Magnetic bead for eliminating anti-CD47 monoclonal antibody drug, preparation method of reagent and detection method

Abstract

The invention discloses a magnetic bead for eliminating an anti-CD47 monoclonal antibody drug, a preparation method of a reagent and a detection method, the immunomagnetic bead comprises a carboxyl magnetic bead located in the immunomagnetic bead and a human CD47 recombinant protein wrapping the periphery of the carboxyl magnetic bead, and the particle size of the immunomagnetic bead is 100 nanometers; according to the method, only one round of adsorption is needed, the serum magnetic bead mixed solution can be placed on the magnetic frame for magnetic separation after full vortex mixing, interference can be completely removed, the time is very short, and detection can be completed within 1 min.

Description

Technical Field

[0001] The invention belongs to the field of anti-human globulin detection in blood transfusion compatibility detection, and particularly relates to a preparation method and a detection method of magnetic beads and reagents for eliminating anti-CD47 monoclonal antibody drugs. Background Art

[0002] CD47 is a new target for cancer immune checkpoint therapy. The recently developed Hu5F9-G4, TJC4, HX009, and IBI188 are all anti-CD47 monoclonal antibody drugs (referred to as anti-CD47 mAbs) for the treatment of lymphoma and advanced malignant tumors. However, since blood samples from patients containing anti-CD47 mAbs need to undergo an indirect anti-human globulin test, false positives will be generated, interfering with blood type antibody detection and cross-matching experiments, affecting the safety and efficiency of blood transfusions.

[0003] Anti-human globulin test: also known as anti-human globulin test, has been widely used to detect irregular blood type antibodies on the surface of red blood cells or irregular blood type antibodies in serum. It is used to diagnose autoimmune hemolytic diseases, transfusion reaction investigations and cross-matching experiments. The principle of the anti-human globulin test is to add anti-human globulin reagent to the saline suspension of red blood cells that have been sensitized with irregular antibodies. The irregular blood type antibodies on the surface of red blood cells react specifically with the antibodies in the anti-human globulin reagent and agglutinate. Anti-human globulin tests can be divided into direct tests and indirect tests. The purpose of the direct anti-human globulin test is to check for irregular blood type antibodies on the surface of red blood cells, and the purpose of the indirect anti-human globulin test is to check whether there are free irregular blood type antibodies in the serum.

[0004] Anti-CD47 monoclonal antibody drugs: In recent years, tumor treatment has tended to study targeted and precise treatment plans. Among them, immunotherapy, such as the use of immune checkpoint inhibitors to target specific cell surface molecules (such as CTLA-4, PD-1, CD38, etc.) has been successfully used in the clinical treatment of various tumors and has shown strong therapeutic effects. Studies have shown that almost all tumor cells and tissues highly express CD47, which is three times that of corresponding normal cells and tissues. CD47 is a "self" signal, which means "don't eat me." Through the CD47 signal, tumor cells effectively avoid the phagocytic effect of macrophages. Anti-CD47 monoclonal antibodies entered the Phase III clinical trial stage in China in September 2020, which means that more patients in my country will use this antibody, and according to the National Drug Clinical Trial Publicity Information Platform, clinical trials of this drug are still underway. Recently developed humanized anti-CD47 monoclonal antibody drugs such as Hu5F9-G4, TJC4, HX009, IBI188, etc. can block this "don't eat me" signal, allowing macrophages to perform phagocytosis, thereby promoting the clearance of tumor cells. Since CD47 molecules are also expressed in large quantities on the red blood cell membrane, anti-CD47 monoclonal antibodies can bind to CD47 molecules on RBCs. After the subjects receive anti-CD47 monoclonal antibody treatment, the drug will bind to the patient's red blood cells or reagent red blood cells, interfering with the results of irregular antibody screening tests. At the same time, there is a risk of clinically significant blood type antibodies being masked, thereby affecting pre-transfusion compatibility testing, affecting the timeliness and effectiveness of transfusion therapy, and threatening the transfusion safety of patients receiving anti-CD47 monoclonal antibody treatment.

[0005] As the research and development of anti-CD47 monoclonal antibody drugs gradually advances, finding and establishing methods to eliminate their interference in transfusion compatibility testing has become a reality and urgent task for transfusion medicine. The main response strategies are as follows. Some studies have reported that the use of antiglobulin reagents Gamma-clone and Immucor capture-R solid phase agglutination method that lacks the detection of IgG4 is a faster method, which can better remove Hu5F9-G4 and other interferences in pre-transfusion testing and cross-matching. If the anti-CD47 monoclonal antibody used by the patient is not IgG4, then this method cannot effectively eliminate the interference of anti-CD47 monoclonal antibody drugs on transfusion compatibility testing. There are also studies reporting the use of red blood cell and platelet adsorption methods to eliminate the adverse interference of anti-CD47 monoclonal antibody drugs on blood type antibody screening and cross-matching laboratory tests, but it needs to be repeatedly absorbed three to four times, which is not only time-consuming and laborious, but also red blood cells and platelets themselves are relatively precious resources, resulting in a waste of blood resources. In summary, the advantages and disadvantages of the current methods for eliminating CD47 monoclonal antibody interference in transfusion compatibility testing are outlined.

[0006] 1) Red blood cell absorption method: Generally, red blood cells of blood donors with O type and other Rh antigen typing as CCDee, ccDEE, and ccdee are used to absorb CD47 monoclonal antibody in the plasma of the subject. Generally, 3 to 4 times of adsorption are required. If the patient has other blood type antibodies, the blood type antibodies will also be absorbed, resulting in missed detection. Moreover, this operation is time-consuming and labor-intensive, delaying the patient's blood use, and is not conducive to the development of the hospital's blood transfusion department.

[0007] 2) Platelet absorption method: Generally, multiple O-type platelets are collected by machine and mixed with the same volume of plasma of the subject to absorb the anti-CD47 monoclonal antibody. Although platelets can be used to absorb anti-CD47 monoclonal antibodies in serum, irregular antibodies can be avoided from being missed, but platelets can absorb other platelet antibodies, so platelet antibody testing should be completed before absorption. Since it is difficult to obtain a large number of platelets, when the titer of drug antibodies is too high, it is difficult to completely remove the interference effect.

[0008] 3) Gamma-clone and solid phase agglutination method: Hu5F9-G4 monoclonal antibody belongs to IgG4 type. Using Gamma-clone, which lacks the anti-human globulin test for IgG4 antibody detection, or Immucor Capture-R solid phase agglutination method can better remove the interference of this drug. Although there are fewer IgG4 type irregular antibodies, there is also a risk of missing IgG4 type irregular antibodies. And with the development of anti-CD47 monoclonal antibody drugs, there will be more other types of anti-CD47 monoclonal antibody drugs. However, this method is only applicable to IgG4 type anti-CD47 monoclonal antibody drugs. Summary of the invention

[0009] The purpose of the present invention is to provide a preparation method and a detection method of magnetic beads and reagents for eliminating anti-CD47 monoclonal antibody drugs, so as to solve the problem that blood samples of patients containing anti-CD47 monoclonal antibodies produce false positives in anti-human globulin tests, interfere with blood type antibody detection and cross-matching experiments, and affect the safety and efficiency of blood transfusion.

[0010] The present invention adopts the following technical scheme: an immunomagnetic bead for eliminating interference in anti-CD47 monoclonal antibody drug detection, comprising: carboxyl magnetic beads located inside and human CD47 recombinant proteins wrapped around the carboxyl magnetic beads, and the particle size of the immunomagnetic beads is 100 nanometers.

[0011] The invention discloses a storage solution for immunomagnetic beads, wherein the storage solution is a PBS storage buffer solution containing 0.05% sodium azide and 0.05% ProClin 300.

[0012] An immunomagnetic bead reagent comprises immunomagnetic beads and a storage solution for the immunomagnetic beads.

[0013] A method for preparing an immunomagnetic bead reagent, comprising:

[0014] Step 1: Weigh the carboxyl magnetic beads and place them in a centrifuge tube. Wash the carboxyl magnetic beads with MES buffer and discard the supernatant.

[0015] Step 2: Add MES buffer and vortex to mix.

[0016] Step 3: Add EDC activator and place the centrifuge tube on a temperature-controlled shaker to maintain room temperature for reaction.

[0017] Step 4: Add human CD47 recombinant protein and quickly place in a temperature-controlled shaker for incubation.

[0018] Step 5: Add blocking agent for blocking.

[0019] Step 6: Add preservation solution to wash the magnetic beads, discard the supernatant, and use the preservation solution to make up the volume of the immunomagnetic beads.

[0020] Further, among them,

[0021] The reaction conditions of step 3 are: 24°C-26°C, rotation speed 250 rpm, reaction time 40 min;

[0022] The reaction conditions of step 4 are: 24°C-26°C, rotation speed 250rpm, reaction time 12h,

[0023] The sealing conditions of step 5 are: 24°C-26°C, rotation speed 250 rpm, reaction time 4h.

[0024] Furthermore, the mass ratio of carboxyl magnetic beads: human CD47 recombinant protein is 200:1; the blocking agent is 3% BSA + 3% PEG 2000;

[0025] The pH of the MES buffer is 7.4, and the storage time in step 7 is 14 days.

[0026] Further, steps 3 and 4 are both performed by placing the support on a temperature-controlled shaker, and placing the support includes:

[0027] The lower end of the fixed clamping block is clamped by the temperature-controlled shaker, so that the placement bracket moves under the drive of the temperature-controlled shaker.

[0028] The lower side of the fixed plate is fixedly connected to the upper end of the fixed clamping block.

[0029] The placing frame is tilted downward from left to right and is formed by four connecting rods connected end to end. The four connecting rods are a left connecting rod, a right connecting rod, a front connecting rod, and a rear connecting rod. The front connecting rod and the rear connecting rod are fixedly connected to the two ends of the fixing plate respectively. A plurality of limiting holes are opened on the left connecting rod from left to right, and a plurality of limiting grooves are evenly opened on the upper side of the right connecting rod.

[0030] A plurality of positioning rods are arranged through corresponding limiting holes, and the right ends of the positioning rods are fixedly connected with sealing blocks. Each sealing block is arranged in a one-to-one correspondence with each limiting groove, and the area between the sealing block and each limiting groove is used to place the centrifuge tube, so that the bottom of the centrifuge tube is against the limiting groove, and the cover of the centrifuge tube is against the sealing block, and the sealing block generates a rightward thrust on the cover of the centrifuge tube to prevent the cover from loosening when moving on the shaking table, so that the centrifuge tube moves in an inclined state to prevent liquid from leaking from the top of the centrifuge tube.

[0031] Furthermore, placing the stent also includes:

[0032] Multiple centrifuge support cylinders are evenly spaced along the direction of the fixed plate and corresponding to the limit grooves. They are formed by an arc-shaped support plate on the left and a circular support cylinder on the right, which are integrally connected. The arc-shaped support plate and the circular support cylinder are coaxially arranged. The bottom of the arc-shaped support plate is fixed on the upper side of the fixed plate, and the right end of the circular support cylinder is fixedly connected to the left side of the right connecting rod. Each arc-shaped support plate is used to support the middle part of the centrifuge tube, and each circular support cylinder is used to limit the bottom of the centrifuge tube, thereby preventing the centrifuge tube from moving in the vertical direction.

[0033] Furthermore, each positioning rod is threadedly connected with a positioning bolt, and each positioning bolt is used to fix the positioning rod after the sealing block is against the top of the centrifuge tube to prevent the positioning rod from moving left and right.

[0034] A method for eliminating interference in anti-human globulin detection of blood, comprising:

[0035] Take a predetermined amount of immunomagnetic bead reagent and place it in a centrifuge tube.

[0036] The supernatant was separated by magnetic separation and discarded.

[0037] Add serum sample to the centrifuge tube containing only immunomagnetic beads and mix thoroughly.

[0038] After 1 min of magnetic separation, serum samples were taken for cassette anti-human globulin test using anti-human globulin reagent;

[0039] The concentration of the immunomagnetic beads in the immunomagnetic bead reagent used was 1 mg / 10 μL serum sample.

[0040] The beneficial effects of the present invention are:

[0041] 1. The present invention labels human CD47 recombinant protein on carboxyl magnetic beads, so the immunomagnetic beads can specifically bind to free anti-CD47 monoclonal antibodies in serum, thereby achieving the effect of eliminating its interference. In addition, the present invention only requires one round of adsorption. After sufficient vortex mixing, the serum magnetic bead mixed solution can be placed on a magnetic rack for magnetic separation to completely remove interference. In addition, the time is very short, and the detection can be completed in 1 minute.

[0042] 2. Compared with the previous methods, the present invention is easy to operate and does not require centrifugation. The steps are quick and more importantly, the free anti-CD47 monoclonal antibody is bound to the solid phase magnetic beads and completely separated from the test serum. This eliminates it more thoroughly and avoids the influence of immune complexes on subsequent tests. In addition, the immunomagnetic beads are solid phase and will not have a dilution effect on other tests in the serum, thus avoiding the missed detection of some trace target test objects.

[0043] 3. Due to its specific binding, the present invention does not affect the detection of other blood type antibodies before blood transfusion, such as irregular blood type antibodies, thus avoiding missed detection;

[0044] 4. The human CD47 recombinant protein used for labeling in the immunomagnetic beads of the present invention is easy to commercialize and obtain, and the protein is the extracellular region of the CD47 antigen, which can not only bind to IgG4 antibodies, but also specifically bind to various types of anti-CD47 monoclonal antibodies, making the immunomagnetic beads more widely used. After labeling, the immunomagnetic beads are easy to store and can be used as a reagent for a short period of time;

[0045] 5. The present invention can prevent anti-CD47 monoclonal antibody drugs from interfering with anti-human globulin reaction and avoid false positive test results;

[0046] 6. The immunomagnetic beads of the present invention can specifically bind to the anti-CD47 monoclonal antibody in the serum, thereby achieving the effect of eliminating interference, and then being applied in anti-human globulin detection to avoid false positives caused by anti-CD47 monoclonal antibodies. The immunomagnetic beads can separate the interfering targets from the serum through magnetic separation to avoid the potential influence of the complex formed by the human CD47 recombinant protein and the anti-CD47 monoclonal antibody on subsequent detection. The immunomagnetic bead method can eliminate interference in the solid phase and avoid the dilution effect on the specimen. The immunomagnetic beads can be widely used to eliminate the interference of various anti-CD47 monoclonal antibodies such as Hu5F9-G4, TJC4, HX009, IBI188 and other drugs, as well as not limited to IgG4 monoclonal antibodies, i.e., various IgG-type anti-CD47 monoclonal antibody drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The antibody coupling rate and reagent protein coupling amount of the MES buffer gradient pH value of the present invention;

[0048] Figure 2The results of the anti-human globulin test of the present invention after the magnetic beads labeled with buffers of different pH values ​​absorbed the patient's serum;

[0049] Figure 3 The protein coupling rate and protein coupling amount after different amounts of protein are added to the label of the present invention;

[0050] Figure 4 The results of the anti-human globulin test of the present invention after different amounts of protein-coupled magnetic beads absorbed the patient's serum;

[0051] Figure 5 The effect of the immunomagnetic beads using different blocking agents on the detection of irregular antibodies in the present invention;

[0052] Figure 6 To optimize the storage time of the immunomagnetic beads of the present invention;

[0053] Figure 7 To optimize the usage of the immunomagnetic beads of the present invention;

[0054] Figure 8 To optimize the incubation time between the immunomagnetic beads and the specimen of the present invention;

[0055] Fig. 9 The serological experiment of the present invention is to analyze the interference effect of anti-CD47 monoclonal antibody drugs on the detection of cassette anti-human globulin;

[0056] Fig.10 The present invention is a serological experiment that uses immunomagnetic beads to eliminate the interference of anti-CD47 monoclonal antibody drugs on cassette anti-human globulin detection;

[0057] Fig.11 It is a schematic diagram of the structure of placing the support of the present invention;

[0058] Fig.12 A diagram showing the use of the present invention to place a stent;

[0059] Fig.13 This is an electron microscope observation picture of the immunomagnetic beads prepared by coupling carboxyl magnetic beads with cell line OVCAR3 lysate protein in the present invention;

[0060] Fig.14 The results of indirect immunofluorescence staining of OVCAR3 using CD47 antibody and FITC-binding affinity pure goat anti-mouse IgG in the present invention;

[0061] Fig.15 The results of flow cytometry detection of the expression of OVCAR3, CD47 in red blood cells and platelets in the present invention.

[0062] Among them, 1. fixed clamping block; 2. fixed plate; 3. left connecting rod; 4. right connecting rod; 5. front connecting rod; 6. rear connecting rod; 7. positioning rod; 8. arc-shaped support plate; 9. circular support cylinder; 10. positioning bolt; 11. sealing block; 12. limit groove. DETAILED DESCRIPTION

[0063] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0064] The invention discloses an immunomagnetic bead for eliminating interference in anti-CD47 monoclonal antibody drug detection, comprising: carboxyl magnetic beads located inside and human CD47 recombinant proteins wrapped around the carboxyl magnetic beads, wherein the particle size of the immunomagnetic bead is 100 nanometers.

[0065] The invention also discloses a preservation solution for the immunomagnetic beads, which is a PBS preservation buffer solution containing 0.05% sodium azide and 0.05% ProClin 300.

[0066] The invention also discloses an immunomagnetic bead reagent, comprising immunomagnetic beads and a storage solution for the immunomagnetic beads.

[0067] The present invention also discloses a method for preparing an immunomagnetic bead reagent, comprising:

[0068] Step 1: Weigh the carboxyl magnetic beads and place them in a centrifuge tube. Wash the carboxyl magnetic beads with MES buffer and discard the supernatant.

[0069] Step 2: Add MES buffer and vortex to mix.

[0070] Step 3: Add EDC activator and place the centrifuge tube on a temperature-controlled shaker to maintain room temperature for reaction.

[0071] Step 4: Add human CD47 recombinant protein and quickly place in a temperature-controlled shaker for incubation.

[0072] Step 5: Add blocking agent for blocking.

[0073] Step 6: Add preservation solution to wash the magnetic beads, discard the supernatant, and use the preservation solution to make the volume of the immunomagnetic beads constant.

[0074] The reaction conditions of step 3 are: 24°C-26°C, rotation speed 250rpm, reaction time 40min;

[0075] The reaction conditions of step 4 are: 24°C-26°C, rotation speed 250rpm, reaction time 12h,

[0076] The sealing conditions of step 5 are: 24°C-26°C, rotation speed 250 rpm, reaction time 4h.

[0077] The mass ratio of carboxyl magnetic beads to human CD47 recombinant protein was 200:1; and the blocking agent was 3% BSA+3% PEG2000.

[0078] Wherein, the pH of the MES buffer is 7.4, and the storage time in step 7 is 14 days.

[0079] Among them, steps 3 and 4 are both performed by placing a bracket on the temperature-controlled shaking table, and the bracket includes: a fixed clamping block 1, a fixed plate 2, a placement frame, and multiple positioning rods 7.

[0080] The lower end of the fixed clamping block 1 is clamped by the temperature-controlled shaker, so that the placement bracket moves under the drive of the temperature-controlled shaker. The lower side of the fixed plate 2 is fixedly connected to the upper end of the fixed clamping block 1. The placement frame is arranged downwardly from left to right. The placement frame is composed of four connecting rods connected end to end. The four connecting rods are respectively a left connecting rod 3, a right connecting rod 4, a front connecting rod 5, and a rear connecting rod 6. The front connecting rod 5 and the rear connecting rod 6 are respectively fixedly connected to the two ends of the fixed plate 2. A plurality of limit holes are opened on the left connecting rod 3 from left to right, and a plurality of limit grooves 12 are evenly opened on the upper side of the right connecting rod 4.

[0081] A plurality of positioning rods 7 are arranged through corresponding limiting holes, and the right ends of the plurality of positioning rods 7 are fixedly connected with sealing blocks 11, and each sealing block 11 is arranged one by one with each limiting groove 12, and the area between the sealing block 11 and each limiting groove 12 is used to place the centrifuge tube, so that the bottom of the centrifuge tube is against the limiting groove 12, and the cover of the centrifuge tube is against the sealing block 11, and the sealing block 11 generates a rightward thrust on the cover of the centrifuge tube to prevent the cover from loosening when moving on the shaking table, so that the centrifuge tube moves in an inclined state to prevent liquid from leaking from the top of the centrifuge tube.

[0082] The placement bracket also includes: multiple centrifugal support cylinders, which are evenly spaced along the direction of the fixed plate 2 and corresponding to each limit groove 12, and are formed by an arc-shaped support plate 8 located on the left and a circular support cylinder 9 located on the right, which are integrally connected. The arc-shaped support plate 8 and the circular support cylinder 9 are coaxially arranged, and the bottom of the arc-shaped support plate 8 is fixed on the upper side of the fixed plate 2. The right end of the circular support cylinder 9 is fixedly connected to the left side of the right connecting rod 4. Each arc-shaped support plate 8 is used to support the middle part of the centrifuge tube, and each circular support cylinder 9 is used to limit the bottom of the centrifuge tube, thereby preventing the centrifuge tube from moving in the vertical direction.

[0083] Each positioning rod 7 is threadedly connected with a positioning bolt 10, and each positioning bolt 10 is used to fix the positioning rod 7 after the sealing block 11 is against the top of the centrifuge tube to prevent the positioning rod 7 from moving left and right.

[0084] The present invention also discloses a method for eliminating interference in anti-human globulin detection of blood, comprising:

[0085] Take a predetermined amount of immunomagnetic bead reagent and place it in a centrifuge tube.

[0086] The supernatant was separated by magnetic separation and discarded.

[0087] Add serum sample to the centrifuge tube containing only immunomagnetic beads and vortex to mix.

[0088] After 1 min of magnetic separation, serum samples were taken for cassette anti-human globulin test using anti-human globulin reagent;

[0089] The concentration of the immunomagnetic beads in the immunomagnetic bead reagent used was 1 mg / 10 μL serum sample.

[0090] The anti-human globulin test of the present invention is referred to as an indirect anti-human globulin test.

[0091] Carboxyl magnetic beads were purchased from Wuxi Biomag Biotechnology Co., Ltd.

[0092] Human CD47 recombinant protein was purchased from Beijing Sino Biological Technology Co., Ltd.

[0093] Prepare MES buffer: prepare 1 L of MES buffer with a pH of 6 and a concentration of 0.05 M. Weigh 9.75 g of MES and dissolve it in 500 mL of ultrapure water. Then adjust the pH to 7.4 with 1 M NaOH solution overnight and make up to 1 L.

[0094] Prepare 10 mg / mL EDC activator: weigh 10 mg EDC and dissolve it in 1 mL MES buffer.

[0095] Prepare the blocking agent: weigh 0.3 g bovine serum albumin (BSA) and 300 μL PEG2000 and add 9.7 mL MES buffer to dissolve. Prepare the preservation solution: weigh 0.05 g sodium azide and 50 μL ProClin 300 and add 100 mL PBS buffer to dissolve.

[0096] The above chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0097] 1. Determination of the optimal conditions for coupling carboxyl magnetic beads to antibodies

[0098] Protein coupling efficiency is an important indicator to measure the quality of immunomagnetic beads. The coupling efficiency is closely related to the type of carboxyl magnetic beads and the important parameters involved in the binding process of the nanoparticle antibody complex, such as the antibody concentration during the binding process between the antibody and the carboxyl magnetic beads, the choice of coupling buffer during binding, the pH of the buffer, the blocking conditions, etc. At the same time, the optimization of these conditions plays an important role in the formation of stable immunomagnetic beads, and the appropriate coupling efficiency is conducive to the elimination of subsequent interference.

[0099] 2. Choice of protein coupling method

[0100] Selection of protein coupling method. There are usually two methods for coupling proteins to carboxyl groups, non-covalent coupling method (physical adsorption method) and covalent coupling method (chemical coupling method). In the physical adsorption method, antibodies are coated by electrostatic adsorption and hydrophobic interaction with the surface of nanoparticles; in the chemical coupling method, chemical groups on the surface of nanoparticles react with groups on antibodies to form chemical bonds for coating. The present invention selects a more stable chemical coupling method, and the complete labeling process after specific optimization is as follows:

[0101] Shake the magnetic beads and pipette 10mg of carboxyl magnetic beads into a 2mL centrifuge tube. Pipette 0.5ml of MES buffer to wash the magnetic beads, separate the magnetic liquid on the magnetic stand, discard the supernatant, repeat three times. Pipette 0.5ml of MES buffer and vortex to mix. Pipette 0.5ml of EDC activator, place it on the stand and put it on the temperature-controlled shaker to keep the mixing state at room temperature (24℃-26℃), 250rpm, and react for 40min. Pipette quickly add 50μg of human CD47 recombinant protein, quickly put it on the temperature-controlled shaker, keep room temperature (24℃-26℃), 250rpm, and react for 12h. Pipette 0.5ml of blocking agent, shake the same conditions as before, and react for 4h. The magnetic beads were washed with 0.5 ml of preservation solution, separated by magnetic stand and magnetic liquid, and the supernatant was discarded, and repeated three times; the volume was adjusted to 1 mL with preservation solution and stored at 4° C. to obtain 10 mg / mL immunomagnetic beads.

[0102] The immunomagnetic beads of the present invention can be used to adsorb free anti-CD47 monoclonal antibody drugs in serum. The immunomagnetic beads of the present invention were characterized by transmission electron microscopy. The electron microscopy image showed that the particle size of the immunomagnetic beads was distributed at 100 nm and had good dispersibility, such as Fig.13 shown.

[0103] 3. Determination of pH value of MES buffer

[0104] Take 10 mg of carboxyl magnetic beads, add MES buffer with different pH values ​​(pH6.0, 7.0, 7.4, 8.0, 8.4, 9.0) respectively, and mix. Pipette 0.5 mL of MES buffer with different pH to wash the magnetic beads, separate the magnetic liquid on the magnetic stand, remove the supernatant, repeat three times, pipette and add 0.5 mL of MES buffer, vortex and mix. Then label the antibody according to the above steps. Calculate the coupling rate and coupling amount of carboxyl magnetic beads coupled to CD47 recombinant protein at different pH. After absorbing 30 μL of patient serum with 100 μL of 10 mg / mL immunomagnetic beads with different pH buffers, perform anti-human globulin experiments and compare the elimination effect of CD47 antibodies at different pH values. Thus, the most suitable buffer pH value for protein coupling is screened out.

[0105] Carboxyl magnetic beads have a high negative charge on their surface and are easily bound to antibodies through charge interaction. Therefore, the isoelectric point of the protein and the pH of the solution affect the directional coupling of the protein on the surface of the nanoparticles. This experiment selected MES buffers with different pH values ​​to observe the coupling efficiency of proteins in different buffers and the elimination effect of labeled immunomagnetic beads on CD47 antibodies in patient serum. Figure 1 shown.

[0106] Depend on Figure 1 It can be seen that in the pH 6.0-7.4 stage, as the pH value of the MES buffer increases, the amount of protein coupled to the surface of the carboxyl magnetic beads and the coupling efficiency also gradually increase, and the coupling efficiency rises from 68% to 85%. However, in the pH 7.4-9.0 stage, the amount of protein coupled to the surface of the carboxyl magnetic beads and the coupling efficiency gradually decrease, and the coupling efficiency decreases from 85% to 36%. Figure 2 As shown in the figure, this result was further confirmed by the effect of the cassette anti-human globulin test after the immunomagnetic beads absorbed the free CD47 antibody in the patient's serum. When the same amount of immunomagnetic beads labeled with different pH values ​​were used to absorb the interference of anti-CD47 monoclonal antibodies in the same amount of serum, the effect of eliminating interference gradually improved with the increase of pH. When the pH was raised to pH>7.4, the effect of eliminating interference was not good. This may be because the stability and coupling efficiency of carboxyl magnetic beads and recombinant proteins were affected to a certain extent when the pH was too high. In summary, the pH of MES buffer was selected as 7.4.

[0107] 4. Determination of the amount of recombinant protein input

[0108] In order to determine the optimal antibody input amount in the carboxyl magnetic beads-CD47 recombinant protein coupling process, 10μg, 20μg, 50μg, 100μg and 150ng of CD47 recombinant protein were added to 10mg of carboxyl magnetic beads, and the other labeling steps were the same as described above. The coupling rate and coupling amount of carboxyl magnetic beads coupled to antibodies under different CD47 recombinant protein input amounts were calculated. After absorbing 100μL of patient serum with 100μL of 10mg / mL immunomagnetic beads with different protein input amounts, an anti-human globulin experiment was performed to compare the CD47 antibody elimination effect with different protein input amounts. Thus, the most suitable protein coupling amount was screened out.

[0109] The different protein strengths on the surface of immunomagnetic beads will affect the degree of binding of free monoclonal antibody CD47 in the serum, and thus affect the effect of eliminating interference. The increase in nano-ion surface protein will increase the binding sites of carboxyl magnetic beads and monoclonal antibodies, thereby enhancing the effect of eliminating interference. Therefore, the input amount of CD47 recombinant protein in the labeled carboxyl magnetic beads is crucial. Therefore, the present invention adds 10μg, 20μg, 50μg, 100μg and 150μg of CD47 recombinant protein to 10mg of carboxyl magnetic beads, respectively, to determine the optimal ratio of protein input amount and carboxyl magnetic beads coupling. Figure 3 It can be seen that with the increase in protein input, the antibody coupling amount also increases, but the increase in the 50-150μg stage is relatively gentle compared to the 10-50μg stage; compared to its coupling rate, the 10-50μg stage has little change at around 85%, and the 50-150μg stage drops from 85% to 32%, that is, with the further increase in antibody input (above 50μg), the antibody coupling rate did not increase, but began to decline. This phenomenon is consistent with the report of researchers Thobhanis et al., who found that equal amounts of nano-ions and excessively high concentrations of antibodies would not have a beneficial effect. This phenomenon is mainly attributed to the steric hindrance caused by the excess of antibodies, such as proteins or antibodies. Figure 4 As shown in the figure, this result was further confirmed by the results of the anti-human globulin test to eliminate interference. That is, as the amount of protein input increases, the elimination effect is enhanced. When the protein input is less than 50μg, the labeled immunomagnetic beads cannot completely eliminate the interference in the serum. However, when the protein input is greater than 50μg, the excessive protein input is consistent with the interference elimination effect of 50μg labeled immunomagnetic beads. In summary, the optimal protein input amount was finally determined to be 50μg.

[0110] 5. Selection of blocking agents in immunomagnetic beads and the effect of immunomagnetic beads on irregular antibody detection

[0111] After the immunomagnetic beads react with antibodies, the surface sites will not be completely bound. When the immunomagnetic beads are incubated with serum, these unbound sites may react with other components in the sample other than the analyte, that is, in the present invention, nonspecific adsorption may occur to other irregularities in the serum, resulting in false negative results in other subsequent tests. Therefore, in order to avoid the impact on other tests, it is necessary to select appropriate blocking agents to block these binding sites, and they will not bind to the labeled proteins on the surface of the magnetic beads, nor will they cross-react with the analyte in the serum.

[0112] 6. Screening of sealants

[0113] The present invention uses irregular antibodies to add to healthy human serum to establish a simulated sample of irregular antibodies. Different concentrations of irregular antibodies including anti-K, anti-D, anti-Fy are added to healthy human serum (from the blood transfusion department of the Second Affiliated Hospital of Air Force Medical University). b , anti-Jk a (All purchased from Shanghai Blood Biopharmaceutical Co., Ltd.) to establish a simulated specimen. The magnetic beads and the simulated specimen were mixed and incubated. By comparing the irregular antibody detection results before and after incubation, it was confirmed that the blocking agent was screened to avoid non-specific binding of the immunomagnetic beads in eliminating interference, thereby producing false negative results. The following blocking agents were screened:

[0114] Blocking agent 1 is composed of 3% BSA + 3% calf serum. Blocking agent 2 is composed of 3% BSA. Blocking agent 3 is composed of 3% BSA + 3% PEG 2000. Each blocking agent is blocked for 4 hours. 100 μL of immunomagnetic beads with different blocking agents are used to absorb 100 μL of the established irregular antibody simulated serum, and then the anti-human globulin experiment is performed to compare the effects of immunomagnetic beads labeled with different blocking agents on irregular antibody detection, so as to screen out the best blocking agent.

[0115] The results are as follows Figure 5 As shown, a simulated specimen with an agglutination strength of 3+ was first established. Then, the three types of immunomagnetic beads blocked by three types of blocking agents were mixed and incubated separately, and the anti-human globulin test was performed again to detect the agglutination strength, and further detect whether the immunomagnetic beads had an effect on the detection of irregular antibodies in the subsequent serum. The results showed that blocking agent 1 and blocking agent 2 had a certain effect on the detection of irregular antibodies in the subsequent serum. Although no false negative results occurred, the agglutination strength became 2+, which was weakened. The reason is speculated to be that there are sites on the carboxyl magnetic beads that are not completely blocked, and these sites non-specifically bind to the irregular antibodies to be detected in the serum, resulting in the subsequent detection of these antibodies being affected. The immunomagnetic beads blocked by blocking agent 3 had no effect on the agglutination strength of irregular antibodies after incubation with the simulated specimen. Therefore, the blocking condition of blocking agent 3 for 4 hours was screened.

[0116] 7. Effect of immunomagnetic beads on detection of irregular red blood cell antibodies and blood type antibodies

[0117] The immunomagnetic beads prepared by the present invention show effective elimination of interference with anti-CD47 monoclonal antibodies in serum. Since clinical patients are prone to produce irregular antibodies during repeated blood transfusions, it is necessary to explore whether the immunomagnetic beads have an impact on the detection results of irregular antibodies and other blood type antibodies. Therefore, the present invention screens the specimens after medication, adds irregular antibodies in the table to the serum of healthy people to establish a simulated specimen with an agglutination strength of 2+, and then adds a simulated anti-CD47 monoclonal antibody specimen with an agglutination strength of 1+, and the agglutination strength is 2+ after detection. SThen, immunomagnetic beads were added to each group of samples for incubation to eliminate the interference of anti-CD47 monoclonal antibody. After magnetic separation, serum was taken for cassette anti-human globulin test. The comparison of the test results of immunomagnetic bead treatment and untreatment is shown in Table 1.

[0118] Table 1 Effect of immunomagnetic beads on detection of irregular red blood cell antibodies and blood type antibodies

[0119]

[0120] Result analysis: The experiment confirmed that all added irregular antibodies were detected. This shows that the immunomagnetic beads effectively eliminated the interference of anti-CD47 monoclonal antibodies in serum, and did not affect the detection reactivity of anti-human globulins such as anti-D, anti-K, anti-Fyb, anti-Jka, and ABO system anti-B antibodies.

[0121] 8. Determination of the storage time of the preservation solution:

[0122] Use 0.05% sodium azide and 0.05% ProClin 300 PBS storage buffer to resuspend the immunomagnetic beads, that is, add 1 mL to resuspend 10 mg of immunomagnetic beads, store in a 4°C refrigerator, and perform interference elimination tests on the simulated serum after CD-47 monoclonal antibody administration for 3 days, 7 days, 10 days, and 14 days. The specific operation process is: take 100 μL serum (agglutination strength 4+) of the simulated specimen after medication and add it to a 1.5 mL centrifuge tube. After fully mixing with the immunomagnetic beads, place the centrifuge tube on a magnetic rack for magnetic separation for 1 minute. Take 25 μL of serum for cassette anti-human globulin detection, and observe whether the interference elimination effect in the anti-human globulin test changes.

[0123] Adding a small amount of sodium azide mainly achieves the antibacterial effect by blocking the electron transfer of cells and inhibiting the degradation of labeled proteins. Adding ProClin 300 is mainly because it acts on four key positions of the tricarboxylic acid cycle: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase and NADH dehydrogenase, and can maintain broad-spectrum antibacterial properties for a long time. Figure 6 As shown, the stored magnetic beads were taken at 3 days, 7 days, 10 days and 14 days respectively to carry out CD47 monoclonal antibody interference elimination experiment of patient serum. The results showed that the CD47 monoclonal antibody interference elimination effect was good in the anti-human globulin test within 14 days, that is, the immunomagnetic beads were stored at 4°C in PBS storage buffer containing 0.05% sodium azide and 0.05% ProClin 300.

[0124] 9. Serological tests:

[0125] 9.1. Effect of using immunomagnetic beads to eliminate the interference of anti-CD47 monoclonal antibody simulated serum samples in the cassette anti-human globulin test

[0126] The main process of anti-human globulin experiment:

[0127] Use DG gel Coombs cards to collect proportionally diluted plasma samples for plasma antibody screening, add 50 μL of Serascan Diana cells and 25 μL of plasma samples with different agglutination strengths to the gel test card, and then incubate in a blood type card incubator at 37°C for 30 min, then centrifuge at 900 rpm for 2 min, and centrifuge at 1500 rpm for 3 min in a blood type card centrifuge, and observe the agglutination strength results.

[0128] Optimization of the dosage of immunomagnetic beads:

[0129] Take 30μL (0.3mg), 50μL (0.5mg), 100μL (1mg), and 150μL (1.5mg) of immunomagnetic beads respectively, add them to a 1.5mL centrifuge tube and place them on a magnetic rack for magnetic separation, and discard the supernatant. Take 100μL of plasma from the simulated sample after medication and add it to a 1.5mL centrifuge tube, mix it thoroughly with the immunomagnetic beads, and incubate it in a 37℃ water bath for 30min. After incubation, place the centrifuge tube on a magnetic rack for magnetic separation for about 1min, and take 25μL of serum for cassette anti-human globulin detection.

[0130] Compare the differences in interference elimination effects after adding different amounts of immunomagnetic beads. Compare the results of interference elimination with those of Gama-clone reagent to screen out the optimal amount of immunomagnetic beads.

[0131] The purpose of the immunomagnetic beads in the present invention is to completely eliminate the interference of anti-CD47 antibodies in anti-human globulin tests. Therefore, the amount of immunomagnetic beads used is also particularly important. Optimize the amount of immunomagnetic beads used, such as Figure 7 As shown, the concentration is 10mg / ml labeled immunomagnetic beads. When the volume of immunomagnetic beads is 30μL (immunomagnetic beads dosage is 0.3mg) and 50μL (immunomagnetic beads dosage is 0.5mg), the interference of the patient's specimen with 4+ agglutination strength cannot be eliminated. When the volume is 100μL (immunomagnetic beads dosage is 1mg), the interference is completely eliminated. When the immunomagnetic beads dosage is greater than 1mg, the elimination effect is consistent. The Gama-clone reagent produced by the authoritative blood transfusion reagent company Immucor is used as a control for complete elimination of interference to prove the validity of the immunomagnetic bead results. Therefore, it is finally determined that the optimal dosage of immunomagnetic beads at this concentration is 1mg.

[0132] Optimization of incubation time for immunomagnetic beads to eliminate interference:

[0133] Different concentrations of anti-CD-47 monoclonal antibody Magrolimab (purchased from MCE Biotech Co., Ltd.) were added to healthy human serum (from the Blood Transfusion Department of the Second Affiliated Hospital of the Air Force Medical University) to establish simulated specimens with different agglutination strengths after medication. 100 μL of 10 mg / mL immunomagnetic beads were added to a 1.5 mL centrifuge tube and placed on a magnetic rack for magnetic separation, and the supernatant was discarded. 100 μL of plasma, a simulated specimen after medication, was added to a 1.5 mL centrifuge tube, mixed thoroughly with the immunomagnetic beads, and placed in a 37 ° C water bath for incubation for 30 min, 15 min, 5 min, and 0 min. After incubation, the centrifuge tube was placed on a magnetic rack for magnetic separation for 1 min, and 25 μL of serum was taken for cassette anti-human globulin detection to determine the optimal incubation time.

[0134] The experimental results are as follows Figure 8 As shown in the figure, the incubation time was shortened from 30 minutes to 0 minutes without incubation. The sample and magnetic beads only needed to be fully mixed before magnetic separation for 1 minute, and the serum sample was taken for testing. The results showed that sufficient mixing could completely eliminate interference, greatly shortening the experimental steps. It was finally determined that immunomagnetic beads did not need to be incubated at 37°C to eliminate interference, and interference could be eliminated after sufficient mixing.

[0135] Comparison with existing methods verifies the effectiveness of immunomagnetic beads in eliminating interference:

[0136] First, three groups of simulated serum specimens after medication (three specimens in each group) were prepared and processed separately. The first group was directly tested with cassette anti-human globulin as a positive control. The two groups were treated to eliminate interference before cassette anti-human globulin testing. The second group used immunomagnetic beads to remove interference from CD47 monoclonal antibodies. The specific operation process was as follows: 100 μL of plasma from the simulated specimen after medication was added to a 1.5 mL centrifuge tube, and after being fully mixed with the immunomagnetic beads, the centrifuge tube was placed on a magnetic rack for magnetic separation for 1 min, and 25 μL of serum was taken for cassette anti-human globulin testing; the third group was treated with Gama-clone (Immucor, USA) reagent as a control for the results of the treated specimens.

[0137] Table 2 Effect of cassette anti-human globulin test on the screening of irregular blood group antibodies by immunomagnetic beads

[0138] Serial number Unprocessed specimen test results Immunomagnetic bead-treated specimen test results Gamma-clone processed specimen test results 1 4+ - - 2 3+ - - 3 4+ - -

[0139] The results showed that the irregular blood group antibody screening (i.e., indirect anti-human globulin test) in the untreated group was positive, while the experimental result in the immunomagnetic bead-treated group was negative. This was because the human CD47 recombinant protein labeled on the immunomagnetic beads specifically bound to the free anti-CD47 monoclonal antibody drug in the serum, thereby eliminating the interference of the anti-human globulin card-type irregular blood group antibody screening. The results were consistent with the results after Gamma-clone reagent treatment. The results are shown in Table 2.

[0140] The immunomagnetic beads of the present invention have been shown to effectively eliminate the interference of anti-CD47 monoclonal antibodies in anti-human globulin detection through experiments. The operation is simple, the interference elimination process takes only 1 minute, and does not affect the detection of other antibody antigens in serum. In addition, human CD47 recombinant protein has been commercialized and is easy to purchase, and the preparation process has high repeatability, so that the immunomagnetic beads prepared by the present invention have stable performance and can target various types of anti-CD47 monoclonal antibodies in clinical practice.

[0141] The immunomagnetic beads of the present invention are convenient for clinical use, and the most effective Gamma-clone reagent for eliminating the interference currently comes from the American Immucor Company. The immunomagnetic beads of the present invention can enhance my country's strength in the field of eliminating anti-CD47 monoclonal antibody drugs, so that tumor patients can effectively use such therapeutic drugs, and prevent blood transfusion-related tests of patients taking the drugs from being affected and misjudged, thereby ensuring the timeliness and effectiveness of blood transfusions for patients.

[0142] 9.2 Effectiveness of immunomagnetic beads in eliminating interference of anti-CD47 monoclonal antibody clinical serum specimens in cassette anti-human globulin test

[0143] Serum specimens from multiple myeloma patients (from the Department of Blood Transfusion, the Second Affiliated Hospital of Xi'an Jiaotong University) were collected before and after treatment with the anti-CD47 monoclonal antibody Lemzoparlimab (clinical drug number TJ011133) for anti-human globulin card-type irregular blood group antibody screening (card-type anti-human globulin test). 50 μL of SerascanDiana cells and 25 μL of plasma samples before and after treatment were added to the card using a DG gel Coombs card, and then incubated at 37°C in a card incubator for 30 min, then centrifuged at 900 rpm for 2 min, and centrifuged at 1500 rpm for 3 min using a blood type card centrifuge.

[0144] The specimens were divided into two groups. The first group was set up with three test samples before and after medication. The irregular antibody anti-screen cell and anti-human globulin test cards used in the test were purchased from Diagnostic Grifols, SA, Spain. The second group of specimens were treated with immunomagnetic beads to remove the interference of CD47 monoclonal antibodies. The specific operation process was to take 100 μL of plasma from the patient's specimen and add it to a 1.5 mL centrifuge tube. After fully mixing with the immunomagnetic beads, the centrifuge tube was placed on a magnetic rack for magnetic separation for 1 minute, and 25 μL of serum was taken for card-type anti-human globulin test; at the same time, the Gama-clone method was used for interference elimination as a control.

[0145] Result analysis: The results of the first group of specimens showed that before taking the medicine, the patient's anti-human globulin irregular blood type antibody screening test was negative; after using the anti-CD47 monoclonal antibody, the patient's anti-human globulin card irregular blood type antibody screening result was positive agglutination intensity 4+. Fig. 9 The results of the second group of samples were positive for cassette anti-human globulin when they were not treated, and negative after immunomagnetic bead treatment, which was consistent with the results after Gamma-clone reagent treatment. Fig.10 shown.

[0146] Among them, human CD47 recombinant protein can be selected from the cell line OVCAR3 which highly expresses CD47 protein in cells. The specific steps are as follows:

[0147] A cell line OVCAR3 (purchased from Wuhan Pronocell Life Science Co., Ltd.) that highly expresses CD47 protein in cells was screened out based on the Human Protein Atlas (HPA) database.

[0148] According to the database data, the expression of CD47 in the cell line was detected by flow cytometry, and the high expression of CD47 in the cell line was confirmed by immunofluorescence staining. Therefore, the OVCAR3 cell line lysate protein was selected and labeled simultaneously in the subsequent magnetic bead-labeled protein. After the OVCAR3 cell line was lysed with RIPA lysis buffer, it was quantified using the BCA protein quantification kit and used in the subsequent magnetic bead-labeled protein experiment. Fig.13 shown.

[0149] like Fig.14 As shown, indirect immunofluorescence staining of OVCAR3 was performed using CD47 antibody and FITC-conjugated affinity-pure goat anti-mouse IgG. The cells were then incubated with CD47 antibody at 4°C overnight. The cells were subsequently incubated with 3% bovine serum albumin in PBS for 30 min as a blocking agent. The samples were then incubated with FITC-conjugated mouse IgG in a dark room at 20°C for 1 h. Finally, OVCAR3 was observed with DAPI anti-fluorescence quenching reagent, and the expression of CD47 was detected under a fluorescence microscope. As shown Fig.14 As shown, the blue color is DAPI-stained cell nuclei, and the green fluorescence is the labeled CD47 protein. The results show that CD47 is expressed on the cell membrane and inside the OVCAR3 cells.

[0150] like Fig.15 As shown, flow cytometry was used to detect the expression of CD47 in OVCAR3, red blood cells and platelets. 10,000 cells were collected and stained with FITC-conjugated mouse anti-human CD47 antibody and analyzed by flow cytometry. The results showed that the expression of CD47 protein in OVCAR3 cell line was higher than that in red blood cells and platelets in blood.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An immunomagnetic bead for eliminating interference in anti-CD47 monoclonal antibody drug detection, characterized in that: include: The carboxyl magnetic beads are located inside and the human CD47 recombinant protein is wrapped around the carboxyl magnetic beads. The particle size of the immunomagnetic beads is 100 nanometers.

2. A storage solution for immunomagnetic beads, characterized in that: The storage solution is PBS storage buffer containing 0.05% sodium azide and 0.05% ProClin 300. The immunomagnetic beads are the immunomagnetic beads as described in claim 1.

3. An immunomagnetic bead reagent, characterized in that: It comprises immunomagnetic beads and a storage solution for the immunomagnetic beads as claimed in claim 2.

4. A method for preparing an immunomagnetic bead reagent, characterized in that: include: Step 1: Weigh the carboxyl magnetic beads and place them in a centrifuge tube. Wash the carboxyl magnetic beads with MES buffer and discard the supernatant. Step 2: Add MES buffer and vortex to mix. Step 3: Add EDC activator and place the centrifuge tube on a temperature-controlled shaker to maintain room temperature for reaction. Step 4: Add human CD47 recombinant protein and quickly place in a temperature-controlled shaker for incubation. Step 5: Add blocking agent for blocking. Step 6: Add preservation solution to wash the magnetic beads, discard the supernatant, and use the preservation solution to make the volume of the immunomagnetic beads constant.

5. The method for preparing an immunomagnetic bead reagent according to claim 4, characterized in that: in, The reaction conditions of step 3 are: 24°C-26°C, rotation speed 250 rpm, reaction time 40 min; The reaction conditions of step 4 are: 24°C-26°C, rotation speed 250rpm, reaction time 12h, The sealing conditions of step 5 are: 24°C-26°C, rotation speed 250 rpm, reaction time 4h.

6. The method for preparing an immunomagnetic bead reagent according to claim 4, characterized in that: The mass ratio of the carboxyl magnetic beads to the human CD47 recombinant protein is 200:1; the blocking agent is 3% BSA + 3% PEG 2000; The pH of the MES buffer is 7.4, and the storage time in step 7 is 14 days.

7. The method for preparing an immunomagnetic bead reagent according to claim 4, characterized in that: Wherein steps 3 and 4 are both performed by placing a bracket on a temperature-controlled shaking table, wherein the bracket placement comprises: The lower end of the fixed clamping block (1) is clamped by the temperature-controlled shaking table, so that the placement bracket moves under the drive of the temperature-controlled shaking table. A fixing plate (2), the lower side of which is fixedly connected to the upper end of the fixing clamping block (1). The placement frame is arranged downwardly from left to right and is formed by four connecting rods connected end to end. The four connecting rods are respectively a left connecting rod (3), a right connecting rod (4), a front connecting rod (5), and a rear connecting rod (6). The front connecting rod (5) and the rear connecting rod (6) are respectively fixedly connected to the two ends of the fixing plate (2). A plurality of limiting holes are opened from left to right on the left connecting rod (3), and a plurality of limiting grooves (12) are evenly opened on the upper side of the right connecting rod (4). A plurality of positioning rods (7) are arranged through corresponding limiting holes, and the right ends of the positioning rods are all fixedly connected with sealing blocks (11). Each of the sealing blocks (11) is arranged in a one-to-one correspondence with each of the limiting grooves (12), and the area between the sealing blocks (11) and each of the limiting grooves (12) is used to place a centrifuge tube, so that the bottom of the centrifuge tube is against the limiting groove (12), and the cover of the centrifuge tube is against the sealing block (11), and the sealing block (11) generates a rightward thrust on the cover of the centrifuge tube to prevent the cover from loosening when moving on the shaking table, so that the centrifuge tube moves in an inclined state to prevent liquid from leaking from the top of the centrifuge tube.

8. The method for preparing an immunomagnetic bead reagent according to claim 7, characterized in that: The placement stent also includes: A plurality of centrifugal support cylinders are evenly spaced along the direction of the fixed plate (2) and are arranged corresponding to the limit grooves (12). The arc-shaped support plate (8) located on the left and the circular support cylinder (9) located on the right are integrally connected. The arc-shaped support plate (8) and the circular support cylinder (9) are coaxially arranged. The bottom of the arc-shaped support plate (8) is fixed to the upper side of the fixed plate (2). The right end of the circular support cylinder (9) is fixedly connected to the left side of the right connecting rod (4). Each of the arc-shaped support plates (8) is used to support the middle part of the centrifugal tube, and each of the circular support cylinders (9) is used to limit the bottom of the centrifugal tube, thereby preventing the centrifugal tube from moving in the vertical direction.

9. The method for preparing an immunomagnetic bead reagent according to claim 8, characterized in that: Each of the positioning rods (7) is threadedly connected with a positioning bolt (10), and each of the positioning bolts (10) is used to fix the positioning rod (7) after the sealing block (11) abuts against the top of the centrifuge tube to prevent the positioning rod (7) from moving left and right.

10. A method for eliminating interference in anti-human globulin detection of blood, characterized in that: include: Take a predetermined amount of immunomagnetic bead reagent and place it in a centrifuge tube. The supernatant was separated by magnetic separation and discarded. Add serum sample to the centrifuge tube containing only immunomagnetic beads and mix thoroughly. After 1 min of magnetic separation, serum samples were taken for cassette anti-human globulin test using anti-human globulin reagent; The concentration of the immunomagnetic beads in the immunomagnetic bead reagent used was 1 mg / 10 μL serum sample.

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