CN108855003B - Immunoadsorbent for removing inflammatory factors in blood and preparation method thereof - Google Patents
Immunoadsorbent for removing inflammatory factors in blood and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an immunoadsorbent for removing inflammatory factors in blood and a preparation method thereof, wherein the immunoadsorbent is characterized in that an affinity ligand of a small molecular polypeptide with 5-18 amino acids is designed by a computer-aided drug design method according to various intermolecular forces among antigenic epitopes of the inflammatory factors such as IL-6, TNF-alpha, IL-1 beta and IL-8 and corresponding receptors thereof, specifically, vinyl acetate-triallyl isocyanurate is used as a basic skeleton, a mixture of ethyl acetate and alkane is used as a pore-forming agent, synthetic resin using benzoyl peroxide as an initiator is used as a carrier, and the small molecular polypeptide is grafted after alcoholysis activation to obtain the small molecular ligand immunoadsorbent without adsorbing macromolecular proteins and the like. The immunoadsorbent is simple to prepare, has high adsorption capacity, high selectivity, good biological and blood compatibility and relatively low cost, and provides a new treatment method for removing excessive pathogenic inflammatory factors in a patient body.
Description
The technical field is as follows:
the invention belongs to the technical field of biological medicines. In particular to a novel medical immunoadsorbent, in particular to an immunoadsorbent which is suitable for removing inflammatory factors in vivo through blood perfusion and a preparation method thereof.
Technical background:
inflammation is one of the most basic pathological changes, and during the development of inflammation, inflammatory cells (such as neutrophils, macrophages, endothelial cells and the like) of a body can be stimulated to release various cytokines, so that a systemic inflammatory response is caused, and sepsis and septic shock are even caused. More than 1800 million people worldwide suffer from sepsis, up to millions of patients suffer from sepsis per year in our country due to antibiotic abuse, air quality deterioration, water pollution, etc., and data show that the fatality rate of sepsis has exceeded myocardial infarction, becoming a leading cause of death in non-cardiac patients in intensive care units.
The plasma perfusion technology can effectively remove a large amount of toxins and cytokines (IL-6, TNF-alpha, IL-1 beta, IL-8 and the like) accumulated in blood, and is a new way for treating inflammation. The blood perfusion is a treatment technology for removing endogenous or exogenous toxicants or pathogenic substances in the blood of a patient by means of extracorporeal circulation of the blood and an adsorbent with a special adsorption function in a blood perfusion device so as to achieve blood purification. The blood perfusion technology has weak anaphylactic reaction, wide adaptability and low treatment cost, and becomes a blood purification technology with great development potential. The core of the blood perfusion technology is the use of an adsorbing material, and the adsorbing material not only meets the use requirements of human body on safety, no toxicity, no anaphylactic reaction, no heat source, stable chemical property, no breakage, no easy shedding, good blood compatibility and the like, but also has the development of the adsorbing material to determine the application and popularization of the blood perfusion device. Therefore, the immunoadsorbent capable of effectively removing the pathogenic toxin and the inflammatory factor in the blood is developed, has relatively low cost, can improve the life quality of patients and reduce the economic burden, and can bring huge economic effect and social value.
At present, the adsorbents which are successfully applied to clinical removal of inflammatory factors abroad comprise a polymyxin B adsorbent in Japan and a Cytosorb adsorbent in America, but the polymyxin B adsorbent is high in product price and has the problems of ligand shedding and toxicity, the Cytosorb adsorbent has a good removal effect on medium-molecular cytokines (such as IL-6, IL-1 beta, IL-8 and the like), but has a low removal effect on TNF-alpha with a large molecular weight, and both the adsorbents do not enter the market of China. In China, related patents such as 201310593832.4 refer to a nano-composite microsphere adsorbent prepared by using cellulose, agarose or polyvinyl alcohol spheres as carriers and hydrophobic alkylaniline as ligands, and a patent 201610880603.4 refers to a nano-composite microsphere adsorbent prepared by using nano calcium carbonate-styrene-divinylbenzene as a basic skeleton. Although the two adsorbents have good in-vitro adsorption effect, the toxin is cleared mainly by hydrophobic effect, the defect of low selectivity exists, and substances beneficial to the body can be cleared away while the toxin is cleared away.
In view of the above, there is a need to develop an immunoadsorbent with specific selectivity, high adsorption performance and low cost for blood perfusion.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a novel immunoadsorbent which has good blood, biocompatibility and higher adsorption performance, can effectively avoid nonspecific adsorption due to high selectivity of a ligand, has relatively low cost, can be used for removing excessive pathogenic inflammatory factors in a patient body through hemoperfusion, and a preparation method thereof.
The immunoadsorbent for removing inflammatory factors in blood provided by the invention is an immunoadsorbent consisting of a hydrophilic carrier and a ligand, and is prepared by taking polyvinyl alcohol microspheres as the carrier, small molecular polypeptides as affinity ligands and epichlorohydrin as a cross-linking agent, and covalently coupling the ligands and the carrier. In the immunoadsorbent, the epoxy content of the carrier is 80-120 mu moL/mL, preferably 90-110 mu moL/mL; the immobilization amount of the small molecular polypeptide is 50-90 mu moL/mL, preferably 60-80 mu moL/mL.
The hydrophilic polyvinyl alcohol carrier is spherical, and the particle size is between 100 and 500 mu m, preferably 200 and 400 mu m; the specific surface area is 20-100m2Between/g, preferably 30-80m2(ii)/g; the pore volume is 1.05-6.8cm3Between/g, preferably 1.89-4.85cm3/g。
The composition of the small molecule polypeptide comprises at least one of phenylalanine (F) and isoleucine (I) which provide hydrophobic interaction; including at least one of histidine (H), tyrosine (Y), serine (S) and threonine (T) that provide hydrogen bonding; comprising providing at least one of aspartic acid (N) and glutamic acid (E) for electrostatic interaction, and simultaneously connecting different interaction sites with alanine (A) of different lengths, wherein the affinity ligand of the small molecule polypeptide to be screened comprises: FAHAN, IAHAN, FIAHYANN, FFAAHHAE, IIAAHHAE, FFAAYYAAEE, IIAAYYAAEE, FAIAHAYASATANAE or FIAAHHAAYYAANNAAEE, etc., preferably FAHAN, FFAAYYAAEE or FIAAHHAAYYAANNAAEE.
The invention provides a preparation method of an immunoadsorbent for removing inflammatory factors in blood, which comprises the following steps:
uniformly mixing vinyl acetate monomer and triallyl isocyanurate serving as a crosslinking agent according to the weight ratio of 1.5-5.5:1, and adding a pore-foaming agent which is 1.2-5 times of the vinyl acetate monomer and the triallyl isocyanurate serving as the crosslinking agent according to the volume ratio, wherein the mass ratio of the pore-foaming agent to the mass ratio of (0.2-1): 1: 0.6-1 of ethyl acetate, n-heptane and n-hexane, adding 0.3-1% of initiator benzoyl peroxide by mass of the total mass of the reaction monomer, the cross-linking agent and the pore-forming agent, and stirring to uniformly mix the two to obtain an oil phase; then adding the oil phase system solution into a water phase of polyvinyl alcohol with the mass percent of 0.5-3%, wherein the water phase contains 1-10% of NaCl; wherein the mass ratio of the oil phase to the water phase is 1: 1.5-3, adjusting the stirring rate to disperse the system into uniform small oil droplets, heating to 50-60 ℃, reacting for 3-6h, then continuously heating to 70-80 ℃, reacting for 3-6h, filtering, performing post-treatment on the reaction system through filtering and hot water washing, then extracting for 12-36h by using methanol, removing a pore-forming agent, performing vacuum drying for 24-48h to obtain white copolymer microspheres, adding the white copolymer microspheres into a NaOH/methanol solution with the mass fraction of 1-5%, performing ester exchange for 24-72h at 40-60 ℃, filtering, extracting for 12-36h by using methanol, and naturally airing to obtain polyvinyl alcohol carrier microspheres;
epoxidizing the polyvinyl alcohol carrier microsphere obtained in the previous step by using a known reagent and a known method, washing the epoxidized polyvinyl alcohol carrier microsphere to be neutral by using ethanol and deionized water, and storing the epoxidized polyvinyl alcohol carrier microsphere at 4 ℃ for later use to obtain the epoxidized polyvinyl alcohol carrier microsphere, wherein the cyclic oxygen content of the carrier microsphere is 80-120 mu moL/mL, and preferably 90-110 mu moL/mL;
dissolving small molecular polypeptide in Na (0.1M, pH 9.8)2CO3-NaHCO3And (3) adding the epoxidized polyvinyl alcohol carrier microspheres obtained in the step (2) into a buffer solution, wherein the mass of the micromolecular polypeptide is as follows: volume of buffer: the mass of the polyvinyl alcohol carrier microspheres is 0.1-0.8 g: 1.5 mL: 0.1 g; oscillating and reacting for 12-24h at 37-50 ℃, filtering, and leaching with deionized water to be neutral. Then added to a 60.6mg/mL Tris solution, wherein the volume of the Tris solution is: the mass of the polyvinyl alcohol carrier microspheres is 2.2-3.5 mL: 1g, carrying out oscillation reaction at 37-50 ℃ for 5-8h, carrying out suction filtration, rinsing to neutrality by using deionized water, and storing at 4 ℃ for later use, thereby obtaining the immunoadsorbent for removing inflammatory factors for hemoperfusion, wherein the immobilization amount of the small molecular polypeptide is 50-90 mu moL/mL, preferably 60-80 mu moL/mL.
The epoxidation of the polyvinyl alcohol microspheres is realized by activating and coupling by a method known in literature and reaction conditions, and then ligand is immobilized after the epoxy activation.
The immunoadsorbent provided by the invention can be used for removing excessive pathogenic inflammatory factors in a patient body through plasma or whole blood perfusion; the inflammatory factors include IL-6, TNF-alpha, IL-1 beta and IL-8.
Advantages and advantageous effects of the invention
The invention designs the affinity ligand of the small molecular polypeptide with selective 5-18 amino acids according to the antigen epitopes of cytokines such as IL-6, TNF-alpha, IL-1 beta and IL-8 and various intermolecular forces between the receptors corresponding to the antigen epitopes, and by a computer-aided drug design method, and prepares the small molecular polypeptide ligand immunoadsorbent for effectively and specifically removing the inflammatory factors in the blood. The immunoadsorbent has good blood and biocompatibility, high adsorption performance, low cost and capacity of eliminating excessive pathogenic inflammation factor in patient body via blood perfusion, and can avoid nonspecific adsorption (such as macromolecular protein) effectively owing to the high selectivity of the ligand.
Drawings
FIG. 1 adsorption assay of adsorbents for static IL-6, TNF- α, IL-1 β, IL-8 and total protein in patient sera.
Detailed Description
Example 1
(1) Preparation of polystyrene microspheres
Adding 53g of vinyl acetate monomer and 10g of triallyl isocyanurate serving as a crosslinking agent into a 250mL beaker, uniformly mixing, adding 35.2g of ethyl acetate, 35.9g of n-heptane, 23.4g of n-hexane and 0.6g of benzoyl peroxide, stirring to dissolve, and fully and uniformly mixing after complete dissolution to obtain an oil phase. Adding the system solution into 252mL of 2.8% polyvinyl alcohol solution (containing 1.2% of NaCl), adjusting the stirring rate to disperse the system into uniform small oil beads, heating to 52 ℃, reacting for 5.5h, then continuously heating to 79 ℃ to react for 3.5h, filtering, washing the reaction system with hot water, processing, extracting with methanol for 16h, removing pore-forming agent, vacuum drying for 36h to obtain white copolymer microspheres, adding the white copolymer microspheres into 1.2% NaOH/methanol solution, performing ester exchange for 72h at 40 ℃, filtering, extracting with methanol for 12h, and naturally drying to obtain the alcoholyzed polyvinyl alcohol microsphere carrier. The particle size is between 200 and 400 mu m, the specific surface area is 58m2Per g, pore volume 4.26cm3/g。
(2) Epoxidation of carrier microspheres
And (2) taking 20g (dry weight) of the polyvinyl alcohol carrier microspheres prepared in the step (1), adding the polyvinyl alcohol carrier microspheres into a three-neck flask provided with a stirrer and a thermometer, adding 200mL of dimethyl sulfoxide, 160mL of epoxy chloropropane and 10mL of 3moL/L NaOH solution into a reactor, carrying out oscillation reaction at 40 ℃ for 5 hours, carrying out suction filtration, washing with ethanol and deionized water to be neutral, storing at 4 ℃ for later use, and controlling the cyclic oxygen content of the carrier microspheres to be 106.8 mu moL/mL.
(3) Epoxidized carrier microsphere coupling ligands
Adding 10g of the epoxidized polyvinyl alcohol carrier microspheres obtained in the step (2) into 150mL of LFAHAN solution (10g of FAHAN dissolved in 150mL of 0.1M Na with pH of 9.8)2CO3-NaHCO3Buffer solution), oscillating and reacting for 24 hours at 37 ℃, filtering, and rinsing with deionized water to be neutral. Then adding into 33mL of 60.6mg/mL Tris solution, oscillating and reacting at 50 ℃ for 5h, filtering, rinsing with deionized water to neutrality, and cooling to 4 DEG CAnd storing for later use to obtain the immunoadsorbent for removing inflammatory cytokines for blood perfusion, wherein the polypeptide immobilization amount is 75.4 mu moL/mL and is numbered as PVA-1.
Example 2
(1) Preparation of polystyrene microspheres
62.6g of vinyl acetate monomer and 17.4g of triallyl isocyanurate as a crosslinking agent are added into a 500mL beaker and uniformly mixed, 84.5g of ethyl acetate, 153.6g of n-heptane, 145.9g of n-hexane and 4.64g of benzoyl peroxide are added into the mixture, the mixture is stirred to be dissolved, and after the mixture is completely dissolved, the mixture is fully mixed to obtain an oil phase. Adding the system solution into 1299.2mL of 0.65% polyvinyl alcohol solution (containing 9.6% of NaCl), adjusting the stirring rate to disperse the system into uniform small oil droplets, heating to 59 ℃, reacting for 3.5h, then continuously heating to 72 ℃, reacting for 5h, filtering, washing the reaction system with hot water, processing, extracting with methanol for 28h, removing pore-forming agent, vacuum drying for 24h to obtain white copolymer microspheres, adding into 4.9% NaOH/methanol solution, performing ester exchange for 24h at 60 ℃, filtering, extracting with methanol for 36h, and naturally airing to obtain the alcoholyzed polyvinyl alcohol microsphere carrier. The particle size is between 200 and 400 mu m, and the specific surface area is 72.3m2Per g, pore volume 4.52cm3/g。
(2) Epoxidation of carrier microspheres
And (2) taking 20g (dry weight) of the polyvinyl alcohol carrier microspheres prepared in the step (1), adding the polyvinyl alcohol carrier microspheres into a three-neck flask provided with a stirrer and a thermometer, adding 200mL of dimethyl sulfoxide, 160mL of epoxy chloropropane and 10mL of 3moL/L NaOH solution into a reactor, carrying out oscillation reaction at 40 ℃ for 5 hours, carrying out suction filtration, washing the mixture to be neutral by using ethanol and deionized water, and storing the mixture at 4 ℃ for later use to obtain the epoxidized polyvinyl alcohol carrier microspheres, wherein the epoxy oxygen content is 102.4 mu moL/mL.
(3) Epoxidized microsphere coupling ligands
Adding 5g of the epoxidized polyvinyl alcohol carrier microspheres obtained in the step (2) into 75mL of LFFAAYYAAEE solution (26g of FFAAYYAAEE dissolved in 75mL of 0.1M Na with pH of 9.8)2CO3-NaHCO3Buffer solution), oscillating and reacting for 16h at 45 ℃, filtering, and leaching with deionized water until the temperature is reachedAnd (4) the product is neutral. And then adding the mixture into 12.5mL of 60.6mg/mL Tris solution, carrying out oscillation reaction for 8 hours at 37 ℃, carrying out suction filtration, eluting the mixture to be neutral by using deionized water, and storing the mixture for later use at 4 ℃ to obtain the immunoadsorbent of the cell factor for blood perfusion, wherein the polypeptide immobilization amount is 73.9 mu moL/mL and the number is PVA-2.
Example 3
(1) Preparation of polystyrene microspheres
After 49.2g of vinyl acetate monomer and 30.8g of triallyl isocyanurate as a crosslinking agent are added into a 500mL beaker and uniformly mixed, 29.6g of ethyl acetate, 118.2g of n-heptane, 92.2g of n-hexane and 2.08g of benzoyl peroxide are added into the beaker, stirred to be dissolved, and fully mixed to obtain an oil phase after the ethyl acetate and the n-heptane are completely dissolved. Adding the system solution into 740.8mL of 1.7% polyvinyl alcohol solution (containing 5.2% of NaCl), adjusting the stirring rate to disperse the system into uniform small oil droplets, heating to 55 ℃, reacting for 4.5h, then continuously heating to 76 ℃, reacting for 4.5h, filtering, washing the reaction system with hot water, treating, extracting with methanol for 36h, removing pore-forming agent, vacuum drying for 12h to obtain white copolymer microspheres, adding the white copolymer microspheres into 2.6% NaOH/methanol solution, performing ester exchange for 48h at 50 ℃, filtering, extracting with methanol for 24h, and naturally drying to obtain the alcoholyzed polyvinyl alcohol carrier microspheres. The particle size is between 200 and 400 mu m, and the specific surface area is 68.2m2Per g, pore volume 4.15cm3/g。
(2) Epoxidation of carrier microspheres
And (2) taking 20g (dry weight) of the polyvinyl alcohol carrier microspheres prepared in the step (1), adding the polyvinyl alcohol carrier microspheres into a three-neck flask provided with a stirrer and a thermometer, adding 200mL of dimethyl sulfoxide, 160mL of epoxy chloropropane and 10mL of 3moL/L NaOH solution into a reactor, carrying out oscillation reaction at 40 ℃ for 5 hours, carrying out suction filtration, washing the mixture to be neutral by using ethanol and deionized water, and storing the mixture at 4 ℃ for later use to obtain the epoxidized polyvinyl alcohol carrier microspheres, wherein the epoxy oxygen content is 95.2 mu moL/mL.
(3) Epoxidized microsphere coupling ligands
Adding 8g of the epoxidized polyvinyl alcohol carrier microspheres obtained in step (2) into 120mL of FIAAHHAAYYAANNAAEE solution (60g of FIAAHHAAYYAANNAAEE dissolved in 120mL of 0.1M, pH 9.8 Na2CO3-NaHCO3Buffer solution), oscillating and reacting for 12h at 50 ℃, filtering, and leaching with deionized water to be neutral. And then adding the mixture into 22.4mL of 60.6mg/mL Tris solution, carrying out oscillation reaction for 6.5h at 45 ℃, carrying out suction filtration, rinsing the mixture to be neutral by using deionized water, and storing the mixture for later use at 4 ℃ to obtain the immunoadsorbent of the cell factor for blood perfusion, wherein the polypeptide immobilization amount is 68.4 mu moL/mL, and the number is PVA-3.
Example 4
Static adsorption test of adsorbent for adsorbing mixed cytokines in serum of sepsis patient
0.3mL of each of the adsorbents PVA-1, PVA-2 and PVA-3 of examples 1, 2 and 3 was used as an experimental group and Cytosorb (commercially available from Cytosorbens, USA) resin as a positive control group, 3mL of a serum sample of a sepsis patient (IL-6 concentration of about 1258.34pg/L, TNF- α concentration of about 514.98pg/L, IL-1 β concentration of about 1092.41pg/L and IL-8 concentration of about 3149.59pg/L in the sample) was added to a centrifuge tube, the centrifuge tube was sealed with a sealing film, and the sample was adsorbed in an air shaker at 37 ℃ for 2 hours with shaking. After the adsorption, the solution was centrifuged at 1000rpm/min for 1min, and the supernatant was collected to determine the concentration.
The adsorption results are shown in figure 1.
Example 5
Determination of total protein in serum of sepsis patient by adsorbent
0.3mL of each of the adsorbents PVA-1, PVA-2 and PVA-3 of examples 1, 2 and 3 was taken as an experimental group and a positive control group, and 3mL of a serum sample (total protein concentration in the sample was about 62.74mg/L) from a sepsis patient was added to each of the centrifuge tubes, and the mixture was sealed with a sealing film and adsorbed by shaking in an air shaker at 37 ℃ for 2 hours. After the adsorption, the solution was centrifuged at 1000rpm/min for 1min, and the supernatant was collected to determine the concentration.
The adsorption results are shown in figure 1.
Example 6
Adhesion Performance test of the adsorbent to platelets
Weighing 0.1g of adsorbent in the embodiments 1, 2 and 3, adding 1mL of platelet-rich rabbit blood plasma after fully washing with sodium chloride injection, taking 20uL of the platelet-rich rabbit blood plasma after water bath at 37 ℃ for 1h, and measuring the change of platelet indexes in a blood cell analyzer respectively, wherein the results are shown in the following table 1:
table 1: platelet adhesion Performance test of adsorbents
From the results in the table, it can be seen that: the adsorbents obtained by the invention have the advantages of adsorbing the platelets less than 5 percent, having good blood compatibility, meeting the national biomedical material standard and being capable of being used for removing inflammatory factors by blood perfusion.
Claims (6)
1. An immunoadsorbent for removing inflammatory factors in blood is characterized in that the adsorbent is an immunoadsorbent consisting of a hydrophilic carrier and a ligand, polyvinyl alcohol microspheres are used as a carrier, micromolecular polypeptide is used as an affinity ligand, epichlorohydrin is used as a cross-linking agent, the ligand and the carrier are coupled through covalent bonding, the cyclic oxygen content of the carrier in the immunoadsorbent is 80-120 mu moL/mL, and the immobilization amount of the micromolecular polypeptide is 50-90 mu moL/mL; the composition of the small molecule polypeptide comprises at least one of phenylalanine F and isoleucine I which provide hydrophobic interaction; including at least one of histidine H, tyrosine Y, serine S, and threonine T, which provide hydrogen bonding; comprises providing at least one of aspartic acid N and glutamic acid E for electrostatic interaction, and simultaneously connecting different interaction sites by alanine A with different lengths;
the preparation method of the immunoadsorbent comprises the following steps:
step 1, synthesizing polyvinyl alcohol carrier microspheres;
uniformly mixing vinyl acetate monomer and triallyl isocyanurate serving as a crosslinking agent according to the weight ratio of 1.5-5.5:1, and adding a pore-foaming agent which is 1.2-5 times of the vinyl acetate monomer and the triallyl isocyanurate serving as the crosslinking agent according to the volume ratio, wherein the mass ratio of the pore-foaming agent to the mass ratio of (0.2-1): 1: 0.6-1 of ethyl acetate, n-heptane and n-hexane, adding 0.3-1% of initiator benzoyl peroxide by mass of the total mass of the reaction monomer, the cross-linking agent and the pore-forming agent, and stirring to uniformly mix the two to obtain an oil phase; then adding the oil phase system solution into a water phase of polyvinyl alcohol with the mass percent of 0.5-3%, wherein the water phase contains 1-10% of NaCl; wherein the mass ratio of the oil phase to the water phase is 1: 1.5-3, adjusting the stirring rate to disperse the system into uniform small oil droplets, heating to 50-60 ℃, reacting for 3-6h, then continuously heating to 70-80 ℃, reacting for 3-6h, filtering, performing post-treatment on the reaction system through filtering and hot water washing, then extracting for 12-36h by using methanol, removing a pore-forming agent, performing vacuum drying for 24-48h to obtain white copolymer microspheres, adding the white copolymer microspheres into a NaOH/methanol solution with the mass fraction of 1-5%, performing ester exchange for 24-72h at 40-60 ℃, filtering, extracting for 12-36h by using methanol, and naturally airing to obtain polyvinyl alcohol carrier microspheres;
step 2, epoxidation of the carrier microspheres;
epoxidizing the polyvinyl alcohol carrier microsphere obtained in the previous step by using a known reagent and a known method, washing the epoxidized polyvinyl alcohol carrier microsphere to be neutral by using ethanol and deionized water, and storing the epoxidized polyvinyl alcohol carrier microsphere at 4 ℃ for later use to obtain the epoxidized polyvinyl alcohol carrier microsphere, wherein the cyclic oxygen content of the carrier microsphere is 80-120 mu moL/mL;
step 3, coupling the epoxy microspheres with ligands;
dissolving small molecular polypeptide in Na (0.1M, pH 9.8)2CO3-NaHCO3And (3) adding the epoxidized polyvinyl alcohol carrier microspheres obtained in the step (2) into a buffer solution, wherein the mass of the micromolecular polypeptide is as follows: volume of buffer: the mass of the polyvinyl alcohol carrier microspheres is 0.1-0.8 g: 1.5 mL: 0.1 g; oscillating and reacting for 12-24h at 37-50 ℃, filtering, and leaching with deionized water to be neutral; then added to a 60.6mg/mL Tris solution, wherein the volume of the Tris solution is: the mass of the polyvinyl alcohol carrier microspheres is 2.2-3.5 mL: 1g, carrying out oscillation reaction at 37-50 ℃ for 5-8h, carrying out suction filtration, rinsing to neutrality by using deionized water, and storing at 4 ℃ for later use, thereby obtaining the immunoadsorbent for removing inflammatory factors for hemoperfusion, wherein the immobilization amount of the small molecular polypeptide is 50-90 mu moL/mL.
2. The immunoadsorbent of claim 1, wherein said carrier is polyvinyl alcohol microparticlesSpheres with a particle size of between 100 and 500 μm; the specific surface area is 20-100m2Between/g; the pore volume is 1.05-6.8cm3Between/g.
3. The immunoadsorbent of claim 2, wherein said support has a particle size of 200-400 μm; the specific surface area is 30-80m2(ii)/g; the pore volume is 1.89-4.85cm3/g。
4. The immunoadsorbent of claim 1, wherein the affinity ligands for the small molecule polypeptides being screened comprise: FAHAN, IAHAN, FIAHYANN, FFAAHHAE, IIAAHHAE, FFAAYYAAEE, IIAAYYAAEE, FAIAHAYASATANAE or FIAAHHAAYYAANNAAEE.
5. The immunoadsorbent of claim 4, wherein said affinity ligand for said small molecule polypeptide is: FAHAN, FFAAYYAAEE or FIAAHHAAYYAANNAAEE.
6. Use of the immunoadsorbent of any one of claims 1 to 5 for the elimination of inflammatory factors from the blood, said inflammatory factors including IL-6, TNF- α, IL-1 β and IL-8, with good elimination.
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