Immunomagnetic bead, preparation method and detection reagent

Abstract

The invention provides an immunomagnetic bead, a preparation method and a detection reagent. The preparation method comprises the following steps: preparing a modified magnetic bead containing a dibenzocyclooctyne group; protein and a coupling agent containing azide groups are sequentially dissolved in a second coupling buffer solution to react, modified protein containing azide groups is obtained, the final concentration of the reacted protein is 0.5-5 mg / mL, and the final concentration of the coupling agent is 0.05-5 mM; and mixing the modified magnetic beads and the modified protein according to an addition ratio of 1mg: (1-40) nmol, and reacting to obtain the immunomagnetic beads. According to the present invention, the immunomagnetic bead preparation method has advantages of high coupling efficiency, small inter-batch difference, strong specificity and difficult aggregation during the preparation process, such that the accurate detection of the antigen concentration is achieved so as to solve the defects of low coupling efficiency, large inter-batch difference and easy aggregation existing in the immunomagnetic bead preparation method in the related technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunodetection, and particularly relates to an immunomagnetic bead, a preparation method and a detection reagent. BACKGROUND

[0002] Immunomagnetic beads (IMB) are a kind of functional material combining magnetic particles and antibodies, which are composed of carrier magnetic beads and antibodies. The core of the carrier magnetic beads is usually made of superparamagnetic material (such as ferroferric oxide or ferric oxide), which has the characteristics of fast response under an external magnetic field. The shell is made of high molecular material (such as polystyrene or dextran), and the surface is provided with active functional groups (such as amino, carboxyl, hydroxyl, etc.), which are convenient for combination with antibodies. The antibodies include antigens, antibodies, agglutinins, etc., which can specifically recognize and combine with target molecules.

[0003] The particle size of the immunomagnetic beads can be designed to be nanoscale to micrometer scale according to different purposes. By adjusting the preparation materials and methods, the surface properties of the immunomagnetic beads can be endowed with hydrophobicity / hydrophilicity, polarity / non-polarity, positive charge / negative charge, etc. In addition, functional components such as pigments, dyes, fluorescent substances, radioactive labels or magnetic iron oxide can be added to the inside of the magnetic beads to meet various detection and separation needs.

[0004] The shell of the magnetic beads is usually made of high molecular material such as polystyrene or dextran, and the surface can be activated to generate functional groups such as amino (-NH2), carboxyl (-COOH) or hydroxyl (-OH). These groups can be coupled with antigens, antibodies and other antibodies through coupling reaction, so that the magnetic beads become the carrier of antigens or antibodies. The antigens / antibodies on the surface of the magnetic beads can combine with specific antibodies / antigens to form an immune complex, which has high specificity. Combined with the separation ability of an external magnetic field, the immunomagnetic beads are widely used in the fields of immunodetection, protein separation and purification, cell screening, etc.

[0005] At present, the commonly used immunomagnetic beads in immunodetection mainly include amino magnetic beads and carboxyl magnetic beads. The carboxyl magnetic beads usually use ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent to realize the coupling of carboxyl with antigens or antibodies. However, EDC has poor stability in aqueous solution, which easily leads to low coupling efficiency, large batch-to-batch difference, etc. The amino magnetic beads usually use glutaraldehyde as a coupling agent, but the aldehyde groups at both ends of the molecule are easy to covalently react with amino groups (-NH2), which leads to the coupling of the magnetic beads, antigens or antibodies themselves, resulting in non-specific adsorption, and further causing the aggregation of the magnetic beads, inaccurate detection results and large batch-to-batch difference, etc., and the coupling efficiency is low. SUMMARY

[0006] The purpose of the present application is to provide an immunomagnetic bead, a preparation method and a detection reagent. The preparation method of the immunomagnetic beads provided in the present application has high coupling efficiency, small batch-to-batch variability, strong specificity and is not easy to aggregate during the preparation process, so as to achieve accurate detection of antigen concentration and solve the problems of low coupling efficiency, large batch-to-batch variability and easy aggregation in the method for preparing immunomagnetic beads in the related art.

[0007] According to the first aspect of the present application, a method for preparing immunomagnetic beads is proposed, comprising the following steps:

[0008] The washed amino magnetic beads are resuspended in a first coupling buffer to obtain a magnetic bead suspension with a concentration of 1-10 mg / mL; a coupling agent A containing a dibenzocyclooctyne group is added to the magnetic bead suspension for reaction for 15-60 minutes, wherein the final reaction concentration of the coupling agent A is 0.05-5 mM, to obtain modified magnetic beads containing a dibenzocyclooctyne group;

[0009] After adding the protein to the second coupling buffer, the azide-containing coupling agent B is added and reacted for 1-8 hours; the final reaction concentration of the protein is 0.5-5 mg / mL; the final reaction concentration of the coupling agent B is 0.05-5 mM; and a modified protein containing an azide group is obtained;

[0010] The modified magnetic beads and the modified protein are mixed at an addition ratio of 1 mg:1-40 nmol, and reacted for 2-12 hours to obtain immunomagnetic beads, and the immunomagnetic beads are washed and stored.

[0011] In some embodiments, the amino magnetic beads are washed at least three times with a washing buffer and rinsed at least three times with the first coupling buffer before being resuspended;

[0012] and / or, the washing buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl, 0.5-10 mM EDTA;

[0013] and / or, the washing buffer further comprises 0.01%-0.5% Tween 20;

[0014] and / or, the pH of the washing buffer is 6.0-8.0;

[0015] and / or, the first coupling buffer comprises 10mM-50mM PBS, 100-500mM NaCl, and 0.5-10mM EDTA;

[0016] And / or, the pH of the first coupling buffer is 6.0-8.0.

[0017] In some embodiments, the coupling agent A is DBCO-R1-NHS ester;

[0018] wherein R1 is one of (PEG)n, (CH2)n, S-S, NHCO-(PEG)n, and n is 1-24

[0019] In some embodiments, the coupling agent A and the magnetic bead suspension are reacted at 4-30°C at a rotation speed of 500-2000 rpm for 15-60 min to obtain a reaction system A.

[0020] And / or, the reaction system A is removed after being adsorbed by a magnetic stand to obtain the modified magnetic beads, and the modified magnetic beads are cleaned with the first coupling buffer.

[0021] In some embodiments, the protein is one of an antibody and an antigen.

[0022] And / or, the second coupling buffer comprises 10mM-50mM PBS, 100-500mM NaCl, and 0.5-10mM EDTA.

[0023] And / or, the pH of the second coupling buffer is 6.5-9.0.

[0024] And / or, the coupling agent B is an azide-labeled carboxyl or succinimidyl ester derivative, and the coupling agent B is N3-R2-COOH or N3-R2-NHS ester; wherein R2 is one of (PEG)n, (CH2)n, S-S, NHCO-(PEG)n, and n is 1-24.

[0025] The protein and the coupling agent B are reacted at 4-30°C at a rotation speed of 50-70 rpm to obtain the modified protein.

[0026] In some embodiments, the modified magnetic beads and the modified protein are reacted in a third coupling buffer at 4-30°C at a rotation speed of 500-2000 rpm for 2-12 h to obtain a reaction system C.

[0027] The reaction system C is removed after being adsorbed by a magnetic stand to obtain the immunomagnetic beads, and the immunomagnetic beads are cleaned at least 3 times and resuspended to a concentration of 1-10 mg / mL using a magnetic bead preservation solution for preservation.

[0028] In some embodiments, the magnetic bead preservation solution is 10-100mM Tris-HCl, and the pH is 6.0-8.0.

[0029] And / or, the third coupling buffer is one of a phosphate buffer, Tris-HCl, MES (2-morpholinoethanesulfonic acid), and a citrate buffer.

[0030] And / or, the pH of the third coupling buffer is 6.5-9.0.

[0031] According to the second aspect of the present application, an immunomagnetic bead is proposed, which is obtained using the preparation method described in any of the above embodiments.

[0032] According to the third aspect of the present application, a detection reagent is proposed, comprising the immunomagnetic bead working solution described in any one of the above embodiments.

[0033] According to a fourth aspect of the present application, a detection kit is proposed, characterized in that it includes the detection reagent described in any one of the above embodiments.

[0034] The beneficial effects of the present application are as follows: the present application couples amino magnetic beads with antibodies and antigens through paired coupling agents to prepare immunomagnetic beads, and applies them to enzyme-linked immunosorbent assay platforms and chemiluminescence platforms. In the method for preparing immunomagnetic beads disclosed in the present application, the primary amino group of the amino magnetic beads specifically reacts with DBCO-R1-NHS ester under neutral or weakly alkaline conditions to form a stable covalent bond to obtain modified magnetic beads containing DBCO groups. The R1 spacer arm provides a long and flexible connection, effectively reduces steric hindrance, and can also improve the water solubility of the molecule. The DBCO group can undergo a ring strain-promoted alkyne-azide cycloaddition reaction (SPAAC) with azide-modified proteins or fluorescent molecules under mild conditions. The reaction is fast, efficient, stable and reliable, and significantly improves the coupling efficiency of the modified magnetic beads with proteins. The present application can complete the reaction under mild activation and coupling conditions, effectively retaining the biological activity of the modified substance, thereby improving the efficiency and sensitivity of the immunomagnetic beads in immune reactions.

[0035] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings.

[0037] Figure 1 This is the synthesis reaction formula of the immunomagnetic beads proposed in one embodiment of the present application;

[0038] Figure 2 The results of NT-proBNP immunomagnetic bead detection with different coating concentrations proposed in an embodiment of the present application are as follows;

[0039] Figure 3 These are the test results of serum samples detected by different PSA detection kits proposed in an embodiment of the present application. DETAILED DESCRIPTION

[0040] The embodiments of the present application are described below in detail with examples shown in the drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0041] For the purpose, technical solutions and advantages of the present application to be clearer, further detailed description of the present application is made below in combination with specific embodiments and with reference to the drawings.

[0042] According to the first aspect of the present application, a preparation method of immunomagnetic beads is provided, comprising the following steps:

[0043] S1: resuspend the washed amino magnetic beads with a first coupling buffer to obtain a magnetic bead suspension with a concentration of 1-10 mg / mL; add a coupling agent A containing a dibenzocyclooctyne group to the magnetic bead suspension and react for 15-60 min, the final reaction concentration of the coupling agent A is 0.05-5 mM, to obtain modified magnetic beads containing a dibenzocyclooctyne group;

[0044] S2: add a protein to a second coupling buffer, then add a coupling agent B containing an azido group and react for 1-8 h; the final reaction concentration of the protein is 0.5-5 mg / mL; the final reaction concentration of the coupling agent B is 0.05-5 mM; to obtain modified proteins containing an azido group;

[0045] S3: mix the modified magnetic beads and the modified proteins according to an addition ratio of 1 mg: 1-40 nmol, and react for 2-12 h to obtain immunomagnetic beads, and then wash and store the immunomagnetic beads.

[0046] In the present application, the washed amino magnetic beads are resuspended with a first coupling buffer to obtain a magnetic bead suspension with a concentration of 1-10 mg / mL, wherein the washed amino magnetic beads are first rinsed with the first coupling buffer for 3 times and then resuspended, wherein the washing buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl and 0.5-10 mM EDTA, with a pH of 6.0-8.0. In some embodiments, the washing buffer further comprises 0.01%-0.5% Tween 20, and the amino magnetic beads are washed with the washing buffer for at least 3 times, then rinsed with the first coupling buffer for at least 3 times, and then resuspended with the first coupling buffer to obtain a magnetic bead suspension with a concentration of 1-10 mg / mL. The first coupling buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl and 0.5-10 mM EDTA, with a pH of 6.0-8.0. For example, the first coupling buffer has a pH of 7.5, and comprises 20 mM PBS, 200 mM NaCl and 5 mM EDTA.

[0047] The example of adding the coupling agent A containing the DBCO group to the magnetic bead suspension with a concentration of 1-10 mg / mL, wherein the coupling agent A is DBCO-R1-NHS ester, the concentration of the coupling agent A is 0.05-5 mM; R1 is one of (PEG)n, (CH2)n, S-S, NHCO-(PEG)n groups, and n is 1-24. In this application, the coupling agent A and the magnetic bead suspension are reacted at 4-30°C and a rotation speed of 500-2000 rpm for 15-60 min to obtain a reaction system A; the reaction system A is adsorbed by using a magnetic stand, and then the reaction solution is removed to obtain modified magnetic beads, and the modified magnetic beads are washed 3 times by using the first coupling buffer to obtain the modified magnetic beads containing the DBCO group. In addition, for the convenience of subsequent use of the modified magnetic beads, the modified magnetic beads containing the dibenzocyclooctyne group are resuspended by using the third coupling buffer, and then stored at 4°C for standby.

[0048] In the process, the coupling agent A is DBCO-PEG 12 -NHS ester is taken as an example, -PEG 12 The spacer wall can increase the distance between the amino magnetic beads and the conjugate, reduce the steric hindrance effect, enhance the specific recognition ability of the antigen and the antibody, effectively avoid the non-specific adsorption of the immunomagnetic beads and the aggregation in the preparation process, and thus improve the efficiency, accuracy and sensitivity of the immune reaction.

[0049] In S2, the protein is added to the second coupling buffer, and then the coupling agent B containing the azido group is added. The protein is one of an antibody and an antigen. In this application, the protein is first added to the second coupling buffer for dissolution. The pH of the second coupling buffer is 6.5-9.0; the second coupling buffer includes a phosphate buffer with a concentration of 10 mM-50 mM, 100-500 mM NaCl, and 0.5-10 mM EDTA. The pH of the example of the second coupling buffer is 8.5, and the second coupling buffer includes 20 mM PBS, 200 mM NaCl, and 5 mM EDTA.

[0050] When the protein in this embodiment is dissolved in the second coupling buffer, the coupling agent B containing the azido group is added to the mixture formed by the protein and the second coupling buffer. The coupling agent B is an azido-labeled carboxyl or succinimidyl ester derivative, for example, the coupling agent B containing the azido group is N3-R2-COOH or N3-R2-NHS ester; wherein R2 is one of (PEG)n, (CH2)n, S-S, NHCO-(PEG)n groups, and n is 1-24. In this embodiment, the protein and the coupling agent B are reacted in the second coupling buffer at 4-30°C and a rotation speed of 50-70 rpm for 1-8 h, wherein the final concentration of the protein is 0.5-5 mg / mL; the concentration of the coupling agent B is 0.05-5 mM; and a modified protein containing the azido group is obtained.

[0051] In S3, the modified magnetic beads obtained in S1 and the modified protein obtained in S2 are mixed in a third coupling buffer at an addition ratio of 1 mg: 1-40 nmol, and the reaction system C is obtained by reacting at a temperature of 4-30°C and a rotation speed of 500-2000 rpm for 2-12 h. The third coupling buffer has a pH of 6.5-9.0 and is one of a phosphate buffer, Tris-HCl, MES (2-morpholinoethanesulfonic acid), and a citrate buffer.

[0052] The reaction system C is adsorbed by using a magnetic stand, and then the reaction solution is removed to obtain the immunomagnetic beads. The immunomagnetic beads are washed at least three times by using a washing buffer, and then the washed immunomagnetic beads are resuspended by using a magnetic bead storage solution and stored at 4°C. The immunomagnetic beads are resuspended to a concentration of 1-10 mg / mL by using the magnetic bead storage solution for storage. The magnetic bead storage solution is 10-100 mM Tris-HCl with a pH of 6.0-8.0. In this embodiment, the washing buffer is the same as the washing buffer in the above-mentioned embodiments, and thus is not described again.

[0053] The immunomagnetic beads provided in the present application have high specificity and small batch-to-batch difference, and have the advantages of high coupling efficiency, high specificity, high accuracy and high sensitivity when an immune response occurs. The preparation method of the immunomagnetic beads provided in the present application can be performed under mild reaction conditions in the process of antigen activation and preparation of immunomagnetic beads, thereby effectively ensuring the biological activity of the antigen and being more conducive to improving the efficiency, accuracy and sensitivity of the immunomagnetic beads in the immune response.

[0054] According to a second aspect of the present application, an immunomagnetic bead is provided, which is obtained by using the preparation method in any one of the above-mentioned embodiments.

[0055] According to a third aspect of the present application, a detection reagent is provided, which comprises the immunomagnetic beads in any one of the above-mentioned embodiments and an acridine working solution.

[0056] According to a fourth aspect of the present application, a detection kit is provided, which comprises the detection reagent in any one of the above-mentioned embodiments.

[0057] In the following examples, the specific experimental steps or conditions are not specified, and the operations or conditions can be performed according to the conventional experimental steps described in the literature in the art. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0058] Example 1

[0059] The present embodiment provides an immunomagnetic bead, which is prepared by using the preparation method provided in the present embodiment. Figure 1 The preparation method comprises the following steps:

[0060] (1) Amino magnetic beads and coupling agent DBCO-PEG 12 - NHS ester reaction:

[0061] Coupling agent A: DBCO-PEG 12 - NHS ester was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 30 mM.

[0062] Take 1 mL of amino polymer magnetic microspheres (amino magnetic beads, MP0201, purchased from Changzhou Boyi Biotechnology Co., Ltd.) with a concentration of 30 mg / mL, wash with washing buffer for 3 times and then wash with first coupling buffer for 3 times. The washing buffer includes 20 mM PBS, 150 mM NaCl, 0.02% Tween 20 and 1 mM EDTA, pH 7.5; the first coupling buffer: 20 mM PBS, 150 mM NaCl and 1 mM EDTA, pH 7.5.

[0063] Resuspend the washed amino magnetic beads in the first coupling buffer to a concentration of 10 mg / mL to obtain a magnetic bead suspension; add DBCO-PEG 12 - NHS ester to the magnetic bead suspension, mix thoroughly, then place in a constant temperature mixer at 1200 rpm, room temperature 25°C, activate for 30 min to obtain reaction system A; the final concentration of coupling agent A after reaction is 0.2 mM. After adsorption, remove the reaction solution to adsorb reaction system A to obtain DBCO modified magnetic beads, i.e. magnetic beads-DBCO. Wash the obtained magnetic beads-DBCO three times with the first coupling buffer, resuspend with the third coupling buffer to make the concentration of magnetic beads-DBCO 10 mg / mL to obtain magnetic beads-DBCO suspension, and store at 4°C for standby.

[0064] (2) Antibody and azide coupling agent reaction:

[0065] Dissolve the antibody NT-proBNP antibody (purchased from Haipai Biotechnology (Shanghai) Co., Ltd.) in the second coupling buffer to a concentration of 0.5 mg / mL to obtain NT-proBNP antibody solution. The second coupling buffer includes 20 mM PBS, 200 mM NaCl and 5 mM EDTA, pH 6.5.

[0066] The coupling agent B is a coupling agent N3-(CH2)5-NHS ester, which is dissolved in dimethyl sulfoxide (DMSO) to a concentration of 30 mM, and then the coupling agent B is added into the NT-proBNP antibody solution, and mixed thoroughly to obtain a reaction system B. The reaction system B is activated at 30°C and a rotation speed of 70 rpm for 60 minutes. The reaction final concentration of the coupling agent B is 0.2 mM. The azide-modified NT-proBNP antibody, i.e., NT-proBNP Ab-N3, is obtained by centrifugation of the 50K ultrafiltration tube.

[0067] (3) Preparation of the immunomagnetic beads

[0068] The magnetic bead-DBCO suspension prepared in step (1) and the NT-proBNP Ab-N3 prepared in step (2) are added into a third coupling buffer, and the mass molar ratio of the magnetic bead-DBCO to the NT-proBNP Ab-N3 is 1 mg:5 nmol to obtain a reaction system C. The reaction system C is reacted at 4°C on a shaker at a rotation speed of 1200 rpm for 10 hours to obtain the immunomagnetic beads. The reaction system C is adsorbed by a magnetic stand, and the reaction solution is removed to obtain the NT-proBNP antibody-coated immunomagnetic beads. The immunomagnetic beads are washed with the washing buffer for 3 times, resuspended to a concentration of 10 mg / mL of the magnetic bead storage solution, and stored at 4°C. The magnetic bead storage solution is 10-100 mM Tris-HCl with a pH of 6.0-8.0. The third coupling buffer is one of a phosphate buffer, a Tris-HCl buffer, a MES (2-morpholinoethanesulfonic acid) buffer, and a citrate buffer. The pH of the third coupling buffer is 6.5-9.0.

[0069] In this embodiment, an NT-proBNP detection kit is provided, which comprises a universal NT-proBNP immunomagnetic bead working solution, a biotin-labeled NT-proBNP antibody working solution, an NT-proBNP calibrator, an SBG solution, and an RGP substrate solution. The preparation method comprises the following steps: the preparation of the immunomagnetic bead suspension is the same as that in the embodiment 1. The immunomagnetic bead suspension is diluted to a concentration of 0.2 mg / mL by using a magnetic bead diluent containing 50-200 mM Tris-HCl, 0.5-20 mM EDTA, 10-50 g / L BSA, and a pH of 7.4 to obtain the NT-proBNP immunomagnetic bead working solution.

[0070] Preparation of the biotin-labeled antibody working solution: the Thermo Fisher EZ-Link TMBiotinylate the paired NT-proBNP detection antibody using the NHS-PEG4-Biotin Biotinylation Kit and dilute to 0.3 μg / mL to obtain the NT-proBNP detection antibody working solution. Purchase SBG concentrate from Quanterix and dilute to 50 pM using the accompanying diluent.

[0071] Preparation of NT-proBNP calibrators: Dilute the NT-proBNP antigen to concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 pg / mL using sample diluent containing 50 mM HEPES, 150 mM NaCl, and 1 g / L BSA, pH 7.4. Use the diluted NT-proBNP antigen as the working calibrator for NT-proBNP detection.

[0072] Method for testing the coupling efficiency of NT-proBNP immunomagnetic beads: Method for testing the accuracy of NT-proBNP immunomagnetic beads: Pipette 100μL of NT-ProBNP working calibrator or serum sample into reaction well 1, add 50μL of magnetic bead working solution, add 50μL of detection antibody working solution, react at 37°C for 60 minutes, wash by magnetic separation, add 100uL of SBG solution, incubate at 37°C for 30 minutes, wash by magnetic separation, and add 100uL of RGP solution to read the fluorescence value.

[0073] Coupling efficiency test According to the coupling effect, the lower the concentration of the labeled antibody in the supernatant, the more antibody substances are coupled to the surface of the immunomagnetic beads. As can be seen from Table 1, the coupling efficiency of the immunomagnetic beads is higher when the ratio of magnetic beads to antibodies is 1 mg:5 nmol.

[0074] Table 1 Antibody concentration test results in the coupled supernatant (mg / mL)

[0075]

[0076]

[0077] The accuracy test results are shown in Table 2 and Figure 2 :

[0078] Table 2 Results of NT-proBNP in serum samples detected by different kits (pg / mL)

[0079] Sample No. Comparison system The present kit Sample No. Comparison system The present kit 1 56.86 58.35 31 85.46 83.86 2 46.25 68.64 32 64.46 68.95 3 48.37 26.38 33 23.51 24.58 4 59.25 62.85 34 46.28 44.39 5 79.82 78.94 35 35.88 38.62 6 101.56 103.54 36 69.94 72.92 7 125.68 183.85 37 78.56 75.86 8 98.64 95.86 38 85.22 83.2 9 38.95 45.94 39 95.46 85.2 10 365.78 347.95 40 1258.62 1062.3 11 95.68 93.85 41 56.46 54.62 12 35.45 33.4 42 78.46 76.58 13 698.46 684.1 43 95.16 98.62 14 892.42 873.5 44 37.69 35.62 15 186.58 185.64 45 16.85 15.52 16 136.58 132.68 46 95.68 94.63 17 234.65 210.54 47 179.65 178.53 18 246.85 272.64 48 192.5 188.65 19 62.46 91.58 49 95.64 93.65 20 78.69 97.65 50 32.52 31.44 21 95.42 93.62 51 798.64 776.2 22 23.46 22.62 52 146.35 148.8 23 25.05 17.61 53 958.64 965.2 24 18.49 15.55 54 258.35 274.6 25 32.54 35.94 55 135.64 132.22 26 85.49 86.95 56 100.64 103.25 27 64.88 42.58 57 105.64 101.25 28 98.75 108.89 58 359.75 365.2 29 85.25 83.85 59 951.42 1073.5 30 98.64 75.64 60 645.35 523.5

[0080] Note: The comparison system is the proBNP II pro-brain natriuretic peptide diagnostic kit purchased from Roche.

[0081] Table 2 and Figure 1The results show that the NT-proBNP immunomagnetic beads of the embodiment are used as the NT-proBNP detection kit component, the linear equation of the detection results of the NT-proBNP concentration in 60 serum samples and the reference system concentration is y = 0.9531x + 5.6682, the correlation coefficient is R 2 = 0.9832, the detection result accuracy is better, and the NT-proBNP in the serum can be determined.

[0082] Example 3: PSA detection kit (chemiluminescence method) and preparation thereof

[0083] The embodiment provides a PSA detection kit, which comprises PSA immunomagnetic bead working solution, acridinium ester-labeled PSA antibody working solution, PSA calibrant, pre-priming solution and priming solution, and a preparation method thereof comprises the following steps:

[0084] Preparation of PSA immunomagnetic bead working solution: the preparation of the immunomagnetic bead suspension is the same as that in Example 1, and the immunomagnetic bead suspension is diluted to a concentration of 0.2 mg / mL by using a magnetic bead diluent containing 50 mM Tris, 150 mM NaCl and 10 mg / mL BSA with a pH of 7.4 to obtain the PSA immunomagnetic bead working solution.

[0085] Preparation of acridinium ester-labeled PSA detection antibody working solution: the PSA detection antibody is dissolved in a labeling buffer, and is uniformly mixed with acridinium ester at a molar ratio of 1:15 at room temperature and in the dark for 1 hour; then blocking buffer (glycine with a final concentration of about 1%) is added and uniformly vortexed, and is reacted at room temperature and in the dark for 30 minutes; after the reaction, purification treatment is performed through an ultrafiltration tube, so that the acridinium ester-labeled PSA antibody is obtained, and the PSA detection antibody working solution is obtained by diluting the acridinium ester-labeled PSA antibody to 0.5 ug / mL.

[0086] Preparation of PSA calibrant: the PSA antigen is diluted to concentrations of 1.56, 3.125, 6.25, 12.5, 25, 50 and 100 pg / mL by using a calibrant diluent containing 50 mM Tris, 100 mM NaCl and 10 mg / mL BSA with a pH of 8.5, and the diluted PSA antigen and the calibrant diluent are used as the PSA working calibrant.

[0087] Detection method of PSA immunomagnetic bead coupling efficiency: detection method of PSA immunomagnetic bead accuracy: 100 μL of the PSA working calibrant or serum sample is taken into reaction well 1, 50 μL of the magnetic bead working solution and 50 μL of the acridinium ester-labeled PSA antibody working solution are added, 37°C reaction is performed for 60 minutes, magnetic separation is performed for cleaning, 100 μL of the pre-priming solution and the priming solution are added, and the light signal intensity is detected by using a chemiluminescence detector.

[0088] The detection results are as follows: the coupling efficiency is detected according to the coupling effect, the lower the concentration of the antibody to be labeled in the supernatant, the more the antibody substance coupled to the surface of the immunomagnetic beads. As can be seen from Table 3, when the concentration ratio of the magnetic beads to the antibody is 1 mg:5 nmol, the coupling efficiency of the immunomagnetic beads is higher.

[0089] Table 3: Detection results of antibody concentration in the coupling supernatant (mg / mL)

[0090] Ratio of magnetic beads and antibody 1 mg: 3 nmol 1 mg: 5 nmol 1 mg: 8 nmol Test 1 (mg / mL) 0.102 0.105 0.204 Test 2 (mg / mL) 0.096 0.125 0.198 Test 3 (mg / mL) 0.093 0.118 0.195 Average (mg / mL) 0.097 0.116 0.199 Coupling efficiency 78％ 85％ 83％

[0091] The detection results of the accuracy are as shown in Table 4 and Figure 3

[0092] Table 4: Detection results of PSA in serum samples (ng / mL) of different kits

[0093]

[0094]

[0095] Note: The comparison system is a total PSA detection kit purchased from Roche.

[0096] The results of Table 4 and Figure 3 indicate that, when the PSA immunomagnetic beads of the embodiment are used as the PSA detection kit component, the linear equation fitted by the concentration of the PSA detection results of 60 serum samples and the concentration of the reference system is y=0.959x+0.0877, the correlation coefficient R 2 =0.9874, the detection results have good accuracy, and can be applied to the determination of PSA in serum.

[0097] In the description of the present application, the terms "first", "second" are only used for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise specifically limited.

[0098] In the present application, unless otherwise specifically defined and limited, the terms "mounting", "connection", "connection", "fixing" and the like should be understood in a broad sense, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrated; it can be mechanically connected, or it can be electrically connected or in communication with each other; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise specifically limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. ​

[0099] In this application, unless otherwise explicitly specified and limited, a first feature "on" or "under" a second feature can mean that the first and second features are in direct contact, or that the first and second features are in indirect contact with an intermediate medium. Also, a first feature "over", "above" and "on top of" a second feature can mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is horizontally higher than the second feature. A first feature "under", "below" and "underneath" a second feature can mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is horizontally lower than the second feature.

[0100] In this application, the terms "one embodiment", "some embodiments", "an example", "specific examples" or "some examples" mean that a particular feature, structure, material or characteristic is included in at least one embodiment or example of the present application. Exemplary expressions of the above terms do not necessarily refer to the same embodiment or example in this specification, and the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in this specification and features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction, if possible.

[0101] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for preparing immunomagnetic beads, characterized in that: The steps include: The washed amino magnetic beads are resuspended in a first coupling buffer to obtain a magnetic bead suspension with a concentration of 1-10 mg / mL; a coupling agent A containing a dibenzocyclooctyne group is added to the magnetic bead suspension for reaction for 15-60 minutes, wherein the final reaction concentration of the coupling agent A is 0.05-5 mM, to obtain modified magnetic beads containing a dibenzocyclooctyne group; After adding the protein to the second coupling buffer, the azide-containing coupling agent B is added and reacted for 1-8 hours; the final reaction concentration of the protein is 0.5-5 mg / mL, and the final reaction concentration of the coupling agent B is 0.05-5 mM to obtain a modified protein containing an azide group; The modified magnetic beads and the modified protein are mixed at an addition ratio of 1 mg:1-40 nmol, and reacted for 2-12 hours to obtain immunomagnetic beads, and the immunomagnetic beads are washed and stored.

2. The preparation method according to claim 1, characterized in that The amino magnetic beads are washed at least three times with a washing buffer, and rinsed at least three times with the first coupling buffer before being resuspended; and / or, the washing buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl and 0.5-10 mM EDTA; and / or, the washing buffer further comprises 0.01%-0.5% Tween 20; and / or, the pH of the washing buffer is 6.0-8.0; and / or, the first coupling buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl, and 0.5-10 mM EDTA; And / or, the pH of the first coupling buffer is 6.0-8.

0.

3. The preparation method according to claim 1, characterized in that The coupling agent A is DBCO-R1-NHS ester; Wherein, R1 is one of (PEG)n, (CH2)n, SS, and NHCO-(PEG)n groups, and the value of n is 1-24.

4. The preparation method according to claim 1, characterized in that The coupling agent A and the magnetic bead suspension are reacted at 4-30° C. and a rotation speed of 500-2000 rpm for 15-60 min to obtain a reaction system A; And / or, the reaction system A is adsorbed using a magnetic stand, and then the reaction solution is removed to obtain the modified magnetic beads, and the modified magnetic beads are washed using the first coupling buffer.

5. The preparation method according to claim 1, characterized in that The protein is one of an antibody and an antigen; and / or, the second coupling buffer comprises 10 mM-50 mM PBS, 100-500 mM NaCl, and 0.5-10 mM EDTA; and / or, the pH of the second coupling buffer is 6.5-9.0; and / or, the coupling agent B containing an azide group is an azide-labeled carboxyl or succinimide ester derivative; And / or, the coupling agent B containing an azide group is N3-R2-COOH or N3-R2--NHS ester; wherein R2 is one of (PEG)n, (CH2)n, SS, and NHCO-(PEG)n groups, and the value of n is 1-24; The protein and the coupling agent B react at 4-30° C. and a rotation speed of 50-70 rpm to obtain the modified protein.

6. The preparation method according to claim 1, characterized in that The modified magnetic beads and the modified protein react in a third coupling buffer at 4-30° C. and a rotation speed of 500-2000 rpm to obtain a reaction system C; The reaction system C is adsorbed using a magnetic stand, and then the reaction solution is removed to obtain the immunomagnetic beads. The immunomagnetic beads are washed at least 3 times and resuspended in a magnetic bead storage solution to a concentration of 1-10 mg / mL for storage.

7. The preparation method according to claim 6, characterized in that The magnetic bead storage solution is 10-100 mM Tris-HCl, pH 6.0-8.0; And / or, the third coupling buffer is one of phosphate buffer, Tris-HCl, MES (2-morpholineethanesulfonic acid), and citrate buffer; And / or, the pH of the third coupling buffer is 6.5-9.

0.

8. An immunomagnetic bead, characterized in that The compound is obtained by the preparation method described in any one of claims 1 to 7.

9. A detection reagent, characterized in that The invention comprises the immunomagnetic bead working solution according to claim 8.

10. A detection kit, characterized in that The method comprises the detection reagent according to claim 9.

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