Red cell membrane with complete structure as well as preparation method and application of red cell membrane

Abstract

The invention belongs to the technical field of cell engineering and biochemical detection, and particularly relates to an erythrocyte membrane with a complete structure and a preparation method and application thereof. The red blood cells are split by limiting the components of the release liquid, the conditions are mild, protein components such as hemoglobin in the red blood cells can be released, the cell attributes are removed, the integrity of the internal skeleton structure and the activity of the membrane surface antigen are reserved, the complete structure of the red cell membrane is kept, meanwhile, the key blood group antigen on the surface is reserved, and the effect is good. The problem of antigenicity reduction caused by erythrocyte membrane fragmentation is avoided. After the erythrocytes are lysed, the nanoparticles are coupled to the surface of the erythrocyte membrane, so that the agglutination reaction of the erythrocyte membrane under the induction of the antibody can be visualized. The engineering red blood cell biological membrane is preserved by adopting a fixed program, so that the damage of ice crystals to the engineering red blood cell biological membrane can be effectively avoided, the antigenicity of the membrane is not influenced, the membrane can be preserved for a long time at normal temperature, and the shelf life can be prolonged to two years or more.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and biochemical detection, and specifically relates to a red blood cell membrane with a complete structure and a preparation method and application thereof. Background Art

[0002] Red blood cell reagents are indispensable core chemical reagents in hematological testing and are widely used in a variety of clinical diagnostic projects such as blood typing, antibody titer determination, neonatal hemolytic disease screening, and platelet antibody testing. They can effectively identify blood type antigens and antibodies in the blood, help medical staff determine the patient's blood type matching, detect potential immune responses, and provide rapid diagnostic evidence in emergency situations, especially in key fields such as transfusion medicine and perinatal medicine. However, the storage conditions of existing red blood cell reagents are very harsh, usually requiring cold chain storage, and their shelf life is short, usually only 3 to 6 months. And with the increase of storage time, the antigenicity of the red blood cell membrane gradually decreases, affecting the accuracy of the test results. In addition, the cell components in the red blood cell reagents are relatively fragile, and hemolysis and denaturation problems often occur during storage, which further shortens their service life.

[0003] To solve the above problems, the prior art proposes a strategy of replacing fresh red blood cell reagents with engineered red blood cells, such as Chinese patents CN200510017117.1, CN200910158168.4 and CN201220684879.2, etc., respectively using different types of nanoparticles or solid microspheres as carriers, coating the red blood cell membrane antigens on the surface of these carriers to simulate the red blood cell membrane antigens. However, these technologies usually break the red blood cell membrane and make it into fragments to adapt to the surface coating of nanoparticles. Although this fragmentation process can maintain some activity of red blood cell membrane antigens to a certain extent, it also brings a series of potential problems. First, the mechanical force such as ultrasound or lysis reagents required to prepare the broken red blood cell membrane usually cause irreversible damage to the red blood cell membrane, causing the red blood cell to lose its integrity and lose the number of blood type antigens. At the same time, its spatial structure may also change, resulting in reduced antigen activity, affecting the sensitivity and accuracy of blood type antibody detection, especially for weakly agglutinated samples, which may lead to misjudgment of blood type. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art, maintain the integrity of the red blood cell membrane structure, retain the key blood type antigens on its surface, avoid the problem of reduced antigenicity caused by red blood cell membrane fragmentation, and prepare an engineered red blood cell biomembrane that visualizes the agglutination reaction induced by antibodies, has high sensitivity for blood type reverse typing detection, and achieves long-term storage at room temperature.

[0005] In order to achieve the above-mentioned object, the present invention provides a structurally intact erythrocyte membrane, which is obtained by separating and lysing erythrocytes using a release solution; the release solution comprises 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum.

[0006] Preferably, the release solution further comprises 10 mM KHCO3 and / or 0.1 mM Na2EDTA.

[0007] Preferably, the pH value of the release liquid is 7.2-7.4; and the solvent of the release liquid is water.

[0008] The present invention also provides a method for preparing the erythrocyte membrane described in the above technical solution, comprising the following steps: uniformly mixing the release solution and erythrocytes, lysing for 5 to 10 minutes, separating the solid and the liquid, collecting the precipitate and washing it to obtain the erythrocyte membrane.

[0009] Preferably, the volume ratio of the red blood cells to the release fluid is 1×10 10 Piece: 1mL.

[0010] Preferably, the solid-liquid separation comprises centrifugation, the centrifugation speed is 7000 rpm, and the time is 10 min; The washing process uses an aqueous solution containing 2% v / v fetal bovine serum; the washing process is repeated 3 times, and in each washing process, the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the red blood cell release solution is 1:1 The present invention also provides an engineered erythrocyte biomembrane, comprising an erythrocyte membrane and nanoparticles coupled to the surface of the erythrocyte membrane; the erythrocyte membrane is the erythrocyte membrane described in the above technical solution.

[0011] Preferably, the mass ratio of the red blood cell membrane to the nanoparticles is 1:5.

[0012] The present invention also provides the use of the engineered erythrocyte biofilm described in the above technical solution in one or more of the following: (1) Preparation of blood typing reverse typing test products; (2) Preparation of platelet antibody detection products; (3) Preparation of low-titer O-type whole blood detection products; (4) Preparation of irregular antibody detection products; (5) Preparation of IgM anti-A and anti-B antibody detection products in whole blood and / or plasma; (6) Preparation of IgG anti-A and anti-B antibody detection products in whole blood and / or plasma; (7) Preparation of neonatal hemolysis detection products; (8) Preparation of blood type antibody adsorption products.

[0013] The present invention also provides a method for preserving the engineered red blood cell biofilm described in the above technical solution, comprising the following steps: The engineered red blood cell biofilm is stored at a constant temperature of 0.0 Pa and 4.0° C. for 10 minutes to obtain a first treated sample; The first treated sample was kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -50.0° C. to obtain a second treated sample; The second treated sample is kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -30.0° C. to obtain a third treated sample; The third treated sample is kept at a constant temperature for 120 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -45.0° C. to obtain a fourth treated sample; The fourth treated sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 12.0 Pa and a temperature of -45.0° C. to obtain a fifth treated sample; Under the conditions of a vacuum degree of 8.0 Pa for 60 minutes, the temperature of the fifth treated sample was raised to -30.0°C, and the sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 8.0 Pa and a temperature of -30.0°C to obtain a sixth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 30 minutes, the temperature of the sixth treated sample was raised to -20.0° C., and the sample was kept at a constant temperature of -20.0° C. for 60 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0 Pa to obtain a seventh treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 minutes, the temperature of the seventh treated sample was raised to -10.0°C, and the sample was kept at a constant temperature of -10.0°C for 30 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0°C, to obtain an eighth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 min, the temperature of the eighth treated sample was raised to 0.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain a ninth treated sample; Under the conditions of vacuum degree of 3.0 Pa for 10 min, the temperature of the ninth treated sample was raised to 100.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of vacuum degree of 3.0 Pa and temperature of 10.0° C., to obtain a tenth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 20 minutes, the temperature of the tenth treated sample is lowered to 0.0° C., and the sample is kept at a constant temperature for 40 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain an eleventh treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 20 minutes, the temperature of the eleventh treated sample is raised to 10.0° C., and the sample is kept at a constant temperature of 10.0° C. for 40 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0° C., to obtain a twelfth treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 10 minutes, the temperature of the twelfth treated sample is raised to 25° C., and the sample is kept at a constant temperature of 25.0° C. for 60 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 3.0° C. to obtain a thirteenth treated sample; The thirteenth processed sample is kept at a constant temperature of 25.0° C. and a vacuum degree of 2.0 Pa for 60 minutes to obtain a fourteenth processed sample; The fourteenth treated sample was kept at a constant temperature of 0.0 Pa in vacuum and 25.0° C. for 240 minutes.

[0014] Beneficial effects: The present invention provides a structurally complete red blood cell membrane, which is obtained by separating red blood cells after lysing them with a release solution; the release solution includes 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum. The present invention limits the components of the release solution, and the conditions are mild, so that protein components such as hemoglobin inside the red blood cells can be released, and the cell properties can be removed, while retaining the integrity of the internal skeleton structure and the activity of the membrane surface antigens. While maintaining the complete structure of the red blood cell membrane, the key blood type antigens on its surface are retained, and the problem of reduced antigenicity caused by red blood cell membrane fragmentation is avoided.

[0015] Furthermore, the present invention couples nanoparticles to the surface of the erythrocyte membrane, which can visualize the agglutination reaction of the erythrocyte membrane under antibody induction, and obtain an engineered erythrocyte biomembrane. Afterwards, the engineered erythrocyte biomembrane obtained by a certain procedure of preservation technology is preserved, which can greatly reduce the intracellular water content, effectively avoid the damage of ice crystal formation to the engineered erythrocyte biomembrane, ensure that the antigenicity of the erythrocyte biomembrane is not affected, and can be stored for a long time at room temperature, and the shelf life can be extended to two years, maintaining its basic blood type reverse typing and blood type antibody titer one-step determination function, greatly improving its feasibility and practicality in clinical and laboratory applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is a graph showing the effect of different red blood cell release solutions on the agglutination strength of engineered red blood cell biofilms; Figure 2 For RBC, RBC bm and RBC bm@Fluo's actual image under bright field and UV fluorescence; Figure 3 For RBC, RBC bm and RBC bm @Fluo's cytoskeleton results under a confocal microscope; Figure 4 For RBC, RBC bm and RBC bm @Fluo's results of CD47 molecule expression on phospholipid bilayer and red blood cell surface under confocal microscope; Figure 5 This is a graph showing the results of a reverse blood typing test; Figure 6 This is a graph showing the results of blood type antibody titer testing; Figure 7 Liquid, lyophilized and rehydrated RBCs bm @Fluo's actual image under bright field and UV fluorescence; Figure 8 RBC in liquid and freeze-dried form bm @Fluo Curve chart of the change of coagulation effect with storage time; Fig. 9 The platelet antibody test results for negative samples N1~N3; Fig.10 These are the platelet antibody test results for positive samples P1 to P6; Fig.11 is the detection limit of platelet antibody test in positive sample P1; Fig.12 These are the repeatability test results for platelet antibody detection in negative sample N1 and positive sample P1. DETAILED DESCRIPTION

[0018] The invention provides a structurally complete red blood cell membrane, which is obtained by separating and lysing red blood cells using a release solution; the release solution comprises 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum.

[0019] As an embodiment, the concentration of Ca(NO3)2 in the release solution of the present invention is 15 mM. As an embodiment, the concentration of fetal bovine serum in the release solution of the present invention is 2% v / v. As an embodiment, the release solution of the present invention further comprises 10 mM KHCO3 and / or 0.1 mM Na2EDTA; as another embodiment, the release solution of the present invention further comprises 10 mM KHCO3 and 0.1 mM Na2EDTA. As an embodiment, the pH value of the release solution of the present invention is 7.2~7.4; as another embodiment, the pH value of the release solution of the present invention is 7.2. As an embodiment, the solvent of the release solution of the present invention is water. Improvements on the formulation of the release solution, that is, other prescriptions obtained by adjusting, improving or changing the formulation without creativity, should also be regarded as the protection content of the present invention. These improvements or changes can optimize or adjust the formulation or its application field without changing the basic technical idea of ​​the present invention, and are also within the protection scope of the present invention.

[0020] The present invention provides a method for preparing the erythrocyte membrane described in the above technical solution, comprising the following steps: uniformly mixing the release solution and erythrocytes, lysing for 5 to 10 minutes, separating the solid and the liquid, collecting the precipitate and washing it to obtain the erythrocyte membrane.

[0021] As an embodiment, the volume ratio of the red blood cells to the release fluid in the present invention is 1×10 10 : 1mL. As an embodiment, the solid-liquid separation of the present invention includes centrifugation, the rotation speed of the centrifugation is 7000rpm, and the time is 10min. As an embodiment, the washing of the present invention uses an aqueous solution containing 2% v / v fetal bovine serum. As an embodiment, the number of washings of the present invention is 3 times, and in each washing, the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the release liquid is 1:1. As an embodiment, the present invention uses a vortex oscillator to fully mix the release liquid and red blood cells.

[0022] The release solution of the present invention has mild ingredients, and the release solution can be used to release protein components such as hemoglobin inside red blood cells for lysis, remove their cellular properties, retain the integrity of their internal skeleton structure and the activity of membrane surface antigens, and prepare red blood cell biofilms. The red blood cell release solution of the present invention retains the key blood type antigens on its surface while maintaining the intact structure of the red blood cell membrane, avoiding the problem of reduced antigenicity caused by red blood cell membrane fragmentation.

[0023] The present invention also provides an engineered erythrocyte biomembrane, comprising an erythrocyte membrane and nanoparticles coupled to the surface of the erythrocyte membrane; the erythrocyte membrane is the erythrocyte membrane described in the above technical solution.

[0024] As an embodiment, the mass ratio of the red blood cell membrane and the nanoparticles of the present invention is 1:5. As an embodiment, the nanoparticles of the present invention include one or more of fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superparamagnetic) magnetic iron oxide nanoparticles and aggregation-induced luminescence nanoparticles; as another embodiment, the nanoparticles of the present invention are fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superparamagnetic) magnetic iron oxide nanoparticles or aggregation-induced luminescence nanoparticles. As an embodiment, the fluorescent nanoparticles of the present invention are Fluo. As an embodiment, the aggregation-induced luminescence nanoparticles of the present invention are AIE. As an embodiment, the latex nanoparticles of the present invention include one or more of red latex nanoparticles (RLNP), black latex nanoparticles (BLNP) and purple latex nanoparticles (PLNP); as another embodiment, the latex nanoparticles of the present invention include red latex nanoparticles (RLNP), black latex nanoparticles (BLNP) or purple latex nanoparticles (PLNP). As an embodiment, the (superparamagnetic) magnetic iron oxide nanoparticles of the present invention include Fe3O4 magnetic nanoparticles.

[0025] The present invention couples the nanoparticles to the surface of the erythrocyte membrane, which can ensure the stable binding of the nanoparticles to the erythrocyte membrane, so that it emits a detectable signal. The labeled erythrocyte membrane can be effectively tracked and used for subsequent agglutination reaction monitoring. The present invention has no strict requirements on the method of coupling the nanoparticles to the surface of the erythrocyte membrane, and the operation can be performed using conventional methods in the art, such as click chemistry.

[0026] In view of the advantages of the engineered erythrocyte biofilm, the application of the engineered erythrocyte biofilm in one or more of the following also falls within the scope of protection of the present invention: (1) preparation of blood type reverse typing detection products; (2) preparation of platelet antibody detection products; (3) preparation of low titer O-type whole blood detection products; (4) preparation of irregular antibody detection products; (5) preparation of IgM anti-A and anti-B antibody detection products in whole blood and / or plasma; (6) preparation of IgG anti-A and anti-B antibody detection products in whole blood and / or plasma; (7) preparation of neonatal hemolysis detection products; (8) preparation of blood type antibody adsorption products. As an embodiment, the product of the present invention includes a reagent.

[0027] The present invention is described in the embodiments using the preparation of human erythrocyte membranes as an example, but this should not be construed as the entire protection scope of the present invention. The preparation of erythrocyte membranes of animals such as cats, dogs, cows, and monkeys is also applicable.

[0028] The present invention also provides a method for preserving the engineered red blood cell biofilm described in the above technical solution, comprising the following steps: The engineered red blood cell biofilm is stored at a constant temperature of 0.0 Pa and 4.0° C. for 10 minutes to obtain a first treated sample; The first treated sample was kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -50.0° C. to obtain a second treated sample; The second treated sample is kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -30.0° C. to obtain a third treated sample; The third treated sample is kept at a constant temperature for 120 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -45.0° C. to obtain a fourth treated sample; The fourth treated sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 12.0 Pa and a temperature of -45.0° C. to obtain a fifth treated sample; Under the conditions of a vacuum degree of 8.0 Pa for 60 minutes, the temperature of the fifth treated sample was raised to -30.0°C, and the sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 8.0 Pa and a temperature of -30.0°C to obtain a sixth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 30 minutes, the temperature of the sixth treated sample was raised to -20.0° C., and the sample was kept at a constant temperature of -20.0° C. for 60 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0 Pa to obtain a seventh treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 minutes, the temperature of the seventh treated sample was raised to -10.0°C, and the sample was kept at a constant temperature of -10.0°C for 30 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0°C, to obtain an eighth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 min, the temperature of the eighth treated sample was raised to 0.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain a ninth treated sample; Under the conditions of vacuum degree of 3.0 Pa for 10 min, the temperature of the ninth treated sample was raised to 100.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of vacuum degree of 3.0 Pa and temperature of 10.0° C., to obtain a tenth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 20 minutes, the temperature of the tenth treated sample is lowered to 0.0° C., and the sample is kept at a constant temperature for 40 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain an eleventh treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 20 minutes, the temperature of the eleventh treated sample is raised to 10.0° C., and the sample is kept at a constant temperature of 10.0° C. for 40 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0° C., to obtain a twelfth treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 10 minutes, the temperature of the twelfth treated sample is raised to 25° C., and the sample is kept at a constant temperature of 25.0° C. for 60 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 3.0° C. to obtain a thirteenth treated sample; The thirteenth processed sample is kept at a constant temperature of 25.0° C. and a vacuum degree of 2.0 Pa for 60 minutes to obtain a fourteenth processed sample; The fourteenth treated sample was kept at a constant temperature of 0.0 Pa in vacuum and 25.0° C. for 240 minutes.

[0029] The preservation method provided by the present invention adopts a certain procedure, which can greatly reduce the intracellular water content, effectively avoid the damage of ice crystal formation to the engineered erythrocyte biomembrane, and ensure that the antigenicity of the membrane is not affected. The engineered erythrocyte biomembrane treated by the preservation method of the present invention can be stored for a long time at room temperature, without complicated cold chain transportation and, is suitable for large-scale production and long-distance transportation, and the shelf life can be extended to two years, maintaining its basic blood type reverse typing and blood type antibody titer one-step determination function, greatly improving its feasibility and practicality in clinical and laboratory applications.

[0030] In order to further illustrate the present invention, a structurally complete red blood cell membrane, a preparation method and application thereof provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1 An erythrocyte release solution consisting of 15 mM Ca(NO3)2, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.

[0032] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g of Ca(NO3)2 and 20 mL of FBS, dissolve them in 850 mL of H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0033] Example 2 An erythrocyte releasing solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.

[0034] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g Ca(NO3)2, 1.0012 g KHCO3 and 20 mL FBS, dissolve them in 850 mL H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0035] Example 3 An erythrocyte release solution consisting of 15 mM Ca(NO3)2, 0.1 mM Na2EDTA, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH of 7.2.

[0036] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g of Ca(NO3)2, 0.0336 g of Na2EDTA and 20 mL of FBS, dissolve them in 850 mL of H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0037] Example 4 An erythrocyte releasing solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 0.1 mM Na2EDTA, 2% v / v fetal bovine serum (FBS) and the balance water, with a pH value of 7.2.

[0038] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g Ca(NO3)2, 1.0012 g KHCO3, 0.0336 g Na2EDTA and 20 mL FBS, dissolve them in 850 mL H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0039] Comparative Example 1 An erythrocyte release solution consisting of 15 mM Ca(NO3)2 and the balance water, with a pH of 7.2.

[0040] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g of Ca(NO3)2 and dissolve it in 850 mL of H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0041] Comparative Example 2 A red blood cell releasing solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3 and the balance water, with a pH of 7.2.

[0042] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g of Ca(NO3)2 and 1.0012 g of KHCO3, dissolve them in 850 mL of H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0043] Comparative Example 3 A red blood cell releasing solution consisting of 15 mM Ca(NO3)2, 0.1 mM Na2EDTA and the balance water, with a pH of 7.2.

[0044] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g of Ca(NO3)2 and 0.0336 g of Na2EDTA, dissolve them in 850 mL of H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0045] Comparative Example 4 A red blood cell releasing solution consisting of 15 mM Ca(NO3)2, 10 mM KHCO3, 0.1 mM Na2EDTA and the balance water, with a pH value of 7.2.

[0046] Taking the preparation of 1000 mL of the red blood cell release solution as an example, the specific preparation method is as follows: weigh 1.6650 g Ca(NO3)2, 1.0012 g KHCO3 and 0.0336 g Na2EDTA, dissolve them in 850 mL H2O, adjust the pH value to 7.2 with K2CO3, then add H2O to 1000 mL, and store at 4°C.

[0047] Example 5 Preparation of Fluoro Nanoparticle-labeled Red Blood Cell Biofilm 1. Isolation and Washing of Red Blood Cells Separate red blood cells from A / B / O type whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 minutes to obtain concentrated red blood cells. Wash the concentrated red blood cells with physiological saline for 3 times, discard the supernatant, and keep the red blood cell pellet for later use.

[0048] 2. Lysis of Red Blood Cells The red blood cell precipitate obtained in step 1 and the red blood cell releasing solution were respectively used at a rate of 1×10 10 The mixture was mixed in a quantity-volume ratio of 1:1 mL, thoroughly mixed by vortex oscillator, and centrifuged at 7000 rpm for 10 min. The supernatant was discarded and the red blood cell membrane precipitate (RBC) was collected.

[0049] 3. Preparation of Red Blood Cell Biofilm The red blood cell membrane precipitate obtained in step 2 was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution in step 2 was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete red blood cell biomembrane (RBC bm ).

[0050] 4. Preparation of Nanoparticle-labeled Red Blood Cell Biofilm The erythrocyte biomembrane prepared in step 3 was mixed with fluorescent nanoparticles (Fluorescent Nanoparticles, Fluo, particle size 200nm, excitation emission wavelength: Ex / Em (nm) 365 / 615) in a quantitative ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain nanoparticle-labeled erythrocyte biomembrane, i.e., engineered erythrocyte biomembrane (RBC bm @Fluo).

[0051] Test Example 1 Red blood cell membrane agglutination strength test Taking the engineered red blood cell biofilm obtained by using different red blood cell releasing fluids in step 4 of Example 5 as the test sample, the different test samples were reacted with anti-A / B IgM antibodies of different titers (1:256, 1:128, 1:64, 1:32, 1:16, 1:8 and 1:4), and the reaction results of each titer were scored with reference to the red blood cell agglutination scoring standard of the "AABB Technical Manual". The agglutination scores corresponding to the seven titers were added to obtain the final agglutination score. The agglutination score reflects the strength of the antigen-antibody reaction: the higher the score, the more obvious the agglutination reaction, the higher the antigen-antibody binding strength, and thus the higher the strength of the red blood cell membrane antigen. The specific agglutination scoring standard is as follows, and the scoring results are as follows: Figure 1 And as shown in Table 1.

[0052] 0 points: no agglutination, the solution is uniform, and there is no visible red blood cell clump or agglutination phenomenon; 3 points: Suspicious agglutination, very weak reaction, only a small amount of tiny particles or uncertain agglutination phenomenon in the solution; 5 points: weak agglutination, small and rare red blood cell agglutination groups can be seen in the solution, and the background is relatively turbid; 8 points: Moderate agglutination. Medium-sized red blood cell agglutination groups can be seen in the solution, and the background is clear.

[0053] 10 points: Strong agglutination. Large red blood cell agglutination groups can be seen in the solution, with a clear background and no free red blood cells.

[0054] 12 points: Very strong agglutination, the red blood cells are completely agglutinated into a large mass, the solution is clear and there are no free red blood cells.

[0055] Table 1 Agglutination strength of different red blood cell release solutions on engineered red blood cell biofilm

[0056] Note: Compared with comparative example 1, * in the table indicates P＜ 0.05; ** indicates P＜ 0.01; *** indicates P＜ 0.001.

[0057] according to Figure 1 As can be seen from Table 1, the agglutination score of Comparative Example 1 using a single reagent (Ca(NO3)2) is low, with an average of 33.00, indicating that it may have a certain negative impact on the agglutination result when lysing red blood cells. After the introduction of other reagents (such as KHCO3 or Na2EDTA), although the agglutination score improved, the difference was not significant, and the average remained at around 32.80. This shows that the promotion effect of adding these reagents alone on the agglutination reaction is limited. In contrast, the addition of FBS significantly increased the agglutination score of red blood cells. Example 1 (Ca(NO3)2+FBS), Example 2 (Ca(NO3)2+KHCO3+FBS), and Example 3 (Ca(NO3)2+Na2EDTA+FBS) all showed obvious agglutination reaction enhancement effects, with averages of 49.40, 52.20, and 53.20, respectively. Example 4 (Ca(NO3)2+KHCO3+Na2EDTA+FBS) performed the best, with an average agglutination score of 59.60 and a standard deviation of only 0.89, showing a high stability and consistency of the agglutination effect. Therefore, the introduction of FBS plays a key role in promoting the agglutination reaction of red blood cell membranes, especially when used in combination with other chemical reagents (such as KHCO3 and Na2EDTA). The synergistic effect of the four components in Example 4 may significantly enhance the stability of the red blood cell membrane and optimize the conditions of the agglutination reaction, thereby achieving the best agglutination effect.

[0058] Test Example 2 Integrity test 1. Take the red blood cell membrane precipitate (RBC) and the intact red blood cell biomembrane (RBC) obtained by using the red blood cell release solution in Example 4 in Example 5. bm ) and engineered red blood cell biofilms (RBC bm @Fluo, respectively, under bright field (BF) and ultraviolet fluorescence (UVFL), the results showed that the engineered red blood cell biofilm (RBCbm @Fluo) emits bright red fluorescence under ultraviolet fluorescence, and the fluorescence is evenly distributed ( Figure 2 ).

[0059] 2. Take the red blood cell membrane precipitate (RBC) and the intact red blood cell biomembrane (RBC) obtained by using the red blood cell release solution in Example 4 in Example 5. bm ) and engineered red blood cell biofilms (RBC bm @Fluo), detected under a confocal microscope, red is actin, green is tubulin, and yellow is a merged image of red and green. The results show that RBC, RBC bm and RBC bm @Fluo's red blood cell membranes remain intact, and the cytoskeleton structure is partially preserved ( Figure 3 ).

[0060] 3. Take the red blood cell membrane precipitate (RBC) and the intact red blood cell biomembrane (RBC) obtained by using the red blood cell release solution in Example 4 in Example 5. bm ) and engineered red blood cell biofilms (RBC bm @Fluo, using conventional methods, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL) was used to label the phospholipid bilayer, and PE-labeled anti-human CD47 antibody was used to label red blood cells. The expression of CD47 molecules on the surface of phospholipid bilayer (PBL) and red blood cells was detected using confocal microscopy. The results showed that the entire labeling process had little effect on the phospholipid bilayer, and the red blood cell membrane remained intact, not red blood cell membrane fragments ( Figure 4 ), based on the conclusion of step 2, it can be determined that the engineered erythrocyte biomembrane obtained by the present invention can maintain the integrity of erythrocyte membrane antigens.

[0061] Test Example 3 Performance Testing 1. Take Example 5 to obtain the engineered red blood cell biofilm (RBC bm @Fluo) and red blood cell membrane sediment (RBC), respectively, using human ABO reverse typing red blood cell reagent (purchased from Shanghai Blood Biomedicine Co., Ltd., denoted as RBC), according to the product instructions for blood type reverse typing test, the results are as follows Figure 5 As shown. Figure 5 It can be seen that RBC and RBC bm @Fluo The agglutination phenomenon caused by blood type antigen-antibody reaction is similar between them.

[0062] 2. Take the B-type engineered red blood cell biofilm and B-type red blood cell membrane precipitate obtained in Example 5, and use anti-A IgM antibody as a negative control to detect B-type red blood cells (denoted as RBC) and B-type RBCbm@Fluo (denoted as RBC bm @Fluo) with different titers (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) of anti-B IgM antibodies. The results are as follows Figure 6 As shown; among them, Figure 6 The middle (-) is the negative control (anti-A IgM antibody).

[0063] according to Figure 6 It can be seen that RBC and RBC bm The agglutination scores caused by blood group antigen-antibody reactions were consistent between @Fluo.

[0064] 3. Using Bio-Layer Interferometry (BLI), the red blood cell membrane precipitate (RBC) and the intact red blood cell biomembrane (RBC) of Example 5 were obtained by using the red blood cell release solution of Example 4. bm ) and type B engineered red blood cell biofilms (RBC bm The dissociation rate constant Kon, association rate constant Koff, and equilibrium dissociation constant (affinity) KD of the antigen-antibody reaction between IgG1@Fluo) and anti-B IgM antibody (purchased from Shanghai Blood Biomedicine Co., Ltd.) are shown in Table 2.

[0065] Table 2 Antigen-antibody reaction test results

[0066] According to Table 2, RBC, RBC bm、 RBC bm @Fluo's equilibrium dissociation constant is 10 -8 Order of magnitude, RBC bm The preparation and Fluo labeling process did not affect the affinity of the antigen-antibody reaction.

[0067] Example 6 Freeze-dried storage According to the procedure in Table 3, the engineered red blood cell biofilm (RBC bm @Fluo) for freeze-drying. Freeze-dried RBC bm @Fluo appears as a white, uniform, loose powder, and its average moisture content is reduced to (4.37±0.32)%, which meets the requirements for effective freeze-drying operation. Gently shake the freeze-dried RBC in sterile deionized water. bm@Fluo rehydration, the lyophilized preparation will dissolve into a clear liquid without unexpected coagulation, and the rehydration time is about 10s.

[0068] Table 3 Freeze-drying procedure and temperature control steps for freeze-dried storage

[0069] Test Example 4 Performance Testing 1. Take the liquid (i.e. before freeze-drying) of Example 6, freeze-dried and rehydrated engineered red blood cell biofilm (RBC bm @Fluo, respectively, under bright field (BF) and ultraviolet fluorescence (UVFL), the results showed that the engineered red blood cell biofilms in different states showed uniform fluorescence under ultraviolet light ( Figure 7 ).

[0070] 2. Take the engineered red blood cell biofilm (RBC) in liquid state (i.e. before freeze-drying) of Example 6 bm @Fluo) was stored at 4°C, and the lyophilized engineered red blood cell biofilm (RBC bm @Fluo), and stored at room temperature and 4°C, monitoring RBCs in different states during storage bm @Fluo's agglutination results are as follows Figure 8 As shown. Figure 8 It can be seen that the agglutination score of samples stored in liquid state at 4°C decreased by about 20% within 3 months, and significantly decreased by 70% after 6 months, basically losing the detection function. After 8 months, further monitoring was stopped; the freeze-dried state was stored at room temperature and re-dissolved within the two-year storage period, and the agglutination score did not change significantly compared with before freeze-drying.

[0071] Test Example 5 Clinical trials for reverse typing of blood The clinical microcolumn gel method was used as a comparative method to rehydrate the RBCs after freeze-drying and storage for 7 days in Example 6. bm @Fluo tested 641 clinical plasma samples, and the test results are shown in Tables 4 and 5.

[0072] Table 4 Qualitative results of 641 blood typing

[0073] Table 5 Results of clinical trials of blood type reverse typing test

[0074] According to Tables 4 and 5, it can be seen that using RBC bm@Fluo detected 188 cases of type A, 143 cases of type B, 48 cases of type AB, and 262 cases of type O, with a compliance rate of 100%. The experimental results show that RBC bm @Fluo has shown good accuracy in reverse blood typing testing.

[0075] Example 7 Preparation of IgG-sensitized erythrocyte biofilm labeled with AIE nanoparticles 1. Preparation of IgG-sensitized red blood cells Add 2 drops of RhD(+) packed red blood cells and 2 drops of IgG anti-D blood typing reagent to a clean test tube, mix well, and incubate at 37°C for 30 minutes, mixing continuously. Wash with saline 6 to 8 times (1000×g, 1min), centrifuge for the last time, discard the supernatant, and use absorbent paper to absorb the residual liquid at the tube mouth to make sensitized packed red blood cells. Take another clean test tube, add 1mL of saline, and then add 30μL of sensitized packed red blood cells to prepare a 3% IgG antibody sensitized red blood cell saline suspension.

[0076] 2. Release of IgG-sensitized erythrocyte biofilm Using the red blood cell release solution of Example 4, the 3% IgG antibody-sensitized red blood cell saline suspension obtained in step 1 and the red blood cell release solution were mixed at a rate of 1×10 10 The mixture was mixed at a volume ratio of 100 red blood cells: 1 mL, and after being thoroughly mixed by vortex oscillator, it was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected; The precipitate was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete red blood cell biofilm (RBC bm ).

[0077] 3. Preparation of IgG-sensitized erythrocyte biofilm labeled with AIE nanoparticles The erythrocyte biofilm prepared in step 2 was mixed with aggregation-induced emission nanoparticles (AIE, purchased from the Institute for Advanced Studies of Shenzhen University) at a ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain the nanoparticle-labeled erythrocyte biofilm, i.e., AIE nanoparticle-labeled IgG-sensitized erythrocyte biofilm (AIE@RBC bm ).

[0078] Test Example 6 Platelet antibody test 1. The experimental sample information is shown in Table 6. The experimental environment is: temperature 26°C, relative humidity 47%.

[0079] Table 6 Experimental sample information

[0080] 2. Platelet Antibody Detection Validation Steps 1) Remove the microplate strips from the pouch as needed. Unused microplate strips can be stored in the pouch with desiccant. 2) Use a plastic pipette to add one drop (50 μL) of platelet antigen screening to each well (patient serum well). Each microplate strip is tested for the platelet antibody test kit positive control (platelet antibody test kit positive control well, denoted as PC) and negative control (platelet antibody test kit negative control well, denoted as NC). 3) Centrifuge at 50 rcf for 5 min to fix the platelets to the microplate surface (uninterrupted centrifugation). 4) Manually wash the plate three times with 150 μL of PBS / Tween 0.005% to remove unbound platelets. Use a multichannel pipette to add the wash solution dropwise and let stand for 10 seconds. After each wash, slowly pour and gently tap to remove the PBS / Tween. 5) Add 2 drops (100 μL) of platelet antibody test solution (LISS solution) to each well. 6) Add 1 drop (50 μL) of positive or negative control of the platelet antibody detection kit to the appropriate wells. The positive control detects whether the donor platelets are sufficient. 7) Add 1 drop (50 μL) of patient serum to the other wells. 8) Incubate the microplate at 37°C for 30 minutes (seal the microwells during the incubation process). 9) Wash the plate manually 5 times with 150 μL of PBS / Tween according to step 4). 10) Immediately after washing, add 1 drop of anti-IgG reagent of the platelet antibody detection kit to each well. 11) Add 1 drop (50 μL) of indicator red blood cells of the platelet antibody detection kit and AIE@RBCbm obtained in Example 7 to each well and shake gently. 12) Centrifuge the microplate at 200 rcf for 5 minutes (uninterrupted centrifugation). 13) Read the results visually or using a reader.

[0081] 3. Reference product compliance test (1) The three negative samples N1, N2 and N3 in Table 6 were used as negative references and tested according to step 2. The results showed that the detection results of the three negative samples N1, N2 and N3 were all negative, and the negative reference compliance rate was 100% ( Fig. 9 ).

[0082] (2) The three strong positive samples P1, P2 and P3 and the three weak positive samples P4, P5 and P6 in Table 6 were used as positive references. Among them, P1, P3 and P6 all contained HLA+HPA antibodies. The test was performed according to step 2. The results showed that the test results of P1, P3 and P6 were all positive, and the compliance rate of the positive reference was 100% ( Fig.10 ).

[0083] 4. Detection limit test The P1 sample containing HLA+HPA antibodies was used as a positive reference, and was diluted in multiples. The test was performed according to step 2. The results showed that the P1 sample was still positive after 32-fold dilution ( Fig.11 ).

[0084] 5. Repeatability test The P1 sample containing HLA+HPA antibodies was used as a positive reference, and the N1 sample was used as a negative reference. The test was repeated 10 times according to step 2. The experiment showed that the test results of the N1 sample were all negative, and the test results of the P1 sample were all positive, and the intensity was consistent ( Fig.12 ).

[0085] According to the above content, it can be seen that the AIE@RBC obtained in Example 7 bm It can replace indicator red blood cells for platelet antibody detection and ensure the accuracy of platelet antibody detection.

[0086] Example 8 Preparation of RLNP nanoparticle-labeled type A red blood cell biofilm 1. Isolation and Washing of Red Blood Cells Separate red blood cells from type A whole blood sample. Centrifuge the whole blood at 1000 rpm for 5 min to obtain concentrated red blood cells. Wash the concentrated red blood cells with physiological saline for 3 times, discard the supernatant, and retain the red blood cell pellet for later use.

[0087] 2. Release of Type A Red Blood Cell Biofilm The red blood cell precipitate obtained in step 1 and the red blood cell releasing solution were mixed at a rate of 1×10 10 The mixture was mixed at a volume ratio of 100 red blood cells: 1 mL, and after being thoroughly mixed by vortex oscillator, it was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected; The precipitate was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete type A red blood cell biofilm (RBC bm ).

[0088] 3. Preparation of Nanoparticle-labeled Type A Red Blood Cell Biofilm The A-RBC biofilm prepared in step 2 was mixed with red latex nanoparticles (RLNP) at a ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain the nanoparticle-labeled erythrocyte biofilm, i.e., the engineered erythrocyte biofilm (A-RBC bm @RLNP).

[0089] Example 9 Preparation of BLNP nanoparticle-labeled B-type red blood cell biofilm 1. Isolation and Washing of Red Blood Cells Separate red blood cells from type B whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 minutes to obtain concentrated red blood cells. Wash the concentrated red blood cells with physiological saline for 3 times, discard the supernatant, and retain the red blood cell pellet for later use.

[0090] 2. Release of Type B Red Blood Cell Biofilm The red blood cell precipitate obtained in step 1 and the red blood cell releasing solution were mixed at a rate of 1×10 10 The mixture was mixed at a volume ratio of 100 red blood cells: 1 mL, and after being thoroughly mixed by vortex oscillator, it was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected; The precipitate was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete type B red blood cell biofilm (RBC bm ).

[0091] 3. Preparation of Nanoparticle-labeled B-type Red Blood Cell Biofilm The B-type erythrocyte biomembrane prepared in step 2 was mixed with black latex nanoparticles (BLNP) at a ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain the nanoparticle-labeled erythrocyte biomembrane, i.e., the engineered erythrocyte biomembrane (B-RBC bm @BLNP).

[0092] Test Example 7 Low Titer O Whole Blood (LTOWB) Test (1) According to the American low titer O type whole blood (LTOWB) titer standard (1:128), the A-RBC obtained in Example 8 was used. bm @RLNP replaces type A red blood cells to detect plasma titer. The results show that using A-RBC bm When @RLNP replaces type A red blood cells for plasma titer detection, the results are highly consistent with the traditional type A red blood cell detection results. When the titer is higher than 1:128, agglutination reaction visible to the naked eye is observed; when the titer is lower than 1:128, no agglutination phenomenon is observed. This shows that A-RBC bm @RLNP showed good substitution performance in blood type antibody detection, providing a reliable and effective solution for blood type antibody detection.

[0093] (2) According to the American low titer O type whole blood (LTOWB) titer standard (1:128), the B-RBC obtained in Example 9 was used. bm @BLNP replaces B-type red blood cells for plasma titer detection. The results show that using B-RBC bm When @RLNP replaces type A red blood cells for plasma titer testing, the results are highly consistent with the traditional type B red blood cell test results. When the titer is higher than 1:128, agglutination reaction visible to the naked eye is observed; when the titer is lower than 1:128, no agglutination phenomenon is observed. This shows that B-RBC bm @RLNP showed good substitution performance in blood type antibody detection, providing a reliable and effective solution for blood type antibody detection.

[0094] Example 10 Preparation of PLNP nanoparticle-labeled erythrocyte biofilm 1. Isolation and Washing of Red Blood Cells Separate red blood cells from A / B / O type whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 minutes to obtain concentrated red blood cells. Wash the concentrated red blood cells with physiological saline for 3 times, discard the supernatant, and keep the red blood cell pellet for later use.

[0095] 2. Release of red blood cell biofilm The red blood cell precipitate obtained in step 1 and the red blood cell releasing solution were mixed at a rate of 1×10 10 The mixture was mixed at a volume ratio of 100 red blood cells: 1 mL, and after being thoroughly mixed by vortex oscillator, it was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected; The precipitate was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete red blood cell biofilm (RBC bm ).

[0096] 3. Preparation of Nanoparticle-labeled Red Blood Cell Biofilm The erythrocyte biofilm prepared in step 2 was mixed with purple latex nanoparticles (PLNP) at a ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain the nanoparticle-labeled erythrocyte biofilm, i.e., the engineered erythrocyte biofilm (PLNP@RBC bm ).

[0097] Test Case 8 Irregular antibody detection Utilize the PLNP@RBC obtained in Example 10 bm Replace the traditional anti-screening red blood cell reagent for irregular antibody detection and compare with the conventional anti-screening red blood cell reagent. The antibodies detected include but are not limited to anti-D, C, E, c, e, JK a , JK b ,M,N,S,s,Fy a , Fy b , K, k, Kp a , Kp b ,Le a ,Le b , P1, Xg a 、Lu a 、Lu b 、Di a The results are shown in Tables 7 and 8.

[0098] Table 7 Results of conventional anti-red blood cell (human red blood cell) irregular antibody test

[0099] Note: In the table, “+” indicates positive, and “-” indicates negative, the same below.

[0100] Table 8 PLNP@RBC bm Irregular antibody test results

[0101] According to Tables 7 and 8, PLNP@RBC bm-I, II, III (PI, P-II, P-III) still retain D, C, E, c, e, JK a , JK b ,M,N,S,s,Fy a , Fy b , K, k, Kp a , Kp b ,Le a ,Le b , P1, Xg a 、Lu a 、Lu b 、Di a The activity of blood group antigens was consistent with that of conventional anti-red blood cell reagents, which verified its feasibility as an alternative reagent in irregular antibody detection.

[0102] Embodiment 11 Preparation of MNP nanoparticle-labeled erythrocyte biofilm 1. Isolation and Washing of Red Blood Cells Separate red blood cells from type A / B whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 minutes to obtain concentrated red blood cells. Wash the concentrated red blood cells with saline for 3 times, discard the supernatant, and keep the red blood cell pellet for later use.

[0103] 2. Release of red blood cell biofilm The red blood cell precipitate obtained in step 1 and the red blood cell releasing solution were mixed at a rate of 1×10 10 The mixture was mixed at a volume ratio of 100 red blood cells: 1 mL, and after being thoroughly mixed by vortex oscillator, it was centrifuged at 7000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected; The precipitate was mixed with pure water containing 2% v / v fetal bovine serum (FBS); the volume ratio of the pure water containing 2% v / v fetal bovine serum (FBS) to the red blood cell release solution was 1:1, and the mixture was washed repeatedly for 3 times to further remove the residual free hemoglobin, and finally a complete red blood cell biofilm (RBC bm ).

[0104] 3. Preparation of Nanoparticle-labeled Red Blood Cell Biofilm The erythrocyte biomembrane prepared in step 2 was mixed with Fe3O4 magnetic nanoparticles (MNP) at a ratio of 1:5, and the nanoparticles were coupled to the surface of the erythrocyte membrane using click chemistry to obtain the nanoparticle-labeled erythrocyte biomembrane, i.e., the engineered erythrocyte biomembrane A-RBC. bm @MNP and B-RBC bm@MNP, A-RBC bm @MNP and B-RBC bm @MNPs were mixed in a 1:1 mass ratio to obtain engineered red blood cell biofilm RBC bm @MNP.

[0105] Test Example 9 IgM / IgG anti-A and anti-B antibody titer testing 2 mg of RBC obtained in Example 11 bm @MNPs were incubated with 2 mL of whole blood or plasma at room temperature for 5 minutes to form blood type antibodies and RBC bm @Specific binding of antigens on the surface of MNP. In this process, IgM / IgG anti-A and anti-B antibodies were effectively removed after magnetic adsorption. Then, the titers of IgM / IgG anti-A and anti-B antibodies in whole blood and plasma before and after adsorption were detected using the microcolumn gel method. The results are shown in Table 9.

[0106] Table 9 IgM / IgG anti-A and anti-B antibody titers in whole blood and plasma

[0107] As can be seen from Table 9, before adsorption, the titers of IgM / IgG anti-A and anti-B antibodies in the 6 plasma samples were higher than 1:128; the titers of IgM / IgG anti-A and anti-B antibodies in the 6 whole blood samples were higher than 1:128. bm After immunoadsorption treatment with @MNP, the titers of IgM / IgG anti-A and anti-B antibodies in all samples decreased significantly and dropped below 1:32, indicating that this method can effectively remove blood type antibodies.

[0108] Test Example 10 Neonatal hemolysis test Anti-human globulin test card (incomplete antibody test for neonatal hemolytic disease fetus (infant)), add A-RBC in the 1st and 4th wells respectively bm @50μL of MNP red blood cell membrane suspension; add B-RBC to the 2nd and 5th wells respectively bm @50 μL of MNP red blood cell membrane suspension; add O-RBC to the 3rd and 6th wells respectively bm @MNP red blood cell membrane suspension 50μL each. Add 50μL of neonatal plasma to wells 1-3, and 50μL of neonatal red blood cell suspension to wells 4-6. Place the test card in a 37℃ incubator for 15 minutes. Centrifuge for 5 minutes using a microcolumn gel card centrifuge (900rpm for 2 minutes, 1500rpm for 3 minutes), remove the test card, and read the results. If RBC bm@MNP agglutination is located on the surface of the gel or in the gel, which is positive, indicating that the subject's serum has antibodies corresponding to its antigen or the blood cells have been sensitized; if RBC bm @If the MNP completely settles to the bottom of the gel and forms a red blood cell button at the bottom of the gel tube, it is negative, indicating that no antibodies induced by the corresponding antigen are detected in the serum, or the blood cells are not sensitized by the antibodies.

[0109] Based on the above content, it can be seen that the technical solution provided by the present invention can maintain the intact structure of the red blood cell membrane, retain the key blood type antigens on its surface, avoid the problem of reduced antigenicity caused by red blood cell membrane fragmentation, and the prepared engineered red blood cell biomembrane can visualize the agglutination reaction induced by antibodies and can be stored for a long time at room temperature.

[0110] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative improvement, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A structurally intact erythrocyte membrane, characterized in that: The erythrocytes are lysed and separated by using a release solution; the release solution includes 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum.

2. The erythrocyte membrane according to claim 1, characterized in that The release solution also includes 10 mM KHCO3 and / or 0.1 mM Na2EDTA.

3. The erythrocyte membrane according to claim 1, characterized in that The pH value of the release liquid is 7.2-7.4; the solvent of the release liquid is water.

4. The method for preparing red blood cell membrane according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: uniformly mixing the release solution and red blood cells, separating the solid and the liquid after lysing for 5 to 10 minutes, collecting the precipitate and washing it to obtain the red blood cell membrane.

5. The method for preparing red blood cell membrane according to claim 4, characterized in that: The volume ratio of the red blood cells to the release fluid is 1×10 10 Piece: 1mL.

6. The method for preparing red blood cell membrane according to claim 4, characterized in that: The solid-liquid separation includes centrifugation, the centrifugation speed is 7000 rpm, and the time is 10 min; The washing uses an aqueous solution containing 2% v / v fetal bovine serum; the washing is performed 3 times, and in each washing, the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the red blood cell release solution is 1:

1.

7. An engineered red blood cell biofilm, characterized in that: It comprises a red blood cell membrane and nanoparticles coupled to the surface of the red blood cell membrane; the red blood cell membrane is the red blood cell membrane described in any one of claims 1 to 3 or the red blood cell membrane prepared by the preparation method described in any one of claims 4 to 6.

8. The engineered erythrocyte biofilm according to claim 7, characterized in that The mass ratio of the red blood cell membrane to the nanoparticles is 1:

5.

9. Use of the engineered erythrocyte biofilm according to claim 7 or 8 in one or more of the following: (1) Preparation of blood typing reverse typing test products; (2) Preparation of platelet antibody detection products; (3) Preparation of low-titer O-type whole blood detection products; (4) Preparation of irregular antibody detection products; (5) Preparation of IgM anti-A and anti-B antibody detection products in whole blood and / or plasma; (6) Preparation of IgG anti-A and anti-B antibody detection products in whole blood and / or plasma; (7) Preparation of neonatal hemolysis detection products; (8) Preparation of blood type antibody adsorption products.

10. The method for preserving the engineered erythrocyte biofilm according to claim 7 or 8, characterized in that: The steps include: The engineered red blood cell biofilm is stored at a constant temperature of 0.0 Pa in vacuum and 4.0° C. for 10 minutes to obtain a first treated sample; The first treated sample is kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -50.0° C. to obtain a second treated sample; The second treated sample is kept at a constant temperature for 60 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -30.0° C. to obtain a third treated sample; The third treated sample was kept at a constant temperature for 120 minutes under the conditions of a vacuum degree of 0.0 Pa and a temperature of -45.0° C. to obtain a fourth treated sample; The fourth treated sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 12.0 Pa and a temperature of -45.0° C. to obtain a fifth treated sample; Under the conditions of a vacuum degree of 8.0 Pa for 60 minutes, the temperature of the fifth treated sample was raised to -30.0°C, and the sample was kept at a constant temperature for 360 minutes under the conditions of a vacuum degree of 8.0 Pa and a temperature of -30.0°C to obtain a sixth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 30 minutes, the temperature of the sixth treated sample was raised to -20.0° C., and the sample was kept at a constant temperature of -20.0° C. for 60 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0 Pa to obtain a seventh treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 minutes, the temperature of the seventh treated sample was raised to -10.0° C., and the sample was kept at a constant temperature of -10.0° C. for 30 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 5.0° C. to obtain an eighth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 10 min, the temperature of the eighth treated sample was raised to 0.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain a ninth treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 10 min, the temperature of the ninth treated sample was raised to 100.0° C., and the sample was kept at a constant temperature for 30 min under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0° C., to obtain a tenth treated sample; Under the conditions of a vacuum degree of 5.0 Pa for 20 minutes, the temperature of the tenth treated sample is lowered to 0.0° C., and the sample is kept at a constant temperature for 40 minutes under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0° C., to obtain an eleventh treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 20 minutes, the temperature of the eleventh treated sample is raised to 10.0° C., and the sample is kept at a constant temperature of 10.0° C. for 40 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0° C., to obtain a twelfth treated sample; Under the conditions of a vacuum degree of 3.0 Pa for 10 minutes, the temperature of the twelfth treated sample is raised to 25° C., and the sample is kept at a constant temperature of 25.0° C. for 60 minutes under the conditions of a vacuum degree of 3.0 Pa and a temperature of 3.0° C. to obtain a thirteenth treated sample; The thirteenth processed sample is kept at a constant temperature of 25.0° C. and a vacuum degree of 2.0 Pa for 60 minutes to obtain a fourteenth processed sample; The fourteenth treated sample was kept at a constant temperature of 0.0 Pa in vacuum and 25.0° C. for 240 minutes.

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