CN104525150A - IgG1 adsorbent, and preparation method and application thereof - Google Patents

Abstract

The adsorbent is cross-linked by ethylenediaminetetraacetic anhydride, 1,4-diphenylenediaminetetraacetic anhydride or 1,4-dibenzyldiaminetetraacetic anhydride, activated agarose or cellulose, and then mixed with tyrosine or Prepared by reaction of tryptophan solution. The carrier of the present invention has good blood compatibility and mechanical properties, and the cross-linking activation step used is simple and safe to prepare. The cross-linking activation reagent has dual purposes of spacer arms and adsorption functional groups; The interaction and hydrophobic interaction realize the relatively specific adsorption of immunoglobulin IgG1, while the adsorption rate of IgM and IgA is low, the adsorption rate IgG1/IgM(IgA)=2-4, this adsorbent can be used for the adsorption of IgG1 in blood and Clearance of Population Reactive Antibodies (PRA) Clinically Associated with IgG1 in the Field of Organ Transplantation.

Description

A kind of IgG1 adsorbent and its preparation method and application

technical field

The invention relates to a medical field, in particular to an IgG1 adsorbent that can be used for hemoperfusion and its preparation method and application.

Background technique

The existence of population reactive antibody (PRA) in kidney transplant recipients and the degree of sensitization of the antibody are closely related to the occurrence of acute rejection after transplantation, and also have a significant impact on the survival rate of transplanted organs. Patients with high immunosensitivity (PRA>80%), that is, patients with higher PRA levels, have poorer kidney transplantation outcomes, and are prone to hyperacute, accelerated, and acute rejection reactions, which directly lead to graft failure or shorten renal survival time after transplantation. Studies have found that only IgG anti-HLA antibodies are the antibodies that really affect the survival of organ transplants.

Currently, preconditioning patients with high PRA levels for kidney transplantation is a global challenge. Commonly used clinical methods and their limitations are as follows: (1) Good HLA matching can prevent or reduce the incidence of acute rejection, but waiting for a suitable matching will delay the operation time. (2) Intravenous injection of low-dose immune globulin (1vIG) or combined plasma exchange (PP) for desensitization therapy is also often used, but the incidence of postoperative acute rejection reaction (AHR) is high. (3) Plasma exchange can remove part of the PRA antibody, but the loss of beneficial substances, especially this method itself is a way that is likely to cause sensitization in the human body, and its use is limited. (4) Use of new immunosuppressants such as Neoral, MMF, FK506, etc., whose anti-rejection function is recognized, but in addition to expensive drugs and long-term use, the toxic and side effects on the human body cannot be ignored.

Immunoadsorption (IA) is currently the most clinically applied and effective method, and the most representative one is the first application in 1986 of anti-transplant rejection protein A immunosorbent. The therapeutic effectiveness of protein A adsorbents on various immune diseases has been proven and approved by the US FDA. However, protein A adsorption materials also have some defects, which limit its promotion. First, the high price of protein A has brought a heavy economic burden to ordinary patients; second, protein A is easily denatured as a protein ligand, which brings inconvenience to the production, transportation, storage and use of adsorption materials; third, protein A A is a biologically derived macromolecule, which can easily bring risks to patients if it falls off.

In addition, the carrier activation method used by protein A adsorption column and other immunosorbents generally adopts the epichlorohydrin method, and the arm molecule has only four atoms, and the chain link is short, so it is difficult to match the demand for the space position of the adsorption of IgG macromolecules . If you want to extend the arm molecule, the most common method is to first connect the hydroxyl group of the polysaccharide carrier to the diamine spacer arm with epichlorohydrin, and then couple with the ligand after aldehydelation (such as glutaraldehyde) to form a Greek molecule. The Schiff base, and finally reduce the Schiff base double bond to obtain the adsorption material. In the preparation method, the activation steps are cumbersome, and after multi-step reactions, the active functional groups on the carrier for immobilizing ligands are continuously reduced. Therefore, the preparation cost of the immunosorbent is increased, and its adsorption performance is affected.

Therefore, how to overcome these defects of protein A adsorption materials, design and construct new immunoadsorbents with considerable IgG adsorption capacity, stable physicochemical properties, and relatively low cost are of great importance. Research value and practical significance.

Contents of the invention

The first purpose of the present invention is to overcome the defects of the adsorption material and provide a safe, effective, low-cost, stable and reliable IgG1 adsorbent. The second object of the present invention is to provide a method for preparing the above-mentioned IgG1 adsorbent. The third object of the present invention is to provide the application of the above-mentioned IgG1 adsorbent, the application of the IgG1 adsorption in the blood and the clinical removal of IgG1-related population reaction antibodies (PRA) in the field of organ transplantation.

The first aspect of the present invention is to provide an IgG1 adsorbent, which uses agarose microspheres or cellulose microspheres cross-linked by acid anhydrides as a carrier, and the acid anhydrides are ethylenediaminetetraacetic anhydride, 1,4-diphenyl Diaminetetraacetic anhydride or 1,4-dibenzyldiaminetetraacetic anhydride, the ligand is tyrosine or tryptophan;

The chemical structural formula of the agarose microspheres or cellulose microspheres after the acid anhydride crosslinking is as follows:

The chemical structural formula of the IgG1 adsorbent is as follows:

in,

W is agarose microsphere or cellulose microsphere,

X is the agarose microspheres or cellulose microspheres cross-linked by acid anhydride,

Y is ethylene, p-phenylene or p-xylylene,

R is p-hydroxyphenyl or β-indolyl.

Preferably, the effective amount of acid anhydride to activate the support is 110-180 μmol/ml.

Preferably, the amount of ligand immobilized on the carrier is 70-130 μmol/ml.

The second aspect of the present invention provides the preparation method of the IgG1 adsorbent described in the first aspect of the present invention, comprising the following steps:

(a) Cross-linking of agarose microspheres or cellulose microspheres: react agarose microspheres or cellulose microspheres, acid anhydride and liquid organic base at 30-40°C for 4-10h, filter, and wash the product with water, wherein , the volume ratio of agarose microspheres or cellulose microspheres, acid anhydride and liquid organic base is 1: (0.01-0.2): (20-50); the reaction formula is as follows:

Wherein, W is agarose microsphere or cellulose microsphere, Y is ethylene, p-phenylene or p-xylylene;

(b) Activation of the carrier: react the carrier obtained after crosslinking with acid anhydride and liquid organic base at 20-30°C for 4-12h, filter, and wash the product with water, wherein the carrier obtained after crosslinking, acid anhydride and liquid organic base The volume ratio of the base is 1: (0.01-0.2): (20-50); the reaction formula is as follows:

Wherein, X is the agarose microsphere or the cellulose microsphere after acid anhydride cross-linking;

(c) Immobilization of the ligand: react the activated carrier with the ligand and the liquid organic base at 0-40°C for 4-10 hours, and wash the product with water, wherein the activated carrier, the ligand, and the liquid organic base The dosage ratio is 1mL: (0.05-0.2g): (40-60mL); the reaction formula is as follows:

Wherein, R is p-hydroxyphenyl or β-indolyl.

Preferably, in step a, the volume ratio of agarose microspheres or cellulose microspheres, acid anhydride and liquid organic base is 1:(0.05-0.1):30.

Preferably, in step b, the volume ratio of the carrier obtained after crosslinking, the acid anhydride and the liquid organic base is 1:(0.05-0.1):30.

Preferably, in step c, the dosage ratio of activated carrier, ligand and liquid organic base is 1 mL:0.1 g:50 mL.

Preferably, the liquid organic base is one selected from pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N,N-diisopropylethylamine, diisopropylamine and quinoline or Various.

Wherein, the agarose microspheres can be prepared by methods known in the art, for example, reverse phase suspension embedding method or membrane emulsification method can be used.

Wherein, the cellulose microspheres can be prepared by methods known in the art, for example, emulsification-solidification method, spray drying method or coacervation method can be used.

Preferably, the agarose microspheres are specifically prepared according to the following steps: dissolving agarose powder in water to form an agarose solution with a mass percent concentration of 4-15%; adding the dissolved agarose solution to the In the oil phase containing dispersant, stir and disperse at 45-65°C for about 0.5-1.5h to form agar into balls, cool down, stop stirring, discharge and wash to obtain agarose microspheres.

Preferably, the mass percent concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: (1-5).

Wherein, the oil phase can adopt the oil phase commonly used in the reverse phase suspension embedding method, for example, it can be one of salad oil, epoxy soybean oil, aromatic oil, castor oil, 200# solvent oil, liquid paraffin and n-hexane or more.

Preferably, the oil phase is one or more of salad oil, 200# solvent naphtha and n-hexane.

Wherein, the dispersant can be a dispersant commonly used in the reversed-phase suspension embedding method, such as Span, Tween and the like.

Preferably, the dispersant is one or more of Span 85, Span 80, Tween 80 and Tween 20.

Preferably, the cellulose microspheres are specifically prepared according to the following steps: dissolving the cellulose resin in an organic solvent to prepare a cellulose solution with a concentration of 6-20% by mass, then adding a porogen, and stirring After uniformity, add to the water phase containing dispersant, stir at 25-35°C for 4-10 hours, stop stirring, discharge and wash to obtain cellulose microspheres.

Preferably, the volume ratio of the cellulose solution and the porogen is (100-200): (120-250); the volume ratio of the total volume of the cellulose solution and the porogen to the aqueous phase is (1-5):( 1-5).

Wherein, the cellulose resin of the present invention is one or more of natural cellulose and its esterified and etherified derivatives, such as cellulose, cellulose nitrate, cellulose acetate, Cellulose acetate butyrate and cellulose xanthate, methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, cyanoethylcellulose, hydroxypropylcellulose and hydroxypropylcellulose One or more of methyl cellulose, etc.

Preferably, the cellulose resin is cellulose diacetate.

Wherein, the organic solvent can be an organic solvent commonly used in the preparation of existing cellulose microspheres, such as halogenated hydrocarbons (dichloromethane, etc.), edible vegetable oil, silicone oil, etc.

Wherein, the porogen can be a porogen commonly used in the preparation of existing cellulose microspheres, such as methanol, ethanol, 1,4-butanediol, dimethyl phthalate, dipropylene phthalate One or more of esters, ethyl acetate, polyvinyl alcohol, etc.

Preferably, the porogen consists of dodecyl alcohol, ethanol and dimethyl phthalate in a volume ratio of (50-85):(15-35):(70-120).

Wherein, the dispersant can be a dispersant commonly used in the preparation of cellulose microspheres, such as gelatin, polyvinyl alcohol, polyglycerol monostearate, polyvinylpyrrolidone, and the like.

Preferably, the dispersant is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the two is (7-11):1.

The third aspect of the present invention is to provide an application of the IgG1 adsorbent described in the first aspect in the preparation of IgG1 adsorption products in blood and/or in the removal of IgG1-related group reaction antibody products in the field of organ transplantation .

The present invention uses agarose or cellulose gel rich in hydroxyl groups, blood compatibility and good mechanical properties as the carrier, and amino acids that are safe, cheap, stable, easy to store and disinfect as ligands, and adopts an efficient and simple Ways to prepare a low-cost IgG1 adsorbent that has relatively specific adsorption properties for immunoglobulin IgG1 and can effectively remove quorum reactive antibodies (PRA).

Compared with protein A biomacromolecules, amino acid small molecule compounds have significant advantages in cost and physical and chemical stability. The tyrosine or tryptophan used in the present invention is a ligand, which itself is an essential amino acid for the human body. Negative charges can achieve highly selective adsorption of IgG1.

The carrier cross-linking and activating reagents used in the present invention adopt ethylenediaminetetraacetic anhydride, 1,4-diphenylenediaminetetraacetic anhydride and 1,4-dibenzyldiaminetetraacetic anhydride. Such molecules have moderate length and atomic number At 13-17, the steric requirements for adsorption of IgG class protein molecules can be met. At the same time, there is a aminoacetic acid fragment on the arm molecule. The carboxyl group of aminoacetic acid is negatively charged in the neutral pH environment of the human body, and can achieve the adsorption of positively charged IgG antibodies through the action of additional charges. In addition, the acid radicals on it Ions can play an anticoagulant effect. When this product is used in hemoperfusion, the use of anticoagulant EDTA, sodium citrate, etc. can be exempted.

Therefore, the adsorbent of the present invention can realize the adsorption of positively charged IgG protein macromolecules through the space selective interaction between the long-chain arm and the ligand, the charge adsorption of the negative charge of the carboxyl group, and the hydrophobic interaction of the aromatic group of the ligand.

Human leukocyte antigen (HLA) class Ⅰ and class Ⅱ IgG antibodies mainly lead to the increase of PRA level, and among these antibodies, IgG1, which accounts for 60-70% of the total IgG, has the highest correlation. The IgG1 adsorbent of the present invention can effectively reduce the PRA level of highly sensitized organ transplantation patients while adsorbing immunoglobulin antibodies. Therefore, the IgG1 adsorbent involved in the present invention has a wide range of indications, and can be applied to autoimmune diseases including myasthenia gravis, Guillain-Barre syndrome, systemic lupus erythematosus, rheumatoid arthritis, etc., and can also be applied to hypersensitivity Organ transplant patients, patients with organ transplant rejection, etc.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) Using cheap, safe, stable and modifiable amino acid small molecules as ligands to replace the commonly used protein A to prepare immunosorbents, which can avoid the defects of protein A immunosorbents that are expensive, easy to fall off, and not high in safety.

(2) It is proposed to use ethylenediaminetetraacetic anhydride, 1,4-diphenylenediaminetetraacetic anhydride or 1,4-dibenzyldiaminetetraacetic anhydride to activate the carrier, and then directly immobilize the amino acid ligand to prepare the immunosorbent The research idea can greatly simplify the preparation process of the adsorbent, achieve a high ligand immobilization capacity, and realize the adsorption of IgG1 through the synergistic process of space selection, charge interaction and hydrophobic interaction.

(3) In vitro plasma adsorption test data show that the IgG1 adsorbent of the present invention has high specificity for IgG1 adsorption in human plasma, and can be used for the separation of such antibodies or the treatment of autoimmune diseases.

(4) The biomimetic immunosorbent of the present invention has a good clearance effect on population reactive antibodies (PRA) in highly sensitized organ transplant patients, and is expected to be applied in organ transplant fields such as highly sensitized organ transplant patients and post-organ transplant rejection patients.

Description of drawings

Fig. 1 is a summary diagram of the detection results of the adsorption performance of the IgG1 adsorbent provided by the present invention.

Detailed ways

The IgG1 adsorbent provided by the present invention uses agarose microspheres or cellulose microspheres cross-linked by acid anhydride as a carrier, and the acid anhydride is ethylenediaminetetraacetic anhydride, 1,4-diphenylenediaminetetraacetic anhydride or 1,4-dibenzyldiaminetetraacetic anhydride, the ligand is tyrosine or tryptophan;

The chemical structural formula of the agarose microspheres or cellulose microspheres after the acid anhydride crosslinking is as follows:

The chemical structural formula of the IgG1 adsorbent is as follows:

in,

W is agarose microsphere or cellulose microsphere,

X is the agarose microspheres or cellulose microspheres cross-linked by acid anhydride,

Y is ethylene, p-phenylene or p-xylylene,

R is p-hydroxyphenyl or β-indolyl.

Preferably, the effective amount of acid anhydride to activate the support is 110-180 μmol/ml.

Preferably, the amount of ligand immobilized on the carrier is 70-130 μmol/ml.

The IgG1 adsorbent provided by the present invention can be prepared by the following steps:

(a) Cross-linking of agarose microspheres or cellulose microspheres: react agarose microspheres or cellulose microspheres, acid anhydride and liquid organic base at 30-40°C for 4-10h, filter, and wash the product with water, wherein , the volume ratio of agarose microspheres or cellulose microspheres, acid anhydride and liquid organic base is 1: (0.01-0.2): (20-50), preferably 1: (0.05-0.1): 30;

(b) Activation of the carrier: react the carrier obtained after crosslinking with acid anhydride and liquid organic base at 20-30°C for 4-12h, filter, and wash the product with water, wherein the carrier obtained after crosslinking, acid anhydride and liquid organic base The volume ratio of the base is 1:(0.01-0.2):(20-50), preferably 1:(0.05-0.1):30;

(c) Immobilization of the ligand: react the activated carrier with the ligand and the liquid organic base at 0-40°C for 4-10 hours, and wash the product with water, wherein the activated carrier, the ligand, and the liquid organic base The dosage ratio is 1 mL: (0.05-0.2 g): (40-60 mL), preferably 1 mL: 0.1 g: 50 mL.

Wherein the liquid organic base participates in the reaction on the one hand, and acts as a solvent on the other hand, preferably selected from pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N,N-diisopropylethylamine, diisopropylethylamine, One or more of isopropylamine and quinoline.

Wherein, the agarose microspheres can be prepared by methods known in the art, for example, reverse phase suspension embedding method or membrane emulsification method can be used. The cellulose microspheres can be prepared by methods known in the art, for example, emulsification-solidification method, spray drying method or coacervation method can be used.

For example, the agarose microspheres are specifically prepared according to the following steps: dissolving agarose powder in water to form an agarose solution with a concentration of 4-15% by mass; adding the dissolved agarose solution to the In the oil phase of the dispersant, stir and disperse at 45-65°C for about 0.5-1.5 hours to form agar into balls, cool down, stop stirring, discharge and wash to obtain agarose microspheres. Preferably, the mass percent concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: (1-5). Wherein, the oil phase can adopt the oil phase commonly used in the reverse phase suspension embedding method, for example, it can be one of salad oil, epoxy soybean oil, aromatic oil, castor oil, 200# solvent oil, liquid paraffin and n-hexane or more. Preferably, the oil phase is one or more of salad oil, 200# solvent naphtha and n-hexane. Wherein, the dispersant can be a dispersant commonly used in the reversed-phase suspension embedding method, such as Span, Tween and the like. Preferably, the dispersant is one or more of Span 85, Span 80, Tween 80 and Tween 20. It should be understood that this is only a preferred example of the preparation method of the agarose microspheres, and those skilled in the art can also prepare the agarose microspheres by using other methods known in the art.

For example, the cellulose microspheres are specifically prepared according to the following steps: dissolving the cellulose resin in an organic solvent to form a cellulose solution with a concentration of 6-20% by mass, then adding a porogen and stirring evenly After that, add it into the water phase containing dispersant, stir at 25-35°C for 4-10 hours, stop stirring, discharge and wash to obtain cellulose microspheres. Preferably, the volume ratio of the cellulose solution and the porogen is (100-200): (120-250); the volume ratio of the total volume of the cellulose solution and the porogen to the aqueous phase is (1-5):( 1-5). Wherein, the organic solvent can be an organic solvent commonly used in the preparation of existing cellulose microspheres, such as halogenated hydrocarbons (dichloromethane, etc.), edible vegetable oil, silicone oil, etc. Wherein, the porogen can be a porogen commonly used in the preparation of existing cellulose microspheres, such as methanol, ethanol, 1,4-butanediol, dimethyl phthalate, dipropylene phthalate One or more of esters, ethyl acetate, polyvinyl alcohol, etc. Preferably, the porogen consists of dodecyl alcohol, ethanol and dimethyl phthalate in a volume ratio of (50-85):(15-35):(70-120). Wherein, the dispersant can be a dispersant commonly used in the preparation of cellulose microspheres, such as gelatin, polyvinyl alcohol, polyglycerol monostearate, polyvinylpyrrolidone, and the like. Preferably, the dispersant is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the two is (7-11):1. It should be understood that this is only a preferred example of the method for preparing cellulose microspheres, and those skilled in the art can also use other methods known in the art to prepare cellulose microspheres.

Wherein, the cellulose resin of the present invention is one or more of natural cellulose and its esterified and etherified derivatives, such as cellulose, cellulose nitrate, cellulose acetate, Cellulose acetate butyrate and cellulose xanthate, methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, cyanoethylcellulose, hydroxypropylcellulose and hydroxypropylcellulose One or more of methyl cellulose, etc., preferably cellulose diacetate.

Referring to the accompanying drawings, the present invention will be further described in conjunction with specific embodiments, so as to better understand the present invention.

Example 1

(a) Preparation of agarose gel

Dissolve agar powder in water to make a 5.5% agarose solution. Add the dissolved agarose solution to 2 times the volume of 200# solvent oil containing 2% Tween 20 dispersant, stir and disperse at 55°C for about 1 hour to form the agarose into balls. When the temperature drops to room temperature or below, stop stirring, discharge and wash to obtain agarose microspheres.

(b) Cross-linking of agarose microspheres

Soak the wet agarose microspheres in ethanol, dry them, take 2mL agarose microspheres and soak them with pyridine several times, then add 0.1mL EDTA and 50mL pyridine to react in a three-neck flask at 35°C 6h. Filter and wash the product with water until there is no smell of pyridine.

(c) Activation of the agarose carrier

After soaking the cross-linked carrier with ethanol, dry it, take 2 mL carrier and soak it several times with pyridine, add 0.2 mL ethylenediaminetetraacetic anhydride and 60 mL pyridine into a three-necked flask, and react at 20 ° C for 10 h. After filtration, the product was washed with water until there was no smell of pyridine, and then washed with ethanol for later use.

(d) Preparation of adsorbent

Soak 2 mL of the activated carrier in anhydrous tetrahydrofuran for 3-5 times, then add 0.2 g of tyrosine and 100 mL of anhydrous pyridine, and react at 30° C. for 10 h. The adsorbent was washed with water and stored at 4°C. The content of tyrosine (absorbance at 275 nm) in the tyrosine solution was tested with an ultraviolet spectrophotometer, and the immobilized amount of the ligand on the adsorbent was calculated according to the change of the content of tyrosine before and after the reaction. The test results showed that the immobilized amount of tyrosine on the adsorbent was 97.0 μmol/mL.

(e) Adsorption performance test

The in vitro static adsorption method was used to test the adsorption performance of the adsorbent on human immunoglobulin IgG, IgG1, IgA and IgM. The specific results are shown in Table 1 and Figure 1.

The in vitro static adsorption method was used to test the adsorption performance of the adsorbent on the population reactive antibody (PRA) in the plasma of highly sensitized kidney transplant patients:

That is, 1 mL of adsorbent was taken, and the thawed plasma was added according to the ratio of adsorbent:plasma=1:4 (V/V). The sample was placed in a water bath constant temperature shaker at 37°C and shaken at 60rpm for 2h. After the adsorption is completed, the upper layer of plasma is taken out and sent for testing. Among them, the detection method of immunoglobulin is immunoturbidimetric method, using Roche automatic biochemical analyzer and immunoglobulin detection kit, and the operation method is carried out according to the kit instructions. The specific results are shown in Table 1; PRA was detected using the mixed antigen plate (LATM) of the American Laimde Company, and the specific operation was carried out according to the kit instructions, and the plasma used was the plasma of highly sensitized kidney transplant patients. The specific results are shown in Table 2.

Example 2

(a) Preparation of agarose gel

Dissolve agar powder in water to make a 5.0% agarose solution. Add the dissolved agarose solution into 2.5 times the volume of n-hexane containing 3% Span-80 dispersant, stir and disperse at 65°C for about 0.5h to form the agarose into balls. When the temperature drops to room temperature or below, stop stirring, discharge and wash to obtain agarose microspheres.

(b) Cross-linking of agarose microspheres

Soak the wet agarose carrier with ethanol, dry it, take 2mL carrier and soak it with pyridine for several times, then add 0.2mL1,4-dibenzyldiaminetetraacetic anhydride and 50mL pyridine to react in a three-necked flask at 40°C 4h. Filter and wash the product with water until there is no smell of pyridine.

(c) Activation of the agarose carrier

Soak the carrier obtained by cross-linking with ethanol, dry it, take 2mL of the carrier and soak it several times with pyridine, add 0.15mL of dibenzyldiaminetetraacetic anhydride and 60mL of pyridine into a three-necked flask, and react at 20°C for 10h. After filtration, the product was washed with water until there was no smell of pyridine, and then washed with ethanol for later use.

(d) Preparation of adsorbent

Soak the activated 2mL carrier with anhydrous tetrahydrofuran for 3-5 times, then add 0.2g tryptophan and 100mL anhydrous pyridine, and react at 25°C for 10h. The adsorbent was washed with water and stored at 4°C. The content of tryptophan (absorbance at 275nm) in the tryptophan solution was tested with an ultraviolet spectrophotometer, and the immobilized amount of ligand on the adsorbent was calculated according to the change of tryptophan content before and after the reaction. The test results showed that the immobilized amount of tryptophan on the adsorbent was 105.3 μmol/mL.

(e) Adsorption performance test

The adsorption performance of the adsorbent on human immunoglobulin IgG, IgG1, IgA, and IgM and the adsorption performance on population reactive antibodies (PRA) were detected according to the method in Example 1. The specific results are shown in Table 1, Figure 1 and Table 2, respectively.

Example 3

(a) Preparation of cellulose gel

Dissolve 15g of cellulose diacetate in 120mL of dichloromethane, then add a porogen solution containing 58.5mL of dodecanol, 19mL of ethanol and 82.5mL of dimethyl phthalate, stir well and then add to the solution containing 2% (gelatin: PVA=9:1W/W) in 1L water phase, stirred at 35°C for 6 hours, stopped stirring and discharged, washed with 75% alcohol and water in sequence to obtain cellulose microspheres.

Get 2mL carrier, add a certain volume of 75% alcohol and 10wt% NaOH solution to the Erlenmeyer flask according to the ratio of V _{wet cellulose} : V _{75% alcohol} : V _{10% NaOH} = 1:2:3, saponify at room temperature for 10 Hour. After the saponification is completed, the carrier is sieved, washed with water, and stored in a wet state at room temperature.

(b) Cross-linking of cellulose microspheres

After soaking the wet fiber carrier with ethanol, dry it, take 2mL carrier and soak it several times with pyridine, then add 0.1mL ethylenediaminetetraacetic anhydride and 50mL pyridine to react in a three-necked flask at 35°C for 6h. Filter and wash the product with water until there is no smell of pyridine.

(c) Activation of cellulose support

Soak the cross-linked carrier in ethanol, dry it, take 2mL carrier and soak it several times with pyridine, add 0.2mL EDTA and 60mL pyridine into a three-necked flask, and react at 25°C for 6h. After filtration, the product was washed with water until there was no smell of pyridine, and then washed with ethanol for later use.

(d) Preparation of adsorbent

Soak 2 mL of the activated carrier in anhydrous tetrahydrofuran for 3-5 times, then add 0.2 g of tyrosine and 100 mL of anhydrous pyridine, and react at 30° C. for 10 h. The adsorbent was washed with water and stored at 4°C. The content of tyrosine (absorbance at 275 nm) in the tyrosine solution was tested with an ultraviolet spectrophotometer, and the immobilized amount of the ligand on the adsorbent was calculated according to the change of the content of tyrosine before and after the reaction. The test results showed that the immobilized amount of tyrosine on the adsorbent was 114.8 μmol/mL.

(e) Adsorption performance test

The adsorption performance of the adsorbent on human immunoglobulin IgG, IgG1, IgA, and IgM and the adsorption performance on population reactive antibodies (PRA) were detected according to the method in Example 1. The specific results are shown in Table 1, Figure 1 and Table 2, respectively.

Example 4

(a) Preparation of cellulose gel

Dissolve 15g of cellulose diacetate in 200mL of dichloromethane, then add a porogen solution containing 55.0mL of dodecanol, 20.0mL of ethanol and 90.0mL of dimethyl phthalate, stir well and add Dispersant (gelatin: PVA=9:1W/W) in 1L water phase, stirred at 32°C for 10 hours, stopped stirring and discharged, washed with 75% alcohol and water in sequence to obtain cellulose balls. Get 2mL carrier, add a certain volume of 75% alcohol and 10wt% NaOH solution to the Erlenmeyer flask according to the ratio of V _{wet cellulose} : V _{75% alcohol} : V _{10% NaOH} = 1:2:3, saponify at room temperature for 10 Hour. After the saponification is completed, the carrier is sieved, washed with water, and stored in a wet state at room temperature.

(b) Cross-linking of cellulose microspheres

Soak the wet agarose carrier in ethanol, dry it, take 2mL of the carrier and soak it with pyridine for several times, then add 0.1mL of 1,4-diphenylenediaminetetraacetic anhydride and 50mL of pyridine to react in a three-necked flask at 30°C 10h. Filter and wash the product with water until there is no smell of pyridine.

(c) Activation of cellulose support

After soaking the carrier obtained by cross-linking with ethanol, dry it, take 2 mL of the carrier and soak it several times with pyridine, add 0.2 mL of 1,4-diphenylenediaminetetraacetic anhydride and 60 mL of pyridine into a three-necked flask, Reaction 6h. After filtration, the product was washed with water until there was no smell of pyridine, and then washed with ethanol for later use.

(d) Preparation of adsorbent

Soak the activated 2mL carrier with anhydrous tetrahydrofuran for 3-5 times, then add 0.2g tryptophan and 100mL anhydrous pyridine, and react at 30°C for 10h. The adsorbent was washed with water and stored at 4°C. The content of tryptophan (absorbance at 275nm) in the tryptophan solution was tested with an ultraviolet spectrophotometer, and the immobilized amount of ligand on the adsorbent was calculated according to the change of tryptophan content before and after the reaction. The test results showed that the tryptophan immobilized on the adsorbent was 88.2 μmol/mL.

(e) Adsorption performance test

The adsorption performance of the adsorbent on human immunoglobulin IgG, IgG1, IgA, and IgM and the adsorption performance on population reactive antibodies (PRA) were detected according to the method in Example 1. The specific results are shown in Table 1, Figure 1 and Table 2, respectively.

Table 1 Adsorption performance of adsorbent to immunoglobulin

It can be seen from Table 1 and Fig. 1 that the adsorbent for blood purification of the present invention has excellent adsorption properties for immunoglobulin IgG and IgG1, and also has high adsorption capacity for IgA and IgM. Compared with the adsorption amount of IgG1/IgG, it is in the range of 0.76-0.80, and the proportion of normal IgG1 in IgG is generally 60-70%, so the adsorbent has certain specificity for the adsorption of IgG1 in IgG proteins. Comparing the adsorption ratio IgG1/IgA(IgM), which is in the range of 2.0-3.7, it can be concluded that the adsorption of IgG1 is relatively specific compared to IgA and IgM.

Table 2. Adsorption properties of adsorbents for quorum reactive antibodies (PRA)

As can be seen from the test results in Table 2, the IgG1 adsorbent used for blood purification of the present invention also has a higher adsorption capacity for quorum reactive antibodies (PRA), and Examples 1 and 2 can even directly turn PRA positive plasma into negative . It is reported in the literature that the PRA antibodies that cause hyperacute rejection are mainly IgG1 antibodies. The data in Table 1 show that the adsorbent has a relatively specific adsorption capacity for IgG1. After IgG1 is removed, the PRA value decreases significantly; while IgM has no direct evidence. It has an impact on causing PRA rejection, and IgA is also beneficial to organ transplantation, so the relatively specific removal of IgG1 can achieve the purpose of purifying the internal environment of patients waiting for organ transplantation and improving the survival rate of transplanted organs.

It can be seen from the above examples that the present invention provides IgG1 adsorbents with different polycarboxy amino acids as spacer arms and grafted with different amino acids as ligands. Tests on their adsorption properties show that while adsorbing immunoglobulin antibodies, It can also effectively reduce the level of PRA in highly sensitized organ transplant patients. Therefore, the IgG1 immunoadsorbent involved in the present invention has a wide range of indications, and can be applied not only to autoimmune diseases but also to the field of organ transplantation.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims (10)

1. The IgG1 adsorbent is characterized in that an anhydride crosslinked agarose microsphere or cellulose microsphere is used as a carrier, the anhydride is ethylenediamine tetraacetic anhydride, 1, 4-diphenyldiamine tetraacetic anhydride or 1, 4-dibenzyldiamine tetraacetic anhydride, and the ligand is tyrosine or tryptophan;

the chemical structural formula of the agarose microspheres or cellulose microspheres after anhydride crosslinking is as follows:

the chemical structural formula of the IgG1 adsorbent is shown as follows

Wherein,

w is agarose microsphere or cellulose microsphere,

x is agarose microsphere or cellulose microsphere after acid anhydride crosslinking,

y is ethylene, p-phenylene or p-xylylene,

r is p-hydroxyphenyl or beta-indolyl.

2. A method for preparing IgG1 adsorbent according to claim 1, wherein the effective amount of acid anhydride for activating the carrier is 110-180 μmol/ml and the amount of the ligand carried on the carrier is 70-130 μmol/ml.

3. A method of making the IgG1 adsorbent of claim 1, comprising the steps of:

(a) crosslinking of agarose microspheres or cellulose microspheres: agarose microspheres or cellulose microspheres, acid anhydride and liquid organic alkali react for 4 to 10 hours at the temperature of between 30 and 40 ℃, and are filtered, and products are washed by water, wherein the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic alkali is 1 to (0.01 to 0.2) to (20 to 50);

(b) activation of the carrier: reacting the carrier obtained after crosslinking with acid anhydride and liquid organic base at the temperature of 20-30 ℃ for 4-12h, filtering, and washing a product with water, wherein the volume ratio of the carrier obtained after crosslinking to the acid anhydride to the liquid organic base is 1: 0.01-0.2: 20-50;

(c) immobilization of the ligand: reacting the activated carrier with the ligand and the liquid organic alkali at 0-40 ℃ for 4-10h, and washing the product with water, wherein the dosage ratio of the activated carrier, the ligand and the liquid organic alkali is 1mL to (0.05-0.2g) to (40-60 mL).

4. The preparation method of claim 3, wherein in the step a, the volume ratio of the agarose microspheres or the cellulose microspheres to the acid anhydride to the liquid organic base is 1: 0.05-0.1: 30; in the step b, the volume ratio of the carrier, the acid anhydride and the liquid organic alkali obtained after cross-linking is 1 to (0.05-0.1) to 30; in the step c, the dosage ratio of the activated carrier, the ligand and the liquid organic base is 1 mL: 0.1 g: 50 mL.

5. The method of claim 3, wherein the liquid organic base is selected from one or more of pyridine, triethylamine, trimethylamine, triethanolamine, diethanolamine, N-diisopropylethylamine, diisopropylamine, and quinoline.

6. The preparation method according to claim 3, wherein the agarose microspheres are prepared by an inverse suspension embedding method or a membrane emulsification method; the cellulose microsphere is prepared by an emulsification-solidification method, a spray drying method or an agglomeration method.

7. The preparation method according to claim 6, wherein the agarose microspheres are prepared by the following steps:

dissolving the agarose powder in water to prepare 4-15% agarose solution; adding the dissolved agarose solution into an oil phase containing a dispersing agent, stirring and dispersing at 45-65 ℃ for about 0.5-1.5h to make the agar ball, cooling, stopping stirring, discharging and washing to obtain the agarose microspheres;

wherein the oil phase is one or more of salad oil, 200# solvent oil and n-hexane; the dispersant is one or more of span 85, span 80, tween 80 and tween 20, and the mass percentage concentration of the dispersant in the oil phase is 0.5-5.0%; the volume ratio of the agarose solution to the oil phase is 1: 1-5.

8. The preparation method according to claim 6, wherein the cellulose microspheres are prepared by the following steps:

dissolving cellulose resin in an organic solvent to prepare a cellulose solution with the mass percentage concentration of 6-20%, then adding a pore-foaming agent, uniformly stirring, adding the mixture into a water phase containing a dispersing agent, stirring for 4-10 hours at 25-35 ℃, stopping stirring, discharging, washing, and then performing saponification treatment to obtain cellulose microspheres;

wherein the volume ratio of the cellulose solution to the pore-foaming agent is (100-200) to (120-250); the volume ratio of the total volume of the cellulose solution and the pore-forming agent to the water phase is (1-5) to (1-5).

9. The method of claim 8, wherein the porogen consists of dodecanol, ethanol, and dimethyl phthalate in a volume ratio of (50-85) to (15-35) to (70-120); the dispersing agent is a mixture of gelatin and polyvinyl alcohol, and the mass ratio of the gelatin to the polyvinyl alcohol is (7-11) to 1.

10. The production method according to claim 3 or 8, wherein the cellulose resin from which the cellulose microspheres are produced is cellulose diacetate.