Composite antibody immunomagnetic beads with annular magnetic attraction structure, preparation method and application thereof

Abstract

The application relates to the technical field of immunomagnetic bead preparation, in particular to a composite antibody immunomagnetic bead with a ring-shaped magnetic attraction structure and a preparation method and application thereof. After being cleaned and activated, the magnetic bead is coupled with at least two kinds of composite anti-red blood cell antibodies with different antigen epitopes, and after being sealed, free anti-red blood cell antibodies are added to prepare a vacuum freeze-dried product; through a matched ring-shaped magnetic attraction structure, a plurality of trapezoidal permanent magnets are assembled according to a Halbach array, the periphery is fixed through a ring-shaped stainless steel sleeve, and a uniform radiation ring-shaped magnetic field is formed; when applied, a mixed solution of whole blood and the magnetic bead is placed in the center hole of the structure and is magnetically attracted for 10-90 seconds; the composite antibody immunomagnetic bead with the ring-shaped magnetic attraction structure and the preparation method and application thereof can significantly improve the red blood cell capturing and separation efficiency, shorten the separation time, reduce the magnetic bead consumption, facilitate plasma suction, cover most clinical scenes, and reduce the industrialization cost.

Description

Technical Field

[0001] This invention relates to the field of immunomagnetic bead preparation technology, specifically to composite antibody immunomagnetic beads with a ring magnetic attraction structure, their preparation method, and applications. Background Technology

[0002] Immunomagnetic bead sorting is a highly efficient and simple method for the separation and purification of immune cells, and it has been widely used in various cell sorting processes. In clinical examinations, blood samples need to be centrifuged to obtain serum or plasma because red blood cells can interfere with the capture of antibody antigens in immunological detection, and the intracellular material released by the rupture of red blood cells can reduce sample quality and detection accuracy.

[0003] The mainstream clinical centrifugation method has drawbacks, including the need for specialized equipment, the risk of blood cell rupture, and processing times exceeding 5 minutes, making it difficult to meet the rapid detection needs of acute conditions such as inflammation and myocardial infarction. Existing immunomagnetic bead separation technology for red blood cells also has significant shortcomings, generally involving large quantities of magnetic beads and excessively long separation times, hindering industrial application. Furthermore, the magnetic field devices used in existing technologies cannot form a uniform radial magnetic field, leading to localized accumulation of magnetic beads during magnetic adsorption, resulting in incomplete red blood cell separation. Additionally, plasma aspiration after magnetic adsorption is inconvenient, failing to meet the demands for rapid and efficient blood sample processing in the detection of acute conditions such as inflammation and myocardial infarction.

[0004] Therefore, in view of the above situation, there is an urgent need to develop composite antibody immunomagnetic beads with ring magnetic attraction structure, as well as their preparation methods and applications, in order to overcome the shortcomings in current practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide composite antibody immunomagnetic beads with a ring magnetic attraction structure, their preparation method and application, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Composite antibody immunomagnetic beads with a ring magnetic attraction structure include composite antibody immunomagnetic beads and a ring magnetic attraction structure; The composite antibody immunomagnetic beads are magnetic beads coupled with at least two anti-erythrocyte antibodies with different antigenic epitopes, and free anti-erythrocyte antibodies are added before freeze-drying. The annular magnetic attraction structure is assembled from multiple trapezoidal permanent magnets in a Heilbeck array. The trapezoidal permanent magnets are surrounded by annular stainless steel sleeves, and a circular hole is opened in the center of the annular magnetic attraction structure.

[0007] As a further aspect of the present invention: the particle size of the magnetic beads is .

[0008] As a further aspect of the present invention, the free anti-erythrocyte antibody and the composite anti-erythrocyte antibody have different antigenic epitopes.

[0009] As a further aspect of the present invention: the diameter of the central circular hole of the annular magnetic structure matches the outer diameter of a commonly used blood reaction tube.

[0010] A method for preparing composite antibody immunomagnetic beads with a ring magnetic attraction structure as described above includes the following steps: (1) Cleaning of magnetic beads: The magnetic beads are cleaned with a coupling buffer solution and the supernatant is discarded after strong magnetic separation; (2) Activation of magnetic beads: Add activator solution to the cleaned magnetic beads to carry out the activation reaction, and obtain activated magnetic beads after strong magnetic separation; (3) Antibody conjugation: At least two anti-erythrocyte antibodies with different antigenic epitopes are added to the activated magnetic beads to carry out a conjugation reaction, and the antibody-conjugated magnetic beads are obtained after strong magnetic separation; (4) Magnetic bead blocking: A blocking agent is added to the antibody-conjugated magnetic beads to carry out the blocking reaction, and the blocked immunomagnetic beads are obtained after strong magnetic separation; (5) Vacuum freeze-drying: The blocked immunomagnetic beads were dispersed in freeze-drying buffer, free anti-erythrocyte antibodies were added, and after aliquoting, they were freeze-dried under vacuum to obtain composite antibody immunomagnetic beads; (6) Assembly of the ring magnetic structure: Multiple trapezoidal permanent magnets are arranged in a Heilbeck array and covered with a ring stainless steel sleeve to obtain the ring magnetic structure.

[0011] As a further aspect of the present invention: in step (2), the activator is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate, or chloramine-T; In step (4), the sealing agent is at least one of bovine serum albumin solution, casein solution or glycine solution.

[0012] As a further aspect of the present invention: in step (2), the mass ratio of the activator to the magnetic beads is 1:1-100; In step (3), the mass ratio of the composite anti-erythrocyte antibody to the magnetic beads is 1:1-50; In step (5), the mass ratio of the free anti-erythrocyte antibody to the magnetic beads is 1:1-100.

[0013] A method for applying composite antibody immunomagnetic beads with a ring magnetic attraction structure as described above includes the following steps: (1) Mix the whole blood sample with the composite antibody immunomagnetic beads to ensure that the magnetic beads are in full contact with the whole blood sample; (2) Insert the reaction tube of the mixture into the central circular hole of the annular magnetic structure for magnetic separation; (3) After magnetic attraction is completed, the plasma in the central area of ​​the reaction tube is drawn to complete the red blood cell separation.

[0014] As a further aspect of the present invention: the dosage of the composite antibody immunomagnetic beads is 1-2 mg, and the volume of the whole blood sample is... .

[0015] As a further aspect of the present invention: in step (2), the magnetic separation time is 10-90s.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention prepares immunomagnetic beads by using at least two composite anti-erythrocyte antibodies with different antigenic epitopes, and adds free anti-erythrocyte antibodies before freeze-drying, which effectively improves the capture efficiency of immunomagnetic beads for erythrocytes. The free anti-erythrocyte antibodies can pre-enrich erythrocytes before the immune reaction, further improving the separation efficiency. The annular magnetic attraction structure designed in this invention works synergistically with composite antibody immunomagnetic beads. The structure consists of multiple trapezoidal permanent magnets arranged in a Heilbeck array and fixed by an annular stainless steel sleeve. It can form a uniform and outwardly radiating annular magnetic field, which significantly improves the speed and uniformity of magnetic attraction separation and avoids the problem of incomplete separation caused by local accumulation of magnetic beads. Meanwhile, the central circular hole design of the annular magnetic suction structure can match the outer diameter of commonly used blood reaction tubes, making it convenient to directly draw the separated plasma from the center of the reaction tube after magnetic suction, greatly simplifying the operation steps; This invention requires only 1-2 mg of composite antibody immunomagnetic beads and can complete the process within 10-90 seconds. For the separation of red blood cells from whole blood samples, only 1mg of magnetic beads is needed to process whole blood samples with a hematocrit of ≤50%, covering most clinical application scenarios. The low amount of magnetic beads used and high separation efficiency reduce industrialization costs and meet the rapid detection needs of acute disease testing. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the process flow and magnetic field arrangement for separating red blood cells using composite antibody immunomagnetic beads in an embodiment of the present invention.

[0018] Figure 2 This is an example diagram of the use of the ring magnet in an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0021] Please see Figure 1 and Figure 2 This invention provides a composite antibody immunomagnetic bead with a ring magnetic structure, its preparation method, and its application. By modifying the magnetic beads with composite anti-erythrocyte antibodies and adding free anti-erythrocyte antibodies before freeze-drying, combined with the ring magnetic structure, rapid and efficient separation of erythrocytes in whole blood samples can be achieved, solving the problems of large amount of magnetic beads and long separation time in the prior art.

[0022] I. Preparation of Composite Antibody Immunomagnetic Beads 1.1 Raw material preparation Magnetic beads: Selected with a particle size of Microspheres coated with magnetic materials, wherein the surface of the microspheres has at least one functional group selected from amino, carboxyl, or hydroxyl groups; preferably, the particle size is [missing information]. Carboxylated magnetic beads, with a particle size range, offer the advantages of fast separation speed and high plasma recovery during erythrocyte separation.

[0023] Activator: at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate and chloramine-T.

[0024] Composite anti-erythrocyte antibodies: Contain at least two anti-erythrocyte antibodies with different antigenic epitopes, used to improve the capture efficiency of magnetic beads for erythrocytes.

[0025] Magnetic bead blocking agent: at least one of 0.1-10wt% bovine serum albumin solution, 0.01-5wt% casein solution and 1-50mM glycine.

[0026] Lyophilization buffer: Used to disperse magnetic beads and ensure the stability of the magnetic beads after lyophilization.

[0027] Free anti-erythrocyte antibodies: Anti-erythrocyte antibodies with different antigenic epitopes from those of the complex anti-erythrocyte antibodies are selected for pre-enrichment of erythrocytes.

[0028] 1.2 Preparation steps (1) Cleaning of magnetic beads Take carboxyl magnetic beads with a concentration of 1.0 wt% (10 mg / mL), wash the magnetic beads with 0.1 MMES coupling buffer (pH=6), perform strong magnetic separation after washing, discard the supernatant, and repeat the washing operation twice to remove impurities on the surface of the magnetic beads.

[0029] (2) Activation of magnetic beads Add an activator solution to the cleaned magnetic beads, with a mass ratio of activator to magnetic beads of 1:1-100; specifically, add 0.1 mL of 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and 0.1 mL of 10 mg / mL N-hydroxysuccinimide solution, and add coupling buffer solution to bring the total volume to 1.0 mL, and mix thoroughly.

[0030] Keep the magnetic beads in suspension and activate them at room temperature for 5-30 minutes. After the reaction, separate them with strong magnetic force and remove the supernatant to obtain activated magnetic beads. This step activates the functional groups on the surface of the magnetic beads with an activator, providing reaction sites for subsequent antibody conjugation.

[0031] (3) Antibody conjugation A composite anti-erythrocyte antibody was added to activated magnetic beads, with a mass ratio of composite anti-erythrocyte antibody to magnetic beads of 1:1-50; specifically, it was added... Anti-erythrocyte antibody 1 and Add anti-erythrocyte antibody 2 to the conjugate buffer solution to a total volume of 1.0 mL and mix thoroughly.

[0032] The magnetic beads were kept in suspension and reacted at room temperature for 3-7 hours. After the reaction, they were separated by strong magnetic separation and the supernatant was removed to obtain antibody-conjugated magnetic beads. By modifying the magnetic beads with anti-erythrocyte antibodies with two different antigenic epitopes, multi-target binding to erythrocytes can be achieved, thereby improving the capture efficiency of erythrocytes by the magnetic beads.

[0033] (4) Magnetic bead sealing Add magnetic bead blocking agent to the antibody-conjugated magnetic beads, specifically add 1 mL of magnetic bead conjugation blocking reagent (50 mM Tris + 1% BSA + 0.1% Tween 20, pH 7.4), mix thoroughly, keep the magnetic beads in suspension, and react at room temperature for 5-20 h.

[0034] After the reaction is complete, strong magnetic separation is performed, and the supernatant is discarded to obtain the blocked immunomagnetic beads. This step uses a blocking agent to cover the sites on the surface of the magnetic beads that have not bound to antibodies, avoiding non-specific adsorption of impurities by the magnetic beads and ensuring the specificity of subsequent separation.

[0035] (5) Vacuum freeze drying Add the blocked immunomagnetic bead precipitate to the lyophilization buffer, disperse evenly, and adjust the immunomagnetic bead concentration to 10 mg / mL; according to each sample... (i.e., 1mg magnetic beads) are dispensed, and free anti-erythrocyte antibody is added to the dispensed magnetic beads at a mass ratio of 1:1-100; specifically, the following steps are performed: Free anti-erythrocyte antibody 3 has a different antigenic epitope from both anti-erythrocyte antibody 1 and anti-erythrocyte antibody 2.

[0036] After dispensing, the product is freeze-dried under vacuum for 30 hours to obtain the lyophilized composite antibody immunomagnetic beads. Free anti-erythrocyte antibodies are added before freeze-drying to pre-enrich erythrocytes during subsequent erythrocyte separation, thereby further improving separation efficiency.

[0037] II. Composition and Characteristics of Ring-Shaped Magnetic Structures The annular magnetic attraction structure of the present invention, which works synergistically with composite antibody immunomagnetic beads to achieve rapid magnetic separation of red blood cells, has the following specific composition and characteristics: (1) Structural composition The annular magnetic attraction structure consists of multiple trapezoidal permanent magnets and an annular stainless steel sleeve. The multiple trapezoidal permanent magnets are assembled according to the arrangement order of the Hellbeck array, and the magnetic field directions of adjacent trapezoidal permanent magnets are set at a specific angle to form a uniform and outwardly radiating annular magnetic field. The annular stainless steel sleeve covers the periphery of the trapezoidal permanent magnets to fix the assembly structure of the permanent magnets and prevent the permanent magnets from shifting during the magnetic attraction process.

[0038] The annular magnetic structure has a central hole with a diameter that matches the outer diameter of a commonly used blood reaction tube, which allows for stable insertion of the reaction tube and ensures full contact between the tube wall and the annular magnetic field.

[0039] Specifically, the trapezoidal permanent magnet can have 8 or 12 petals, with an outer circumference diameter of 30.00 mm and an inner circumference diameter of 10.00 mm; the height of the trapezoidal permanent magnet is not less than 10 mm, with a typical value of 20 mm; the bottom width of the trapezoidal surface is 44 mm, the bottom width is 85 mm, and the height is 84 mm, making it suitable for 1.5 ml centrifuge tubes or tubular containers with a diameter of 10 mm.

[0040] (2) Structural characteristics This ring-shaped magnetic attraction structure, based on the arrangement of a Hellbeck array, can significantly improve the uniformity and intensity of the magnetic field, which is distributed radially outward. When the reaction tube is inserted into the central hole, the magnetic field can act evenly on the whole blood sample inside the reaction tube, causing the complex of the composite antibody immunomagnetic beads and red blood cells to be uniformly adsorbed on the inner wall of the reaction tube, avoiding local accumulation and thus improving the magnetic attraction speed and uniformity. At the same time, the design of the central hole can directly expose the central area of ​​the reaction tube, facilitating subsequent plasma aspiration operations.

[0041] III. Application of Composite Antibody Immunomagnetic Beads Combined with Ring Magnetic Structures 3.1 Application Steps Pick Add 1-2 mg of lyophilized composite antibody immunomagnetic beads to a whole blood sample, fully reconstitute and mix, ensuring the magnetic beads are in full contact with the whole blood sample.

[0042] The mixed reaction tube is inserted into the central hole of the annular magnetic structure, and magnetic separation is performed using the annular magnetic field. The magnetic attraction time is 10-90 seconds. Under the action of the annular magnetic field, the complex of magnetic beads and red blood cells can be quickly and uniformly adsorbed on the inner wall of the reaction tube, avoiding the problem of incomplete separation caused by local aggregation of magnetic beads in traditional magnetic fields.

[0043] After magnetic suction is completed, use a pipette to directly aspirate the separated plasma in the center area of ​​the reaction tube to complete the red blood cell separation operation of the whole blood sample.

[0044] 3.2 Application Advantages In this application scheme, the multi-target binding characteristics of the composite antibody immunomagnetic beads, the pre-enrichment of free anti-erythrocyte antibodies, and the highly efficient magnetic attraction characteristics of the ring magnetic structure form a synergistic effect; when the particle size of the composite antibody immunomagnetic beads is When only 1mg of magnetic beads is needed, whole blood samples with hematocrit ≤50% can be separated within 10-90s, covering most clinical whole blood sample usage scenarios; when hematocrit >50%, the amount of magnetic beads can be appropriately increased to meet the separation requirements. For particle size , and The composite antibody immunomagnetic beads can achieve rapid separation within 10-90 seconds when the hematocrit is ≤40%; when the hematocrit is ≥45%, the magnetic adsorption time needs to be appropriately extended or the amount of magnetic beads used needs to be increased to ensure the separation effect. Compared with the existing technology, this solution significantly shortens the separation time (from minutes to seconds), reduces the amount of magnetic beads used, and lowers the industrialization cost.

[0045] IV. Effect Verification Example 1: Preparation and application verification of composite antibody immunomagnetic beads Prepare composite antibody immunomagnetic beads according to the steps in 1.2 above, and select the appropriate particle size. The free anti-erythrocyte antibody used was anti-erythrocyte antibody 3; the prepared magnetic beads were combined with a ring magnetic attraction structure for the following verification: (1) Verification of plasma test results The same whole blood sample was taken and divided into two groups: one group was centrifuged (4℃, 2500 rpm / min, 6 min), and the other group was... 1 mg of composite antibody immunomagnetic beads were added to whole blood and magnetically attracted for 90 seconds using a ring magnetic structure. Plasma from both groups was collected for chemiluminescence detection of procalcitonin (PCT) and interleukin-6 (IL-6). The tests were performed in parallel three times. The results are shown in Tables 1 and 2.

[0046] Table 1. Chemiluminescence detection results of procalcitonin (PCT) Table 2. Chemiluminescence detection results of interleukin-6 (IL-6) As shown in Tables 1 and 2, the plasma test results of whole blood samples treated with immunomagnetic beads combined with the ring magnetic adsorption method were not significantly different from those obtained by centrifugation, indicating that the plasma obtained by this method can meet the accuracy requirements of clinical testing.

[0047] (2) Separation and verification of different hematocrit samples Pick Whole blood samples with different hematocrits were each treated with 1 mg of a composite antibody immunomagnetic bead. The beads were then magnetically separated using a ring-shaped magnetic structure, and the separation was recorded and recovered. The plasma separation time and results are shown in Table 3.

[0048] Table 3 Separation time for samples with different hematocrit levels As shown in Table 3, the separation time increases slightly with the increase of hematocrit, but it is still within 90 seconds, which can meet the needs of rapid clinical testing.

[0049] Examples 2-4: Comparison of the effects of magnetic beads with different particle sizes In Example 1 Carboxylated magnetic beads replaced with , , Carboxyl magnetic beads, with other preparation and application conditions remaining unchanged, were used. Whole blood samples with different hematocrits were separated and verified. The separation time and plasma recovery volume were recorded. The results are shown in Table 4.

[0050] Table 4. Comparison of separation effects of magnetic beads with different particle sizes As shown in Table 4, the particle size is Magnetic beads (belonging to) The preferred particle size range showed the best separation effect in all hematocrit samples, with the separation time remaining stable within 10-90s and the plasma recovery volume remaining stable, fully meeting the magnetic adsorption time range specified in claim 10.

[0051] Particle size , and The magnetic beads can achieve rapid separation within 10-90 seconds when the hematocrit is ≤40%; however, when the hematocrit is ≥45%, the separation time is significantly prolonged and the plasma recovery volume decreases. Therefore, , and The application of magnetic beads in samples with high hematocrit requires adjustment of magnetic adsorption time or amount of magnetic beads according to actual needs.

[0052] Comparative Examples 1-2: Effects of Monoclonal Antibody Magnetic Beads Comparative Example 1 was an immunomagnetic bead obtained by conjugating carboxyl magnetic beads only with anti-erythrocyte antibody 1, and Comparative Example 2 was an immunomagnetic bead obtained by conjugating carboxyl magnetic beads only with anti-erythrocyte antibody 2; [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] 1 mg of magnetic beads were added to whole blood samples with different hematocrit values. The beads were then magnetically attracted for 90 seconds using a ring-shaped magnetic structure. The plasma recovery volume was recorded. The results are shown in Table 5.

[0053] Table 5. Comparison of the effects of monoclonal antibody magnetic beads and composite antibody magnetic beads. As shown in Table 5, the recovery of plasma from monoclonal antibody magnetic beads was significantly reduced in samples with high hematocrit, while the recovery of composite antibody magnetic beads was stable. The plasma indicates that the multi-target binding properties of the composite anti-erythrocyte antibody can effectively improve the capture efficiency of magnetic beads for erythrocytes.

[0054] Comparative Example 3: Effects of not adding free anti-erythrocyte antibodies In the preparation steps of the magnetic beads in Comparative Example 3, free anti-erythrocyte antibody 3 was not added before freeze-drying, and other conditions were the same as in Example 1; 1 mg of magnetic beads were added to whole blood samples with different hematocrit values. The beads were then magnetically attracted for 90 seconds using a ring-shaped magnetic structure. The plasma recovery volume was recorded. The results are shown in Table 6.

[0055] Table 6. Effect of free anti-erythrocyte antibodies on separation efficiency As shown in Table 6, the amount of plasma recovered by magnetic beads without the addition of free anti-erythrocyte antibodies was significantly reduced in samples with hematocrit ≥ 40%, and in samples with hematocrit ≥ 50%, separation of erythrocytes and plasma could not even be achieved. This indicates that adding free anti-erythrocyte antibodies before freeze-drying can further improve the separation efficiency by pre-enriching erythrocytes.

[0056] In summary, the composite antibody immunomagnetic beads with a ring magnetic attraction structure of the present invention achieve rapid and efficient separation of red blood cells from whole blood samples by modifying the magnetic beads with composite anti-erythrocyte antibodies and adding free anti-erythrocyte antibodies before lyophilization, combined with the synergistic effect of the ring magnetic attraction structure. The invention has the advantages of low magnetic bead usage, short separation time and stable plasma recovery, which can meet the needs of rapid clinical testing and is suitable for industrial application.

[0057] It should be noted that, in this invention, although the specification describes the embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A composite antibody immunomagnetic bead with a ring-shaped magnetic attraction structure, characterized in that, Including composite antibody immunomagnetic beads and ring magnetic attraction structures; The composite antibody immunomagnetic beads are magnetic beads coupled with at least two anti-erythrocyte antibodies with different antigenic epitopes, and free anti-erythrocyte antibodies are added before freeze-drying. The annular magnetic attraction structure is assembled from multiple trapezoidal permanent magnets in a Heilbeck array. The trapezoidal permanent magnets are surrounded by annular stainless steel sleeves, and a circular hole is opened in the center of the annular magnetic attraction structure.

2. The composite antibody immunomagnetic bead with ring magnetic attraction structure according to claim 1, characterized in that, The particle size of the magnetic beads is .

3. The composite antibody immunomagnetic bead with annular magnetic attraction structure according to claim 1, characterized in that, The free anti-erythrocyte antibody and the complex anti-erythrocyte antibody have different antigenic epitopes.

4. The composite antibody immunomagnetic bead with annular magnetic attraction structure according to claim 1, characterized in that, The diameter of the central circular hole in the annular magnetic structure matches the outer diameter of a commonly used blood reaction tube.

5. The method for preparing the composite antibody immunomagnetic beads with annular magnetic attraction structure according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Cleaning of magnetic beads: The magnetic beads are cleaned with a coupling buffer solution and the supernatant is discarded after strong magnetic separation; (2) Activation of magnetic beads: Add activator solution to the cleaned magnetic beads to carry out the activation reaction, and obtain activated magnetic beads after strong magnetic separation; (3) Antibody conjugation: At least two anti-erythrocyte antibodies with different antigenic epitopes are added to the activated magnetic beads to carry out a conjugation reaction, and the antibody-conjugated magnetic beads are obtained after strong magnetic separation; (4) Magnetic bead blocking: A blocking agent is added to the antibody-conjugated magnetic beads to carry out the blocking reaction, and the blocked immunomagnetic beads are obtained after strong magnetic separation; (5) Vacuum freeze-drying: The blocked immunomagnetic beads were dispersed in freeze-drying buffer, free anti-erythrocyte antibodies were added, and after aliquoting, they were freeze-dried under vacuum to obtain composite antibody immunomagnetic beads; (6) Assembly of the ring magnetic structure: Multiple trapezoidal permanent magnets are arranged in a Heilbeck array and covered with a ring stainless steel sleeve to obtain the ring magnetic structure.

6. The method of claim 5, wherein the magnetic beads are prepared by mixing the antibody with the magnetic material in a solution, and then drying the mixture. In step (2), the activator is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate, or chloramine-T; In step (4), the sealing agent is at least one of bovine serum albumin solution, casein solution or glycine solution.

7. The method for preparing composite antibody immunomagnetic beads with a ring-shaped magnetic attraction structure according to claim 5, characterized in that, In step (2), the mass ratio of the activator to the magnetic beads is 1:1-100; In step (3), the mass ratio of the composite anti-erythrocyte antibody to the magnetic beads is 1:1-50; In step (5), the mass ratio of the free anti-erythrocyte antibody to the magnetic beads is 1:1-100.

8. A method for applying composite antibody immunomagnetic beads with a ring-shaped magnetic attraction structure as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Mix the whole blood sample with the composite antibody immunomagnetic beads to ensure that the magnetic beads are in full contact with the whole blood sample; (2) Insert the reaction tube of the mixture into the central circular hole of the annular magnetic structure for magnetic separation; (3) After magnetic attraction is completed, the plasma in the central area of ​​the reaction tube is drawn to complete the red blood cell separation.

9. The method according to claim 8, wherein the method is characterized by, The amount of the composite antibody immunomagnetic beads is 1-2 mg, and the volume of the whole blood sample is .

10. The method of claim 8, wherein the magnetic beads are conjugated with a magnetic ring structure. In step (2), the time for the magnetic separation is 10-90 s; the particle size of the composite antibody immunomagnetic beads is .

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