A material for adsorbing lipoprotein in blood and its preparation method and application

Abstract

The present application relates to the field of bioseparation engineering, and specifically discloses a material for adsorbing lipoprotein in blood, a preparation method thereof, and an application thereof. A material for adsorbing lipoprotein in blood comprises a microsphere carrier and a lipoprotein antibody covalently coupled to the microsphere carrier. The adsorbing material of the present application can be used to adsorb different types of lipoprotein in blood, and the adsorbing material of the present application can be used in combination with gel exclusion chromatography or ultracentrifugation to purify extracellular vesicles in blood.

Description

Technical Field

[0001] The present application relates to the field of biological separation engineering, and more specifically, to an adsorption material for lipoprotein in blood, a preparation method thereof, and an application thereof. Background Art

[0002] Extracellular vesicles (EVs) refer to vesicles with double-layer membrane structures that fall off the cell membrane or are secreted by cells, with diameters ranging from 30nm to 1000nm. Extracellular vesicles are mainly composed of microvesicles (MVs) and exosomes. Exosomes mainly refer to extracellular vesicles with diameters between 30nm and 100nm, while microvesicles mainly refer to extracellular vesicles with diameters between 100nm and 1000nm. Extracellular vesicles are widely present in various body fluids, carrying a variety of cell-derived proteins, lipids, DNA, mRNA, miRNA, etc., and are involved in processes such as intercellular communication, cell migration, angiogenesis, and immune regulation.

[0003] Due to these characteristics of extracellular vesicles, they have the characteristics of being a detection target. Therefore, early screening of diseases can be performed by detecting biomarkers in extracellular vesicles, such as early screening of tumors or early screening of nervous system diseases. In addition, due to their good targeting, extracellular vesicles can be used as natural drug carriers, that is, drugs can be encapsulated in extracellular vesicles to achieve the purpose of targeted drug delivery.

[0004] In the application of extracellular vesicles, it is necessary to purify the extracellular vesicles, among which the most widely used are the purification of exosomes and some microvesicles. Among all the purification techniques of extracellular vesicles, ultracentrifugation and gel exclusion chromatography are the two most commonly used techniques. However, in the purification process of extracellular vesicles, lipoprotein contamination is very likely to occur.

[0005] Lipoproteins are spherical particles composed of a hydrophobic core rich in sterols and triglycerides and an outer shell composed of proteins, phospholipids, cholesterol, etc. Lipoproteins play an important role in the packaging, storage, transportation and metabolism of extracellular lipids in mammals. Abnormal lipoprotein metabolism (usually accompanied by changes in lipid and protein components) is closely related to atherosclerosis, diabetes, obesity and tumorigenesis.

[0006] Lipoproteins can be divided into chylomicrons, very low-density lipoproteins, intermediate-density lipoproteins, low-density lipoproteins and high-density lipoproteins according to their density.

[0007] Since lipoproteins are complex in types and have many similarities in size and density with extracellular vesicles, it is difficult to separate lipoproteins when purifying extracellular vesicles, which leads to lipoprotein contamination during the purification of extracellular vesicles.

[0008] The concentration of lipoprotein in the blood is relatively high, with the concentration of high-density lipoprotein and low-density lipoprotein being approximately 1015 particles / ml of blood, while the concentration of extracellular vesicles in the blood is approximately 107-109 vesicles / ml of blood. Since the concentration of extracellular vesicles is much lower than that of lipoproteins, it is very difficult to remove lipoprotein contamination during the purification of extracellular vesicles.

[0009] Currently, the following methods are often used to reduce lipoprotein contamination during the purification of extracellular vesicles.

[0010] First, through the control of sampling time and method

[0011] Since the metabolism of lipoprotein is closely related to eating, data show that the concentration of chylomicrons increases by 2 times within 3-4 hours after eating. In order to reduce the contamination of lipoprotein, the method of pre-screening blood samples is adopted. The blood sampling group is required to control their diet before sampling, or to collect samples within a certain time after eating. This method is very difficult in clinical applications and reduces the feasibility of reagent application. Moreover, even if the sampling time is controlled, it can only reduce the amount of some lipoproteins such as chylomicrons, but cannot eliminate the contamination of lipoproteins during the purification process. In addition, this method is difficult to implement in actual clinical applications, and the test personnel cannot determine the dietary conditions of the patients to be tested, so this method can only be used in specific research projects.

[0012] Second, combine purification methods with different principles

[0013] The commonly used purification techniques include ultracentrifugation + gel exclusion chromatography, that is, the blood sample is separated from the extracellular vesicles in the conventional ultracentrifugation method, and the ultracentrifugation is carried out at a centrifugal force of >100,000×g for more than 10 hours, and then the corresponding extracellular vesicle layer is collected, and the collected extracellular vesicles are separated and purified according to the particle size by gel exclusion chromatography. By combining two different purification methods, the contamination of lipoproteins in extracellular vesicles can be greatly reduced.

[0014] The above purification process can also be carried out in different orders. Some researchers first use gel exclusion chromatography to separate extracellular vesicles from blood samples, and then ultracentrifuge the collected extracellular vesicles. Ultracentrifugation at a centrifugal force of 100,000×g for 20 hours can purify extracellular vesicles with higher purity.

[0015] This method has the following problems: 1. It is time-consuming. The operation time after combining these two purification methods often exceeds 24 hours, which is very long;

[0016] 2. The loss of extracellular vesicles during the purification process is relatively large because of multiple sample transfers, resulting in a large loss and a low recovery rate.

[0017] 3. Even if the two methods are combined, the lipoprotein pollution can only be reduced to a limited extent.

[0018] Third, adsorbent adsorption

[0019] At present, the adsorbents for lipoprotein in blood mainly adsorb and remove low-density lipoprotein in blood. Commonly used low-density lipoprotein adsorption and removal technologies mainly include the following methods:

[0020] 1. Use dialysis membrane as carrier and modify ligand on the carrier for adsorption. Dialysis membrane includes poly(ethylene glycol) membrane, polyether membrane, etc. The adsorption ligand is mainly heparin.

[0021] 2. Using microspheres as carriers, modifying ligands on the carriers for adsorption. This form of adsorption material is of many types and is the mainstream form of adsorbent. Microspheres mainly include chitosan, cellulose or polyvinyl alcohol microspheres. Commonly used adsorption ligands include heparin, dextran sulfate, acidic amino acids, etc. There are also ways to modify ligands such as sulfonic acid groups and phosphate groups for adsorption.

[0022] This method of eliminating lipoprotein pollution has the following disadvantages:

[0023] 1. The current lipoprotein adsorbent can only adsorb a certain type of lipoprotein. The reason is that the current lipoprotein adsorbent is mainly used in the field of blood purification, and the main purpose is to treat hyperlipidemia. It is used to treat hyperlipidemia that is insensitive to lipid-lowering drugs. Therefore, the current lipoprotein adsorbent mainly targets low-density lipoprotein LDL in the blood, and the purpose of adsorption is to reduce the concentration of low-density lipoprotein in the blood.

[0024] 2. The current lipoprotein adsorption materials have poor specificity

[0025] Current lipoprotein adsorption materials mainly rely on the attraction and combination of the charge properties of lipoproteins and the charge of the adsorption material under different pH environments for adsorption. However, under the same environment, there are multiple components in the blood that can bind to the adsorption material, resulting in poor specificity of the adsorption material.

[0026] 3. The current lipoprotein adsorption material has a low binding capacity and a long reaction time

[0027] For current lipoprotein adsorption materials, the interaction time between blood and lipoprotein adsorption materials is usually more than one hour. In addition, the lipoprotein binding capacity of current adsorption materials is usually low, and the blood purification reaction time is long. The purification scenario of blood extracellular vesicles described in the present invention requires a faster binding speed and binding efficiency.

[0028] Based on the above problems, the present application proposes an adsorption material for lipoprotein in blood and a preparation method and application thereof. Summary of the invention

[0029] In order to solve the above problems, the present application provides an adsorption material for lipoprotein in blood, a preparation method and application thereof.

[0030] In the first aspect, the present application provides a material for adsorbing lipoprotein in blood, which adopts the following technical solution:

[0031] A material for adsorbing lipoprotein in blood comprises a microsphere carrier and a lipoprotein antibody covalently coupled to the microsphere carrier.

[0032] Preferably, the microsphere carrier includes but is not limited to agarose gel microspheres, cellulose microspheres, polystyrene microspheres or polyvinyl alcohol microspheres.

[0033] Preferably, the preparation method of the polystyrene resin microspheres is as follows:

[0034] A1. dissolving a stabilizer and a surfactant in deionized water to form a uniform solution to obtain an aqueous phase; A2. dissolving a functional monomer and a crosslinking agent in a porogen, and adding an initiator to prepare an oil phase;

[0035] A3. The oil phase prepared in A2 is added to the water phase prepared in A1 to carry out a polymerization reaction. After the polymerization reaction is completed, the obtained substance is filtered and washed to remove unreacted residues, and finally polystyrene microspheres are obtained.

[0036] Preferably, in step A1, the stabilizer includes but is not limited to polyvinyl pyrrolidone and polyvinyl alcohol, and the mass percentage concentration of the stabilizer is 1%-10%, and the surfactant includes but is not limited to copolymers of polyoxyethylene and polyoxypropylene and fatty acid esters, and the mass percentage concentration of the surfactant is 1%-5%;

[0037] In step A2, the molar ratio of the functional monomer to the cross-linking agent is 2:1-8:1, the usage ratio of the functional monomer, the cross-linking agent and the porogen is 1:1-1:5 of the total volume of the functional monomer and the cross-linking agent to the volume of the porogen, and the molar number of the initiator is 1%-10% of the total molar number of the functional monomer and the cross-linking agent;

[0038] In step A3, the volume ratio of the oil phase to the water phase is 1:2-1:10; the polymerization reaction temperature is 20°C-100°C; and the polymerization reaction time is 1-20h.

[0039] Preferably, the particle size of the microsphere carrier is 50 μm-600 μm.

[0040] Preferably, the particle size of the microsphere carrier is 100 μm-500 μm.

[0041] Preferably, the pore size of the microsphere carrier is between 50nm and 3000nm.

[0042] Preferably, the pore size of the microsphere carrier is 150nm-1000nm.

[0043] Preferably, the lipoprotein antibody comprises one or more of an apolipoprotein AI antibody, an apolipoprotein B100 antibody, and an apolipoprotein B48 antibody.

[0044] Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0045] In a second aspect, the present application provides a method for preparing a material for adsorbing lipoprotein in blood, using the following technical solution:

[0046] A method for preparing a material for adsorbing lipoprotein in blood comprises the following steps:

[0047] S1, preparing microsphere carrier;

[0048] S2, activated microsphere carrier;

[0049] S3. dissolving Staphylococcus aureus protein A or Streptococcus protein G in a modification buffer to obtain a second mixed solution;

[0050] S4. mixing the first mixed solution and the second mixed solution and reacting at an appropriate temperature to obtain a reaction product, and washing the reaction product with a modification buffer after the reaction is completed;

[0051] S5. The reaction product after washing in S4 is blocked with a blocking buffer, and after blocking, it is resuspended with a modification buffer to obtain a first composite solution;

[0052] S6. dissolving the lipoprotein antibody in a coupling buffer to obtain a second complex solution;

[0053] S7. The first composite solution and the second composite solution are mixed for reaction, and after the reaction, the reaction product is washed and then resuspended with a coupling buffer;

[0054] S8. Dissolve suberic acid bis(N-hydroxysuccinimide ester) in dimethyl sulfoxide, and then add the resultant to the solution obtained in step S7 to react. The product obtained by the reaction is first washed with an elution buffer, and then washed with a storage buffer and stored in the storage buffer to obtain a product.

[0055] Preferably, the microsphere carrier is a polystyrene microsphere, the microsphere carrier prepared in S2 is added to a modification buffer, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide are added to the modification buffer for reaction, after the reaction is completed, the microsphere carrier is washed several times and resuspended with the modification buffer to obtain an activated microsphere carrier, the modification buffer is 10-100mM MES buffer, and its pH is 5.5-7.5, and the amount of modification buffer, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide is: every 100mg of microsphere carrier is suspended in 5-10ml modification buffer and mixed with 10-120mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-120 mg N-hydroxysuccinimide are reacted at a temperature of 20°C-30°C; the amount of modification buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide is as follows: every 100 mg of microsphere carrier is suspended in 5-10 ml of modification buffer and reacted with 10-120 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10-120 mg N-hydroxysulfosuccinimide at a temperature of 20°C-30°C;

[0056] Preferably, in step S3, the molar ratio of Staphylococcus aureus protein A or Streptococcus protein G to the ligand is 1:1-10:1, and the modification buffer is 10-100 mM MES buffer, and its pH is 5.5-7.5;

[0057] In step S4, the reaction temperature is 20°C-30°C, the reaction time is 2-4h, the modification buffer is 10-100mM MES buffer, and its pH is 5.5-7.5;

[0058] In step S5, the blocking buffer is 20-120 mM Tris-HCl buffer, pH 7.5-8.5, and the modification buffer is 10-100 mM MES buffer, pH 5.5-7.5;

[0059] In step S6, the coupling buffer is a phosphate buffer with a concentration of 50mM-100mM and a pH of 6.5-8.5, and the amount of antibody is: 50-1000ug of antibody is coupled to every 100ug of the microsphere carrier obtained in step S5;

[0060] In step S7, the reaction temperature is 20°C-30°C, and the reaction time is 30-60min;

[0061] In step S8, the concentration of suberic acid bis(N-hydroxysuccinimide ester) in dimethyl sulfoxide is 1mM-5mM, and the final concentration of suberic acid bis(N-hydroxysuccinimide ester) after mixing suberic acid bis(N-hydroxysuccinimide ester), dimethyl sulfoxide and the product obtained in S7 is 100-500uM, the reaction temperature is 20°C-30°C, and the reaction time is 30-60min; the elution buffer is 50-150mM glycine buffer, pH 2.0-3.0, and the storage buffer is 50mM-60mM phosphate buffer, pH 7.0-7.5.

[0062] In a third aspect, the present application provides an application of a lipoprotein adsorption material, using the following technical solution:

[0063] The invention discloses an application of a lipoprotein adsorption material, wherein the adsorption material is used to adsorb different types of lipoproteins in the blood, wherein the lipoproteins include but are not limited to high-density lipoproteins, low-density lipoproteins and chylomicrons.

[0064] The invention discloses an application of a lipoprotein adsorption material, which combines the adsorption material with gel exclusion chromatography or ultracentrifugation to purify extracellular vesicles in blood.

[0065] In summary, this application has the following beneficial effects:

[0066] 1. The lipoprotein adsorption material of the present invention can bind to different types of lipoproteins including high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein and chylomicrons.

[0067] 2. The lipoprotein adsorption material of the present invention can specifically bind to different types of lipoproteins, but has no non-specific binding to other components in the blood.

[0068] 3. The lipoprotein adsorption material of the present invention adopts a directional covalent coupling technology, which greatly improves the binding efficiency of the adsorption material compared with conventional coupling technology.

[0069] 4. The lipoprotein adsorption material of the present invention is applied to the purification process of extracellular vesicles in blood, which can greatly reduce the contamination of blood lipoproteins and quickly remove lipoproteins in blood, thus greatly saving purification time. Thus, the overall purification time of extracellular vesicles can be greatly saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a nanoparticle tracking analysis (NTA) diagram of the purified product prepared by Application Example 1 and Application Comparative Example 1 of the present application;

[0071] Figure 2This is a Western blot test diagram of the purified products prepared in Application Example 1 and Comparative Example 1 of the present application using CD9 antibody;

[0072] Figure 3 Western blot detection diagram of the purified products prepared in Application Example 1 and Comparative Example 1 of the present application using Apo A antibody;

[0073] Figure 4 It is an electron microscope detection image of the purified product prepared by Application Example 1 and Application Comparative Example 1 of the present application;

[0074] Figure 5 This is a graph showing the detection results of the lipoprotein detection system of the purified products prepared in Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0075] The present application is further described in detail below with reference to the embodiments. The serum used in the present application is prepared from blood by centrifugation, and the blood is derived from the blood sample of the same person; the ultracentrifuge in the present application is Beckman's Optima MAX-XP ultracentrifuge, the rotor model is MLA-55, and the centrifuge tube is a polycarbonate centrifuge tube model Beckman355630; polyvinyl pyrrolidone PVP is sigma 81420; P123 is McLean Biochemical T832531; the functional monomer methyl methacrylate MMA is Sigma M55909; ethylene glycol dimethacrylate EGDMA is Merck 52465; streptococcal protein G is Sigma 08062, and the serum preparation method and source in the application examples and comparative examples of the present application are the same as those in Application Example 1 - that is, the serum is prepared by centrifugation from the blood sample of the same person; UC+SEC in the present application refers to the purified product obtained by ultracentrifugation combined with gel exclusion chromatography in Comparative Example 1; Method 1 refers to the purified product prepared in Application Example 1 of the present application.

[0076] Example

[0077] Example 1

[0078] S1. Preparation of microsphere carriers: A1, accurately weigh 30 g of polyvinyl pyrrolidone and 10 g of P123 into 500 mL of deionized water, stir until uniform and transparent to form a uniform solution to obtain an aqueous phase;

[0079] A2: 10 ml of functional monomer methyl methacrylate and 5 ml of cross-linking agent ethylene glycol dimethacrylate are dissolved in 60 ml of porogen dichloromethane, and then 1 ml of azobisisobutyronitrile is added to prepare an oil phase, wherein the total molar number of the functional monomer and the cross-linking agent is 0.125 mmol;

[0080] A3: Take 76 ml of the oil phase prepared in A2, add it to 400 ml of the water phase prepared in A1, stir slowly, and control the temperature at 80°C. React for 12 hours. After the reaction is completed, filter out the liquid and dry to obtain polymethyl methacrylate resin microspheres.

[0081] S2. Activation of microsphere carrier: 100 mg of polymethyl methacrylate resin microspheres prepared in S1 were suspended in 50 mM pH 6.0 MES buffer to obtain a reaction solution, 100 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 100 mg of N-hydroxysulfosuccinimide were added to the reaction solution and reacted at 25° C. for 15 min. After the reaction, the microspheres were washed three times with 50 mM pH 6.0 MES buffer and resuspended with 5 ml of 50 mM pH 6.0 MES buffer to obtain a first mixed solution.

[0082] S3. Dissolve 60 mg of Streptococcus protein G in 5 ml of 50 mM MES buffer (pH 6.0) to obtain a second mixed solution.

[0083] S4. The first mixed solution and the second mixed solution were mixed and reacted at 25°C for 3 hours. After the reaction, the reaction product was washed three times with 50 mM MES buffer (pH 6.0).

[0084] S5. The reaction product after washing in step S4 was blocked with 100 mM Tris-HCl buffer at pH 8.0 at 25°C for 1 hour. After blocking, it was washed three times with 100 mM phosphate buffer at pH 7.2 and resuspended with 5 ml of the same buffer to obtain a first complex solution.

[0085] S6. Dissolve 10 mg of anti-human apolipoprotein B100 monoclonal antibody, 2 mg of human apolipoprotein B48 monoclonal antibody and 2 mg of anti-human apolipoprotein AI monoclonal antibody in 2 ml of 100 mM pH 7.2 phosphate buffer to obtain a second complex solution.

[0086] S7. Take 1 ml of the first complex solution obtained in step S5 and mix it with the second complex solution obtained in step S8 and block the reaction at 25° C. for 45 min. The product obtained by the reaction is washed three times with 100 mM pH 7.2 phosphate buffer and then resuspended with 300 ul of the same buffer.

[0087] S8. Dissolve 0.2 mg of bis(N-hydroxysuccinimide) suberate in 217 ul of dimethyl sulfoxide to obtain a 2.5 mM DSS solution. Then take 100 ul of the 2.5 mM DSS solution, add it to the solution obtained in step S7, and react at 25° C. for 45 min. After the reaction, wash 3 times with 100 mM pH 2.8 glycine buffer elution buffer, then wash 3 times with 100 mM pH 7.2 phosphate buffer and store in 100 mM pH 7.2 phosphate buffer to obtain the product.

[0088] Based on the above embodiments, the present application further proposes a blood lipoprotein adsorption column, which is prepared by using the lipoprotein adsorption material prepared in the above embodiments.

[0089] Specifically, the adsorption column includes a column tube, an adsorption ball sieve plate and a plug. In this embodiment, both ends of the column tube are set to be open, and the column tube is made of polypropylene. The volume can be selected from 1ml, 3ml, 6ml, 12ml and other specifications of column tubes. This preparation example selects a 6ml column tube. There are two sieve plates, which are arranged in sequence along the length direction of the column tube, and the sieve plate close to the bottom of the column tube is the lower sieve plate, and the other sieve plate is the upper sieve plate. The upper sieve plate and the lower sieve plate are arranged at intervals to form an accommodation for the adsorption balls, which are the lipoprotein adsorption materials in the above embodiments.

[0090] Two plugs are provided, which are an upper plug and a lower plug respectively along the direction of the upper sieve plate close to the lower sieve plate. Both plugs are located on the side of the corresponding side sieve plate away from the adsorption ball to block the opening of the column tube.

[0091] The assembly method of the adsorption column in this embodiment is as follows:

[0092] a1. Wash the column tube, sieve plate, etc. with deionized water;

[0093] a2. Install the lower sieve plate into the column tube until it reaches the bottom;

[0094] a3. The adsorption balls were loaded into the column tube. In this embodiment, the loading amount of the lipoprotein adsorption material was 3 ml;

[0095] a4. Install the upper sieve plate into the column tube;

[0096] a5. Install the upper and lower plugs on the column tube.

[0097] Example 2

[0098] S1. Preparation of agarose microspheres. In this embodiment, the agarose microspheres used are commercially available NHS-modified agarose microspheres;

[0099] S2. Take 5 ml of NHS-modified agarose microspheres, filter and wash them with 1 mM hydrochloric acid three times, add them into 100 mM MOPS buffer and shake them at 4°C for 4 hours to obtain a first mixed solution buffer containing 0.1 M NaCl and a pH of 7.2.

[0100] S3. Dissolve 100 mg of Streptococcal protein G in 10 ml of 100 mM MOPS buffer, pH 7.2, containing 0.1 M NaCl to obtain a second mixed solution.

[0101] S3. The solution obtained from the first mixed solution and the second mixed solution in step S3 was mixed and reacted at 25° C. for 3 hours. After the reaction, the reaction product was washed three times with 100 mM, pH 7.2 MOPS buffer containing 0.1 M NaCl.

[0102] S4. The reaction product after washing in step S3 is blocked with 50 mM pH 8.0 ethanolamine buffer at 25° C. for 1 hour. After blocking, it is washed three times with 100 mM pH 7.2 phosphate buffer and resuspended with 5 ml of the same buffer to obtain a first composite solution. The preparation method of phosphate buffer is public in the industry and is known to technicians in the industry.

[0103] S5. Dissolve 10 mg of anti-human apolipoprotein B100 monoclonal antibody, 2 mg of human apolipoprotein B48 monoclonal antibody, and 2 mg of anti-human apolipoprotein AI monoclonal antibody in 2 ml of 100 mM pH 7.2 phosphate buffer to obtain a second complex solution.

[0104] S6. Take 1 ml of the first complex solution obtained in step S4 and mix it with the second complex solution obtained in step S5 and block the reaction for 45 min at 25° C. The product obtained by the reaction is washed three times with 100 mM pH 7.2 phosphate buffer and then resuspended with 300 ul of the same buffer.

[0105] S7. Dissolve 0.2 mg of bis(N-hydroxysuccinimide) suberate in 217 ul of dimethyl sulfoxide to obtain a 2.5 mM DSS solution. Then take 100 ul of 2.5 mM DSS solution, add it to the solution obtained in step S6, and react at 25°C for 45 min. After the reaction, wash 3 times with 100 mM pH 2.8 glycine buffer elution buffer, then wash 3 times with 100 mM pH 7.2 phosphate buffer and store in 100 mM pH 7.2 phosphate buffer to obtain the product.

[0106] Based on the above embodiments, the present application further proposes a blood lipoprotein adsorption column, which is prepared by using the lipoprotein adsorption material prepared in the above embodiments.

[0107] Specifically, the adsorption column includes a column tube, an adsorption ball sieve plate and a plug. In this embodiment, both ends of the column tube are set to be open, and the column tube is made of polypropylene. The volume can be selected from 1ml, 3ml, 6ml, 12ml and other specifications of column tubes. This preparation example selects a 6ml column tube. There are two sieve plates, which are arranged in sequence along the length direction of the column tube, and the sieve plate close to the bottom of the column tube is the lower sieve plate, and the other sieve plate is the upper sieve plate. The upper sieve plate and the lower sieve plate are arranged at intervals to form an accommodation for the adsorption balls, which are the lipoprotein adsorption materials in the above embodiments.

[0108] Two plugs are provided, which are an upper plug and a lower plug respectively along the direction of the upper sieve plate close to the lower sieve plate. Both plugs are located on the side of the corresponding side sieve plate away from the adsorption ball to block the opening of the column tube.

[0109] The assembly method of the adsorption column in this embodiment is as follows:

[0110] a1. Wash the column tube, sieve plate, etc. with deionized water;

[0111] a2. Install the lower sieve plate into the column tube until it reaches the bottom;

[0112] a3. The adsorption balls were loaded into the column tube. In this embodiment, the loading amount of the lipoprotein adsorption material was 3 ml;

[0113] a4. Install the upper sieve plate into the column tube;

[0114] a5. Install the upper and lower plugs on the column tube.

[0115] Application Examples

[0116] Application Example 1

[0117] s1. Take 3.6 ml of serum and add 0.4 ml of 1 M phosphate buffer with a pH of 7.4 and mix well;

[0118] s2. The adsorption column prepared in Example 1 was washed three times with 100 mM pH 7.4 phosphate buffer;

[0119] s3. The diluted serum obtained in s1 was slowly added to the lipoprotein adsorption column in step s2, and the adsorption column was washed with 4 ml of 100 mM phosphate buffer at pH 7.4, and the flow-through was collected;

[0120] s4. The flow-through collected in s3 was diluted to 8 ml with 100 mM pH 7.4 phosphate buffer; ultracentrifuged at 100,000 × g, 4 ° C for 70 min, the supernatant was removed, and the precipitate was resuspended with 8 ml of 100 mM pH 7.4 phosphate buffer to obtain a purified product, and the exosome content, lipoprotein content and other indicators of the purified product were detected. The purification time of this method is 40 min, and the overall operation time is 60 min.

[0121] Application Example 2

[0122] s1. Take 3.6 ml of serum and add 0.4 ml of 1 M phosphate buffer pH 7.4 and mix well;

[0123] s2. The lipoprotein adsorption column prepared in Example 1 was washed three times with 100 mM pH 7.4 phosphate buffer;

[0124] s3. Slowly add the diluted serum obtained in s1 to the lipoprotein adsorption column in step s2, wash the adsorption column with 4 ml of 100 mM pH 7.4 phosphate buffer, and collect the flow-through.

[0125] s4. The flow-through collected in s3 was loaded onto an exosome-specific gel exclusion chromatography column (iZONqEVcolumn), and the qEV column was washed with 100 mM pH 7.4 phosphate buffer in advance. All subsequent steps were carried out according to the product instructions.

[0126] s5. The adsorption column obtained in s4 was eluted with 100 mM pH 7.4 phosphate buffer, with 0.5 ml per tube, and the eluate was collected. The exosome content, lipoprotein content and other indicators in the eluate were detected. The purification time of this method was 26 min, and the overall operation time was 50 min.

[0127] Application Example 3

[0128] s1. Take 4 ml of serum and add 0.4 ml of 1 M phosphate buffer pH 7.4 and mix well;

[0129] s2. The lipoprotein adsorption column prepared in Example 1 was washed three times with 100 mM pH 7.4 phosphate buffer;

[0130] s3. Slowly add the diluted serum obtained in s1 to the lipoprotein adsorption column in step s2, wash the adsorption column with 4 ml of 100 mM pH 7.4 phosphate buffer, and collect the flow-through.

[0131] s4. The flow-through collected in s3 was loaded onto an exosome-specific gel exclusion chromatography column (iZONqEVcolumn), and the qEV column was washed with 100 mM pH 7.4 phosphate buffer in advance. All subsequent steps were carried out according to the product instructions.

[0132] s5. The adsorption column obtained in s4 was eluted with 100 mM pH 7.4 phosphate buffer, with 0.5 ml per tube, and the eluate was collected. The exosome content, lipoprotein content and other indicators in the eluate were detected. The purification time of this method was 30 min, and the overall operation time was 50 min.

[0133] Application Example 4

[0134] s1. Take 3.6 ml of serum and add 0.4 ml of 1 M phosphate buffer pH 7.4 and mix well;

[0135] s2. The lipoprotein adsorption column prepared in Example 2 was washed three times with 100 mM pH 7.4 phosphate buffer;

[0136] s3. Slowly add the diluted serum obtained in s1 to the lipoprotein adsorption column in step s2, wash the adsorption column with 4 ml of 100 mM pH 7.4 phosphate buffer, and collect the flow-through.

[0137] s4. The flow-through collected in s3 was loaded onto an exosome-specific gel exclusion chromatography column (iZONqEVcolumn), and the qEV column was washed with 100 mM pH 7.4 phosphate buffer in advance. All subsequent steps were carried out according to the product instructions.

[0138] s5. The adsorption column obtained in s4 was eluted with 100 mM pH 7.4 phosphate buffer, with 0.5 ml per tube, and the eluate was collected. The exosome content, lipoprotein content and other indicators in the eluate were detected. The purification time of this method was 28 min and the overall operation time was 57 min.

[0139] Application comparison

[0140] Application Comparative Example 1

[0141] s1. Take 4 ml of serum sample and dilute to 8 ml with 100 mM pH 7.4 phosphate buffer.

[0142] s2. The serum samples were centrifuged at 100,000 × g for 120 min at 4 ° C using an ultracentrifuge. The supernatant was removed and the precipitate was resuspended in 4 ml of 100 mM pH 7.4 phosphate buffer.

[0143] s3. Load the liquid obtained in s2 onto a gel exclusion chromatography column dedicated for exosomes. The gel exclusion chromatography column was pre-washed with 100 mM pH 7.4 phosphate buffer, and the subsequent steps were performed according to the product instructions of the gel exclusion chromatography column;

[0144] s4. The adsorption column obtained in s3 was eluted with 100 mM pH 7.4 phosphate buffer, and the eluate was collected in one tube of 0.5 ml. The exosome content, lipoprotein content and other indicators in the eluate were detected. The purification time of this method was 150 min and the overall operation time was 300 min.

[0145] Performance testing

[0146] Detection Methods

[0147] For lipoprotein detection, the purified products prepared in the corresponding examples and comparative examples were tested for different lipoprotein proportions using the Apo lipoprotein lipoprotein immunoassay system (R&D Systems DAPB00). For specific operations, refer to the manufacturer's instructions, and the test results are recorded in Table 1. The test results of the lipoprotein lipoprotein detection system for the purified products prepared in Example 1 and Comparative Example 1 are shown in FIG. Figure 5 .

[0148] The exosome purification efficiency is expressed by the number of particles per milliliter to indicate the number of exosomes in the purified product. The number of particles is calculated using the classic nanoparticle tracking analysis method (NTA). In this application, the nanoparticle tracking analysis method uses the Nanosight LM10-HS of Malvern, selects the 488nm laser mode and the NTA3.1 software system, and the operation is in accordance with the manufacturer's instructions. The specific results refer to Figure 1 .

[0149] Western Blot detection mainly analyzes two protein markers in the purified product, namely CD9 protein and ApoA1 protein. CD9 protein is a marker protein on the exosome membrane and is used to detect exosomes. ApoA1 is a marker protein of high-density lipoprotein and is used to detect lipoprotein residues. The operation adopts conventional electrophoresis and transfer operation, which is well known to technicians in the industry. The antibodies used are all from Abcam, including rabbit anti-CD9 polyclonal antibody (ab236630), rabbit anti-APOA1 (ab300085), HRP-labeled goat anti-rabbit polyclonal antibody (ab150077), and the specific results are shown in Figure 2 and Figure 3 .

[0150] For transmission electron microscopy (TEM) detection, 2ul of the purified product was dropped onto a polyvinyl acetate-carbon electron microscope grid and incubated at room temperature for 10min. The grid was then washed with pure water for 3 times and then immersed in a pH 7.0 uranyl acetate buffer for 3min. Excess liquid was blotted with absorbent paper and the grid was air-dried at room temperature. The grid was then placed under an electron microscope (JEM-1400PLUS) for detection. For specific results, see Figure 4 .

[0151] Table 1

[0152] Comparison of the proportions of different lipoprotein families in two exosome purification methods

[0153]

[0154] Combining Table 1 and Figure 5It can be seen that the lipoprotein adsorption material described in the present invention and the blood exosome purification method based on the lipoprotein adsorption material described in the present invention far exceeds the existing ultracentrifugation + gel exclusion chromatography method in lipoprotein removal efficiency.

[0155] The results of the Apo lipoprotein lipoprotein detection system showed that after adsorption by the lipoprotein adsorption material described in the present invention and then combined with gel exclusion chromatography, lipoproteins in the product were basically undetectable. In contrast, ultracentrifugation combined with gel exclusion chromatography purified exosomes in blood. Lipoproteins still remained in the product, of which low-density lipoprotein accounted for the highest proportion, exceeding 80%.

[0156] Combination Figure 1 From the NTA results, it can be seen that the concentrations of exosomes obtained by using Example 1 and Comparative Example 1 are both high-concentration exosomes, and the concentrations of the two are comparable. The difference is that the particle size of the exosomes obtained by using the lipoprotein adsorption column combined with gel exclusion chromatography of the present invention is higher than that of ultracentrifugation + gel exclusion chromatography, and the average particle sizes of the two are specifically 117nm and 85nm.

[0157] Combination Figure 2 and Figure 3 From the results of CD9, it can be seen that the blood exosome purification method (Method 1) using the lipoprotein adsorption material described in the present invention and the ultracentrifugation combined with gel exclusion chromatography method (UC+SEC) can both obtain exosomes with a higher concentration, but the ultracentrifugation combined with gel exclusion chromatography method cannot completely eliminate the lipoprotein contamination, and the imprinted band of apolipoprotein A1 ApoA1 can still be displayed in the Western blot.

[0158] Combination Figure 4 From the electron microscopy results, i.e., the electron microscopy images of the two, it can be seen that the blood exosome purification method (Method 1) of the lipoprotein adsorption material described in the present invention and the ultracentrifugation combined with gel exclusion chromatography method (UC+SEC) for purifying blood exosomes can both obtain corresponding exosomes. In comparison, the average particle size of the exosomes obtained by ultracentrifugation+gel exclusion chromatography is slightly smaller, while the average particle size of the exosomes purified and collected by the method described in the present invention is slightly larger. The difference between the exosomes obtained by the two methods is that the peak values ​​of the two methods are different, but the range of the exosome particles is basically the same.

[0159] According to the operation time and purification time records of Application Comparative Example 1 and Application Example 1, the blood exosome purification method of the lipoprotein adsorption material described in the present invention purifies blood exosomes in a shorter time and with higher efficiency.

[0160] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a material for adsorbing lipoprotein in blood, characterized in that: The method comprises the following steps: S1. preparing a microsphere carrier; S2. activating the microsphere carrier; S3. dissolving Staphylococcus aureus protein A or Streptococcus protein G in a modification buffer to obtain a second mixed solution; S4. mixing the activated microsphere carrier with the second mixed solution and reacting at an appropriate temperature to obtain a reaction product, and washing the reaction product with the modification buffer after the reaction is completed; S5. The reaction product after washing in S4 is blocked with a blocking buffer, and after blocking, it is resuspended with a modification buffer to obtain a first composite solution; S6. The lipoprotein antibody is dissolved in a coupling buffer to obtain a second composite solution; S7. mixing the first composite solution and the second composite solution for reaction, washing the reaction product after the reaction is completed, and then resuspending it with a coupling buffer; S8. Dissolve bis(N-hydroxysuccinimide) suberate in dimethyl sulfoxide, and then add the resultant to the liquid obtained in step S7 for reaction. The product obtained by the reaction is first washed with an elution buffer, then washed with a storage buffer, and then stored in the storage buffer to obtain a product. The lipoprotein antibodies include apolipoprotein AI antibodies, apolipoprotein B100 antibodies, and apolipoprotein B48 antibodies.

2. The method for preparing a material for adsorbing lipoprotein in blood according to claim 1, characterized in that: The microsphere carrier includes agarose gel microspheres, cellulose microspheres, polystyrene microspheres or polyvinyl alcohol microspheres.

3. The method for preparing a material for adsorbing lipoprotein in blood according to claim 1, characterized in that: The particle size of the microsphere carrier is 50 μm-600 μm.

4. The method for preparing a material for adsorbing lipoprotein in blood according to claim 1, characterized in that: The pore size of the microsphere carrier is 50nm-3000nm.

5. The method for preparing a material for adsorbing lipoprotein in blood according to claim 1, characterized in that: The particle size of the microsphere carrier is 100 μm-500 μm, and the pore size of the microsphere carrier is 150 nm-1000 nm.

6. The method for preparing a material for adsorbing lipoprotein in blood according to claim 1, characterized in that: In step S3, the molar ratio of Staphylococcus aureus protein A or Streptococcus protein G to the microsphere carrier is 1:1-10:1, the modification buffer is 10-100mM MES buffer, and its pH is 5.5-7.5; In step S4, the reaction temperature is 20°C-30°C, the reaction time is 2-4h, the modification buffer is 10-100mM MES buffer, and its pH is 5.5-7.5; In step S5, the blocking buffer is 20-120mM Tris-HCl buffer, and its pH is 7.5-8.5, and the modification buffer is 10-100mM MES buffer, and its pH is 5.5-7.5; In step S6, the coupling buffer is a phosphate buffer with a concentration of 50mM-100mM, a pH of 6.5-8.5, and the antibody dosage is: 50-1000ug antibody is coupled to every 100ug of the microsphere carrier obtained in step S7; In step S7, the reaction temperature is 20°C-30°C, and the reaction time is 30-60min; in step S8, the concentration of suberic acid bis(N-hydroxysuccinimide ester) in dimethyl sulfoxide is 1mM-5mM, and the final concentration of suberic acid bis(N-hydroxysuccinimide ester) after mixing suberic acid bis(N-hydroxysuccinimide ester), dimethyl sulfoxide and the product obtained in S7 is 100-500uM, the reaction temperature is 20°C-30°C, and the reaction time is 30-60min; the elution buffer is 50-150mM glycine buffer, pH 2.0-3.0, and the storage buffer is 50mM-60mM phosphate buffer, pH 7.0-7.

5.

7. An application of a lipoprotein adsorption material, characterized in that: The adsorption material prepared by the preparation method according to any one of claims 1 to 6 is used to adsorb different types of lipoproteins in the blood, wherein the lipoproteins include high-density lipoproteins, low-density lipoproteins and chylomicrons.

8. An application of a lipoprotein adsorption material, characterized in that: The adsorption material prepared by the preparation method according to any one of claims 1 to 6 is used in combination with gel exclusion chromatography or ultracentrifugation to purify extracellular vesicles in blood.

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