A structurally complete erythrocyte membrane and its preparation method and application

Abstract

The present invention belongs to the technical fields of cell engineering and biochemical detection, and particularly relates to a structurally complete red blood cell membrane, a preparation method thereof, and an application thereof. The present invention lyses red blood cells by defining the components of a release solution, with mild conditions, which can release protein components such as hemoglobin inside the red blood cells, remove their cell attributes, retain the integrity of their internal skeleton structure and the activity of membrane surface antigens, and while maintaining the complete structure of the red blood cell membrane, retain the key blood group antigens on its surface, avoiding the problem of reduced antigenicity caused by fragmentation of the red blood cell membrane. After lysing the red blood cells, nanoparticles are conjugated to the surface of the red blood cell membrane, enabling the agglutination reaction of the red blood cell membrane induced by antibodies to be visualized. Using a fixed procedure to preserve the engineered red blood cell biomembrane can effectively avoid damage to the engineered red blood cell biomembrane by ice crystals, ensure that the antigenicity of the membrane is not affected, and can be stored at room temperature for a long time, with the shelf life extended to two years or more.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and biochemical detection, and particularly relates to a structurally complete red blood cell membrane, a preparation method thereof, and an application thereof. Background Art

[0002] Red blood cell reagents are essential core chemical reagents in hematological tests and are widely used in various clinical diagnostic items such as blood group identification, antibody titer determination, neonatal hemolytic disease screening, and platelet antibody detection. They can effectively identify blood group antigens and antibodies in blood, helping medical staff judge the blood group matching of patients, detect potential immune reactions, and provide rapid diagnostic basis in emergency situations, especially playing an important role in key fields such as transfusion medicine and perinatal medicine. However, the existing red blood cell reagents have very strict storage conditions, usually requiring cold chain storage, and their shelf life is relatively short, usually only 3 - 6 months. Moreover, as the storage time increases, the antigenicity of the red blood cell membrane gradually decreases, affecting the accuracy of test results. In addition, the cell components in red blood cell reagents are relatively fragile, and problems such as hemolysis and denaturation often occur during storage, which further shortens their service life.

[0003] To solve the above problems, the prior art has proposed a strategy of replacing fresh red blood cell reagents with engineered red blood cells. For example, Chinese patents CN200510017117.1, CN200910158168.4, and CN201220684879.2, etc., respectively use different types of nanoparticles or solid microspheres as carriers, and coat the red blood cell membrane antigens on the surfaces of these carriers to simulate the red blood cell membrane antigens. However, these technologies usually perform fragmentation treatment on the red blood cell membrane and make it into fragments to fit the surface coating of the nanoparticles. Although this fragmentation treatment can maintain partial activity of the red blood cell membrane antigens to a certain extent, it also brings a series of potential problems. First of all, mechanical forces such as ultrasound or lysis reagents used for preparing fragmented red blood cell membranes usually cause irreversible damage to the red blood cell membrane, making the red blood cells lose integrity, reducing the number of blood group antigens, and at the same time, their spatial structure may also change, resulting in a decrease in antigen activity, affecting the sensitivity and accuracy of blood group antibody detection, and especially for weakly agglutinated samples, it may lead to misjudgment of blood groups. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art, maintain the complete structure of the red blood cell membrane while retaining the key blood group antigens on its surface, avoid the problem of reduced antigenicity caused by fragmentation of the red blood cell membrane, prepare an engineered red blood cell biomembrane, make the agglutination reaction occurring under the induction of antibodies visual, have high sensitivity in reverse blood group typing detection, and achieve long-term storage at room temperature.

[0005] To achieve the above object, the present invention provides a structurally complete erythrocyte membrane, which is obtained by separating after lysing erythrocytes with a releasing solution; the releasing solution includes 15 - 20 mM Ca(NO 3 ) 2 and 2 - 4% v / v fetal bovine serum.

[0006] Preferably, the releasing solution further includes 10 mM KHCO 3 and / or 0.1 mM Na 2 EDTA.

[0007] Preferably, the pH value of the releasing solution is 7.2 - 7.4; the solvent of the releasing solution is water.

[0008] The present invention also provides a method for preparing the erythrocyte membrane according to the above technical solution, including the following steps: mixing the releasing solution and erythrocytes evenly, lysing for 5 - 10 min and then separating the solid from the liquid, collecting the precipitate and washing it to obtain the erythrocyte membrane.

[0009] Preferably, the number - volume ratio of the erythrocytes to the releasing solution is 1×10 10 cells: 1 mL.

[0010] Preferably, the solid - liquid separation includes centrifugation, the rotation speed of the centrifugation is 7000 rpm, and the time is 10 min;

[0011] The washing is carried out using an aqueous solution containing 2% v / v fetal bovine serum; the number of washing times is 3 times, and each time of washing, the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the erythrocyte releasing solution is 1:1

[0012] The present invention also provides an engineered erythrocyte biomembrane, including an erythrocyte membrane and nanoparticles coupled to the surface of the erythrocyte membrane; the erythrocyte membrane is the erythrocyte membrane according to the above technical solution.

[0013] Preferably, the mass ratio of the erythrocyte membrane to the nanoparticles is 1:5.

[0014] The present invention also provides the application of the engineered erythrocyte biomembrane according to the above technical solution in one or more of the following:

[0015] (1) Preparing a blood group reverse typing detection product;

[0016] (2) Preparing a platelet antibody detection product;

[0017] (3) Preparing a low - titer group O whole blood detection product;

[0018] (4) Preparing an irregular antibody detection product;

[0019] (5)Preparing products for detecting IgM anti-A and anti-B antibodies in whole blood and / or plasma;

[0020] (6)Preparing products for detecting IgG anti-A and anti-B antibodies in whole blood and / or plasma;

[0021] (7)Preparing products for detecting neonatal hemolysis;

[0022] (8)Preparing products for adsorbing blood group antibodies.

[0023] The present invention also provides a preservation method for the engineered erythrocyte biomembrane described in the above technical solution, including the following steps:

[0024] Constantly preserving the engineered erythrocyte biomembrane for 10 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of 4.0 °C to obtain a first processed sample;

[0025] Constantly preserving the first processed sample for 60 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -50.0 °C to obtain a second processed sample;

[0026] Constantly preserving the second processed sample for 60 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -30.0 °C to obtain a third processed sample;

[0027] Constantly preserving the third processed sample for 120 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -45.0 °C to obtain a fourth processed sample;

[0028] Constantly preserving the fourth processed sample for 360 min under the conditions of a vacuum degree of 12.0 Pa and a temperature of -45.0 °C to obtain a fifth processed sample;

[0029] Under the conditions of a vacuum degree of 8.0 Pa and 60 min, raising the temperature of the fifth processed sample to -30.0 °C, and constantly preserving it for 360 min under the conditions of a vacuum degree of 8.0 Pa and a temperature of -30.0 °C to obtain a sixth processed sample;

[0030] Under the conditions of a vacuum degree of 5.0 Pa and 30 min, raising the temperature of the sixth processed sample to -20.0 °C, and constantly preserving it for 60 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of -20.0 °C to obtain a seventh processed sample;

[0031] Under the conditions of a vacuum degree of 5.0 Pa and 10 min, raising the temperature of the seventh processed sample to -10.0 °C, and constantly preserving it for 30 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of -10.0 °C to obtain an eighth processed sample;

[0032] Under the conditions of a vacuum degree of 5.0 Pa for 10 min, raise the temperature of the eighth processed sample to 0.0 °C, and keep it 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 the ninth processed sample;

[0033] Under the conditions of a vacuum degree of 3.0 Pa for 10 min, raise the temperature of the ninth processed sample to 100.0 °C, and keep it 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 the tenth processed sample;

[0034] Under the conditions of a vacuum degree of 5.0 Pa for 20 min, lower the temperature of the tenth processed sample to 0.0 °C, and keep it at a constant temperature for 40 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0 °C to obtain the eleventh processed sample;

[0035] Under the conditions of a vacuum degree of 3.0 Pa for 20 min, raise the temperature of the eleventh processed sample to 10.0 °C, and keep it at a constant temperature for 40 min under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0 °C to obtain the twelfth processed sample;

[0036] Under the conditions of a vacuum degree of 3.0 Pa for 10 min, raise the temperature of the twelfth processed sample to 25 °C, and keep it at a constant temperature for 60 min under the conditions of a vacuum degree of 3.0 Pa and a temperature of 25.0 °C to obtain the thirteenth processed sample;

[0037] Keep the thirteenth processed sample at a constant temperature for 60 min under the conditions of a vacuum degree of 2.0 Pa and a temperature of 25.0 °C to obtain the fourteenth processed sample;

[0038] Keep the fourteenth processed sample at a constant temperature for 240 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of 25.0 °C.

[0039] Beneficial effects:

[0040] The present invention provides a structurally complete red blood cell membrane, which is obtained by lysing red blood cells with a release solution and then separating; the release solution includes 15 - 20 mM Ca(NO 3 ) 2 and 2 - 4% v / v fetal bovine serum. By defining the components of the release solution, the present invention has mild conditions, can release protein components such as hemoglobin inside red blood cells, remove their cell properties, retain the integrity of their internal skeleton structure and the activity of membrane surface antigens, while maintaining the complete structure of the red blood cell membrane, retains the key blood group antigens on its surface, and avoids the problem of reduced antigenicity caused by fragmentation of the red blood cell membrane.

[0041] Furthermore, the present invention conjugates nanoparticles on the surface of the red blood cell membrane, enabling the visualization of the agglutination reaction that occurs on the red blood cell membrane under the induction of antibodies, and obtaining an engineered red blood cell biomembrane. Subsequently, the engineered red blood cell biomembrane obtained by using a certain preservation technique can significantly reduce the intracellular water content, effectively avoid the damage to the engineered red blood cell biomembrane caused by ice crystal formation, ensure that the antigenicity of the red blood cell biomembrane is not affected, can be stored at room temperature for a long time, and the shelf life can be extended to two years, maintaining its basic functions of reverse blood group typing and one-step determination of blood group antibody titer, greatly enhancing its feasibility and practicality in clinical and laboratory applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0043] Figure 1 FIG. Influence of different red blood cell release fluids on the agglutination intensity of engineered red blood cell biomembranes;

[0044] Figure 2 For RBC, RBC bm and RBC bm @Fluo in bright field and ultraviolet fluorescence;

[0045] Figure 3 For RBC, RBC bm and RBC bm @Fluo cytoskeleton results under a confocal microscope;

[0046] Figure 4 For RBC, RBC bm and RBC bm @Fluo results of phospholipid bilayer and CD47 molecule expression on the red blood cell surface under a confocal microscope;

[0047] Figure 5 FIG. Blood group reverse typing test results;

[0048] Figure 6 FIG. Blood group antibody titer test results;

[0049] Figure 7 For liquid, freeze-dried and rehydrated RBC bm @Fluo in bright field and ultraviolet fluorescence;

[0050] Figure 8 For liquid and freeze-dried RBC bm @Fluo curve of agglutination effect change with storage time;

[0051] Figure 9Results of platelet antibody detection for negative samples N1 to N3;

[0052] Figure 10 Results of platelet antibody detection for positive samples P1 to P6;

[0053] Figure 11 Detection limit for platelet antibody detection of positive sample P1;

[0054] Figure 12 Results of the repeatability experiment for platelet antibody detection of negative sample N1 and positive sample P1. Detailed implementation

[0055] The present invention provides a structurally complete erythrocyte membrane, which is obtained by lysing erythrocytes with a release solution and then separating; the release solution includes 15 - 20 mM Ca(NO 3 ) 2 and 2 - 4% v / v fetal bovine serum.

[0056] As an implementation, the concentration of Ca(NO 3 ) 2 in the release solution of the present invention is 15 mM. As an implementation, the concentration of fetal bovine serum in the release solution of the present invention is 2% v / v. As an implementation, the release solution of the present invention further includes 10 mM KHCO 3 and / or 0.1 mM Na 2 EDTA; as another implementation, the release solution of the present invention further includes 10 mM KHCO 3 and 0.1 mM Na 2 EDTA. As an implementation, the pH value of the release solution of the present invention is 7.2 - 7.4; as another implementation, the pH value of the release solution of the present invention is 7.2. As an implementation, the solvent of the release solution of the present invention is water. For the improvement of the release solution formulation, that is, other prescriptions obtained by non - creative adjustment, improvement or change of the formulation should also be regarded as the protected 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.

[0057] The present invention provides a method for preparing the erythrocyte membrane according to the above - mentioned technical solution, including the following steps: mixing the release solution and erythrocytes evenly, lysing for 5 - 10 min and then separating the solid and liquid, washing after collecting the precipitate to obtain the erythrocyte membrane.

[0058] As an implementation, the quantity - volume ratio of the erythrocytes and the release solution in the present invention is 1×10 10Volume: 1 mL. As an implementation, the solid-liquid separation in the present invention includes centrifugation at a rotational speed of 7000 rpm for 10 min. As an implementation, the washing in the present invention uses an aqueous solution containing 2% v / v fetal bovine serum. As an implementation, the number of washing times in the present invention is 3 times. Each time of washing, the volume ratio of the aqueous solution containing 2% v / v fetal bovine serum to the release solution is 1:1. As an implementation, the present invention uses a vortex oscillator to fully mix the release solution and red blood cells.

[0059] The release solution in the present invention has mild components. Using the release solution for lysis can release protein components such as hemoglobin inside red blood cells, remove their cell attributes, retain the integrity of their internal skeleton structure and the activity of membrane surface antigens, and prepare red blood cell biomembranes. While maintaining the complete structure of the red blood cell membrane, the red blood cell release solution in the present invention retains the key blood group antigens on its surface, avoiding the problem of reduced antigenicity caused by fragmentation of the red blood cell membrane.

[0060] The present invention also provides an engineered red blood cell biomembrane, comprising 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 the above technical solution.

[0061] As an implementation, the mass ratio of the red blood cell membrane to the nanoparticles in the present invention is 1:5. As an implementation, the nanoparticles in the present invention include one or more of fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superparamagnetic) iron oxide nanoparticles, and aggregation-induced emission nanoparticles; as another implementation, the nanoparticles in the present invention are fluorescent nanoparticles, photosensitizer nanoparticles, latex nanoparticles, colloidal gold, (superparamagnetic) iron oxide nanoparticles, or aggregation-induced emission nanoparticles. As an implementation, the fluorescent nanoparticles in the present invention are Fluo. As an implementation, the aggregation-induced emission nanoparticles in the present invention are AIE. As an implementation, the latex nanoparticles in the present invention include one or more of red latex nanoparticles (RLNP), black latex nanoparticles (BLNP), and purple latex nanoparticles (PLNP); as another implementation, the latex nanoparticles in the present invention include red latex nanoparticles (RLNP), black latex nanoparticles (BLNP), or purple latex nanoparticles (PLNP). As an implementation, the (superparamagnetic) iron oxide nanoparticles in the present invention include Fe 3 O 4 magnetic nanoparticles.

[0062] In the present invention, nanoparticles are conjugated to the surface of the red blood cell membrane, which can ensure the stable binding of the nanoparticles to the red blood cell membrane and enable it to emit detectable signals. The labeled red blood cell membrane can be effectively tracked and used for subsequent agglutination reaction monitoring. The present invention has no strict requirements for the method of conjugating nanoparticles to the surface of the red blood cell membrane, and conventional methods in the art can be used for operation, such as Click Chemistry.

[0063] In view of the advantages of the engineered red blood cell biomembrane, the application of the engineered red blood cell biomembrane in one or more of the following also falls within the protection scope of the present invention: (1) preparing a reverse blood group typing detection product; (2) preparing a platelet antibody detection product; (3) preparing a low-titer group O whole blood detection product; (4) preparing an irregular antibody detection product; (5) preparing an IgM anti-A and anti-B antibody detection product in whole blood and / or plasma; (6) preparing an IgG anti-A and anti-B antibody detection product in whole blood and / or plasma; (7) preparing a neonatal hemolysis detection product; (8) preparing a blood group antibody adsorption product. As an implementation manner, the product of the present invention includes reagents.

[0064] In the embodiments of the present invention, the preparation of human red blood cell membrane is taken as an example for illustration, but it should not be understood as the entire protection scope of the present invention only. The preparation of red blood cell membranes of animals such as cats, dogs, cows, and monkeys is also applicable.

[0065] The present invention also provides a preservation method for the engineered red blood cell biomembrane described in the above technical solution, including the following steps:

[0066] The engineered red blood cell biomembrane is stored at a constant temperature for 10 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of 4.0 °C to obtain a first processed sample;

[0067] The first processed sample is stored at a constant temperature for 60 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -50.0 °C to obtain a second processed sample;

[0068] The second processed sample is stored at a constant temperature for 60 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -30.0 °C to obtain a third processed sample;

[0069] The third processed sample is stored at a constant temperature for 120 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of -45.0 °C to obtain a fourth processed sample;

[0070] The fourth processed sample is stored at a constant temperature for 360 min under the conditions of a vacuum degree of 12.0 Pa and a temperature of -45.0 °C to obtain a fifth processed sample;

[0071] Under the conditions of a vacuum degree of 8.0 Pa and 60 min, raise the temperature of the fifth processed sample to -30.0 °C, and keep it at a constant temperature for 360 min under the conditions of a vacuum degree of 8.0 Pa and a temperature of -30.0 °C to obtain a sixth processed sample;

[0072] Under the conditions of a vacuum degree of 5.0 Pa and 30 min, raise the temperature of the sixth processed sample to -20.0 °C, and keep it at a constant temperature for 60 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of -20.0 °C to obtain a seventh processed sample;

[0073] Under the conditions of a vacuum degree of 5.0 Pa and 10 min, raise the temperature of the seventh processed sample to -10.0 °C, and keep it at a constant temperature for 30 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of -10.0 °C to obtain an eighth processed sample;

[0074] Under the conditions of a vacuum degree of 5.0 Pa and 10 min, raise the temperature of the eighth processed sample to 0.0 °C, and keep it 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 processed sample;

[0075] Under the conditions of a vacuum degree of 3.0 Pa and 10 min, raise the temperature of the ninth processed sample to 100.0 °C, and keep it 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 processed sample;

[0076] Under the conditions of a vacuum degree of 5.0 Pa and 20 min, lower the temperature of the tenth processed sample to 0.0 °C, and keep it at a constant temperature for 40 min under the conditions of a vacuum degree of 5.0 Pa and a temperature of 0.0 °C to obtain an eleventh processed sample;

[0077] Under the conditions of a vacuum degree of 3.0 Pa and 20 min, raise the temperature of the eleventh processed sample to 10.0 °C, and keep it at a constant temperature for 40 min under the conditions of a vacuum degree of 3.0 Pa and a temperature of 10.0 °C to obtain a twelfth processed sample;

[0078] Under the conditions of a vacuum degree of 3.0 Pa and 10 min, raise the temperature of the twelfth processed sample to 25 °C, and keep it at a constant temperature for 60 min under the conditions of a vacuum degree of 3.0 Pa and a temperature of 25.0 °C to obtain a thirteenth processed sample;

[0079] Keep the thirteenth processed sample at a constant temperature for 60 min under the conditions of a vacuum degree of 2.0 Pa and a temperature of 25.0 °C to obtain a fourteenth processed sample;

[0080] Keep the fourteenth processed sample at a constant temperature for 240 min under the conditions of a vacuum degree of 0.0 Pa and a temperature of 25.0 °C.

[0081] 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 the biological membrane of engineered red blood cells caused by ice crystal formation, and ensure that the antigenicity of the membrane is not affected. The biological membrane of engineered red blood cells treated by the preservation method of the present invention can be stored at room temperature for a long time without complex cold chain transportation, and is suitable for large-scale production and long-distance transportation. The shelf life can be extended to two years, maintaining its basic function of reverse blood group typing and one-step determination of blood group antibody titer, greatly improving its feasibility and practicality in clinical and laboratory applications.

[0082] In order to further illustrate the present invention, the following will combine the drawings and examples to describe in detail a structurally complete red blood cell membrane provided by the present invention, its preparation method and application.

[0083] Example 1

[0084] A red blood cell releasing solution consists of 15 mM Ca(NO 3 ) 2 , 2% v / v fetal bovine serum (FBS) and the balance of water, with a pH value of 7.2.

[0085] Taking the preparation of 1000 mL of the red blood cell releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 and 20 mL of FBS, dissolve them in 850 mL of H 2 O, adjust the pH value to 7.2 using K 2 CO 3 , and then add H 2 O to 1000 mL, and store at 4 °C.

[0086] Example 2

[0087] A red blood cell releasing solution consists of 15 mM Ca(NO 3 ) 2 , 10 mM KHCO 3 , 2% v / v fetal bovine serum (FBS) and the balance of water, with a pH value of 7.2.

[0088] Taking the preparation of 1000 mL of the red blood cell releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 , 1.0012 g of KHCO 3 and 20 mL of FBS, dissolve them in 850 mL of H 2 O, adjust the pH value to 7.2 using K 2 CO 3 and then add H2 0 to 1000 mL, store at 4 °C.

[0089] Example 3

[0090] An erythrocyte releasing solution, which consists of 15 mM Ca(NO 3 ) 2 , 0.1 mM Na 2 EDTA, 2% v / v fetal bovine serum (FBS) and the balance of water, with a pH value of 7.2.

[0091] Taking the preparation of 1000 mL of the erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 , 0.0336 g of Na 2 EDTA and 20 mL of FBS, dissolve them in 850 mL of H 2 O, use K 2 CO 3 to adjust the pH value to 7.2, and then add H 2 O to 1000 mL, store at 4 °C.

[0092] Example 4

[0093] An erythrocyte releasing solution, which consists of 15 mM Ca(NO 3 ) 2 , 10 mM KHCO 3 , 0.1 mM Na 2 EDTA, 2% v / v fetal bovine serum (FBS) and the balance of water, with a pH value of 7.2.

[0094] Taking the preparation of 1000 mL of the erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 , 1.0012 g of KHCO 3 , 0.0336 g of Na 2 EDTA and 20 mL of FBS, dissolve them in 850 mL of H 2 O, use K 2 CO 3 to adjust the pH value to 7.2, and then add H 2 O to 1000 mL, store at 4 °C.

[0095] Comparative Example 1

[0096] An erythrocyte releasing solution, which consists of 15 mM Ca(NO 3 ) 2 and the balance of water, with a pH value of 7.2.

[0097] Taking the preparation of 1000 mL of the said erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 and dissolve it in 850 mL of H 2 O. Use K 2 CO 3 to adjust the pH value to 7.2, then add H 2 O to 1000 mL and store it at 4°C.

[0098] Comparative Example 2

[0099] An erythrocyte releasing solution is composed of 15 mM Ca(NO 3 ) 2 , 10 mM KHCO 3 and the balance of water, with a pH value of 7.2.

[0100] Taking the preparation of 1000 mL of the said erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 and 1.0012 g of KHCO 3 , dissolve them in 850 mL of H 2 O. Use K 2 CO 3 to adjust the pH value to 7.2, then add H 2 O to 1000 mL and store it at 4°C.

[0101] Comparative Example 3

[0102] An erythrocyte releasing solution is composed of 15 mM Ca(NO 3 ) 2 , 0.1 mM Na 2 EDTA and the balance of water, with a pH value of 7.2.

[0103] Taking the preparation of 1000 mL of the said erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 and 0.0336 g of Na 2 EDTA, dissolve them in 850 mL of H 2 O. Use K 2 CO 3 to adjust the pH value to 7.2, then add H 2 O to 1000 mL and store it at 4°C.

[0104] Comparative Example 4

[0105] An erythrocyte releasing solution is composed of 15 mM Ca(NO3 ) 2 、 10 mM KHCO 3 、 0.1 mM Na 2 EDTA and the balance of water, with a pH value of 7.2.

[0106] Taking the preparation of 1000 mL of the above-mentioned erythrocyte releasing solution as an example, the specific preparation method is as follows: Weigh 1.6650 g of Ca(NO 3 ) 2 、 1.0012 g of KHCO 3 and 0.0336 g of Na 2 EDTA, dissolve them in 850 mL of H 2 O, adjust the pH value to 7.2 using K 2 CO 3 , then add H 2 O to 1000 mL, and store at 4 °C.

[0107] Example 5

[0108] Preparation of Fluo Nanoparticle-Labeled Erythrocyte Biomembrane

[0109] 1. Isolation and Washing of Erythrocytes

[0110] Isolate erythrocytes from A / B / O type whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 min to obtain concentrated erythrocytes. Wash the concentrated erythrocytes 3 times with physiological saline, discard the supernatant, and retain the erythrocyte pellet for standby.

[0111] 2. Lysis of Erythrocytes

[0112] Respectively use the erythrocyte releasing solutions of Examples 1 to 4 and Comparative Examples 1 to 4, mix the erythrocyte pellet obtained in Step 1 and the erythrocyte releasing solution at a volume ratio of 1×10 10 cells: 1 mL, fully mix them by a vortex oscillator, then centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the erythrocyte membrane pellet (RBC).

[0113] 3. Preparation of Erythrocyte Biomembrane

[0114] Mix the erythrocyte membrane pellet obtained in Step 2 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 erythrocyte releasing solution in Step 2 is 1:1, wash repeatedly 3 times to further remove residual free hemoglobin, and finally prepare a complete erythrocyte biomembrane (RBC bm ).

[0115] 4. Preparation of Nanoparticle-Labeled Erythrocyte Biomembrane

[0116] Mix the red blood cell biomembrane prepared in Step 3 with fluorescent nanoparticles (Fluorescent Nanoparticles, Fluo, particle size 200 nm, excitation / emission wavelength: Ex / Em (nm) 365 / 615) at a quantity ratio of 1:5. Use click chemistry to conjugate the nanoparticles to the surface of the red blood cell membrane, obtaining nanoparticle-labeled red blood cell biomembrane, namely engineered red blood cell biomembrane (RBC bm @Fluo).

[0117] Test Example 1

[0118] Test for Agglutination Intensity of Red Blood Cell Membrane

[0119] Take the engineered red blood cell biomembranes obtained from different red blood cell release fluids in Step 4 of Example 5 as the test samples. React the different test samples with anti-A / B IgM antibodies at different titers (1:256, 1:128, 1:64, 1:32, 1:16, 1:8, and 1:4). Refer to the red blood cell agglutination scoring criteria in the "AABB Technical Manual" to score the reaction results for each titer. Add up the agglutination scores corresponding to the seven titers to obtain the final agglutination score. The higher the agglutination score, the stronger 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 criteria are as follows, and the scoring results are as Figure 1 shown in Table 1.

[0120] 0 points: No agglutination, the solution is uniform, and there are no visible red blood cell clumps or agglutination phenomena;

[0121] 3 points: Suspected agglutination, very weak reaction, only a small amount of tiny particles or uncertain agglutination phenomena are visible in the solution;

[0122] 5 points: Weak agglutination, small and sparse red blood cell agglutination clusters are visible in the solution, and the background is relatively turbid;

[0123] 8 points: Moderate agglutination, medium-sized red blood cell agglutination clusters are visible in the solution, and the background is clearer.

[0124] 10 points: Strong agglutination, large red blood cell agglutination clusters are visible in the solution, and the background is clear and there are no free red blood cells.

[0125] 12 points: Extremely strong agglutination, the red blood cells are completely aggregated into a large mass, the solution is clear and there are no free red blood cells.

[0126] Table 1 Agglutination Intensity of Engineered Red Blood Cell Biomembranes by Different Red Blood Cell Release Fluids

[0127]

[0128] Note: Compared with Comparative Example 1, * in the table represents P＜ 0.05; ** represents P＜ 0.01; *** represents P＜ 0.001.

[0129] According to Figure 1 and Table 1, it can be seen that in Comparative Example 1, the agglutination score using a single reagent (Ca(NO 3 ) 2 ) was relatively low, with an average value of 33.00, indicating that it may have a certain negative impact on the agglutination result when lysing red blood cells. After introducing other reagents (such as KHCO 3 or Na 2 EDTA), although the agglutination score improved, the difference was not significant, and the average value remained at about 32.80. This indicates 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(NO 3 ) 2 +FBS), Example 2 (Ca(NO 3 ) 2 +KHCO3+FBS), and Example 3 (Ca(NO 3 ) 2 +Na 2 EDTA+FBS) all showed an obvious enhancement effect on the agglutination reaction, with average values reaching 49.40, 52.20, and 53.20 respectively. And Example 4 (Ca(NO 3 ) 2 +KHCO 3 +Na 2 EDTA+FBS) was the most prominent, with an average agglutination score as high as 59.60 and a standard deviation of only 0.89, showing a high degree of stability and consistency in the agglutination effect. Therefore, the introduction of FBS played a key promoting role in the agglutination reaction of the red blood cell membrane, especially when used in combination with other chemical reagents (such as KHCO 3 and Na 2 EDTA). The synergistic effect of the four components in Example 4 may have significantly enhanced the stability of the red blood cell membrane and optimized the conditions of the agglutination reaction, thus achieving the best agglutination effect.

[0130] Test Example 2

[0131] Integrity Detection

[0132] 1. Take the red blood cell membrane precipitate (RBC), intact red blood cell biomembrane (RBC bm ), and engineered red blood cell biomembrane (RBC bm@Fluo), were detected under bright field (BF) and ultraviolet fluorescence (UVFL), respectively. The results showed that the engineered red blood cell biomembrane (RBC bm @Fluo) emitted bright red fluorescence under ultraviolet fluorescence, and the fluorescence was evenly distributed ( Figure 2 ).

[0133] 2. Take the red blood cell membrane precipitate (RBC), intact red blood cell biomembrane (RBC bm ) and engineered red blood cell biomembrane (RBC bm @Fluo) obtained in Example 5 using the red blood cell lysate in Example 4, and detect them under a confocal microscope. Actin is red, tubulin is green, and yellow is the merged image of red and green. The results showed that the RBC, RBC bm and RBC bm @Fluo red blood cell membranes all remained intact, and the cytoskeleton structure was partially retained ( Figure 3 ).

[0134] 3. Take the red blood cell membrane precipitate (RBC), intact red blood cell biomembrane (RBC bm ) and engineered red blood cell biomembrane (RBC bm @Fluo) obtained in Example 5 using the red blood cell lysate in Example 4. In a conventional manner, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DIL) was used to label the phospholipid bilayer, and an anti-human CD47 antibody labeled with PE was used to label red blood cells. The confocal microscope was used to detect the expression of the phospholipid bilayer (Phospholipid Bilayer, PBL) and CD47 molecules on the surface of red blood cells. The results showed that the entire labeling process had a minimal impact on the phospholipid bilayer, the red blood cell membrane remained intact, and it was not a red blood cell membrane fragment ( Figure 4 ). Combining the conclusions of step 2, it can be determined that the engineered red blood cell biomembrane obtained in the present invention can maintain the integrity of red blood cell membrane antigens.

[0135] Test Example 3

[0136] Performance Detection

[0137] 1. Take the engineered red blood cell biomembrane (RBC bm @Fluo) and red blood cell membrane precipitate (RBC) obtained in Example 5, and use human ABO reverse typing red blood cell reagent (purchased from Shanghai Blood Biopharmaceutical Co., Ltd., denoted as RBC) respectively, and refer to the product instruction manual for blood group reverse typing detection. The results are as Figure 5As shown. According to Figure 5 It can be seen that the agglutination phenomenon caused by the blood group antigen-antibody reaction between RBC and RBC bm @Fluo is similar.

[0138] 2. Take the B-type engineered red blood cell biomembrane and B-type red blood cell membrane precipitate obtained in Example 5, use anti-A IgM antibody as the negative control, and detect the agglutination results of B-type red blood cells (denoted as RBC) and B-type RBCbm@Fluo (denoted as RBC bm @Fluo) reacting with anti-B IgM antibody at different titers (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) respectively. The results are as Figure 6 shown; among them, Figure 6 (-) in the figure is the negative control (anti-A IgM antibody).

[0139] According to Figure 6 It can be seen that the agglutination scores caused by the blood group antigen-antibody reaction between RBC and RBC bm @Fluo are consistent.

[0140] 3. Using the Bio-Layer Interferometry (BLI), measure the dissociation rate constant Kon, association rate constant Koff, and equilibrium dissociation constant (affinity) KD of the antigen-antibody reaction of the B-type red blood cell membrane precipitate (RBC), B-type intact red blood cell biomembrane (RBC bm ) and B-type engineered red blood cell biomembrane (RBC bm @Fluo) obtained in Example 5 with anti-B IgM antibody (purchased from Shanghai Blood Biopharmaceutical Co., Ltd.). The results are shown in Table 2.

[0141] Table 2 Detection results of antigen-antibody reaction

[0142]

[0143] According to Table 2, it can be seen that the equilibrium dissociation constants of RBC, RBC bm、 RBC bm @Fluo are all in the order of 10 -8 , and the preparation and Fluo labeling process of RBC bm do not affect the affinity of the antigen-antibody reaction.

[0144] Example 6

[0145] Lyophilized preservation

[0146] According to the procedure in Table 3, the engineered red blood cell biomembrane (RBCbm (@Fluo) was freeze-dried. The freeze-dried RBC bm @Fluo presented as a white, uniform, and loose powdery substance, and its average moisture content decreased to (4.37±0.32)%, meeting the requirements of effective freeze-drying operation. The freeze-dried RBC bm @Fluo was gently shaken in sterile deionized water for rehydration, and the freeze-dried preparation dissolved into a clear liquid without unexpected aggregation. The rehydration time was about 10 s.

[0147] Table 3 Freeze-drying procedures and temperature control steps for freeze-dried preservation

[0148]

[0149] Test Example 4

[0150] Performance detection

[0151] 1. Take the liquid of Example 6 (i.e., before freeze-drying), freeze-dried and rehydrated engineered red blood cell biomembranes (RBC bm @Fluo), and detect them under bright field (BF) and ultraviolet fluorescence (UVFL) respectively. The results show that the engineered red blood cell biomembranes in different states show uniform fluorescence under ultraviolet light ( Figure 7 ).

[0152] 2. Take the engineered red blood cell biomembranes (RBC bm @Fluo) in the liquid state of Example 6 (i.e., before freeze-drying) for low-temperature preservation at 4°C, and take the freeze-dried engineered red blood cell biomembranes (RBC bm @Fluo) of Example 6 for room-temperature preservation and low-temperature preservation at 4°C. Monitor the agglutination of RBC bm @Fluo in different states during the preservation process. The results are as Figure 8 shown. According to Figure 8 it can be seen that the agglutination score of the sample preserved at 4°C in the liquid state decreased by about 20% within 3 months, and decreased significantly by 70% at 6 months, basically losing the detection function. After 8 months, further monitoring was stopped; the freeze-dried state was preserved at room temperature, and there was no significant change in the reconstitution and agglutination score compared with before freeze-drying during the two-year preservation period.

[0153] Test Example 5

[0154] Clinical trial of blood group reverse typing detection

[0155] Taking the clinical microcolumn gel method as the comparison method, 641 clinical plasma samples were detected using the RBC bm @Fluo rehydrated after 7 days of freeze-dried preservation in Example 6. The detection results are shown in Tables 4 and 5.

[0156] Table 4 Qualitative Results of Blood Group Typing of 641

[0157]

[0158] Table 5 Clinical Trial Results of Blood Group Reverse Typing Detection

[0159]

[0160] It can be seen from Table 4 and Table 5 that by using RBC bm @Fluo detection, 188 cases were type A, 143 cases were type B, 48 cases were type AB, and 262 cases were type O, with a coincidence rate of 100%. The experimental results show that RBC bm @Fluo showed good accuracy in blood group reverse typing detection.

[0161] Example 7

[0162] Preparation of IgG-Sensitized Red Blood Cell Biomembrane Labeled with AIE Nanoparticles

[0163] 1. Preparation of IgG-Sensitized Red Blood Cells

[0164] Add 2 drops of RhD(+) packed red blood cells and 2 drops of IgG-type anti-D blood group typing reagent into a clean test tube, mix well, incubate at 37°C for 30 min, and keep mixing constantly during this period. Wash 6 - 8 times with physiological saline (1000×g, 1 min). After the last centrifugation, discard the supernatant, and use absorbent paper to absorb the residual liquid at the tube mouth to prepare sensitized packed red blood cells. Take another clean test tube, add 1 mL of physiological saline, and then add 30 μL of sensitized packed red blood cells to prepare a 3% IgG-type antibody-sensitized red blood cell saline suspension.

[0165] 2. Release of IgG-Sensitized Red Blood Cell Biomembrane

[0166] Using the red blood cell release solution of Example 4, mix the 3% IgG-type antibody-sensitized red blood cell saline suspension obtained in step 1 and the red blood cell release solution at a volume ratio of 1×10 10 red blood cells: 1 mL, mix well by vortex oscillator, then centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the precipitate;

[0167] Mix the precipitate 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) and the red blood cell release solution is 1:1, wash 3 times repeatedly to further remove the residual free hemoglobin, and finally prepare a complete red blood cell biomembrane (RBC bm ).

[0168] 3. Preparation of IgG-Sensitized Red Blood Cell Biomembrane Labeled with AIE Nanoparticles

[0169] Mix the red blood cell biomembrane prepared in step 2 with aggregation-induced emission (AIE) nanoparticles (purchased from the Institute for Advanced Studies, Shenzhen University) at a quantity ratio of 1:5, and couple the nanoparticles to the surface of the red blood cell membrane using Click Chemistry to obtain nanoparticle-labeled red blood cell biomembrane, that is, IgG-sensitized red blood cell biomembrane labeled with AIE nanoparticles (AIE@RBC bm ).

[0170] Test Example 6

[0171] Platelet Antibody Detection

[0172] 1. The experimental sample information is shown in Table 6, and the experimental environment is: temperature 26°C, relative humidity 47%.

[0173] Table 6 Experimental Sample Information

[0174]

[0175] 2. Platelet Antibody Detection Verification Steps

[0176] 1) Take out the microplate strips from the sachet as needed. The unused microplate strips can be stored in the sachet with desiccant. 2) Add one drop (50 μL) of screening platelet antigen to each well (patient serum wells) using a plastic pipette. Perform positive control (positive control well of platelet antibody detection kit, denoted as PC) and negative control (negative control well of platelet antibody detection kit, denoted as NC) tests for each microplate strip using the platelet antibody detection kit. 3) Centrifuge at 50 rcf for 5 min to fix the platelets to the surface of the microplate (continuous centrifugation). 4) Manually wash the plate three times with 150 μL of PBS / Tween 0.005% to remove unbound platelets. Add the washing solution drop by drop using a multi-channel pipette and let it stand for 10 seconds. After each wash, slowly pour out and gently tap to remove the PBS / Tween. 5) Add 2 drops (100 μL) of platelet antibody detection 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 well. The positive control is used to detect 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 min (seal the microplate during incubation). 9) Wash the plate 5 times manually with 150 μL of PBS / Tween according to the method in step 4). 10) Immediately add 1 drop of anti-IgG reagent of the platelet antibody detection kit to each well after washing. 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 gently shake. 12) Centrifuge the microplate at 200 rcf for 5 min (continuous centrifugation). 13) Visually interpret the results or use a reader to interpret the results.

[0177] 3. Detection of reference product compliance

[0178] (1) Using the 3 negative samples N1, N2, and N3 in Table 6 as negative reference products, perform the detection according to the method in step 2. The results show that the detection results of the 3 negative samples N1, N2, and N3 are all negative, and the compliance rate of the negative reference products is 100% ( Figure 9 ).

[0179] (2) Using the 3 strongly positive samples P1, P2, and P3 and the 3 weakly positive samples P4, P5, and P6 in Table 6 as positive reference products. Among them, P1, P3, and P6 all contain HLA + HPA antibodies. Perform the detection according to the method in step 2. The results show that the experimental results of P1, P3, and P6 are all positive, and the compliance rate of the positive reference products is 100% ( Figure 10 ).

[0180] 4. Detection of detection limit

[0181] Using the P1 sample containing HLA + HPA antibodies as a positive reference product, performing serial dilutions, and detecting according to the method in Step 2, the results showed that the P1 sample was still positive after being diluted 32-fold ( Figure 11 ).

[0182] 5. Repeatability test

[0183] Using the P1 sample containing HLA + HPA antibodies as a positive reference product and the N1 sample as a negative reference product, repeating the detection 10 times according to the method in Step 2. The experiments showed that the detection results of the N1 sample were all negative, and the detection results of the P1 sample were all positive, and the intensities were consistent ( Figure 12 ).

[0184] It can be seen from the above that the AIE@RBC obtained in Example 7 bm can replace the indicator red blood cells for platelet antibody detection and ensure the accuracy of platelet antibody detection.

[0185] Example 8

[0186] Preparation of RLNP nanoparticles labeled with A-type red blood cell membrane

[0187] 1. Isolation and washing of red blood cells

[0188] Isolate red blood cells from the A-type 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 3 times with physiological saline, discard the supernatant, and retain the red blood cell pellet for later use.

[0189] 2. Release of A-type red blood cell membrane

[0190] Using the red blood cell releasing solution in Example 4, mix the red blood cell pellet obtained in Step 1 with the red blood cell releasing solution at a volume ratio of 1×10 10 red blood cells: 1 mL. After thoroughly mixing by a vortex oscillator, centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the pellet;

[0191] Mix the pellet 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 releasing solution is 1:1, and wash repeatedly 3 times to further remove the residual free hemoglobin, and finally prepare a complete A-type red blood cell membrane (RBC bm ).

[0192] 3. Preparation of nanoparticles labeled with A-type red blood cell membrane

[0193] Mix the type A red blood cell biomembrane prepared in step 2 with red latex nanoparticles (RLNP) at a quantity ratio of 1:5, and couple the nanoparticles to the surface of the red blood cell membrane using click chemistry to obtain nanoparticle-labeled red blood cell biomembrane, namely engineered red blood cell biomembrane (A-RBC bm @RLNP).

[0194] Example 9

[0195] Preparation of black latex nanoparticle (BLNP)-labeled type B red blood cell biomembrane

[0196] 1. Isolation and washing of red blood cells

[0197] Isolate red blood cells from a type B 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 3 times with physiological saline, discard the supernatant, and retain the red blood cell pellet for later use.

[0198] 2. Release of type B red blood cell biomembrane

[0199] Using the red blood cell release solution of Example 4, mix the red blood cell pellet obtained in step 1 with the red blood cell release solution at a quantity-volume ratio of 1×10 10 red blood cells: 1 mL, mix well by vortex oscillator, then centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the pellet;

[0200] Mix the pellet 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 is 1:1, and wash repeatedly 3 times to further remove residual free hemoglobin, and finally prepare a complete type B red blood cell biomembrane (RBC bm ).

[0201] 3. Preparation of nanoparticle-labeled type B red blood cell biomembrane

[0202] Mix the type B red blood cell biomembrane prepared in step 2 with black latex nanoparticles (BLNP) at a quantity ratio of 1:5, and couple the nanoparticles to the surface of the red blood cell membrane using click chemistry to obtain nanoparticle-labeled red blood cell biomembrane, namely engineered red blood cell biomembrane (B-RBC bm @BLNP).

[0203] Test Example 7

[0204] Detection of Low Titer O Whole Blood (LTOWB)

[0205] (1) According to the titer standard of Low Titer O Whole Blood (LTOWB) in the United States (1:128), the A-RBC obtained in Example 8 bm @RLNP was used to replace A-type red blood cells for the detection of plasma titer. The results showed that when using A-RBC bm @RLNP to replace A-type red blood cells for plasma titer detection, the results were highly consistent with those of traditional A-type red blood cell detection. When the titer was higher than 1:128, visible agglutination reactions were observed; while when the titer was lower than 1:128, no agglutination phenomenon was seen. Thus, A-RBC bm @RLNP demonstrated good substitution performance in blood group antibody detection, providing a reliable and effective solution for blood group antibody detection.

[0206] (2) According to the titer standard of Low Titer O Whole Blood (LTOWB) in the United States (1:128), the B-RBC obtained in Example 9 bm @BLNP was used to replace B-type red blood cells for the detection of plasma titer. The results showed that when using B-RBC bm @RLNP to replace A-type red blood cells for plasma titer detection, the results were highly consistent with those of traditional B-type red blood cell detection. When the titer was higher than 1:128, visible agglutination reactions were observed; while when the titer was lower than 1:128, no agglutination phenomenon was seen. Thus, B-RBC bm @RLNP demonstrated good substitution performance in blood group antibody detection, providing a reliable and effective solution for blood group antibody detection.

[0207] Example 10

[0208] Preparation of PLNP Nanoparticle-Labeled Red Blood Cell Biomembrane

[0209] 1. Isolation and Washing of Red Blood Cells

[0210] Red blood cells were isolated from A / B / O-type whole blood samples. The whole blood was centrifuged at 1000 rpm for 5 min to obtain concentrated red blood cells. The concentrated red blood cells were washed 3 times with physiological saline, the supernatant was discarded, and the red blood cell precipitate was reserved for use.

[0211] 2. Release of Red Blood Cell Biomembrane

[0212] Using the red blood cell release solution of Example 4, the red blood cell precipitate obtained in Step 1 and the red blood cell release solution were mixed at a ratio of 1×10 10Red blood cells: Mix the quantity-volume ratio of 1 mL, and mix well by vortex oscillator. Then centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the precipitate.

[0213] Mix the precipitate 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 lysate is 1:1, and wash repeatedly 3 times to further remove residual free hemoglobin, and finally prepare a complete red blood cell biomembrane (RBC bm ).

[0214] 3. Preparation of nanoparticle-labeled red blood cell biomembrane

[0215] Mix the red blood cell biomembrane prepared in step 2 with purple latex nanoparticles (Purple Latex Nanoparticles, PLNP) at a quantity ratio of 1:5, and couple the nanoparticles to the surface of the red blood cell membrane by click chemistry to obtain nanoparticle-labeled red blood cell biomembrane, that is, engineered red blood cell biomembrane (PLNP@RBC bm ).

[0216] Test Example 8

[0217] Irregular antibody detection

[0218] Use the PLNP@RBC obtained in Example 10 bm to replace the traditional anti-screening red blood cell reagent for irregular antibody detection, and compare it with the conventional anti-screening red blood cell reagent. The detected antibodies 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 , P 1 , Xg a , Lu a , Lu b , Di a and other positive antibody standards, and the results are shown in Tables 7 and 8.

[0219] Table 7 Results of irregular antibody detection of conventional anti-screening red blood cells (human red blood cells)

[0220]

[0221] Note: In the table, "+" indicates positive and "-" indicates negative, the same below.

[0222] Table 8 PLNP@RBC bm Irregular antibody detection results

[0223]

[0224] It can be seen from Table 7 and Table 8 that PLNP@RBC bm -I, II, III (P-I, P-II, P-III) still retain the activities of blood group antigens such as 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 、P 1 、Xg a 、Lu a 、Lu b 、Di a etc., and its agglutination reaction is consistent with that of conventional anti-screening red blood cell reagents, verifying its feasibility as a substitute reagent in the detection of irregular antibodies.

[0225] Example 11

[0226] Preparation of MNP nanoparticle-labeled red blood cell biomembrane

[0227] 1. Isolation and washing of red blood cells

[0228] Isolate red blood cells from A / B type whole blood samples. Centrifuge the whole blood at 1000 rpm for 5 min to obtain concentrated red blood cells. Wash the concentrated red blood cells 3 times with physiological saline, discard the supernatant, and retain the red blood cell precipitate for later use.

[0229] 2. Release of red blood cell biomembrane

[0230] Using the red blood cell release solution of Example 4, mix the red blood cell precipitate obtained in Step 1 with the red blood cell release solution at a volume ratio of 1×10 10 red blood cells: 1 mL, mix well by vortex oscillator, then centrifuge at 7000 rpm for 10 min, discard the supernatant, and collect the precipitate;

[0231] Mix the precipitate 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 is 1:1, and wash repeatedly 3 times to further remove residual free hemoglobin, and finally prepare a complete red blood cell biomembrane (RBC bm ).

[0232] 3. Preparation of Nanoparticle-Labeled Red Blood Cell Biomembrane

[0233] Mix the red blood cell biomembrane prepared in Step 2 with Fe 3 O 4 magnetic nanoparticles (MNP) at a quantity ratio of 1:5, and couple the nanoparticles to the surface of the red blood cell membrane by Click Chemistry to obtain the nanoparticle-labeled red blood cell biomembrane, namely the engineered red blood cell biomembrane A-RBC bm @MNP and B-RBC bm @MNP. Mix A-RBC bm @MNP and B-RBC bm @MNP at a mass ratio of 1:1 to obtain the engineered red blood cell biomembrane RBC bm @MNP.

[0234] Test Example 9

[0235] Detection of IgM / IgG Anti-A and Anti-B Antibody Titers

[0236] Incubate 2 mg of RBC bm @MNP obtained in Example 11 with 2 mL of whole blood or plasma at room temperature for 5 min to form specific binding of blood group antibodies to the surface antigens of RBC bm @MNP. During this process, IgM / IgG anti-A and anti-B antibodies are effectively removed by magnetic pole adsorption. Then, the microcolumn gel method is used to detect the IgM / IgG anti-A and anti-B antibody titers in whole blood and plasma before and after adsorption, and the results are shown in Table 9.

[0237] Table 9 IgM / IgG Anti-A and Anti-B Antibody Titers in Whole Blood and Plasma

[0238]

[0239] As can be seen from Table 9, before adsorption, the titers of IgM / IgG anti-A and anti-B antibodies in the test results of 6 plasma samples were all higher than 1:128; the titers of IgM / IgG anti-A and anti-B antibodies in the test results of 6 whole blood samples were all higher than 1:128. After the immunoadsorption treatment with RBC bm @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 group antibodies.

[0240] Test Example 10

[0241] Neonatal Hemolysis Detection

[0242] Anti-human globulin test card (for detecting incomplete antibodies in fetuses (infants) with hemolytic disease of the newborn), add 50 μL of A-RBC bm @MNP red blood cell membrane suspension to each of wells 1 and 4; add 50 μL of B-RBC bm @MNP red blood cell membrane suspension to each of wells 2 and 5; add 50 μL of O-RBC bm @MNP red blood cell membrane suspension to each of wells 3 and 6. Add 50 μL of neonatal plasma to wells 1 to 3 respectively, and add 50 μL of neonatal red blood cell eluate to wells 4 to 6 respectively. Place the test card in an incubator at 37 °C and incubate for 15 min. Use a special centrifuge for microcolumn gel cards to centrifuge for 5 min (centrifuge at 900 rpm for 2 min and 1500 rpm for 3 min), take out the test card, and interpret the results. If the RBC bm @MNP agglutination clumps are located on the surface or in the gel, it is positive, indicating that there are antibodies corresponding to its antigens in the tested serum or the blood cells have been sensitized; if the RBC bm @MNP completely sediment to the bottom of the gel and form 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 have not been sensitized by antibodies.

[0243] As can be seen from the above content, the technical solution provided by the present invention can maintain the integrity of the red blood cell membrane structure, retain the key blood group antigens on its surface, avoid the problem of reduced antigenicity caused by red blood cell membrane fragmentation, and make the agglutination reaction that occurs in the engineered red blood cell biomembrane under the induction of antibodies visible and can be stored at room temperature for a long time.

[0244] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative improvement, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a structurally intact red blood cell membrane, characterized in that: The method comprises the following steps: uniformly mixing the release solution and the 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; The release solution includes 15-20 mM Ca(NO3)2 and 2-4% v / v fetal bovine serum; The pH value of the release solution is 7.2-7.4; The solvent of the release liquid is water; The volume ratio of the red blood cells to the release fluid is 1×10 10 Piece: 1mL; 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.

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

3. 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 prepared by the preparation method according to claim 1 or 2.

4. The engineered erythrocyte biofilm according to claim 3, characterized in that The mass ratio of the red blood cell membrane to the nanoparticles is 1:

5.

5. Use of the engineered erythrocyte biofilm according to claim 3 or 4 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.

6. The method for preserving the engineered erythrocyte biofilm according to claim 3 or 4, 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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