injure red blood cells.

EXAMPLES

Materials The following reagents were used: sodium chloride, calcium chloride, sodium alginate, EDTA, Tween 20 (Sigma, USA). Chitosan (MB 3.7 kDa) was obtained by chemical destruction of commercial high molecular weight chitosan. Synthetic glycoconjugates B _di- PAA, A _tri- PAA, A _tri- PAA-fluo (MB ~ 2000 kDa) were obtained as described in [Shilova NV, Galanina O.E., Pochechueva T.V., Chinarev AA, Kadykov VA, Tuzikov AA and Bovin NV // Glycoconjugate J. 2005. V.22. P.43-51]. All solutions were prepared with 0.9% NaCl.

Example 1. To obtain Ca-alginate microspheres, a 4% solution of sodium alginate is mixed with a solution of B _di- PAA (2.5 mg / ml) in a ratio of 1: 1 (vol.) And added dropwise to 25 ml of a 2% solution of CaCl ₂ . The experimental setup for the preparation of Ca-alginate microspheres is shown in FIG. 5. A solution of alginate containing B _di- PAA, using a peristaltic pump, is fed into the body of the device 1 (feed rate 0.28 ml / min), equipped with a needle 2 (diameter 0.8 mm), where it is sprayed with a stream of compressed air, and in the form of small drops alginate solution enters a 2% solution of CaCl ₂ . The formed Ca-alginate microspheres are washed 3 times with 0.9% NaCl, transferred to a 0.2% chitosan solution (pH 4.5), incubated for 5 minutes at room temperature and washed again with 0.9% NaCl (see the scheme in FIG. one). The prepared alginate-chitosan microspheres are stored in 0.9% NaCl at 4 ° C. Under these conditions, the average size of the obtained microspheres was 900 μm, and the average membrane thickness was 40 μm.

Example 2. The microspheres are prepared according to example 1, but in this case, to remove calcium ions, the microspheres are treated for 30 minutes with a 0.1 M EDTA solution at room temperature. All further operations are done, as in example 1.

Example 3. The microspheres are prepared according to example 1, but in order to obtain a membrane, the microspheres are incubated in a 0.2% solution of poly-L-lysine (pH 4.5) with a molecular weight of 15,000-30000 Yes, then everything is done as in example 1 .

Example 4. The microspheres are prepared according to example 1, but in order to obtain a membrane, the microspheres are incubated in a 0.2% solution of poly-L-arginine (pH 4.5) with a molecular weight of 10000-40000 Da. All further operations are done, as in example 1.

Example 5. The microspheres are prepared according to example 1, but in order to obtain a membrane, the microspheres are incubated in a 0.2% solution of DEAE-dextran (pH 4.5) with a molecular weight of ~ 6500 Yes, then everything is done as in example 1.

Example 6. The microspheres are prepared according to example 1, but in order to obtain a membrane, the microspheres are incubated in a 0.2% solution of poly-L-ornithine (pH 4.5) with a molecular weight of 15000-25000 Da. All further operations are done, as in example 1.

Tables 1-4 show the values of antibody sorption by microspheres obtained under various conditions using various antigens and polymers. The percentage (%) of adsorbed antibodies was determined as the difference in optical density values before and after incubation of the antibody solution with microspheres.

Table 1 Dependence of the value of antibody sorption by microspheres of various compositions on incubation conditions Type of antigen carrier The number of B _di- PAA included in the microspheres, mg / ml The amount of bound polyclonal anti-B _di (IgG + IgM) antibodies (%) after incubation for 1 h at t, ° C 24 h at t, ° C 24 37 24 37 According to example 1 one 60 65 one hundred one hundred According to example 2 0.6 40 fifty 70 75 For example 3 one fifty fifty 80 85 For example 4 one 45 fifty 70 75 For example 5 one fifty 55 75 80 For example 6 one 45 fifty 70 75

Example 7. The microspheres are prepared according to example 1, but the feed rate of the polymer solution is 3 ml / min. All further operations are done, as in example 1.

Example 8. The microspheres are prepared according to example 1, but the feed rate of the polymer solution is 0.28 ml / min. All further operations are done, as in example 1.

Example 9. The microspheres are prepared according to example 1, but the feed rate of the polymer solution is 0.7 ml / min. All further operations are done, as in example 1.

table 2 Microspheres with antigen on The feed rate of the polymer solution, ml / min Pressure bar The average size of the microspheres, microns % sorption For example 7 3.0 5 1600 twenty For example 8 2,8 5 1400 25 For example 9 0.7 5 1200 70 According to example 1 0.28 5 900 one hundred

Example 10. A mixture for the preparation of microspheres is prepared according to example 1, but the antigen (glycoconjugate) is A _tri- PAA and its amount in the mixture is 2.7 mg (Table 3). All further operations are done, as in example 1.

Example 11. A mixture for the preparation of microspheres is prepared according to example 1, but the antigen is A _tri- PAA and its amount in the mixture is 2.5 mg; the amount of alginate varies (table 3). All further operations are done, as in example 1.

Example 12. A mixture for the preparation of microspheres is prepared according to example 1, but the antigen is A _tri- PAA and its amount in the mixture is 1 mg (Table 3).

Table 3 The amount of alginate in the mixture, mg The amount of antigen in the mixture, mg The amount of bound anti-A _tri (IgM) antibodies after incubation for 1 h,% Example 10 40 2.7 55 Example 11 twenty 2.5 52 Example 12 twenty one 45

Example 13. The microspheres obtained in example 1, incubating in a 0.2% solution of chitosan (pH 4.5) for 10 minutes (table 4).

Example 14. The microspheres obtained in example 1, incubating in a 0.2% solution of chitosan (pH 4.5) for 30 minutes (table 4).

Table 4 Type of antigen carrier The incubation time of the microspheres in chitosan, min Membrane thickness, microns The number of bound anti-B _di (IgG + IgM) after 1 h of incubation,% According to example 1 5 40 fifty Example 13 10 one hundred 25 Example 14 thirty not visible -

Example 15. Characterization of alginate-chitosan microspheres

The size of the microspheres and the thickness of their membranes were determined using a Biolam-P1 light microscope (Lomo, Russia), which has an eyepiece with a scale ruler. For better visualization of the membrane, a suspension of latex particles (600 nm diameter, Sigma, USA) was used. For this, a suspension of latex particles was added to the alginate solution and Ca-alginate microspheres with the included latex particles coated with an alginate-polycationic membrane were prepared as described above. To deposit latex particles on the inner surface of the membrane, the microspheres were incubated in a 0.1 M EDTA solution and then centrifuged (10,000 rpm, 10 min).

The thickness of the membrane of the microspheres obtained after the removal of calcium using EDTA is 40-100 μm (figure 5). The resulting microspheres have high mechanical strength (can withstand centrifugation for 10 min at 10,000 rpm). With an increase in centrifugation time to 30 minutes, the number of deformed microspheres is no more than 5%.

Example 16. The study of the effectiveness of the inclusion of glycoconjugate

The efficiency of incorporating a glycoconjugate into alginate-polycationic microspheres was determined using A _tri- PAA labeled with a fluorescent label. For this, A _tri- PAA-fluo was mixed with a 4% alginate solution in a ratio of 1: 1 (vol.) And used to prepare microspheres according to the method described above. The degree of inclusion of A _tri- PAA-fluo was determined as follows: aliquots of the prepared microspheres (50 μl of a dense precipitate) were dissolved in water by adding NaOH (pH 11.5) and fluorescence (485/535 nm) was measured using Perkin Elmer Instrument, Wallac 1420. The degree of desorption of low molecular weight fractions of A _tri- PAA-fluo from microcapsules during storage in 0.9% NaCl was estimated by the fluorescence of the supernatant.

Example 17. The study of the effectiveness of sorption of antibodies

To study the adsorption of antibodies, aliquots of microspheres and sepharose (50 μl of solid sediment) were incubated with antibody solutions in a ratio of 1:10 (vol.) For 1 or 24 hours. The sorption process was carried out at 24 and 37 ° C. Depending on the type of glycoconjugate incorporated and the class of adsorbed antibodies, polyclonal anti-B _di (IgG + IgM) antibodies (initial concentration of 254 μg / ml) or ascites containing monoclonal antibodies of the IgM class in various dilutions were used for incubation (Table 5).

Table 5 Characteristics of Used Antibodies Sorbent Glycoconjugate Class of adsorbed antibodies Dilution of antibodies for incubation Microspheres (Ca-containing) B _di- paa IgG, IgM 1: 640 Microspheres (after Ca removal) B _di- paa IgG, IgM 1: 640 Microspheres (Ca-containing) A _tri- paa IgM 1: 10000-1: 100000 Sepharose A _tri- paa IgM 1: 10000-1: 100000

The efficiency of sorption of antibodies by microspheres and sepharose sorbent was determined using enzyme-linked immunosorbent assay. 96-well plates (NUNC MaxiSorp, Denmark) were used to adsorb antigens. 100 μl of glycoconjugate (B _di- PAA or A _tri- PAA) with a concentration of 10 μg / ml in 50 mM carbonate buffer at pH 9.6 was added to each well and incubated overnight at 4 ° C. The plate was then washed with distilled water, 100 μl of 1% BSA / PBS was added to each well, incubated for 40 min at 37 ° C and washed again three times with 0.05% Tween 20 / PBS. Then, 200 μl of antibodies were added to the wells (after incubation with microcapsules or with Sepharose, Table 2), a series of twofold dilutions were made in a 0.3% BSA solution and incubated for 1 hour at 37 ° C. The plate was then washed 3 times with a 0.05% Tween 20 / PBS solution and 100 μl per well of anti-human Ig (IgM + IgG) HRP was added at a dilution of 1: 8000 (when working with human antibodies) or anti-mouse IgM-biotin at a dilution of 1: 1000 and then streptavidin-HRP at a dilution of 1: 8000, followed by incubation for 1 hour at 37 ° C (when working with murine antibodies). After washing three times with a 0.05% Tween 20 / PBS solution, 100 μl of o-phenylenediamine (0.4 mg / ml) was added to the wells in phosphate-citrate buffer containing 4 μl of 30% H ₂ O ₂ . After 30 minutes, the reaction was stopped by adding 50 μl of 1M H ₂ SO ₄ . The results were evaluated using a reader (Perkin Elmer Instrument, Wallac 1420) at 492 nm.

Similarly, the removal of antibodies from blood serum by microspheres was carried out: 1) with polyacrylic acid conjugates included in them; 2) with conjugates of a copolymer of polyacrylamide and polyacrylic acid included therein; 3) with the conjugate of Neu5Acα2-3Galβ1-4Glc trisaccharide included in them (carbohydrate portion of GM3 ganglioside); 4) with the sulfated oligosaccharide 3-HSO ₃ -OGal included in them; 5) with the tumor associated Tk tetrasaccharide included in them.

Claims (10)

1. Sorbent for removing anti-carbohydrate antibodies from whole blood, including microspheres 0.2-1.2 mm in size, consisting of antigen and alginic acid or calcium alginate, enclosed in a semi-permeable polycationic polymer membrane, transmitting proteins of molecular weight less than 1000 kDa, where the antigen is a high molecular weight water soluble conjugate of a polymer with a saccharide. 2. The sorbent according to claim 1, characterized in that it comprises chitosan, or poly-L-lysine, or poly-L-arginine, or poly-L-ornithine, or DEAE-dextran as the polycationic polymer. 3. The sorbent according to claim 1, characterized in that the polymer is a polyacrylamide, or polyacrylic acid, or their copolymer, or chitosan. 4. The sorbent according to claim 1, characterized in that the saccharide is an oligosaccharide of blood group A or B, or an oligosaccharide portion of a ganglioside or sulfated oligosaccharide, or a tumor-associated oligosaccharide. 5. A method of producing a sorbent for removing anti-carbohydrate antibodies from whole blood, which is that drops of a solution of sodium alginate and antigen are injected into a solution of calcium salt, and the formed Ca-alginate microspheres are kept in a solution of a polycationic polymer to obtain microspheres with a size of 0.2-1 , 2 mm, coated with a semipermeable alginate-polycationic membrane, transmitting proteins with a molecular weight of less than 1000 kDa. 6. The method according to claim 5, characterized in that the obtained microspheres are treated with complexon to remove calcium ions, for example ethylenediaminetetraacetate. 7. The method according to claim 6, characterized in that chitosan, or poly-L-lysine, or poly-L-arginine, or poly-L-ornithine, or DEAE-dextran is used as the polycationic polymer. 8. The method according to claim 5, characterized in that the high molecular weight water-soluble conjugate of the polymer with a saccharide is used as antigen. 9. The method according to claim 8, characterized in that the polymer used is polyacrylamide, or polyacrylic acid, or their copolymer, or chitosan. 10. The method according to claim 8, characterized in that the saccharide used is an oligosaccharide of blood group A or B, or an oligosaccharide part of a ganglioside or sulfated oligosaccharide, or a tumor-associated oligosaccharide.

Images