Tacrolimus detection kit and application thereof

Abstract

The invention discloses a tacrolimus detection kit and application thereof. The tacrolimus detection kit comprises a reagent R1, a reagent R2, a reagent R3, a standard substance, a reagent tube A, a reagent tube B, a reagent tube C and a freeze-drying bottle, wherein the reagent tube A, the reagent tube B, the reagent tube C and the freeze-drying bottle are respectively used for the reagent R1, the reagent R2, the reagent R3 and the standard substance; the reagent R1 is a magnetic bead antibody working solution; the reagent R2 is an enzyme-labeled antigen working solution; the reagent R3 is a blood sample treatment solution, which is a TBS buffer solution containing EDTA-2Na (Ethylene Diamine Tetraacetic Acid), a zinc sulfate solution, Triton X-100 and ProClin300; the standard substance is a freeze-dried whole blood standard substance containing tacrolimus. According to the tacrolimus detection kit and the application thereof, the general blood sample treating fluid is adopted, so that the reliability and the stability of a detection result of tacrolimus in a blood sample are improved.

Description

Technical Field

[0001] The present invention relates to a tacrolimus detection kit and application thereof, belonging to the technical field of biomedicine detection. Background Art

[0002] Tacrolimus, also known as FK506, is a macrolide antibiotic extracted from the fungus (Streptomyces tsukubaensis) with a molecular weight of 804. It is a powerful new immunosuppressant. Clinical trials have shown that tacrolimus is effective in treating rejection reactions caused by heart, lung, intestinal, and bone marrow transplants, and is also used to treat some autoimmune diseases. Its anti-rejection mechanism of action is that tacrolimus binds to a protein (FKBP-12), thereby blocking T cell signaling pathways, thereby preventing the expression of specific genes such as IL-2, ultimately hindering T cell activation and division, thereby reducing immune activity. Currently, tacrolimus is a commonly used medication in organ transplantation.

[0003] The blood concentration of tacrolimus significantly affects the therapeutic effect. If the blood concentration of tacrolimus is too low, the treatment effect will be poor, while if the blood concentration of tacrolimus is too high, it can easily lead to drug poisoning. At the same time, due to its narrow therapeutic window, the therapeutic dose and the toxic dose are close, so the blood concentration needs to be strictly monitored. Currently, there are no clear guidelines for the specific dosage of tacrolimus. The tolerance of tacrolimus varies greatly among different patients, and the patient's own physiological factors and treatment regimen can affect the drug concentration in the blood.

[0004] Commonly used methods for measuring tacrolimus blood concentrations include liquid chromatography-mass spectrometry (LC-MS), homogeneous enzyme immunoassay (HEIA), and chemiluminescence immunoassay (CLI). LC-MS is the most accurate and sensitive method, but the high cost of instrumentation and equipment, the complex and tedious operation process, and the high skill requirements for testers make these instrumental detection methods unsuitable for screening large numbers of samples. Although the homogeneous enzyme immunoassay (HEIA) is a rapid screening method, it has low sensitivity, poor specificity, and a narrow detection range. The CLI requires a chemiluminescence analyzer to fully automate the detection process and reduce human error. Its sensitivity can reach 0.1 ng / mL, with high accuracy and low detection cost, making it suitable for precise quantitative detection of tacrolimus blood concentrations. However, sample processing is currently highly personalized, test data has poor reproducibility, and large batch variability limits its clinical application. Summary of the Invention

[0005] To address at least one problem existing in the above-mentioned prior art, the present invention provides a tacrolimus detection kit and its application. The detection kit is prepared with a universal blood sample processing solution, is simple to operate, can effectively avoid matrix interference in blood samples, and improves the reliability and stability of tacrolimus detection results in blood samples.

[0006] To achieve the above-mentioned object, the present invention adopts the following technical solution: a tacrolimus detection kit, comprising reagent R1, reagent R2, reagent R3 and a standard, and reagent tube A, reagent tube B, reagent tube C and a lyophilized bottle for R1, reagent R2, reagent R3 and the standard, respectively;

[0007] The reagent R1 is a magnetic bead antibody working solution, which includes a superparamagnetic nanomaterial with tacrolimus antibody cross-linked on the surface, and the particle size is 0.5 to 3 μm;

[0008] The reagent R2 is an enzyme-labeled antigen working solution, which includes tacrolimus antigen labeled with alkaline phosphatase;

[0009] The reagent R3 is a blood sample processing solution, which is a TBS buffer containing EDTA-2Na, zinc sulfate solution, Triton X-100, and ProClin300;

[0010] The standard is a freeze-dried whole blood standard containing tacrolimus.

[0011] Preferably, the blood sample processing solution is a TBS buffer solution with a pH of 7.2 to 7.6, containing 1 to 2 mM EDTA-2Na, 30 to 60 mM zinc sulfate solution, 0.8 to 1.2% Triton X-100, and 0.06 to 0.1% ProClin300.

[0012] Preferably, when the blood to be tested is processed, the volume ratio of the reagent R3 (ie, the blood sample processing solution) to the blood to be tested is 5-6:1.

[0013] Preferably, the magnetic bead antibody working solution further comprises a blocking solution I, and the concentration of the superparamagnetic nanomaterial with tacrolimus antibody cross-linked on the surface is 0.1-1 mg / ml.

[0014] Preferably, the blocking solution I comprises 40-60 mM Tris-HCl, 0.08-0.12% Proclin 300, 0.5-1.5% Tween-20, 0.5-1% BSA, and has a pH of 6.5-8.0.

[0015] Preferably, the enzyme-labeled antigen working solution further comprises blocking solution II, and the concentration of the alkaline phosphatase-labeled tacrolimus antigen is 1 μg / mL.

[0016] Preferably, the blocking solution II comprises 10-50 mM HEPES and 0.08-0.12% Proclin 300, with a pH of 6.8-8.2.

[0017] Preferably, the freeze-dried whole blood standard containing tacrolimus is prepared by freeze-drying whole blood containing tacrolimus.

[0018] The present invention also provides an application method of a tacrolimus detection kit, which comprises the following steps:

[0019] (1) Add reagent R3 to a centrifuge tube containing the blood to be tested, vortex mix, centrifuge, and transfer the centrifuge fluid into an empty sample tube as the sample;

[0020] (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge liquid and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place the standard solution, reagent R1, and reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration, and prepare a standard curve between the standard solution concentration and the luminescence value;

[0021] (3) The sample of step 1 is used to replace the standard solution of step (2) for testing, and the tacrolimus concentration in the sample is calculated based on the standard curve.

[0022] Preferably, in step (1), the volume ratio of the reagent R3 to the blood to be tested is 5 to 6:1, and 20 μL of the blood to be tested is taken.

[0023] Preferably, in step (2), the volume ratio of the standard solution, reagent R1, and reagent R2 is 1:4:10, and 5 μL of the standard solution is taken.

[0024] The beneficial effects of the present invention are as follows: the detection kit of the present invention is designed with a blood sample processing liquid that is more universal for the blood to be tested, is simple to operate, and can effectively avoid matrix interference in the blood sample, thereby improving the reliability and stability of the detection results of tacrolimus in the sample; the detection kit of the present invention can quickly detect the blood sample, and the detection time is short; the reagents in the kit of the present invention have a longer storage time. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the implementation of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0026] A tacrolimus detection kit of the present invention comprises reagent R1, reagent R2, reagent R3 and a standard, as well as reagent tube A, reagent tube B, reagent tube C and a lyophilization bottle for R1, reagent R2, reagent R3 and the standard, respectively;

[0027] Test tube A is filled with reagent R1, which is a magnetic bead antibody working solution: first, the magnetic beads are mixed with EDC and NHS in proportion, 5 μg EDC and 2.5 μg NHS corresponding to 1 mg of magnetic beads, and then placed in a PBS buffer with a pH of 5-6.5 and activated for 15-30 minutes. After the activation is completed, tacrolimus monoclonal antibodies are added and mixed with the activated magnetic beads, 5-50 μg tacrolimus monoclonal antibodies corresponding to 1 mg of magnetic beads, and then the pH is adjusted to 7.2-7.4 for cross-linking for 3 hours. After the cross-linking is completed, the buffer is removed by centrifugation to obtain a superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, with a particle size of 0.5-3 μm. Finally, the superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody is resuspended in a blocking solution I of 6.5-8.0 including Proclin300, Tween-20, BSA, and Tris-HCl to obtain a magnetic bead antibody working solution with a pH of 6.5-8.0. 6.5-8.0, and its specific concentrations are as follows: superparamagnetic nanomaterials with surface cross-linked tacrolimus antibodies 0.1-1 mg / ml, Tris-HCl 40-60 mM, Proclin 300 0.08-0.12%, Tween-20 0.5-1.5%, BSA 0.5-1%;

[0028] Test tube B contains reagent R2, which is an enzyme-labeled antigen working solution: tacrolimus antigen labeled with alkaline phosphatase is used, and the labeling ratio of tacrolimus antigen to alkaline phosphatase is 1:1-10. The alkaline phosphatase-labeled tacrolimus antigen is then resuspended in blocking buffer II containing HEPES and Proclin 300 to obtain an enzyme-labeled antigen working solution with a pH of 6.8-8.2. The specific concentrations of the solution are as follows: alkaline phosphatase-labeled tacrolimus antigen 1 μg / mL, HEPES 10-50 mM, and Proclin 300 0.8-0.12%;

[0029] Test tube C contains reagent R3, which is a blood sample processing solution used to process the blood to be tested. When in use, the volume ratio of reagent R3 to the blood to be tested is 5-6:1. Take 20 μL of the blood to be tested and take 100-120 μL of reagent R3, which is the blood sample processing solution. This blood sample processing solution is a 7.2-7.6 1× TBS buffer and contains 1-2 mM EDTA-2Na, 30-60 mM zinc sulfate, 0.8-1.2% Triton X-100, and 0.06-0.1% ProClin300.

[0030] The freeze-dried bottle contains a standard product, which is a freeze-dried whole blood standard containing tacrolimus, and is prepared by freeze-drying whole blood containing tacrolimus.

[0031] The application method of the tacrolimus detection kit of the present invention comprises the following specific steps:

[0032] (1) Add the above-mentioned reagent R3 to a centrifuge tube containing the blood to be tested, vortex mix, centrifuge, and take the centrifuge liquid into an empty sample tube as the sample;

[0033] The volume ratio of reagent R3 to the blood to be tested is 5-6:1. For example, 20 μL of blood to be tested is taken;

[0034] (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge liquid and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place the standard solution, reagent R1, and reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration, and prepare a standard curve between the standard solution concentration and the luminescence value;

[0035] The volume ratio of standard solution, reagent R1, and reagent R2 is 1:4:10. For example, take 5 μL of standard solution;

[0036] (3) The sample of step 1 is used to replace the standard solution of step (2) for testing, and the tacrolimus concentration in the sample is calculated based on the standard curve.

[0037] Example 1

[0038] A tacrolimus detection kit comprises reagent R1, reagent R2, reagent R3 and a standard, as well as reagent tube A, reagent tube B, reagent tube C and a lyophilized bottle for R1, reagent R2, reagent R3 and the standard, respectively;

[0039] Test tube A is filled with reagent R1, which is a magnetic bead antibody working solution: first, the magnetic beads are mixed with EDC and NHS in proportion, 5 μg EDC and 2.5 μg NHS corresponding to 1 mg of magnetic beads, and then placed in a PBS buffer solution at pH 6 and activated for 30 minutes. After the activation is completed, tacrolimus monoclonal antibodies are added and mixed with the activated magnetic beads, 20 μg tacrolimus monoclonal antibodies corresponding to 1 mg of magnetic beads, and cross-linked for 3 hours. After the cross-linking is completed, the buffer solution is removed by centrifugation to obtain a superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, with a particle size of 0.5 to 3 μm. Finally, the superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody is resuspended with a blocking solution I including Proclin300, Tween-20, BSA, and Tris-HCl to obtain a magnetic bead antibody working solution, pH 7, and its specific concentrations are as follows: 0.2 mg / ml superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, Tris-HCl 50mM, Proclin3000.1%, Tween-20 1%, BSA 0.8%;

[0040] Test tube B contains reagent R2, which is an enzyme-labeled antigen working solution: alkaline phosphatase-labeled tacrolimus antigen is used, and the labeling ratio of tacrolimus antigen to alkaline phosphatase is 1:5. The alkaline phosphatase-labeled tacrolimus antigen is then resuspended in blocking solution II containing HEPES and Proclin 300 to obtain an enzyme-labeled antigen working solution, pH 7.4, with the following specific concentrations: alkaline phosphatase-labeled tacrolimus antigen 1 μg / mL, HEPES 20 mM, Proclin 300 0.1%;

[0041] Test tube C contains reagent R3, which is a blood sample processing solution. The blood sample processing solution is a 7.4% 1× TBS buffer solution and contains 1.5 mM EDTA-2Na, 50 mM zinc sulfate, 1% Triton X-100, and 0.08% ProClin 300.

[0042] The freeze-dried bottle contains a standard product, which is a freeze-dried whole blood standard containing tacrolimus, and is prepared by freeze-drying whole blood containing tacrolimus.

[0043] The application method of the tacrolimus detection kit comprises the following specific steps:

[0044] (1) Add 100 μL of the above-mentioned reagent R3 to a centrifuge tube containing 20 μL of blood to be tested, vortex mix, centrifuge, and transfer the centrifuge fluid to an empty sample tube as the sample;

[0045] (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place 5 μL of standard solution, 20 μL of reagent R1, and 50 μL of reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration to prepare a standard curve of standard solution concentration and luminescence value;

[0046] (3) The sample of step 1 is used to replace the standard solution of step (2) for testing, and the tacrolimus concentration in the sample is calculated based on the standard curve.

[0047] Tacrolimus calibration solution was added to negative samples to prepare test blood with tacrolimus concentrations of 0.2, 5, 15, and 24 ng / mL, respectively. The test was performed using the above-mentioned tacrolimus detection kit and its application method. The test results are shown in Table 1 below.

[0048] Table 1

[0049]

[0050] Example 2

[0051] A tacrolimus detection kit comprises reagent R1, reagent R2, reagent R3 and a standard, as well as reagent tube A, reagent tube B, reagent tube C and a lyophilized bottle for R1, reagent R2, reagent R3 and the standard, respectively;

[0052] Test tube A is filled with reagent R1, which is a magnetic bead antibody working solution: first, the magnetic beads are mixed with EDC and NHS in proportion, 5 μg EDC and 2.5 μg NHS corresponding to 1 mg of magnetic beads, and then placed in a PBS buffer solution at pH 6 and activated for 30 minutes. After the activation is completed, tacrolimus monoclonal antibodies are added and mixed with the activated magnetic beads, 20 μg tacrolimus monoclonal antibodies corresponding to 1 mg of magnetic beads, and cross-linked for 3 hours. After the cross-linking is completed, the buffer solution is removed by centrifugation to obtain a superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, with a particle size of 0.5 to 3 μm. Finally, the superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody is resuspended with a blocking solution I of 7 including Proclin300, Tween-20, BSA, and Tris-HCl to obtain a magnetic bead antibody working solution, pH 7, whose specific concentrations are as follows: 0.2 mg / ml superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, 0.2 mg / ml Tris-HCl 50mM, Proclin3000.1%, Tween-20 1%, BSA 0.8%;

[0053] Test tube B contains reagent R2, which is an enzyme-labeled antigen working solution: alkaline phosphatase-labeled tacrolimus antigen is used, and the labeling ratio of tacrolimus antigen to alkaline phosphatase is 1:5. The alkaline phosphatase-labeled tacrolimus antigen is then resuspended in blocking solution II containing HEPES and Proclin 300 to obtain an enzyme-labeled antigen working solution, pH 7.4, with the following specific concentrations: alkaline phosphatase-labeled tacrolimus antigen 1 μg / mL, HEPES 20 mM, Proclin 300 0.1%;

[0054] Test tube C contains reagent R3, which is a blood sample processing solution. The blood sample processing solution is a 7.4% 1× TBS buffer solution and contains 1 mM EDTA-2Na, 30 mM zinc sulfate, 0.8% Triton X-100, and 0.06% ProClin 300.

[0055] The freeze-dried bottle contains a standard product, which is a freeze-dried whole blood standard containing tacrolimus, and is prepared by freeze-drying whole blood containing tacrolimus.

[0056] The application method of the tacrolimus detection kit comprises the following specific steps:

[0057] (1) Add 100 μL of the above-mentioned reagent R3 to a centrifuge tube containing 20 μL of blood to be tested, vortex mix, centrifuge, and transfer the centrifuge fluid to an empty sample tube as the sample;

[0058] (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge liquid and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place 5 μL of standard solution, 20 μL of reagent R1, and 50 μL of reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration, and prepare a standard curve of standard solution concentration and luminescence value;

[0059] The diluent was 20 mM Tris-NaCl, 0.08% Proclin300, and 0.5% BSA;

[0060] (3) The sample of step 1 is used to replace the standard solution of step (2) for testing, and the tacrolimus concentration in the sample is calculated based on the standard curve.

[0061] Tacrolimus calibration solution was added to negative samples to prepare test blood with tacrolimus concentrations of 0.2, 5, 15, and 24 ng / mL, respectively. The test was performed using the above-mentioned tacrolimus detection kit and its application method. The test results are shown in Table 2 below.

[0062] Table 2

[0063]

[0064]

[0065] Example 3

[0066] A tacrolimus detection kit comprises reagent R1, reagent R2, reagent R3 and a standard, as well as reagent tube A, reagent tube B, reagent tube C and a lyophilized bottle for R1, reagent R2, reagent R3 and the standard, respectively;

[0067] Test tube A is filled with reagent R1, which is a magnetic bead antibody working solution: first, the magnetic beads are mixed with EDC and NHS in proportion, 5 μg EDC and 2.5 μg NHS corresponding to 1 mg of magnetic beads, and then placed in a PBS buffer solution at pH 6 and activated for 30 minutes. After the activation is completed, tacrolimus monoclonal antibodies are added and mixed with the activated magnetic beads, 20 μg tacrolimus monoclonal antibodies corresponding to 1 mg of magnetic beads, and cross-linked for 3 hours. After the cross-linking is completed, the buffer solution is removed by centrifugation to obtain a superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, with a particle size of 0.5 to 3 μm. Finally, the superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody is resuspended with a blocking solution I of 7 including Proclin300, Tween-20, BSA, and Tris-HCl to obtain a magnetic bead antibody working solution, pH 7, whose specific concentrations are as follows: 0.2 mg / ml superparamagnetic nanomaterial with a surface cross-linked tacrolimus antibody, 0.2 mg / ml Tris-HCl 50mM, Proclin3000.1%, Tween-20 1%, BSA0.8%;

[0068] Test tube B contains reagent R2, which is an enzyme-labeled antigen working solution: alkaline phosphatase-labeled tacrolimus antigen is used, and the labeling ratio of tacrolimus antigen to alkaline phosphatase is 1:5. The alkaline phosphatase-labeled tacrolimus antigen is then resuspended in blocking solution II containing HEPES and Proclin 300 to obtain an enzyme-labeled antigen working solution, pH 7.4, with the following specific concentrations: alkaline phosphatase-labeled tacrolimus antigen 1 μg / mL, HEPES 20 mM, Proclin 300 0.1%;

[0069] Test tube C contains reagent R3, which is a blood sample processing solution. The blood sample processing solution is a 7.4% 1× TBS buffer solution and contains 2 mM EDTA-2Na, 60 mM zinc sulfate, 1.2% Triton X-100, and 0.1% ProClin 300.

[0070] The freeze-dried bottle contains a standard product, which is a freeze-dried whole blood standard containing tacrolimus, and is prepared by freeze-drying whole blood containing tacrolimus.

[0071] The application method of the tacrolimus detection kit comprises the following specific steps:

[0072] (1) Add 100 μL of the above-mentioned reagent R3 to a centrifuge tube containing 20 μL of blood to be tested, vortex mix, centrifuge, and transfer the centrifuge fluid to an empty sample tube as the sample;

[0073] (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place 5 μL of standard solution, 20 μL of reagent R1, and 50 μL of reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration to prepare a standard curve of standard solution concentration and luminescence value;

[0074] The diluent was 20 mM Tris-NaCl, 0.08% Proclin300, and 0.5% BSA;

[0075] (3) replacing the standard solution in step (2) with the sample in step 1 for testing, and then calculating the tacrolimus concentration in the sample based on the standard curve;

[0076] Among them, tacrolimus calibration solution was added to the negative sample to prepare blood samples containing tacrolimus concentrations of 0.2, 5, 15, and 24 ng / mL, respectively. The blood samples were tested using the above-mentioned tacrolimus detection kit and its application method. The test results are shown in Table 3 below.

[0077] Table 3

[0078]

[0079] Comparative Example 1

[0080] A tacrolimus detection kit differs from Example 2 in that: test tube C contains reagent R3, which is a blood sample processing solution. The blood sample processing solution is a 7.4% 1×TBS buffer solution containing 1 mM EDTA-2Na, 0.8% Triton X-100, and 0.06% ProClin300. The test results are shown in Table 4 below.

[0081] Table 4

[0082]

[0083] Comparative Example 2

[0084] A tacrolimus detection kit differs from Example 2 in that: test tube C contains reagent R3, which is a blood sample processing solution. The blood sample processing solution is a 7.4% 1×TBS buffer solution containing 1 mM EDTA-2Na, 30 mM zinc sulfate, 0.5% Triton X-100, and 0.06% ProClin300. The test results are shown in Table 4 below.

[0085] Table 5

[0086]

[0087] Through the above Examples 1 to 3 and Comparative Examples 1 to 2 and the corresponding measurement results in Tables 1 to 5, it was found that the tacrolimus detection kit of the present invention and the blood sample processing liquid used in the application have more universality in treating the blood to be tested, can effectively avoid matrix interference in the blood sample, and make the detection results of tacrolimus in the blood more reliable and more accurate.

[0088] In addition, the blood to be tested containing tacrolimus at concentrations of 5 and 15 ng / mL was treated in step 1 of Examples 1 to 3 and Comparative Examples 1 to 2, and then placed at room temperature for 12, 24, and 48 hours before being tested in step 3. The test results are shown in Table 6.

[0089]

[0090] The above results indicate that the tacrolimus detection kit of the present invention and the blood sample processing solution used in its application process the blood to be tested, thereby improving the stability of tacrolimus determination.

[0091] The present invention will reseal each reagent after using it once and store it at 2-8°C for 12 months. The reagents will be taken out every month for testing to verify whether the test results of each reagent are normal and stable. The results are as follows

[0092] As shown in Table 7.

[0093] Long lasting stability Example 1 Less than 10 months Example 2 Less than 9 months Example 3 Less than 10 months Comparative Example 1 Less than 9 months Comparative Example 2 Less than 9 months Comparative Example 3 Less than 9 months

[0094] The above test results show that adding preservatives to the reagents in the detection kit of the present invention can improve the long-term stability of the test results of each liquid and sample storage.

[0095] In summary, the detection kit of the present invention is designed with a blood sample processing solution that is more universal for the blood to be tested, is simple to operate, and can effectively avoid matrix interference in the blood sample, thereby improving the reliability and stability of the detection results of tacrolimus in the sample. In addition, the detection kit of the present invention can quickly test blood samples with a short detection time. In addition, the reagents in the kit of the present invention can be stored for a longer time.

[0096] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit and essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0097] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A tacrolimus detection kit, characterized in that: Including reagent R1, reagent R2, reagent R3 and standard, as well as reagent tube A, reagent tube B, reagent tube C and lyophilized bottle for R1, reagent R2, reagent R3 and standard respectively; The reagent R1 is a magnetic bead antibody working solution, which includes a superparamagnetic nanomaterial with tacrolimus antibody cross-linked on the surface, and the particle size is 0.5 to 3 μm; The reagent R2 is an enzyme-labeled antigen working solution, which includes tacrolimus antigen labeled with alkaline phosphatase; The reagent R3 is a blood sample processing solution, which is a TBS buffer containing EDTA-2Na, zinc sulfate solution, Triton X-100, and ProClin300; The standard is a freeze-dried whole blood standard containing tacrolimus.

2. A tacrolimus detection kit according to claim 1, characterized in that: The blood sample processing solution is a TBS buffer solution with a pH of 7.2 to 7.6 and containing 1 to 2 mM EDTA-2Na, 30 to 60 mM zinc sulfate solution, 0.8 to 1.2% Triton X-100, and 0.06 to 0.1% ProClin300.

3. A tacrolimus detection kit according to claim 1, characterized in that: The magnetic bead antibody working solution also includes a blocking solution I, and the concentration of the superparamagnetic nanomaterial with tacrolimus antibody cross-linked on the surface is 0.1-1 mg / ml.

4. A tacrolimus detection kit according to claim 3, characterized in that: The blocking solution I includes 40-60 mM Tris-HCl, 0.08-0.12% Proclin 300, 0.5-1.5% Tween-20, 0.5-1% BSA, and has a pH of 6.5-8.

0.

5. A tacrolimus detection kit according to claim 1, characterized in that: The enzyme-labeled antigen working solution also includes blocking solution II, and the concentration of the alkaline phosphatase-labeled tacrolimus antigen is 1 μg / mL.

6. A tacrolimus detection kit according to claim 5, characterized in that: The blocking solution II includes 10-50 mM HEPES and 0.08-0.12% Proclin 300, with a pH of 6.8-8.

2.

7. A tacrolimus detection kit according to claim 1, characterized in that: The freeze-dried whole blood standard containing tacrolimus is prepared by freeze-drying whole blood containing tacrolimus.

8. A method for using the tacrolimus detection kit according to any one of claims 1 to 6, characterized in that: The specific steps are as follows: (1) Add reagent R3 to a centrifuge tube containing the blood to be tested, vortex mix, centrifuge, and transfer the centrifuge fluid into an empty sample tube as the sample; (2) Dissolve the standard in pure water, add reagent R3, vortex mix, centrifuge, take the centrifuge liquid and put it into an empty standard tube, and prepare different concentrations as standard solutions. Place the standard solution, reagent R1, and reagent R2 in a reaction tube, shake and mix, incubate at 37°C for 15 minutes, and then use chemiluminescence immunoassay to measure the luminescence value corresponding to the standard solution concentration, and prepare a standard curve between the standard solution concentration and the luminescence value; (3) The sample of step 1 is used to replace the standard solution of step (2) for testing, and the tacrolimus concentration in the sample is calculated based on the standard curve.

9. The application method of the tacrolimus detection kit according to claim 8, characterized in that: In step (2), the volume ratio of the reagent R3 to the blood to be tested is 5 to 6:

1.

10. The application method of the tacrolimus detection kit according to claim 8, characterized in that: The volume ratio of the standard solution, reagent R1, and reagent R2 is 1:4:10.

Images