Protease treatment reagent and application thereof in enzyme treatment of cells

Abstract

The invention relates to a protease treatment reagent and application thereof in enzyme treatment of cells, and the protease treatment reagent is prepared by the following steps: adding L-cysteine, Pluronic, gelatin, a chelating agent and a bacteriostatic agent into a buffer agent, dissolving at room temperature, and obtaining the protease treatment reagent after complete dissolution; the protease treatment reagent is used as a grinding reagent to fully grind protease, the protease treatment reagent is used for fixing the volume, then a microporous filter is used for filtering to obtain a protease solution, and the obtained protease solution is used for treating red blood cells and further used for red blood cell blood group serological detection. The protease treatment reagent has a stable pH buffer environment, the protease activity can be effectively protected, the protease is pretreated by the protease treatment reagent to form a protease solution, and the protease solution can be stably stored for a long time; an original enzyme treatment cell method is converted into a unified, controllable and standardized enzyme treatment cell production mode from a laboratory method, and the utilization rate of protease and the success rate of enzyme treatment cells are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology materials, and particularly relates to a protease treatment reagent and application thereof in enzyme-treated cells. Background Art

[0002] Antibody screening tests are of great clinical significance for guiding compatible blood transfusions and ensuring clinical safety and effectiveness. Alloimmunization against red blood cell antigens can occur during pregnancy, blood transfusion, transplantation, or injection of immunogenic substances, and the frequency of alloimmunization varies greatly among different populations. However, before serological testing, it is often impossible to confirm the presence of unexpected blood type antibodies in the blood. These unexpected antibodies, which react with red blood cell surface antigens, can cause hemolytic transfusion reactions (HTR), hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic anemia (AIHA), and significantly reduce the survival rate of transfused red blood cells. Therefore, screening for unexpected red blood cell antibodies can aid in the diagnosis of certain diseases and ensure the safety and effectiveness of blood transfusions.

[0003] However, during antibody screening and identification, unexpected antibodies with low titer and affinity are often missed. These missed detections cannot be enhanced by controlling cell concentration, increasing serum volume, or extending incubation time. This is because some blood group systems are mostly heterozygous rather than homozygous, or due to insufficient antigen quantity. Homozygous genes typically encode twice the antigen dose of heterozygous genes, resulting in a weak response or missed detection due to the antigen dose effect. Typical examples include the Rh, Kidd, and Lewis blood group systems. Missed detection can cause severe acute or delayed transfusion reactions, which can be life-threatening. Some enzyme-only antibodies, such as Rh anti-E and anti-e, can only be detected in mediators such as papain or pineapple enzymes, or in combination with these antiglobulins. Although these antibodies are generally clinically insignificant, transfusion reactions have been reported.

[0004] Protease-treated red blood cells are generally used for red blood cell blood type serological testing, including antibody screening and antibody identification. The main principle is that the surface of red blood cells carries a large amount of negative charge, mainly due to the presence of N-acetylneuraminic acid on the sialic acid glycoprotein on the surface of red blood cells. N-acetylneuraminic acid is a carboxyl group with a negative charge. Normal red blood cells carry a large amount of negative charge on their surface, which repels like charges and maintains the distance between red blood cells, making it impossible for the IgG immunoglobulin to bind to antigens on the surfaces of two red blood cells at the same time. Proteolytic enzymes can hydrolyze N-acetylneuraminic acid, reduce the negative charge on the surface of red blood cells, and reduce the distance between red blood cells. IgG immunoglobulins can bind to antigens on the surfaces of two different red blood cells at the same time. Commonly used proteases include: papain, bromelain, fig protease, trypsin, chymosin, pronase, etc. Generally, the above-mentioned proteases need to be pretreated to form a protease solution before treating red blood cells, but the existing protease solutions have the following common defects: (1) Instability: The effect will vary depending on the production batch. When preparing the protease solution, its reactivity must be tested and the incubation time must be standardized to achieve the best effect. (2) Storage issues: The prepared protease solution needs to be stored at -20℃. It should be taken out and dissolved when used. It should not be frozen and thawed repeatedly. After thawing, it can be used effectively for no more than 1 hour at room temperature. (3) To prevent the rapid weakening of the protease during preparation or in a short period of use, or to prevent the protease from being completely dissolved, filtered or the supernatant used, the protease concentration will be increased or the mother liquor will be prepared during preparation. Dilution during use will cause false positives due to excessive protease concentration or activity. Summary of the Invention

[0005] The present invention aims to provide a protease treatment reagent having a stable pH buffer environment and capable of effectively protecting protease activity. The protease solution formed after the protease is pretreated with the protease treatment reagent can be stably stored for a long time for 6 months under a refrigerated environment at 2-8°C. The original enzyme-treated cell production method is converted from a laboratory method to a unified, controllable, and standardized enzyme-treated cell production method, thereby greatly improving the utilization rate of the protease and the success rate of the enzyme-treated cell.

[0006] The technical solution adopted by the present invention to solve the above problems is: a protease treatment reagent, comprising the following components: a buffer, gelatin, pluronic, a chelating agent, L-cysteine, and an antibacterial agent.

[0007] Preferably, the buffer is 0.02 mol isotonic phosphate buffer with a pH of 5.5.

[0008] More preferably, the 0.02M isotonic phosphate buffer solution with a pH of 5.5 is prepared as follows: 2.77g of NaH2PO4•H2O is accurately weighed, dissolved in distilled water and diluted to 100ml to obtain solution A; 0.284g of Na2HPO4 is accurately weighed, dissolved in distilled water and diluted to 100ml to obtain solution B; 94ml of solution A is mixed with 6ml of solution B, and then 900ml of 0.9% sodium chloride solution is added to prepare 0.02M phosphate buffer solution, and the pH is measured and adjusted to 5.5.

[0009] Preferably, the chelating agent is EDTA.2Na.

[0010] Preferably, the antibacterial agent is sodium benzoate.

[0011] Preferably, the mass ratio of the gelatin, Pluronic, chelating agent, L-cysteine ​​and antibacterial agent is 0.4-0.6:1.5-2.5:0.9-1.1:0.04-0.06:0.4-0.6.

[0012] More preferably, the mass ratio of the gelatin, pluronic, chelating agent, L-cysteine ​​and antibacterial agent is 0.5:2:1:0.05:0.5.

[0013] Preferably, the protease treatment reagent is prepared by the following steps: weighing L-cysteine, pluronic, gelatin, a chelating agent, and an antibacterial agent and adding them to a buffer; dissolving them at room temperature until they are completely dissolved to obtain the protease treatment reagent; and storing them at 2-8°C.

[0014] Preferably, the protease treatment reagent can be prepared by including ficin, papain or bromelain.

[0015] Another object of the present invention is to provide a use of a protease treatment reagent in enzyme-treated cells, comprising the following steps: (1) Protease pretreatment: Grind the protease repeatedly in a mortar until the particles are fine and there are no obvious large particles. Then add the protease treatment reagent and continue to grind thoroughly. Collect the contents in the mortar and adjust the volume with the protease treatment reagent. After it is completely dissolved, filter it with a 0.22 μm microporous filter membrane to obtain the protease solution. (2) Protease treatment of cells: Wash the red blood cells to be treated with physiological saline three times, discard the supernatant after centrifugation to obtain packed red blood cells, take 0.1 ml of packed red blood cells and add them to 0.5 ml of the above protease solution, mix well; incubate at 37°C for 15 minutes; after the incubation, wash them with physiological saline twice, then wash them once with red blood cell preservation solution, and then use red blood cell preservation solution to prepare a red blood cell suspension with a mass concentration of 3-5%, and store it at 2-8°C for use; (3) Take a clean test tube, add 2 drops of serum to be tested, 1 drop of the above-mentioned enzyme-treated red blood cell preservation solution with a mass concentration of 3-5% and 2 drops of the above-mentioned protease solution into the test tube, mix well; incubate at 37°C for 15 minutes; after the incubation is completed, centrifuge it, take it out and shake it gently to observe whether there is agglutination.

[0016] Quality Control of Protease Solution: (1) Reagent preparation: 1. First, use the protease treatment reagent to prepare ficin, papain, and bromelain into ficin solution, papain solution, and bromelain solution according to the above steps. 2. Anti-Fya: titer ≥4. 3. Control group cells: Fy (a+b-) packed red blood cells washed 3 times. 4. Weak Rh antibodies: such as anti-C, titer ≤16. (2) Fy(a+b-) erythrocytes were incubated with each of the above enzyme solutions at 37°C for 15 minutes, and the Fy(a+b-) erythrocytes treated with and without ficin were detected using the anti-Fya antibody enzyme method combined with the antiglobulin method. If the enzyme-treated Fy(a+b-) still reacted with anti-Fya, the erythrocytes were re-treated, and the incubation time of the erythrocytes with the enzyme was extended by 1 minute. If there was still a reaction, the incubation time was further extended.

[0017] (3) Incubate type O red blood cells with the enzyme solution at 37°C for 15 minutes. Use multiple normal serum samples and test using the enzyme method combined with the antiglobulin method. If more than 2 of the 10 normal sera show a positive reaction, the red blood cells need to be reprocessed and the incubation time of the red blood cells with the ficin enzyme is shortened (the incubation time can be shortened by 1 minute. If there is still a reaction, shorten the incubation time again).

[0018] (4) Low titer (titer ≤ 16) IgG type Rh antibodies (such as anti-C) and heterozygous antigen red blood cells (anti-C selects Cc antigen-positive red blood cells) were used, and the enzyme method combined with the antiglobulin method was used to test the red blood cells treated with and without ficin. Rh antibodies can directly agglutinate red blood cells treated with ficin, but not with red blood cells not treated with ficin. After using indirect antiglobulin reagents, the agglutination of red blood cells treated with ficin is significantly stronger than that of red blood cells not treated with ficin.

[0019] (5) After the red blood cells are treated with ficin, the Fya antigen is destroyed, and the red blood cells treated with ficin will not react with normal serum in a false positive reaction. At the same time, it can also enhance the reaction intensity between the antibodies and antigens of the Rh and Kidd systems.

[0020] A method for determining the potency of a protease solution: (1) Take 11 clean test tubes, add 100 μl of physiological saline to the first 10 test tubes, and add 100 μl of IgG anti-D to the first and 11 test tubes; (2) Pipet the contents of the first test tube to mix, transfer 100 μl of the mixture to the second tube, and pipette to mix; (3) Transfer 100 μl of the contents to the third tube, and dilute the mixture to the tenth tube in the same manner, discarding 100 μl of the mixture. (4) Add 100 μl of the above enzyme solution to the first 10 test tubes and mix. (6) Add 50 μl of 3-5% RhD positive red blood cell suspension to each test tube. (7) Mix well, centrifuge immediately, centrifuge at 1000g for 15 seconds, and take out the result. (8) The result of the 11th test tube must be negative. (9) The enzyme titer is determined and controlled at 32. If the titer is lower than 32, the amount of protease needs to be increased and the enzyme solution needs to be re-prepared. If the titer is higher than 32, the prepared enzyme solution needs to be diluted with enzyme treatment reagent.

[0021] Compared with the prior art, the advantages of the present invention are: (1) The protease treatment reagent of the present invention has a stable pH buffer environment. Gelatin and Pluronic form a stable protective environment. L-cysteine ​​stabilizes enzyme activity and can effectively protect protease activity. The protease solution formed after the protease is pretreated with the protease treatment reagent can be stably stored at 2-8°C for a long time.

[0022] (2) The protease treatment reagent of the present invention is applicable to ficin, papain, and bromelain commonly used in serological laboratories, without the need to adjust the enzyme concentration, usage method, and pH.

[0023] (3) The protease treatment reagent of the present invention transforms the original enzyme-treated cell method from a laboratory method to a unified, controllable, and standardized enzyme-treated cell production method, greatly improving the utilization rate of protease and the success rate of enzyme-treated cells. DETAILED DESCRIPTION

[0024] The present invention is described in further detail below with reference to the examples. Example 1

[0025] A protease treatment reagent is prepared by the following preparation method: (1) Accurately weigh 2.77 g of NaH2PO4•H2O, dissolve it in distilled water and dilute to 100 ml to obtain solution A; accurately weigh 0.284 g of Na2HPO4, dissolve it in distilled water and dilute to 10 ml to obtain solution B; measure 94 ml of solution A and mix it with 6 ml of solution B, then add 900 ml of 0.9% sodium chloride solution to prepare 0.02 M phosphate buffer solution, measure and adjust the pH to 5.5 to obtain 0.02 mol isotonic phosphate buffer solution with a pH of 5.5.

[0026] (2) Accurately weigh 0.05 g L-cysteine, 2 g Pluronic, 0.5 g gelatin, 1 g EDTA.2Na, and 0.5 g sodium benzoate and add them to 1000 ml of 0.02 mol isotonic phosphate buffer (pH 5.5). Dissolve at room temperature and store at 2-8°C until completely dissolved to obtain the protease treatment reagent. Example 2

[0027] Ficin solution: Weigh 0.1 g of ficin and grind it into fine, uniform particles using a mortar. Add 10 ml of the protease treatment reagent from Example 1 and grind thoroughly. Carefully collect the contents of the mortar and adjust the volume to 100 ml with the protease treatment reagent from Example 1. Dissolve the mixture at room temperature for 2 hours, filter through a 0.22 μm microporous filter membrane, and store at 2-8°C.

[0028] After dissolving at room temperature for 2 hours, the solution showed no visible powder. When filtered through a 0.22 μm microporous membrane, the solution filtered smoothly, with no impurities on the membrane. The protease solution was assayed using the aforementioned titer assay method, resulting in a titer of 64. The solution was then diluted 1-fold with protease treatment reagent and stored at 2–8°C. The solution was removed every month for appearance and potency measurement. After 6 months, the solution was a colorless, transparent liquid with no impurities precipitated. The titer was consistently 32, and decreased to 4 at 7 months. Example 3

[0029] Papain solution: Weigh 0.1 g of papain and grind it into fine uniform particles in a mortar. Add 10 ml of the protease treatment reagent in Example 1 and grind thoroughly. Carefully collect the contents in the mortar and then dilute to 100 ml with the protease treatment reagent in Example 1. After dissolution, filter with a 0.22 μm microporous filter.

[0030] After dissolving at room temperature for 2 hours, the solution showed no visible powder. When filtered through a 0.22 μm microporous membrane, the solution filtered smoothly, with no impurities on the membrane. The protease solution was assayed according to the above titer determination method, resulting in a titer of 32. The solution was stored at 2–8°C and removed every month for appearance and potency measurement. After 6 months, the solution was a colorless, transparent liquid with no impurities precipitated. The titer was consistently 32, reaching 16 at 7 months. The enzyme solution was inactivated at 8 months. Example 4

[0031] Bromelain solution Weigh 0.1 g of bromelain and grind it into fine, uniform particles using a mortar. Add 10 ml of the protease treatment reagent from Example 1 and grind thoroughly. Carefully collect the contents in the mortar and then dilute to 100 ml with the protease treatment reagent from Example 1. After dissolution, filter using a 0.22 μm microporous filter.

[0032] After dissolving at room temperature for 2 hours, the solution showed no visible powder. When filtered through a 0.22 μm microporous membrane, the solution filtered smoothly, with no impurities on the membrane. The protease solution was titered according to the above-described titer determination method, resulting in a titer of 128. The solution was then diluted 4-fold with protease treatment reagent and stored at 2-8°C. The solution was removed every month for appearance and potency measurement. After 8 months, the solution was a colorless, transparent liquid with no impurities precipitated. The titer was consistently 32, and at 9 months, it was 16, decreasing to 4. Example 5

[0033] Wash the red blood cells to be treated with normal saline three times, take 0.1 ml of packed red blood cells and add them to 0.5 ml of ficin solution, mix well; incubate at 37°C for 15 minutes; after the incubation, wash them twice with normal saline and once with red blood cell preservation solution; then use red blood cell preservation solution to prepare a red blood cell suspension with a mass concentration of 3-5%, and store it at 2-8°C until use.

[0034] After the cell suspension was treated with the ficin solution, the cells appeared bright red, and the cell membrane was intact without deformation or rupture under a microscope. Every 15 days, the enzyme-treated cells were removed and the supernatant was tested for hemolysis rate using a free hemoglobin detection reagent and enzyme treatment titer (according to the above method: take 11 clean test tubes, add 100 μl of physiological saline to the first 10 test tubes, and add 100 μl of IgG anti-D to the first test tube and the 11th test tube; (2) pipette the contents of the first test tube to mix, transfer 100 μl of the mixture to the second tube, pipette to mix; (3) transfer 100 μl of the contents to the third tube, dilute in the same way to the tenth tube, and discard 100 μl of the mixture. (4) Add 50 μl of the above-mentioned ficin-treated cells to each test tube. (6) Mix well, centrifuge immediately, centrifuge at 1000 g for 15 seconds, and take out the results for interpretation. (8) It is required that only the first 5 test tubes show positive agglutination, and the rest are negative.) The results showed that the cells treated with ficin could be stored at 2-8°C for 2 months with a hemolysis rate of ≤5% and the titer of the enzyme-treated cells maintained at 32. On the 75th day, the hemolysis rate was ≤5%, but the first two tubes of the enzyme-treated cell titer test showed positive reactions. Example 6

[0035] Wash the red blood cells to be treated with normal saline three times, take 0.1 ml of packed red blood cells and add them to 0.5 ml of papain solution, mix well; incubate at 37°C for 15 minutes; after the incubation, wash them twice with normal saline and once with red blood cell preservation solution; then use red blood cell preservation solution to prepare a red blood cell suspension with a mass concentration of 3-5%, and store it at 2-8°C for use.

[0036] After the cell suspension was treated with papain solution, the cells appeared bright red, and the cell membrane was intact without deformation or rupture under a microscope. Every 15 days, the enzyme-treated cells were removed and the supernatant was tested for hemolysis rate using a free hemoglobin detection reagent and enzyme treatment titer (according to the above method: take 11 clean test tubes, add 100 μl of physiological saline to the first 10 test tubes, and add 100 μl of IgG anti-D to the first test tube and the 11th test tube; (2) pipette the contents of the first test tube to mix, transfer 100 μl of the mixture to the second tube, pipette to mix; (3) transfer 100 μl of the contents to the third tube, dilute in this way to the tenth tube, and discard 100 μl of the mixture. (4) Add 50 μl of the above papain-treated cells to each test tube. (6) Mix well, centrifuge immediately, centrifuge at 1000 g for 15 seconds, and take out the results for interpretation. (8) It is required that only the first 5 test tubes show positive agglutination, and the rest are negative.) The results showed that papain-treated cells could be stored at 2-8°C for 2 months with a hemolysis rate of ≤5% and a titer of 32. On day 75, the hemolysis rate was ≥5% and only the first tube showed a positive reaction in the titer test of the enzyme-treated cells. Example 7

[0037] Wash the red blood cells to be treated with normal saline three times, take 0.1 ml of packed red blood cells and add them to 0.5 ml of bromelain solution, mix well; incubate at 37°C for 15 minutes; after the incubation, wash them twice with normal saline and once with red blood cell preservation solution; then use red blood cell preservation solution to prepare a red blood cell suspension with a mass concentration of 3-5%, and store it at 2-8°C until use.

[0038] After the cell suspension was treated with bromelain solution, the cells appeared bright red, and the cell membrane was intact without deformation or rupture under a microscope. Every 15 days, the enzyme-treated cells were removed and the supernatant was tested for hemolysis rate using a free hemoglobin detection reagent and enzyme treatment titer (according to the above method: take 11 clean test tubes, add 100 μl of physiological saline to the first 10 test tubes, and add 100 μl of IgG anti-D to the first test tube and the 11th test tube; (2) pipette the contents of the first test tube to mix, transfer 100 μl of the mixture to the second tube, pipette to mix; (3) transfer 100 μl of the contents to the third tube, dilute in this way to the tenth tube, and discard 100 μl of the mixture. (4) Add 50 μl of the above bromelain-treated cells to each test tube. (6) Mix well, centrifuge immediately, centrifuge at 1000 g for 15 seconds, and take out the results for interpretation. (8) It is required that only the first 5 test tubes show positive agglutination, and the rest are negative.) The results showed that bromelain-treated cells could be stored at 2-8°C for 2 months with a hemolysis rate of ≤5% and a titer of enzyme-treated cells maintained at 32. On day 90, the hemolysis rate was ≥5%, and there was no agglutination reaction with IgG anti-D in the enzyme-treated cell titer assay. Example 8

[0039] Take 11 clean test tubes and add 100 μl of normal saline to each. Add 100 μl of Diagast anti-Lea (protease is required for use) to the first and 11th test tubes. Mix the contents of the first test tube, transfer 100 μl to the second test tube, mix the contents, transfer 100 μl to the third test tube, and continue this process until the 10th test tube is diluted in the same proportions. Remove 100 μl and discard. Then, add 50 μl of a 3-5% suspension of type O (Lea+) red blood cells treated as in Example 5 and 100 μl of the ficin solution from Example 2 to each of the 11 test tubes, mix thoroughly, and incubate at 37°C for 15 min. After incubation, centrifuge, remove, gently shake, and observe for agglutination. The results are: 3+, 3+, 2+, 1+, 1+, 0, 0, 0, 0, 0, 0. Example 9

[0040] Take 11 clean test tubes and add 100 μl of normal saline to each. Add 100 μl of Diagast anti-Lea (protease is required for use) to the first and 11th test tubes. Mix the contents of the first test tube, transfer 100 μl to the second test tube, mix the contents, transfer 100 μl to the third test tube, and continue diluting the contents in this manner up to the tenth test tube. Remove 100 μl and discard. Then, add 50 μl of a 3-5% concentration of type O (Lea+) red blood cell suspension treated as in Example 6 and 100 μl of the papain solution from Example 3 to each of the 11 test tubes, mix thoroughly, and incubate at 37°C for 15 min. After incubation, centrifuge, remove, gently shake, and observe for agglutination. The results are: 2+, 2+, 2+, 2+, 1+, 0, 0, 0, 0, 0, 0. Example 10

[0041] Take 11 clean test tubes and add 100 μl of normal saline to each. Add 100 μl of Diagast anti-Lea (protease is required for use) to the first and 11th test tubes. Mix the contents of the first test tube, transfer 100 μl to the second test tube, mix the contents, transfer 100 μl to the third test tube, and continue this process until the 10th test tube is diluted in multiples. Remove 100 μl and discard. Then, add 50 μl of a 3-5% suspension of type O (Lea+) red blood cells treated as in Example 7 and 100 μl of the bromelain solution from Example 4 to each of the 11 test tubes, mix thoroughly, and incubate at 37°C for 15 min. After incubation, centrifuge, remove, gently shake, and observe for agglutination. The results are: 2+, 2+, 2+, 2+, 1+, 0, 0, 0, 0, 0, 0.

[0042] In addition to the above embodiments, the present invention also includes other implementation methods. Any technical solutions formed by equivalent transformation or equivalent replacement should fall within the scope of protection of the claims of the present invention.

Claims

1. A protease treatment reagent, characterized in that: The invention comprises the following components: buffer, gelatin, pluronic, chelating agent, L-cysteine ​​and antibacterial agent.

2. The protease treatment reagent according to claim 1, characterized in that: The buffer is a 0.02 mol isotonic phosphate buffer with a pH of 5.

5.

3. The protease treatment reagent according to claim 1, characterized in that: The chelating agent is EDTA.2Na.

4. The protease treatment solution according to claim 1, characterized in that: The antibacterial agent is sodium benzoate.

5. The protease treatment reagent according to claim 1, characterized in that: The mass ratio of the gelatin, Pluronic, chelating agent, L-cysteine ​​and antibacterial agent is 0.4-0.6:1.5-2.5:0.9-1.1:0.04-0.06:0.4-0.

6.

6. The protease treatment reagent according to claim 1, characterized in that: The mass ratio of the gelatin, Pluronic, chelating agent, L-cysteine ​​and antibacterial agent is 0.5:2:1:0.05:0.

5.

7. The protease treatment reagent according to claim 1, characterized in that: The protease treatment reagent is prepared by the following preparation steps: weighing L-cysteine, pluronic, gelatin, a chelating agent, and an antibacterial agent, adding them to a buffer, dissolving them at room temperature, and obtaining the protease treatment reagent after complete dissolution, and storing them at 2-8°C.

8. The protease treatment reagent according to claim 1, characterized in that: The protease in the protease treatment reagent includes ficin, papain or bromelain.

9. Use of the protease treatment reagent according to any one of claims 1 to 8 in enzyme-treated cells, characterized in that: The following steps are involved: (1) Protease pretreatment: Grind the protease repeatedly in a mortar until the particles are fine and there are no obvious large particles. Add the protease treatment reagent and continue to grind thoroughly. Collect the contents in the mortar and adjust the volume with the protease treatment reagent. After it is completely dissolved, filter it with a microporous filter to obtain the protease solution. (2) Protease treatment of cells: Wash the red blood cells to be treated with physiological saline three times, discard the supernatant after centrifugation to obtain packed red blood cells, take 0.1 ml of packed red blood cells and add them to 0.5 ml of the above protease solution, mix well; incubate at 37°C for 15 minutes; after the incubation, wash them with physiological saline twice, and then wash them once with red blood cell preservation solution to obtain enzyme-treated red blood cells. The enzyme-treated red blood cells are prepared into a red blood cell suspension with a mass concentration of 3~5% using red blood cell preservation solution, and stored at 2~8°C for use.