A method for eliminating blood cell interference in magnetic particle chemiluminescence detection

Abstract

The present invention provides a method for eliminating the interference of blood cells in magnetic particle chemiluminescence detection. The method comprises mixing whole blood with a whole blood processing buffer to obtain a whole blood sample to be tested, adding a magnetic bead coating and a marker to prepare reacted magnetic beads, washing the reacted magnetic beads with a washing solution for washing the magnetic beads, and finally placing the washed magnetic beads in a luminescent substrate solution for a test reaction. The improved detection method fully prevents non-plasma substances in the whole blood from interfering with the test results.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescence detection, and in particular to a method for eliminating blood cell interference in magnetic particle chemiluminescence detection. Background Art

[0002] The magnetic microparticle chemiluminescence method is a medical testing technology that uses magnetic beads as solid phase carriers, based on the principle of immune reaction, and uses alkaline phosphatase, acridinium ester, luminol, terpyridine ruthenium, etc. as markers. Compared with ELISA, immunofluorescence and other methods, it shows excellent sensitivity and precision.

[0003] At present, in vitro diagnostic reagents using the chemiluminescence method are mainly used in scenarios with large clinical sample volumes and high testing demands, such as the laboratory departments of large hospitals and third-party testing centers. In scenarios such as grassroots hospitals and emergency departments, POCT reagents using the immunofluorescence method are the mainstream.

[0004] With the advancement of biotechnology and the policy inclination to sink medical resources, chemiluminescence POCT reagents have begun to become popular in grassroots hospitals, emergency departments and other scenarios, and there is a trend of gradually replacing traditional fluorescence POCT reagents. However, compared with immunofluorescence POCT reagents, the current chemiluminescence POCT has the following main disadvantages: 1. The price of reagents and instruments is still relatively high. 2. When the sample type is whole blood, the test results may be unreliable. In response to the latter, the present invention proposes a solution for anti-blood cell interference in chemiluminescence reagents.

[0005] In medical tests using chemiluminescence methods, interference when testing whole blood samples primarily comes from blood cells, which primarily include red blood cells (RBCs), white blood cells (WBCs), and platelets. Red blood cells contain hemoglobin, which has peroxide activity. Therefore, the escape of RBC contents during the reaction can directly affect the immune response or catalyze the substrate to produce a light signal. Furthermore, WBCs contain substances such as phosphatases, and the escape of phosphatases during the reaction can directly catalyze the luminescence of the AP enzyme substrate, generating an interfering light signal. Furthermore, platelets have a strong adhesion function. If platelets are activated during the reaction, platelet-bridged aggregates are easily formed, resulting in a large amount of non-specific adsorption, seriously affecting the specificity of the immune response. Summary of the Invention

[0006] To solve the above problems, the present application provides a method for eliminating the interference of blood cells in magnetic particle chemiluminescence detection, which can directly test plasma with whole blood.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for eliminating blood cell interference in magnetic particle chemiluminescence detection, characterized by comprising the steps of:

[0009] S1. Mixing a whole blood sample with a buffer for processing whole blood samples to obtain a whole blood sample to be tested;

[0010] S2. Adding the corresponding reagent magnetic bead coating and the marker to the whole blood sample to be tested to prepare reacted magnetic beads;

[0011] S3, washing the reacted magnetic beads in a magnetic field with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0012] S4. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0013] It should be noted that in step S1, the whole blood sample is mixed with a buffer for processing the whole blood sample in a test tube to obtain a processed whole blood sample to be tested. The buffer for processing the whole blood sample inhibits platelet activity, specifically inactivates white blood cells, and keeps all blood cells dispersed, preventing any blood cells from agglomerating.

[0014] It should also be noted that in step S2, the treated whole blood sample is added to a reagent component containing a magnetic bead coating, etc. The coating on the surface of the magnetic beads will specifically bind to the corresponding analyte in the treated whole blood sample, forming a coating-analyte (including but not limited to antibody-antigen, biotin-avidin, binding protein-small molecule, etc.) complex on the surface of the magnetic beads. At the same time, the magnetic bead coating-analyte complex will also specifically bind to another marker (including but not limited to alkaline phosphatase marker, acridinium ester marker, horseradish peroxidase marker, etc.), forming a post-reaction magnetic bead-analyte-marker complex.

[0015] It should also be noted that the magnetic beads after the reaction in step S2 are taken out and placed in another test tube, and the magnetic beads after the reaction are washed in a magnetic field with a washing solution for washing the magnetic beads. The washing solution for washing the magnetic beads can clean up free markers, substances non-specifically bound to the magnetic beads, unruptured blood cells, miscellaneous proteins after ruptured blood cells, and other substances, thereby cleaning up as much as possible the substances in the test tube except the complex of magnetic beads-test objects-markers.

[0016] It should also be noted that the luminescent substrate solution not only specifically enables alkaline phosphatase to catalyze the substrate to produce photons, but also protects the intact blood cells that were not cleaned in step S2, preventing the blood cells from rupturing in the luminescent substrate solution and releasing their contents to interfere with the test results.

[0017] Furthermore, the preparation steps of the magnetic bead coating are as follows:

[0018] S31, wash the magnetic beads with 50 mM PBS buffer;

[0019] S32, adding 50 mM PBS to resuspend the magnetic beads;

[0020] S33, adding the coating material to the magnetic beads and incubating at 37°C overnight;

[0021] S34. Add a blocking solution containing agarose, lysine, and Tween 20, and block at 37° C. overnight to obtain the magnetic bead coating.

[0022] Furthermore, the buffer for processing the whole blood sample includes: a red blood cell protectant, a platelet inhibitor, a leukocyte lysing agent and a cell dispersant.

[0023] It should be noted that blood cell protectants are used to strengthen red blood cells, allowing them to remain intact during biological reactions and mechanical pressure, preventing their contents from spilling out. Because red blood cells contain hemoglobin, which has peroxide activity, if red blood cells rupture due to external factors, the hemoglobin spills out and specifically binds to the luminescent substrate, resulting in substandard test results.

[0024] It should also be noted that platelet inhibitors inhibit platelet activation and reduce platelet aggregation. Leukocyte lysing agents primarily target and lyse white blood cells, breaking them into fragments. Because white blood cells contain phosphatases and other substances, if they rupture in the presence of a substrate, the phosphatases will directly catalyze the alkaline phosphatase substrate to emit light, generating an interfering signal. Therefore, prior to reacting with the substrate solution, the white blood cells must be lysed to remove the phosphatases within them, resulting in more accurate test results.

[0025] It should also be noted that platelets have a strong adhesion function. If platelets are activated during the reaction, it is very easy to produce aggregates with platelets as the bridging center, causing a large amount of nonspecific adsorption, which seriously affects the specificity of the immune response. Therefore, adding platelet inhibitors, blood cell dispersants, etc. to the buffer for processing whole blood samples can help avoid platelet activation, make blood cells fully dispersed and non-adhesive, and reduce interference with the immune response.

[0026] Furthermore, the material of the magnetic beads is any one of polystyrene, polyacrylamide, polyacrylic acid, and silica.

[0027] Furthermore, the surface of the magnetic beads has a modification group, and the modification group is any one or more of streptavidin, NHS, epoxy, amino, and carboxyl.

[0028] Furthermore, the magnetic bead blocking agent includes: agarose and lysine.

[0029] It should be noted that in order to reduce the degree of platelet activation by the magnetic bead coating, the surface of the magnetic bead coating needs to be specially sealed.

[0030] Furthermore, the cleaning solution for cleaning the magnetic beads includes: a detergent, a membrane protein solubilizer, and a cell membrane solubilizer.

[0031] Furthermore, the detergent is selected from any one of Tween 20, Tween 80, surfactant S9, ceteareth-13, and Brij35;

[0032] Preferably, the detergent formulation is: 0.1 g Tween 20 per 100 mL of cleaning solution.

[0033] It should be noted that the main function of the detergent is to remove substances non-specifically adsorbed on the surface of the magnetic beads after washing, including biofilm fragments, miscellaneous proteins and other fragments after blood cell lysis.

[0034] Furthermore, the membrane protein dissolving agent acts to dissolve membrane proteins in the cell membrane, making blood cells more likely to rupture, thereby releasing the contents of the blood cells.

[0035] Furthermore, the cell membrane dissolving agent acts to dissolve the cell membrane, so that the cell membrane of the blood cells can be fully lysed and the contents can be fully released for easy cleaning.

[0036] It should also be noted that the washing of the magnetic beads after the reaction in the previous step is mainly to clean all blood cells and foreign proteins. However, it is inevitable that some intact blood cells will not be washed away. If these residual blood cells are added to the luminescent substrate solution and rupture, the contents of these blood cells will interfere with the accuracy of the luminescent immunoassay reaction to a certain extent. Therefore, it is necessary to add a blood cell protectant to the luminescent substrate solution. The main function of the blood cell protectant is to ensure the integrity of the blood cell structure in the luminescent substrate solution and prevent these remaining intact blood cells from rupturing.

[0037] Furthermore, the red blood cell protective agent includes: polyoxyethylene hydrogenated castor oil, potassium chloride and sucrose; the platelet inhibitor is L-arginine; the leukocyte lysis agent is leukocidin; and the cell dispersant is polytetraoxyethylene micropowder.

[0038] Preferably, the formula of the red blood cell protectant is: per 100 mL of the buffer solution for treating the whole blood sample contains 6 g of polyoxyethylene hydrogenated castor oil, 1 g of potassium chloride, and 6 g of sucrose; the formula of the platelet inhibitor is: per 100 mL of the buffer solution for treating the whole blood sample contains 0.1 g of L-arginine; the formula of the leukocyte lysis agent is: per 100 mL of the buffer solution for treating the whole blood sample contains 5 mg of leukocidin; and the formula of the cell dispersant is specifically: per 100 mL of the buffer solution for treating the whole blood sample contains 0.2 g of polytetraoxyethylene micropowder.

[0039] Furthermore, the detergent is Tween 20; the membrane protein solubilizing agent is CHAPS; and the cell membrane solubilizing agent is alkyl glycoside.

[0040] Preferably, the formula of the detergent is as follows: 0.1g Tween 20 is contained in every 100mL of the washing solution for washing magnetic beads; the formula of the membrane protein solubilizing agent is as follows: 2g CHAPS is contained in every 100mL of the washing solution for washing magnetic beads; and the formula of the cell membrane solubilizing agent is as follows: 2g alkyl glycoside is contained in every 100mL of the washing solution for washing magnetic beads.

[0041] Furthermore, the luminescent substrate solution includes: a luminescent substrate, a surfactant, and a blood cell protective agent.

[0042] It should be noted that after the reaction, the magnetic bead coating-analyte-label is washed and transferred to the luminescent substrate liquid for detection reaction. The main function of the luminescent substrate liquid is to react with the label to generate photons. Through the photon counter, the luminescence value is proportional or inversely proportional to the amount of the analyte.

[0043] Furthermore, the luminescent substrate is AMPPD or APS-5 or CDP-Star; and the cell protectant is lecithin.

[0044] Preferably, the formula of the cell protectant is: 2g of lecithin per 100mL of luminescent substrate solution.

[0045] The present invention provides a method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection. The method comprises mixing a whole blood sample with a buffer for processing the whole blood sample to obtain a whole blood sample to be tested. Antibody-coated magnetic beads and alkaline phosphatase-labeled antibodies are added to the whole blood sample to prepare treated magnetic beads. The treated magnetic beads are then washed with a cleaning solution for washing the magnetic beads. Finally, the washed magnetic beads are placed in a luminescent substrate for a test reaction. Furthermore, the formulations of the buffer for processing the whole blood sample, the cleaning solution for washing the magnetic beads, and the luminescent substrate solution are improved. The buffer for processing the whole blood sample contains a red blood cell protectant to reinforce the red blood cells and prevent them from rupturing; a platelet inhibitor to inhibit platelet activation and reduce platelet aggregation; a leukocyte lysing agent to target and lyse white blood cells; and a blood cell dispersant to fully disperse the blood cells. The cleaning solution for washing the magnetic beads contains a detergent for washing substances nonspecifically adsorbed on the surface of the magnetic beads; a membrane protein solubilizer for fully dissolving membrane proteins in the cell membrane; and a cell membrane solubilizer for dissolving the cell membrane. The luminescent substrate solution contains not only the luminescent substrate but also a blood cell protectant to ensure the integrity of the blood cell structure. This improved detection method fully prevents interference from non-plasma substances in whole blood on the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a comparative analysis chart of the correlation between plasma and whole blood test results in the cTnI chemiluminescence immunoassay method;

[0047] Figure 2 This is a comparative analysis chart of the correlation between plasma and whole blood test results in the MYO chemiluminescent immunoassay method;

[0048] Figure 3 This is a comparative analysis chart of the correlation between plasma and whole blood test results in the CK-MB chemiluminescence immunoassay detection method. DETAILED DESCRIPTION

[0049] The present invention discloses a method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

[0051] The materials and reagents used in the method for eliminating the interference of blood cells in magnetic particle chemiluminescence detection provided by the present invention can be purchased from the market.

[0052] Example 1

[0053] This embodiment provides a chemiluminescent immunoassay detection and analysis method for cTnI that eliminates the interference of blood cells in magnetic microparticle chemiluminescent detection;

[0054] (1) Preparation of cTnI (cardiac troponin) capture magnetic beads:

[0055] Step 1: Wash the commercial magnetic bead stock solution 3 times with 50mM PBS (pH 7.4)

[0056] Step 2: Add 50mM PBS (pH 7.4) to resuspend the magnetic beads

[0057] Step 3: Add cTnI monoclonal antibody, incubate at 37°C overnight, add blocking solution containing agarose, lysine, and Tween 20, and block at 37°C overnight to obtain magnetic beads captured with cTnI monoclonal antibody.

[0058] Step 4: The magnetic beads captured with the cTnI monoclonal antibody prepared in step 3 were washed three times with 50 mM PBS (pH 7.4) and stored for future use.

[0059] (2) Preparation of cTnI enzyme-labeled antibody: cTnI monoclonal antibody was coupled with alkaline phosphatase using SMCC to obtain cTnI antibody labeled with alkaline phosphatase. The prepared cTnI enzyme-labeled antibody was stored in 50 mM MES (pH 6.0) for future use.

[0060] (3) Comparison of test samples: 40 plasma samples with EDTA as the anticoagulant were selected and each sample was divided into two parts, one for testing plasma and the other for testing whole blood.

[0061] The whole blood testing process is as follows:

[0062] S1. Take 50 μl of the sample to be tested and 50 μl of the buffer for treating whole blood samples into the reaction well for reaction, incubate at 37°C for 3 minutes to prepare the whole blood sample to be tested;

[0063] S2. Take 50 μl of the whole blood sample to be tested, add 50 μl of alkaline phosphatase-labeled cTnI antibody, and incubate at 37°C for 5 minutes to obtain the treated magnetic beads;

[0064] S3, washing the treated magnetic beads three times with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0065] S4. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0066] The testing process for plasma is as follows:

[0067] S1. Adding magnetic beads coated with cTnI monoclonal antibodies and alkaline phosphatase-labeled cTnI antibodies to a plasma sample to prepare treated magnetic beads;

[0068] S2, washing the treated magnetic beads with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0069] S3. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0070] The test results are shown in Table 1 and Figure 1 As shown in the figure, it can be seen that the cTnI antibody detection results in whole blood and plasma have a high correlation.

[0071] Table 1 cTnI homologous plasma and whole blood sample test data

[0072] Sample ID Plasma samples Whole blood sample Sample ID Plasma samples Whole blood sample 1 859919 533651 21 635551 397860 2 86140 56672 22 239820 160292 3 241362 137676 23 113825 63912 4 290588 167963 24 102354 69097 5 106954 60919 25 2588 1478 6 9118 6159 26 42644 23122 7 5878 3922 27 6840 4544 8 335668 171296 28 325537 182080 9 17906 9172 29 392057 269075 10 248384 139169 30 4615 2925 11 6416 4303 31 1212 652 12 437541 271283 32 768910 429164 13 50557 31582 33 8913 5695 14 105415 64308 34 270231 179091 15 290947 170867 35 215927 134722 16 7195 4542 36 139947 76761 17 8927 4738 37 362343 231032 18 2163 1196 38 299776 206670 19 175705 98138 39 317960 215707 20 462885 253591 40 55662 30708

[0073] Example 2

[0074] This embodiment provides a chemiluminescent immunoassay detection and analysis method for MYO that eliminates the interference of blood cells in magnetic microparticle chemiluminescent detection;

[0075] (1) Preparation of MYO (myoglobin) capture magnetic beads:

[0076] Step 1: Wash the commercial magnetic bead stock solution 3 times with 50mM PBS (pH 7.4)

[0077] Step 2: Add 50mM PBS (pH 7.4) to resuspend the magnetic beads

[0078] Step 3: Add MYO monoclonal antibody, incubate at 37°C overnight, add blocking solution containing agarose, lysine, and Tween 20, and block at 37°C overnight to obtain magnetic beads captured with MYO monoclonal antibody.

[0079] Step 4: The magnetic beads captured with the MYO monoclonal antibody prepared in step 3 were washed three times with 50 mM PBS (pH 7.4) and stored for future use.

[0080] (2) Preparation of MYO enzyme label: MYO monoclonal antibody was coupled with alkaline phosphatase using SMCC to obtain MYO antibody labeled with alkaline phosphatase. The prepared MYO enzyme-labeled antibody was stored in 50 mM MES (pH 6.0) for future use.

[0081] (3) Comparison of test samples: 40 plasma samples with EDTA as the anticoagulant were selected and each sample was divided into two parts, one for testing plasma and the other for testing whole blood.

[0082] The whole blood testing process is as follows:

[0083] S1. Take 50 μl of the sample to be tested and 50 μl of the buffer for treating whole blood samples into the reaction well for reaction, incubate at 37°C for 3 minutes to prepare the whole blood sample to be tested;

[0084] S2. Take 50 μl of the whole blood sample to be tested, add 50 μl of alkaline phosphatase-labeled MYO antibody, and incubate at 37°C for 5 minutes to obtain the treated magnetic beads;

[0085] S3, washing the treated magnetic beads three times with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0086] S4. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0087] The testing process for plasma is as follows:

[0088] S1. Adding magnetic beads coated with MYO monoclonal antibodies and MYO antibodies labeled with alkaline phosphatase to a plasma sample to prepare treated magnetic beads;

[0089] S2, washing the treated magnetic beads with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0090] S3. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0091] The test results are shown in Table 2 and Figure 2 As shown, it can be seen that the MYO antibody test results in whole blood and plasma have a high correlation.

[0092] Table 2. MYO homologous plasma and whole blood sample test data

[0093] Sample ID Plasma samples Whole blood sample Sample ID Plasma samples Whole blood sample 1 323478 334345 21 441 436 2 3149 3522 22 45806 40011 3 249854 281255 23 9730 10624 4 4777 4749 24 280923 320449 5 7213 6522 25 760404 650672 6 8842 8646 26 9313 10187 7 78949 72166 27 70865 73941 8 324 325 28 468540 522240 9 1388 1378 29 200515 174782 10 3957 4147 30 421040 469523 11 169065 188730 31 376339 376023 12 100006 90935 32 483596 458639 13 409268 410890 33 785344 766003 14 1835 1963 34 875534 802116 15 6798 6607 35 31241 34488 16 4117 3780 36 1552 1518 17 8682 8002 37 145895 156511 18 2279 2203 38 8461 8730 19 314363 319855 39 8240 7357 20 4753 5037 40 547095 613841

[0094] Example 3

[0095] This embodiment provides a chemiluminescent immunoassay detection and analysis method for CK-MB that eliminates the interference of blood cells in magnetic microparticle chemiluminescent detection;

[0096] (1) Preparation of CK-MB (creatine kinase isoenzyme) capture magnetic beads:

[0097] Step 1: Wash the commercial magnetic bead stock solution 3 times with 50mM PBS (pH 7.4)

[0098] Step 2: Add 50mM PBS (pH 7.4) to resuspend the magnetic beads

[0099] Step 3: Add CK-MB monoclonal antibody, incubate at 37°C overnight, add blocking solution containing agarose, lysine, and Tween 20, and block at 37°C overnight to obtain magnetic beads captured with CK-MB monoclonal antibody.

[0100] Step 4: The magnetic beads captured with CK-MB monoclonal antibody prepared in step 3 were washed three times with 50 mM PBS (pH 7.4) and stored for future use.

[0101] (2) Preparation of CK-MB enzyme label: CK-MB monoclonal antibody was coupled with alkaline phosphatase using SMCC to obtain CK-MB antibody labeled with alkaline phosphatase. The prepared CK-MB enzyme-labeled antibody was stored in 50 mM MES (pH 6.0) for future use.

[0102] (3) Comparison of test samples: 40 plasma samples with EDTA as the anticoagulant were selected and each sample was divided into two parts, one for testing plasma and the other for testing whole blood.

[0103] The whole blood testing process is as follows:

[0104] S1. Take 50 μl of the sample to be tested and 50 μl of the buffer for treating whole blood samples into the reaction well for reaction, incubate at 37°C for 3 minutes to prepare the whole blood sample to be tested;

[0105] S2. Take 50 μl of the whole blood sample to be tested, add 50 μl of alkaline phosphatase-labeled CK-MB antibody thereto, and incubate at 37°C for 5 minutes to obtain the treated magnetic beads;

[0106] S3, washing the treated magnetic beads three times with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0107] S4. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0108] The testing process for plasma is as follows:

[0109] S1. Adding magnetic beads coated with CK-MB monoclonal antibodies and CK-MB antibodies labeled with alkaline phosphatase to a plasma sample to prepare treated magnetic beads;

[0110] S2, washing the treated magnetic beads with a washing solution for washing magnetic beads to obtain washed magnetic beads;

[0111] S3. Place the washed magnetic beads into a luminescent substrate solution to perform a test reaction.

[0112] The test results are shown in Table 3 and Figure 3 As shown, it can be seen that the CK-MB antibody test results in whole blood and plasma have a high correlation.

[0113] Table 3. CK-MB homologous plasma and whole blood sample test data

[0114] Sample ID Plasma samples Whole blood sample Sample ID Plasma samples Whole blood sample 1 392 368 21 611433 632994 2 144526 153715 22 990252 1019312 3 4912 4938 23 5519 5851 4 34 31 24 4733 4557 5 484826 422676 25 1186 1114 6 869392 922636 26 408910 438907 7 6910 6749 27 335997 325925 8 352646 335917 28 9014 7713 9 619911 671145 29 5756 5580 10 703013 671629 30 8292 8572 11 475665 454398 31 9866 10614 12 670038 634659 32 2737 3120 13 407170 423875 33 5992 6036 14 89832 81757 34 8184 8070 15 170938 180867 35 3379 3661 16 6214 6420 36 6996 6180 17 410274 413925 37 6116 5609 18 9750 8632 38 970 1092 19 530852 495714 39 584699 627396 20 479731 465800 40 379710 347686

Claims

1. A method for eliminating blood cell interference in magnetic particle chemiluminescence detection, characterized in that: Including steps: S1. Mixing a whole blood sample with a buffer for processing whole blood samples to obtain a whole blood sample to be tested; S2. Adding a reagent component containing a magnetic bead coating and a marker to the whole blood sample to be tested to obtain a magnetic bead-test substance-marker complex; S3, washing the magnetic bead-analyte-label complex in a magnetic field with a washing solution for washing the magnetic beads to obtain a washed magnetic bead-analyte-label complex; S4, placing the washed magnetic bead-analyte-label complex into a luminescent substrate solution to perform a test reaction; The buffer for processing the whole blood sample comprises: a red blood cell protectant, a platelet inhibitor, a leukocyte lysing agent and a cell dispersant; The cleaning solution for cleaning the magnetic beads includes: detergent, membrane protein solubilizer, cell membrane solubilizer; The luminescent substrate liquid comprises: Luminescent substrate, surfactant, blood cell protective agent.

2. The method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection according to claim 1, characterized in that: The preparation steps of the magnetic bead coating are as follows: S31, wash the magnetic beads with 50 mM PBS buffer; S32, adding 50 mM PBS to resuspend the magnetic beads; S33, adding the coating material to the magnetic beads and incubating at 37°C overnight; S34. Add a blocking solution containing agarose, lysine, and Tween 20, and block at 37° C. overnight to obtain the magnetic bead coating.

3. The method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection according to claim 2, characterized in that: The material of the magnetic beads is any one of polystyrene, polyacrylamide, polyacrylic acid, and silicon dioxide.

4. The method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection according to claim 3, characterized in that: The surface of the magnetic beads has a modification group, and the modification group is any one or more of streptavidin, NHS, epoxy, amino, and carboxyl groups.

5. The buffer for processing a whole blood sample according to claim 1, wherein The red blood cell protective agent comprises: polyoxyethylene hydrogenated castor oil, potassium chloride and sucrose; The platelet inhibitor is L-arginine; The leukocyte lysis agent is leukocidin; The cell dispersant is polytetraoxyethylene powder.

6. The cleaning solution for cleaning magnetic beads according to claim 1, wherein The detergent is Tween 20; The membrane protein solubilizing agent is CHAPS; The cell membrane dissolving agent is an alkyl glycoside.

7. The luminescent substrate liquid according to claim 1, wherein The luminescent substrate is AMPPD or APS-5 or CDP-Star; The blood cell protective agent is lecithin.

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