Composite antibody immunomagnetic bead as well as preparation method and application thereof

Abstract

The invention discloses a composite antibody immunomagnetic bead and a preparation method and application thereof, and the preparation method comprises the following steps: (1) cleaning a magnetic bead, and activating the magnetic bead with an activator; (2) adding a composite anti-erythrocyte antibody into the activated product obtained in the step (1) for antibody coupling; the composite anti-erythrocyte antibody at least comprises two anti-erythrocyte antibodies with different epitopes; (3) adding a magnetic bead sealing agent into the antibody coupling product obtained in the step (2) for sealing, and performing strong magnetic separation after the reaction is finished; and (4) adding a product obtained after strong magnetic separation in the step (3) into a freeze-drying buffer solution, uniformly dispersing, sub-packaging, adding a free red blood cell antibody, and performing vacuum freeze-drying to obtain the composite antibody immunomagnetic beads. The composite antibody immunomagnetic bead provided by the invention has excellent erythrocyte separation efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of immunomagnetic bead preparation, and in particular to a composite antibody immunomagnetic bead and a preparation method and application thereof. Background Art

[0002] Immunomagnetic cell sorting is an efficient and simple method for separating and purifying immune cells, and is currently widely used in various cell sorting. Immunomagnetic cell sorting is based on the ability of cell surface antigens to bind to specific monoclonal antibodies connected to magnetic beads. In an external magnetic field, cells connected to magnetic beads by antibodies are adsorbed and retained in the magnetic field. Cells without such surface antigens cannot bind to specific monoclonal antibodies connected to magnetic beads and therefore have no magnetism and do not stay in the magnetic field, thus allowing the cells to be separated.

[0003] Blood samples in clinical examinations need to be centrifuged to obtain serum or plasma according to the requirements of the test items. The main reason is that the presence of red blood cells will interfere with the antibody capture of antigens in immunological detection methods, and the rupture of red blood cells will cause a large amount of intracellular hemoglobin, enzymes, ions and other cell contents to be released, causing strong interference, greatly reducing the specimen quality and detection accuracy.

[0004] In clinical testing, the main method for separating red blood cells from whole blood to obtain serum or plasma is centrifugation. In addition to requiring centrifugal equipment, centrifugation may also cause blood cells to rupture and reduce sample quality, and the centrifugation time is about 5 minutes or even longer. For many acute disease detection projects such as inflammation, myocardial infarction, etc., rapid and accurate blood sample processing and testing is conducive to gaining a treatment time window for patients.

[0005] The Chinese patent document with application number CN200710305363.6 discloses a method for separating red blood cells from whole blood using magnetic microspheres coated with lectins or anti-red blood cell antibodies, but the separation time takes 10 minutes. The Chinese patent document with application number CN202010490280.4 discloses an immunomagnetic bead for separating red blood cells and its preparation method and application. The patent uses vacuum drying to prepare freeze-dried immunomagnetic beads, and the red blood cell adsorption separation time in a 500uL whole blood sample is 2 minutes. The Chinese patent document with application number 202210965075.8 uses magnetic beads less than 1 micron coupled to antibody red blood cells, and freeze-dries the immunomagnetic beads using liquid nitrogen. The separation time of 100 microliters of whole blood sample using 1mg of immunomagnetic beads is 85.5s. As the volume of whole blood increases, the proportional increase in immunomagnetic bead freeze-dried microspheres leads to a longer separation time.

[0006] In the above prior art, a large amount of magnetic beads is used when separating red blood cells from whole blood. 100 microliters of whole blood requires at least 1 mg of magnetic beads or even more. The magnetic beads are expensive and not conducive to industrialization. In addition, the separation time of red blood cells from whole blood is too long. Summary of the invention

[0007] The invention provides a composite antibody immunomagnetic bead and a preparation method and application thereof. The composite antibody immunomagnetic bead has excellent red blood cell separation efficiency.

[0008] The technical solution of the present invention is as follows: A method for preparing composite antibody immunomagnetic beads comprises the following steps: (1) After washing the magnetic beads, activate them with an activator; (2) adding the composite anti-erythrocyte antibody to the activated product obtained in step (1) for antibody coupling; the composite anti-erythrocyte antibody comprises at least two anti-erythrocyte antibodies with different antigenic epitopes; (3) adding a magnetic bead blocking agent to the antibody coupling product obtained in step (2) for blocking, and performing strong magnetic separation after the reaction is completed; (4) The product after strong magnetic separation in step (3) is added to a freeze-drying buffer and dispersed evenly. After packaging, free anti-erythrocyte antibody is added, and vacuum freeze-dried to obtain composite antibody immunomagnetic beads.

[0009] Preferably, the particle size of the magnetic beads is 1-5 μm.

[0010] More preferably, the magnetic bead particle size is 2-3 μm. When the magnetic bead particle size is 2-3 μm, the red blood cell separation time is shorter and the amount of recovered plasma is larger.

[0011] Preferably, the magnetic beads are microspheres coated with magnetic substances, and have amino groups, carboxyl groups or hydroxyl groups on their surfaces.

[0012] Preferably, the activator is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate and chloramine-T.

[0013] Preferably, in step (1), the mass ratio of the activator to the magnetic beads is 1:1-100.

[0014] Preferably, in step (1), the activation time is 5-30 min.

[0015] Step (1) comprises: washing the magnetic beads, adding an activator solution and mixing evenly, keeping the magnetic beads in a suspended state, activating the beads at room temperature for 5-30 minutes, performing strong magnetic separation after the reaction is completed, and removing the supernatant.

[0016] Preferably, in step (2), the mass ratio of the composite anti-erythrocyte antibody to the magnetic beads is 1:1-50.

[0017] Step (2) comprises: adding the composite anti-erythrocyte antibody to the activated product obtained in step (1) and mixing evenly, keeping the magnetic beads in suspension, reacting at room temperature for 3-7 hours, performing strong magnetic separation, and removing the supernatant.

[0018] Preferably, the magnetic bead blocking agent is at least one of 0.1-10wt% bovine serum albumin solution, 0.01-5wt% casein solution and 1-50mM glycine.

[0019] Step (3) comprises: adding the magnetic bead blocking agent to the antibody coupling product obtained in step (2) and mixing evenly, keeping the magnetic beads in a suspended state, reacting at room temperature for 5-20 hours, performing strong magnetic separation after the reaction is completed, and removing the supernatant.

[0020] In step (4), the mass ratio of free anti-erythrocyte antibody to magnetic beads is 1:1-100.

[0021] The present invention also provides composite antibody immunomagnetic beads prepared by the above preparation method.

[0022] The present invention also provides an application of the composite antibody immunomagnetic beads in separating red blood cells in blood, comprising: adding 1-2 mg of the composite antibody immunomagnetic beads to 500-1000 μL of whole blood sample, mixing evenly, and magnetically attracting in a magnetic field for 10-90 seconds.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses composite anti-erythrocyte antibodies with different antigenic epitopes to prepare composite antibody immunomagnetic beads, thereby improving the efficiency of immunomagnetic beads in capturing erythrocytes; free anti-erythrocyte antibodies are added before freeze-drying, and when separating erythrocytes, the free anti-erythrocyte antibodies pre-enrich erythrocytes before the immune reaction, thereby improving the efficiency of erythrocyte separation.

[0024] The immunomagnetic beads of the present invention are suitable for whole blood samples with a wide range of hematocrit. Using 1 mg of composite antibody immunomagnetic beads can solve the separation problem of hematocrit ≤ 50%. Since the reference value of hematocrit for normal people is: 0.40-0.50 for males and 0.35-0.45 for females, it can cover most of the usage scenarios of whole blood separation of red blood cells. When the hematocrit is greater than 50%, increasing the amount of magnetic beads can also solve the problem. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0026] Example 1

[0027] The method for preparing composite antibody immunomagnetic beads comprises the following steps: (1) Magnetic bead cleaning The original carboxyl magnetic beads had a concentration of 1.0 wt% (10 mg / mL) and a particle size of 2.8 μm. 1 mL was taken and washed with a coupling buffer solution of 0.1 M MES (pH=6) and then subjected to strong magnetic separation. The supernatant was discarded and repeated twice.

[0028] (2) Magnetic bead activation Add 0.1 mL (10 mg / mL, prepared with coupling buffer) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and 0.1 mL (10 mg / mL, prepared with coupling buffer), add coupling buffer to a total volume of 1.0 mL, and mix thoroughly; keep the magnetic beads suspended, activate the reaction at room temperature for 30 minutes, and then remove the supernatant.

[0029] (3) Antibody-coupled magnetic beads Add 100 μg of anti-erythrocyte antibody 1 (Beijing Biolab Technology Co., Ltd., K23981-WKD) and 100 μg of anti-erythrocyte antibody 2 (Beijing Biolab Technology Co., Ltd., K23981), add coupling buffer to a total volume of 1.0 mL, and mix thoroughly; keep the magnetic beads suspended, react at room temperature for 6 hours, and then remove the supernatant.

[0030] (4) Magnetic bead sealing Add 1 mL of magnetic bead coupling blocking reagent (50 mM Tris + 1% BSA + 0.1% Tween 20, pH 7.4), mix thoroughly, keep the magnetic beads suspended, and block overnight at room temperature. After the reaction is completed, use strong magnetic separation, discard the supernatant, and obtain the immunomagnetic beads.

[0031] (5) Magnetic bead freeze-drying Add the freeze-dried buffer solution to the immunomagnetic bead precipitate obtained after the blocking reaction to make the final concentration of the immunomagnetic beads 10 mg / mL, and mix thoroughly. Package the immunomagnetic beads in 100 μL (i.e., 1 mg) portions, add 100 μg of free anti-erythrocyte antibody 3 (Shanghai Xinfan Biotechnology Co., Ltd., XFKT2104) to the packaged magnetic beads, and vacuum freeze-dry for 30 hours to obtain the immunofreeze-dried magnetic beads for red blood cells.

[0032] The antigenic epitopes of anti-erythrocyte antibody 1, anti-erythrocyte antibody 2, and anti-erythrocyte antibody 3 are different.

[0033] (1) Results of procalcitonin (PCT) chemiluminescence detection kit The same whole blood sample was centrifuged (4°C, 2500rpm / min, 6min) and 600μL was added with 1 mg of erythrocyte separation immune freeze-dried magnetic beads to fully dissolve and mix, and then magnetically absorbed in a strong magnetic field for 90s to separate whole blood erythrocytes and take plasma for testing. The signal was detected using a fully automatic chemiluminescence analyzer, and the test was repeated 3 times in parallel, and the luminescence value was recorded. The results are shown in Table 1.

[0034] Table 1

[0035] (2) Detection results of interleukin-6 (IL-6) chemiluminescence detection kit The same whole blood sample was centrifuged (4°C, 2500rpm / min, 6min) and 600μL was added with 1mg of erythrocyte separation immune freeze-dried magnetic beads to fully dissolve and mix, and then magnetically attracted in a strong magnetic field for 90s to separate the whole blood erythrocytes and take plasma for testing. The signal was detected using a fully automatic chemiluminescence analyzer, and the test was repeated 3 times in parallel, and the luminescence value was recorded. The results are shown in Table 2.

[0036] Table 2

[0037] The whole blood samples processed by the present invention have little difference in luminescence value detection results with those processed by the centrifugation method, indicating that the plasma obtained from the whole blood samples processed by the present invention has a good correlation with the plasma obtained by the centrifugation method, and reliable and accurate detection results can be obtained.

[0038] (3) Whole blood separation test with different hematocrits 600 μL of whole blood with different hematocrits was taken, 1 mg of lyophilized magnetic beads for separating red blood cells was added to each portion, the mixture was fully reconstituted and mixed, and the mixture was magnetically attracted in a strong magnetic field. The separation time for recovering 200 μL of plasma was recorded. The results are shown in Table 3.

[0039] Table 3

[0040] Since separation time is affected not only by hematocrit but also by age, whole blood viscosity, individual differences and other components, the relationship between separation time and hematocrit is not linear.

[0041] The present invention uses anti-erythrocyte antibodies with different antigenic epitopes to prepare erythrocyte immunomagnetic beads, thereby improving the efficiency of erythrocyte capture by immunomagnetic beads. Free anti-erythrocyte antibodies with a third antigenic epitope are added before freeze-drying, and the free anti-erythrocyte antibodies pre-enrich erythrocytes before the immune reaction, thereby improving the efficiency of erythrocyte separation.

[0042] Embodiment 2-4 The carboxyl magnetic beads with a particle size of 2.8 μm in Example 1 were replaced with carboxyl magnetic beads with particle sizes of 1 μm, 3 μm, and 5 μm, respectively, and other conditions remained unchanged.

[0043] 600 μL of whole blood with different hematocrits was taken, 1 mg of red blood cell separation immune freeze-dried magnetic beads was added to each portion, fully reconstituted and mixed, and magnetically adsorbed in a strong magnetic field. The separation time and the amount of recovered plasma were recorded. The results are shown in Table 4.

[0044] Table 4

[0045] The 2.8 μm magnetic beads used in the present invention can achieve similar effects using 1 μm and 5 μm magnetic beads, but the separation effect for high hematocrit is not ideal, the separation time will be longer, even up to 10 minutes, and the amount of plasma recovered is less than 200 μL.

[0046] Comparative Example 1-2 Comparative Example 1 is a method of coupling carboxyl magnetic beads with anti-erythrocyte antibody 1 to obtain immunomagnetic beads, and Comparative Example 2 is a method of coupling carboxyl magnetic beads with anti-erythrocyte antibody 2 to obtain immunomagnetic beads.

[0047] The whole blood sample volume was 600 μL. 1 mg of isolated red blood cell immune freeze-dried magnetic beads was added to each sample and fully reconstituted and mixed. The samples were magnetically attracted in a strong magnetic field and the amount of plasma recovered was recorded in 90 seconds. The results are shown in Table 5.

[0048] Table 5

[0049] Comparative Example 3 In step (5), free red blood cell antibody 3 is not added before the magnetic beads are freeze-dried, and other conditions are the same as in Example 1.

[0050] The volume of whole blood sample was 600 μL. 1 mg of separated red blood cell immune freeze-dried composite magnetic beads was added to each sample. The samples were fully reconstituted and mixed, and then magnetically attracted in a strong magnetic field. The amount of plasma recovered in 90 seconds was recorded. "0" represents that red blood cells and plasma were not separated, "+" represents the addition of free anti-red blood cell antibody 3, and "-" represents the non-addition of free anti-red blood cell antibody 3. The results are shown in Table 6.

[0051] Table 6

[0052] The efficiency of capturing red blood cells by the immune magnetic beads prepared in the embodiment of the present invention is improved, and the separation time of the magnetic beads is shortened. Using 1 mg of magnetic beads, 600 μL of whole blood can be separated within 90 seconds to obtain 200 μL of plasma. Multiple projects can be tested simultaneously, which reduces the use of magnetic beads and reduces costs.

[0053] The immunomagnetic beads of the present invention are suitable for a wider range of hematocrit: this patent uses 1 mg of immunomagnetic beads to solve the separation problem of hematocrit ≤ 50%. Since the reference value of normal human hematocrit is: male: 0.40-0.50, female: 0.35-0.45, it can cover most of the whole blood separation red blood cell usage scenarios. When the hematocrit is greater than 50%, increasing the amount of magnetic beads can also solve the problem.

[0054] The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing composite antibody immunomagnetic beads, characterized in that: The following steps are involved: (1) After washing the magnetic beads, activate them with an activator; (2) adding the composite anti-erythrocyte antibody to the activated product obtained in step (1) for antibody coupling; the composite anti-erythrocyte antibody comprises at least two anti-erythrocyte antibodies with different antigenic epitopes; (3) adding a magnetic bead blocking agent to the antibody coupling product obtained in step (2) for blocking, and performing strong magnetic separation after the reaction is completed; (4) The product after strong magnetic separation in step (3) is added to a freeze-drying buffer and dispersed evenly. After packaging, free anti-erythrocyte antibody is added, and vacuum freeze-dried to obtain composite antibody immunomagnetic beads.

2. The method for preparing composite antibody immunomagnetic beads according to claim 1, characterized in that: The particle size of the magnetic beads is 1-5 μm.

3. The method for preparing composite antibody immunomagnetic beads according to claim 1, characterized in that: The activator is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, glutaraldehyde, periodate and chloramine-T; the mass ratio of the activator to the magnetic beads is 1:1-100.

4. The method for preparing composite antibody immunomagnetic beads according to claim 1, characterized in that: In step (2), the mass ratio of the composite anti-erythrocyte antibody to the magnetic beads is 1:1-50.

5. The method for preparing composite antibody immunomagnetic beads according to claim 1, characterized in that: The magnetic bead sealing agent is at least one of 0.1-10wt% bovine serum albumin solution, 0.01-5wt% casein solution and 1-50mM glycine.

6. The method for preparing composite antibody immunomagnetic beads according to claim 1, characterized in that: In step (4), the mass ratio of free anti-erythrocyte antibody to magnetic beads is 1:1-100.

7. A composite antibody immunomagnetic bead, characterized in that: The preparation method is described in any one of claims 1 to 6.

8. Use of the composite antibody immunomagnetic beads according to claim 7 in separating red blood cells from blood.

9. The use according to claim 8, characterized in that: include: Add 1-2 mg of composite antibody immunomagnetic beads to 500-1000 μL of whole blood sample, mix well, and magnetically absorb in a magnetic field for 10-90 seconds.

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