Pore membrane, pore membrane for immobilizing bioactive protein and preparation method and application of pore membrane

Abstract

The invention discloses a porous membrane, a bioactive protein immobilized porous membrane and a preparation method and application thereof, specific materials are treated through a series of chemical reagents to generate specific chemical groups, so that a multilayer porous membrane is prepared, and bioactive proteins can be immobilized by the chemical groups on the multilayer porous membrane through a chemical crosslinking method. The preparation method of the multilayer porous membrane for immobilizing the bioactive protein is very simple and rapid, and the material cross-linked with the antibody can be used after being directly freeze-dried or dried. According to the invention, the pore membrane matrix can generate a chemical group for fixing an antibody only through a simple chemical reaction, and then a specific immune reaction effect can be achieved by adding a specific cross-linked antibody. The cross-linked antibody can keep high biological activity for a long time after being coated, and long-term storage is facilitated.

Description

Technical Field

[0001] The present invention belongs to in vitro diagnostic reagents, and more specifically, relates to a porous membrane, a porous membrane with fixed biologically active proteins, and a preparation method and application thereof. Specifically, a multilayer porous membrane with fixed biologically active proteins is prepared by treating the porous membrane with a series of chemical reagents. Background Art

[0002] Incompatible blood types between blood donors and recipients can lead to severe hemolytic transfusion reactions. Blood type compatibility testing is one of the most important ways to ensure safe blood transfusion. Compatibility means harmonious coexistence, which means that there are no specific antibodies corresponding to the transfused red blood cell antigens in the patient's serum, and no immune response occurs, which is called compatible blood transfusion. On the contrary, it is called incompatible blood transfusion, that is, there are specific antibodies corresponding to the transfused red blood cell antigens in the patient's serum, an immune response occurs, and the transfused red blood cells are damaged and even hemolyzed. The specific antigens on the surface of red blood cells and the antibodies in the serum determine the blood type. The blood type compatibility test before transfusion includes not only ABO positive and negative typing, RhD blood typing, irregular antibody detection in the recipient's serum, detection and identification of clinically significant antibodies, and cross-matching tests.

[0003] The most traditional gold standard method for blood typing is the test tube method, which requires cumbersome centrifugation and washing steps, as well as the preparation of cell suspensions. Other conventional blood typing methods include the slide method, microcolumn gel immunoassay (MGIA), and microplate solid phase assay. However, the above methodologies have great limitations and their use scenarios are relatively limited. It is difficult to apply them in the field, at home, and other scenarios.

[0004] In addition, the prior patent CN110520736B discloses a functionalized biological multilayer porous membrane immune carrier, preparation method, and application, which includes a biological multilayer porous membrane; a specific anti-biological multilayer porous membrane antibody, the specific anti-biological multilayer porous membrane antibody and the biological multilayer porous membrane undergo antigen-antibody specific binding and thus adhere to the surface of the biological multilayer porous membrane; a cross-linked antibody, the cross-linked antibody and the specific anti-biological multilayer porous membrane antibody are coupled together; wherein one Fab end of the cross-linked antibody can be coupled with the specific anti-biological multilayer porous membrane antibody, and the other Fab end can be combined with the specific antibody. This method has the following shortcomings: (1) This method couples the specific anti-biological multilayer porous membrane antibody to the multilayer porous membrane, and then couples the cross-linked antibody with the specific anti-biological multilayer porous membrane antibody. The multilayer porous membrane contains more active raw materials, which will have a larger steric hindrance, which will affect the binding efficiency of the target substance and the cross-linked antibody to a certain extent. (2) The coating effect of the multilayer porous membrane prepared by the method through antigen-antibody binding is greatly affected by the activity of the antigen and antibody, and the material requirements and storage conditions are relatively high. (3) This method uses more antigens and antibodies, the overall cost is high, the production process is complicated, and it is not suitable for mass production. (4) The multilayer porous membrane contains many types of active raw materials (specific anti-biological multilayer porous membrane antibodies, cross-linked antibodies, etc.), which are prone to non-specific binding. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a porous membrane and a preparation method thereof in view of the deficiencies in the prior art, which can be used to make a multilayer porous membrane for fixing biologically active proteins. The target substances such as antigens / antibodies have high binding efficiency with the cross-linked antibodies in the multilayer porous membrane for fixing biologically active proteins and have a long storage time, and are suitable for use in various rapid detection kits.

[0006] The invention also provides a porous membrane for fixing biologically active proteins and a preparation method thereof.

[0007] The invention also provides application of the multi-layer porous membrane.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0009] A porous membrane comprises a porous membrane matrix and a chemical structure fixed on the surface of the porous membrane matrix by chemical reaction, wherein the chemical structure comprises an end R 1 Group, the R 1 The group is

[0010] The chemical structure of the present invention is fixed on the surface of the porous membrane matrix, and the R 1The group can react with the biologically active protein, so that the biologically active protein is stably fixed on the surface of the porous membrane matrix. The heterocyclic ring provided by NHS (N-hydroxysuccinimide) helps to improve the storage stability of blood type detection reagents and test strips, increase the fixed amount of antibody protein, and make the replacement reaction more stable, thereby improving the color development effect. 1 The CO- in the group reacts and combines with the biologically active protein, which can improve the effect of intercepting red blood cells in the biological multi-layer porous membrane immune carrier for subsequent ABO blood type detection.

[0011] The present invention can make the porous membrane matrix produce chemical groups for fixing antibodies only through simple chemical reactions, and then can have the effect of specific immune response after adding specific cross-linked antibodies.

[0012] The processing process of the present invention is simple and rapid, the production process is relatively simple, and the cross-linked antibody can maintain high biological activity for a long time after coating, which is conducive to long-term storage.

[0013] In a preferred embodiment of the present invention, the porous membrane substrate is a porous membrane product containing silicon dioxide; preferably, the porous membrane substrate is a glass cellulose membrane containing silicon dioxide;

[0014] The porous membrane matrix and the R 1 There is R between the groups 2 Group, the R 2 The group is

[0015] The basic material used in the present invention is glass fiber, which is easy to obtain.

[0016] The invention also discloses a porous membrane for fixing biologically active proteins, comprising the porous membrane and a cross-linked antibody / antigen fixed on the surface of the porous membrane.

[0017] The present invention also discloses a method for preparing a porous membrane, comprising the following steps:

[0018] S1. dissolving succinic anhydride and a silanization agent in a solvent to react and form an amide bond;

[0019] S2. Immersing the porous membrane in the solution after the reaction in S1 to cause a silane reaction, taking out the porous membrane, washing it and drying it to obtain a carboxylated porous membrane;

[0020] S3. The carboxylated porous membrane in S2 is reacted with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide dissolved in a solvent to cross-link to generate amino-reactive NHS lipids to obtain a porous membrane.

[0021] In S1, another carboxyl group of succinic anhydride is exposed.

[0022] A silylation reaction occurs in S2, thereby fixing another exposed carboxyl group of succinic anhydride on the multilayer porous membrane.

[0023] The amide bond is specifically -CO-NH-.

[0024] In a preferred embodiment of the present invention, the silanization agent in S1 is one or more of 3-aminopropyltriethoxysilane, silane-polyethylene glycol-carboxyl, and N-trimethylsilylacetamide;

[0025] The solvent in S1 is one or more of N,N-dimethylformamide, dimethyl sulfoxide, methanol, and ethanol;

[0026] The mass of succinic anhydride is 0.1-20 g; the volume of the silanization agent is 0.1-20 mL; the volume of the solvent in S1 is 10-100 mL. Preferably, the mass of succinic anhydride is 10-20 g; the volume of the solvent in S1 is 30-100 mL.

[0027] When the amount of silanization agent 3-aminopropyltriethoxysilane (APTS) is less than 0.1 mL, the color development is very weak; when the amount of APTS is more than 20 mL, the cost increases. When the amount of succinic anhydride is less than 0.1 g, the color development is affected.

[0028] The reaction time in S1 is 3-6 h.

[0029] In a preferred embodiment of the present invention, the reaction time in S2 is 3 to 12 hours;

[0030] The solvent in S3 is one or more of 2-morpholineethanesulfonic acid, disodium hydrogen phosphate, and sodium dihydrogen phosphate;

[0031] The reaction time in S3 is 20 to 120 min;

[0032] The mass concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in S3 is 0.01 to 40 mg / mL;

[0033] The mass concentration of N-hydroxysuccinimide in S3 is 0.01-40 mg / mL;

[0034] The molar concentration of the solvent in S3 is 0.01-0.2 mol / L, the volume is 30-100 mL, and the pH is 5.0-6.3.

[0035] Preferably, the mass concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in S3 is 20-40 mg / mL; the mass concentration of N-hydroxysuccinimide in S3 is 20-40 mg / mL; the molar concentration of the solvent in S3 is 0.01-0.2 mol / L, the volume is 50-80 mL, and the pH is 5.0-6.3.

[0036] When the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is lower than 0.01 mg / mL, the color development will be affected.

[0037] If the pH of 2-morpholineethanesulfonic acid (MES) concentration is too low, it will affect antibody binding, and if the pH of MES concentration is too high, it will affect EDC binding.

[0038] The invention also discloses a method for preparing a porous membrane for fixing biologically active proteins, wherein cross-linked antibodies / antigens are reacted with the porous membrane to generate amide bonds, and the cross-linked antibodies / antigens are fixed on the multi-layer porous membrane.

[0039] In a preferred embodiment of the present invention, a cross-linked antibody / antigen is taken and diluted to a desired concentration using a diluent to prepare a cross-linked antibody / antigen dilution solution, and a porous membrane or a porous membrane prepared by the above method is immersed in the cross-linked antibody / antigen dilution solution and reacted for 30 minutes to 3 hours to obtain a porous membrane with fixed biologically active proteins.

[0040] Preferably, the final concentration of the antigen / antibody dilution solution is 10-1000 μg / mL; wherein the antibody dilution solution is: 0.2-0.3 mol / L boric acid buffer, 1%-10% trehalose, 1%-10% sucrose, 0.05% Proclin-300, and the solution pH is 7.8-8.2. The antigen / antibody dilution solution is used to preserve the antibody and maintain the activity of the antibody.

[0041] The present invention also discloses a blood type detection reagent test paper, comprising a porous membrane for fixing biologically active proteins, or a porous membrane for fixing biologically active proteins prepared by the above method, wherein the cross-linked antibody / antigen is a red blood cell blood type antibody or a sensitized red blood cell blood type antigen.

[0042] Preferably, the red blood cell blood type antibodies include IgM, IgG, and IgM-IgG mixed type antibodies of red blood cell A, B, Rh, MNS, Kell, and Kidd blood types, and the red blood cell blood type antibody titer is greater than 1:32.

[0043] Preferably, the sensitized red blood cell blood type antigens include sensitized A-type red blood cell antigens, sensitized B-type red blood cell antigens, sensitized irregular antibody screening red blood cell antigens, and sensitized red blood cell spectrum cell antigens.

[0044] The blood type detection test paper comprises a flushing pad, a sample pad, a buffer pad, absorbent paper and a plastic bottom plate. The multi-layer porous membrane is arranged on the sample pad. The sample is dripped on the multi-layer porous membrane. The cross-linked antibodies fixed on the multi-layer porous membrane are combined with the blood type antigens on the red blood cells in the blood sample to fix the red blood cells on the multi-layer porous membrane. 100 to 300 μL of flushing liquid is dripped on the flushing pad. The flushing liquid passes through the flushing pad, the sample pad and the buffer pad and finally reaches the absorbent paper to flush away the unfixed red blood cells in the multi-layer porous membrane. The sample pad does not show color, while the combined red blood cells are retained on the sample pad and show red.

[0045] Preferably, the flushing solution is a phosphate buffer solution containing 0.05-1.0 PEG20000, 0.0.5-1.0 PEG6000, 0.05-1.0 polyvinyl pyrrolidone (MW: 40000), and 0.1 mol / mL, and the pH of the flushing solution is 6.8-7.2.

[0046] The flushing solution uses macromolecular chemicals, which can more easily flush out unbound red blood cells.

[0047] The invention also discloses the application of a multi-layer porous membrane for fixing biologically active proteins in blood type detection and analysis of pathogenic microorganism cell antigens and antibodies, bacterial cell antigens and antibodies, tumor cell antigens and antibodies and various cell antigens and antibodies.

[0048] The multilayer porous membrane provided by the present invention can be combined with red blood cell blood type antibodies or sensitized red blood cell type antigens through coupling or antibody-antigen specific binding, so as to be used for blood type detection; the multilayer porous membrane provided by the present invention can also select corresponding specific capture antibodies as needed to carry out immune response or continue immune selection and binding, so as to be used for detecting and analyzing pathogenic microorganism cell antigen antibodies, bacterial cell antigen antibodies, tumor cell antigen antibodies and various cell antigen antibodies.

[0049] Specifically, the method for preparing a porous membrane for fixing biologically active proteins comprises the following steps:

[0050] Take APTS (silane reagent, 3-aminopropyltriethoxysilane), succinic anhydride and DMF (N,N-dimethylformamide), mix the three evenly and fully dissolve them, and place them at room temperature for 3 to 10 hours to allow the amino group on the 3-aminopropyltriethoxysilane reagent to undergo a replacement reaction with the carboxyl group on the succinic anhydride, thereby preparing a silane reagent containing a carboxyl group, which is referred to herein as: glass fiber treatment liquid.

[0051] Take a clean glass cellulose membrane (Fuzhou Tongsheng, 8860), immerse it completely in the glass fiber treatment solution, and place it at room temperature for 3 to 12 hours to allow the silica in the glass fiber to undergo a silane etherification reaction with the carboxyl-containing silane reagent (the reaction of the silane coupling agent with silica will produce an organic substance called silane ether, thereby combining the organic substance with the inorganic surface), thereby fixing the carboxyl group on the glass fiber, and then take out the residual reagent, rinse it with purified water and dry it to obtain a carboxylated glass cellulose membrane.

[0052] Take EDC and NHS, use MES solution to dissolve them, make EDC / NHS mixed solution (glass fiber activation solution), immerse the carboxylated glass cellulose membrane in the EDC / NHS mixed solution for 20 to 120 minutes, use EDC to couple NHS with the carboxyl groups on the carboxylated glass cellulose membrane, thereby generating amino-reactive NHS lipid (the lipid can be stored for a long time at room temperature), then remove the rinsed residual reagent and dry it to obtain a glass cellulose membrane containing amino-reactive NHS lipid, which is referred to herein as a multilayer porous membrane.

[0053] Take red blood cell blood type antibody or sensitized red blood cell blood type antigen, dilute it to the target concentration with diluent, immerse it in the solution with the above multilayer porous membrane, react for 30min to 3h, and obtain the multilayer porous membrane for blood type compatibility detection as described above.

[0054] Compared with the prior art, the present invention has the following beneficial effects: the multilayer porous membrane provided by the present invention can fix more bioactive proteins and has higher detection sensitivity because of its large porous surface area; the pore diameter is large, so it can more easily pass various samples containing large particles and viscosity, and can be used in more application scenarios.

[0055] The multilayer porous membrane prepared by the present invention is a membrane in a dry state, on which is attached an amino-reactive NHS lipid, which is easily degraded in an aqueous solution and requires immediate addition of protein for cross-linking, but the lipid can be stably stored under dry conditions. After being fixed on glass fibers and stored in dry conditions, it can be used as an intermediate in the production of a test kit for long-term storage, which is more conducive to actual production applications.

[0056] The present invention is highly practical and can fix bioactive proteins on inorganic glass cellulose membranes through simple and common chemical reagents, which greatly expands the application scenarios of glass fiber materials, increases the choices of materials for fixing bioactive proteins, and has broad prospects in the preparation of various rapid detection kits.

[0057] The multilayer porous membrane for fixing biologically active proteins provided by the present invention can be used not only for blood type detection, but also for chemical cross-linking by selecting corresponding antibodies according to needs, and then performing immune reaction, and can be used for immune experimental detection and analysis of pathogenic microorganisms, bacteria, tumors and various cell antigen-antibody, including but not limited to, and has a wide range of application scenarios.

[0058] Compared with the prior patent CN110520736B, this patent uses commonly used chemical reagents, which have low storage requirements and are easy to store for a long time, and basically have no expiration problem.

[0059] Compared with the prior patent CN110520736B, which mainly uses active proteins, this method mainly uses chemical reagents, which are cheap and easy to purchase.

[0060] Compared with the prior patent CN110520736B, the cross-linking method used in this method is a combination of chemical bonds, and the prepared intermediate product is also easy to preserve and more suitable for production.

[0061] Compared with the prior patent CN110520736B, the cross-linking method used in this method is a chemical bond, and only the cross-linked antibody is a specific antibody, which greatly reduces the possibility of non-specific binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The A-type blood test result of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Example 1 of the present invention;

[0063] Figure 2 The B-type blood test result of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Example 1 of the present invention;

[0064] Figure 3 The AB blood type detection result of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Example 1 of the present invention;

[0065] Figure 4 The O-type blood test result of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Example 1 of the present invention;

[0066] Figure 5 The blood test result of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Comparative Example 1 of the present invention;

[0067] Figure 6 The blood test results of the biological multi-layer porous membrane immune carrier for human ABO blood type detection prepared in Comparative Example 2 of the present invention;

[0068] Figure 7It is a reaction process in the preparation of glass fiber treatment liquid. Succinic anhydride reacts with 3-aminopropyltriethoxysilane to expose the carboxyl group in succinic anhydride through the amide bond.

[0069] Figure 8 It is the reaction process between the silicon dioxide in the glass fiber cellulose membrane and the effective components of the glass fiber treatment liquid. The carboxyl groups in the glass fiber treatment liquid are fixed on the glass fiber cellulose membrane through the silane etherification reaction.

[0070] Fig. 9 It is a reaction process between EDC, NHS and carboxyl groups to immobilize NHS active ester on the glass cellulose membrane.

[0071] Fig.10 It is the reaction process between active proteins and active groups on the multilayer porous membrane. The active proteins are fixed on the multilayer porous membrane through the amide bonds generated after the reaction.

[0072] Note: Figure 1-Figure 6 The left hole in the middle is the anti-A hole, and the right hole is the anti-B hole. DETAILED DESCRIPTION

[0073] Embodiment 1:

[0074] Preparation of glass fiber treatment liquid: Weigh 10 g of succinic anhydride, add it to 30 mL of N,N-dimethylformamide (DMF), and after it is completely dissolved, add 20 mL of 3-aminopropyltriethoxysilane (APTS) and react at room temperature (15-30°C) for 3 hours.

[0075] Preparation of carboxylated glass fiber: Place the glass fiber into a glass fiber treatment solution, react at room temperature for 5 hours, rinse with purified water after the reaction, and dry.

[0076] Preparation of multilayer porous membrane: prepare 20 mg / mL EDC and NHS solutions, add 500 μL of EDC (20 mg / mL) and 1 mL of NHS (20 mg / mL) respectively to 50 mL of MES (0.05 M) solution, mix thoroughly, add to the prepared carboxylated glass fiber, react for 40 minutes, rinse with purified water after the reaction, put into an oven to dry, and you can get a multilayer porous membrane.

[0077] The obtained multilayer porous membrane was added with commercially available monoclonal anti-A and anti-B with a titer of 128, respectively, and reacted at room temperature for 2 hours. Then, it was placed in a 37°C oven for drying, stored dry, and kept at room temperature for use, thereby obtaining a biological multilayer porous membrane immune carrier for human ABO blood type detection.

[0078] The chemical reactions of the overall preparation process are detailed in Figures 1 to 4 , the test results comparison table is as follows:

[0079]

[0080] The prepared biological multilayer porous membrane immune carrier for ABO blood type detection was used to detect 10 samples of A, B, AB and O blood types. The results are as follows Figure 1-4 shown.

[0081] Comparative Example 1 (without addition of succinic anhydride):

[0082] The membrane of Comparative Example 1 includes the following components and preparation method, which is different from Example 1 in that APTS does not react with succinic anhydride, but directly reacts with glass fiber, and the carboxyl groups on the antibody and the amino groups fixed on the glass fiber undergo a condensation reaction. The specific implementation is as follows:

[0083] Preparation of amino-treated glass fiber: Place the glass fiber into APTS and react at room temperature for 5 hours. After the reaction, rinse with purified water and dry.

[0084] Preparation of multilayer porous membrane: EDC and NHS were added to commercially available monoclonal anti-A or anti-B with a titer of 128, so that the final concentrations of EDC were 1 mg / mL and NHS was 2 mg / mL, and then added to the prepared amino-treated glass fiber, reacted at room temperature for 40 minutes, and placed in an oven for drying to obtain a biological multilayer porous membrane immune carrier for human ABO blood type detection.

[0085] The prepared biological multilayer porous membrane immune carrier for ABO blood type detection detected A, B, and AB blood types respectively, and the results are as follows Figure 5 shown.

[0086] The membrane material obtained by the comparison of the above-mentioned comparative example 1 cannot achieve the effect of retaining red blood cells of Example 1 and cannot develop color.

[0087] Comparative Example 2 (without NHS):

[0088] Preparation of glass fiber treatment liquid: Weigh 10 g of succinic anhydride, add it to 30 mL of N,N-dimethylformamide (DMF), and after it is completely dissolved, add 20 mL of 3-aminopropyltriethoxysilane (APTS) and react at room temperature (15-30°C) for 3 hours.

[0089] Preparation of carboxylated glass fiber: Place the glass fiber into a glass fiber treatment solution, react at room temperature for 5 hours, rinse with purified water after the reaction, and dry.

[0090] Preparation of multilayer porous membrane: EDC was added to commercially available monoclonal anti-A or anti-B with a titer of 128 to a final concentration of 1 mg / mL. After thorough mixing, it was added to the prepared carboxylated glass fiber, reacted at room temperature for 40 minutes, and dried in an oven to obtain a biological multilayer porous membrane immune carrier for human ABO blood type detection.

[0091] The prepared biological multilayer porous membrane immune carrier for ABO blood type detection detects A, B, AB and O blood types respectively. The results are as follows Figure 6 shown.

[0092] The membrane material obtained by the comparison of the above-mentioned comparative example 2 cannot achieve the effect of retaining sufficient red blood cells as in Example 1.

[0093] No glass fiber activation liquid was added, only EDC was added, and the storage stability was low.

[0094] After adding the glass fiber activation solution, the pH value can be adjusted, the fixed amount of antibody protein can be increased, the replacement reaction will be more stable, and the color development effect will be improved.

Claims

1. A porous membrane, characterized in that: It includes a porous membrane matrix and a chemical structure fixed on the surface of the porous membrane matrix by chemical reaction, wherein the chemical structure includes an end R1 group, wherein the R1 group is 2. The porous membrane according to claim 1, characterized in that: The porous membrane substrate is a porous membrane product containing silicon dioxide; preferably, the porous membrane substrate is a glass cellulose membrane containing silicon dioxide; An R2 group is connected between the porous membrane matrix and the R1 group, and the R2 group is 3. A porous membrane for fixing biologically active proteins, characterized in that: The invention comprises the porous membrane according to claim 1 or 2, and a cross-linked antibody / antigen fixed on the surface of the porous membrane.

4. A method for preparing a porous membrane, characterized in that: The following steps are involved: S1. dissolving succinic anhydride and a silanization agent in a solvent to react and form an amide bond; S2. Immersing the porous membrane in the solution after the reaction in S1 to cause a silane reaction, taking out the porous membrane, washing it and drying it to obtain a carboxylated porous membrane; S3. The carboxylated porous membrane in S2 is reacted with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide dissolved in a solvent to cross-link to generate amino-reactive NHS lipids to obtain a porous membrane.

5. The method for preparing a porous membrane according to claim 4, characterized in that: The silanization agent in S1 is one or more of 3-aminopropyltriethoxysilane, silane-polyethylene glycol-carboxyl, and N-trimethylsilylacetamide; The solvent in S1 is one or more of N,N-dimethylformamide, dimethyl sulfoxide, methanol, and ethanol; The mass of succinic anhydride is 0.1-20 g; the volume of the silanization agent is 0.1-20 mL; and the volume of the solvent in S1 is 10-100 mL.

6. The method for preparing a porous membrane according to claim 4, characterized in that: The reaction time in S2 is 3 to 12 hours; The solvent in S3 is one or more of 2-morpholineethanesulfonic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate; The reaction time in S3 is 20 to 120 min; The mass concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in S3 is 0.01 to 40 mg / mL; The mass concentration of N-hydroxysuccinimide in S3 is 0.01-40 mg / mL; The molar concentration of the solvent in S3 is 0.01-0.2 mol / L, and the pH is 5.0-6.

3.

7. A method for preparing a porous membrane for fixing biologically active proteins, characterized in that: The cross-linked antibody / antigen reacts with the porous membrane to generate an amide bond, and the cross-linked antibody / antigen is fixed on the multi-layer porous membrane.

8. The method for preparing a porous membrane for fixing biologically active proteins according to claim 7, characterized in that: Take the cross-linked antibody / antigen, dilute it to the target concentration with a diluent to obtain a cross-linked antibody / antigen dilution solution, immerse the porous membrane in claim 1 or 2 or the porous membrane obtained by the method in claim 4 in the cross-linked antibody / antigen dilution solution, and react for 30 minutes to 3 hours to obtain a porous membrane with fixed biologically active proteins.

9. A blood type detection reagent test paper, characterized in that: The invention comprises the porous membrane for fixing biologically active proteins as claimed in claim 3, or the porous membrane for fixing biologically active proteins obtained by the method as claimed in claim 7 or 8, wherein the cross-linked antibody / antigen is a red blood cell blood group antibody or a sensitized red blood cell blood group antigen.

10. Application of a multi-layer porous membrane with fixed bioactive proteins in blood typing detection and analysis of pathogenic microorganism cell antigens and antibodies, bacterial cell antigens and antibodies, tumor cell antigens and antibodies, and various cell antigens and antibodies.

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