A tacrolimus chemiluminescent immunoassay reagent and its preparation and detection method

Abstract

This invention relates to a chemiluminescent immunoassay reagent for tacrolimus, its preparation, and detection method. The reagent comprises a first reagent, a second reagent, and a magnetic separation reagent. The first reagent is a buffer solution containing a fluorescein-labeled anti-tacrolimus monoclonal antibody at a pH of 6.0–9.0, with the concentration of the fluorescein-labeled anti-tacrolimus monoclonal antibody being 1.0–5.0 μg / mL. The second reagent is a buffer solution containing a tacrolimus-alkaline phosphatase conjugate at a pH of 6.0–9.0, with the concentration of the tacrolimus-alkaline phosphatase conjugate being 0.05–0.20 μg / mL. The magnetic separation reagent is a suspension of magnetic microparticles coated with the anti-fluorescein monoclonal antibody. This invention enables the quantitative detection of tacrolimus with lower cost and higher accuracy and precision.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a tacrolimus chemiluminescent immunoassay reagent and its preparation and detection method. Background Technology

[0002] Therapeutic Drug Monitoring (TDM) is a process guided by clinical pharmacology, pharmacokinetics, and clinical chemistry principles. It utilizes advanced drug analysis techniques to measure drug concentrations in blood or other body fluids, obtaining relevant pharmacokinetic parameters to guide safe and rational drug use in clinical practice. This helps clinicians develop individualized dosing regimens to avoid or reduce side effects, improve treatment efficacy, and increase cure rates. Tacrolimus, a macrolide antibiotic isolated from Streptomyces, is a representative second-generation immunosuppressant. Due to its strong immunosuppressive effect and good selectivity, tacrolimus has been widely used since its market launch for the treatment of acute and chronic rejection in liver, kidney, and bone marrow transplants, becoming a first-line drug for treating rejection after liver and kidney transplants. In addition, it can also be used clinically for corneal and uterine transplants, as well as for the treatment of skin diseases (such as systemic lupus erythematosus, plaque psoriasis, and vitiligo) and nephrotic syndrome. Tacrolimus is mainly absorbed through the small intestine after oral administration. It has low bioavailability and high individual variability in absorption (average bioavailability is 20%, ranging from 5% to 67%). The average half-life is 8.7 hours. It binds to proteins at a rate of over 99%. After absorption, it can be widely distributed in the body. It is mainly metabolized by the liver and excreted in feces via bile, with very little excretion in urine.

[0003] This drug concentration monitoring project primarily serves as an adjunct therapy for tacrolimus in liver and kidney transplant patients. Because tacrolimus has a narrow therapeutic window, its therapeutic dose is close to the toxic dose. Low concentrations can easily cause transplant rejection, while high concentrations can lead to serious adverse reactions (such as liver and kidney toxicity, hypertension, hyperkalemia, diabetes, and neurotoxicity), directly impacting its clinical application. Therefore, careful attention should be paid to the timing of tacrolimus administration and dose adjustments to minimize adverse reactions. Contraindications include children, the elderly, pregnant women, those with liver or kidney dysfunction, anemia, hypoalbuminemia, and a body mass index (BMI) ≥28 kg / m². 2 Patients with tacrolimus should have their whole blood tacrolimus levels monitored regularly and their treatment dose adjusted accordingly. Due to CYP3A5 gene polymorphisms leading to significant individual differences in pharmacokinetic characteristics and bioavailability, close monitoring of blood drug concentrations is crucial when using tacrolimus clinically to avoid individualized variations that could result in drug toxicity or ineffectiveness. When co-administered with cytochrome P450 inducers or inhibitors, blood drug concentrations should be monitored promptly, and the tacrolimus dose adjusted accordingly.

[0004] This invention employs a competitive immunoassay principle, using magnetic microparticles as the solid phase of the immunoassay reaction. It utilizes chemiluminescence immunoassay in conjunction with a fully automated chemiluminescence immunoassay analyzer to determine the tacrolimus content in human samples. The sample, a tacrolimus-alkaline phosphatase conjugate, and magnetic microparticles containing an anti-tacrolimus monoclonal antibody are added to the reaction vessel. Tacrolimus in the sample competes with the tacrolimus-alkaline phosphatase conjugate for a limited number of binding sites on the anti-tacrolimus-specific antibody. After incubation at 37°C in the reaction vessel, the tacrolimus and tacrolimus-alkaline phosphatase conjugate bound to the specific antibody on the solid-phase magnetic beads are held in the magnetic field, while unbound tacrolimus and tacrolimus-alkaline phosphatase conjugate are washed away. Then, substrate solution for the fully automated immunoassay system is injected. The tacrolimus-alkaline phosphatase conjugate bound to the antibody on the magnetic beads is catalytically cleaved, and the intensity of the chemiluminescent photons is detected. The light intensity is inversely proportional to the concentration of tacrolimus in the sample, and the amount of analyte in the sample is determined by a stored multi-point calibration curve. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a tacrolimus chemiluminescent immunoassay reagent and its preparation and detection method. The tacrolimus chemiluminescent immunoassay reagent can be prepared at a low cost and can achieve accurate and highly precise quantitative determination of tacrolimus.

[0006] To solve the above technical problems, the present invention adopts the following technical solution:

[0007] A tacrolimus chemiluminescent immunoassay reagent, comprising an immunoreaction reagent for an immunoreaction step in chemiluminescent immunoassay, particularly comprising a first reagent, a second reagent, and a magnetic separation reagent, wherein:

[0008] The first reagent is a buffer solution containing a fluorescently labeled anti-tacrolimus monoclonal antibody and a pH of 6.0-9.0, wherein the concentration of the fluorescently labeled anti-tacrolimus monoclonal antibody is 1.0-5.0 μg / mL;

[0009] The second reagent is a buffer solution containing tacrolimus-alkaline phosphatase conjugate with a pH of 6.0-9.0, wherein the concentration of the tacrolimus-alkaline phosphatase conjugate is 0.05-0.20 μg / mL;

[0010] The magnetic separation reagent is a suspension of magnetic microparticles coated with anti-fluorescein monoclonal antibodies.

[0011] The tacrolimus-alkaline phosphatase conjugate according to the present invention can be linked using conventional labeling methods. In this invention, a preferred labeling method is to link the two using the cross-linking agent disuccinimide octanoate. The applicant has found that using disuccinimide octanoate (DSS) as a cross-linking agent for the coupling of alkaline phosphatase and tacrolimus antigen exhibits higher coupling efficiency compared to other cross-linking agents, reducing preparation costs while improving detection efficiency. Therefore, the tacrolimus-alkaline phosphatase conjugate of the present invention is preferably composed of alkaline phosphatase and tacrolimus antigen linked by the cross-linking agent disuccinimide octanoate.

[0012] The fluorescein-labeled anti-tacrolimus monoclonal antibody in the first reagent according to the present invention can be easily prepared by a mature and stable process.

[0013] The preparation of the magnetic microparticles coated with anti-fluorescein monoclonal antibodies according to the present invention is relatively easy, and there are mature preparation processes available in the prior art. For example, they can be prepared by conventional physical adsorption or chemical coupling coating methods, without any particular limitations. As a preferred embodiment of the present invention: in the magnetic microparticles coated with anti-fluorescein monoclonal antibodies, the anti-fluorescein monoclonal antibody is chemically coupled to the magnetic microparticles.

[0014] Another technical solution adopted by the present invention is: a method for preparing the above-mentioned tacrolimus chemiluminescent immunoassay reagent, which includes the steps of preparing the first reagent, the second reagent and the magnetic separation reagent respectively, wherein: the preparation process of the second reagent is as follows: ① Tacrolimus antigen and cross-linking agent disuccinimide octanoate are reacted in dimethyl sulfoxide solvent at room temperature to generate a conjugate of triiodothyronine antigen and disuccinimide octanoate, which is stored at 2-8°C for later use;

[0015] ② Mix the buffer solution containing alkaline phosphatase with the solution obtained in step ① at a molar ratio of alkaline phosphatase to the conjugate of triiodothyronine antigen and succinimide dioctanoate at 1:1.1-1.3, and react at room temperature to generate the tacrolimus-alkaline phosphatase conjugate. After the reaction is complete, desalt the reaction solution by passing it through a G-25 gel column, and adjust the concentration and pH value by selecting an appropriate pH buffer to obtain the second reagent.

[0016] Furthermore, the purity of the tacrolimus antigen and the alkaline phosphatase is preferably greater than or equal to 95 wt%, and the specific activity of the alkaline phosphatase exceeds 1000 u / mg. The concentration of the buffer solution for the alkaline phosphatase is preferably 0.5 to 1.5 mg / mL.

[0017] According to one specific aspect, the preparation method of the first reagent is as follows: Prepare a buffer solution containing fluorescein with a pH of 6.0–9.0. Then, according to the molecular ratio of fluorescein to anti-tacrolimus monoclonal antibody of 20–200:1, mix the buffer solution containing fluorescein with a pH of 6.0–9.0 with the buffer solution containing anti-tacrolimus monoclonal antibody of 6.0–9.0. After mixing, allow the mixture to stand at room temperature for reaction. Then, separate the reaction solution through a G-25 gel column to remove the free fluorescein, and obtain a solution containing fluorescein-labeled anti-tacrolimus monoclonal antibody. Then, adjust the concentration and pH with a buffer solution of appropriate pH value to obtain the first reagent.

[0018] According to another specific aspect, the magnetic separation reagent is prepared as follows: magnetic microparticles containing carboxyl active groups are reacted with antifluorescein monoclonal antibodies at room temperature for 2 to 18 hours in the presence of a coupling agent. After the reaction is completed, magnetic separation is performed, the supernatant is removed, and the pH and concentration are adjusted with a buffer solution with an appropriate pH value to obtain the magnetic separation reagent.

[0019] Preferably, the magnetic microparticles are superparamagnetic, with a diameter of 0.5–2 μm, and the content of carboxyl active groups on each gram of magnetic microparticles is not less than 0.4 mmol; the antifluorescein monoclonal antibody is a monoclonal antibody or a polyclonal monoclonal antibody with a purity greater than or equal to 90 wt% and a dilution titer greater than 1:1,000,000; the coupling agent is carbodiimide.

[0020] The buffer solutions described above in this invention can be those commonly used in the art, such as carbonate buffers, phosphate buffers, and TRIS buffers. In actual implementation, the most suitable buffer solution can be selected. For example, in the final stage of preparing the first reagent, the second reagent, and the magnetic separation reagent, a 0.1 mol / L TRIS buffer solution containing 0.5% bovine serum albumin (BSA) and pH 8.0 is typically used.

[0021] The fluorescein described in this invention can be any known fluorescein, such as fluorescein isothiocyanate, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, etc.

[0022] Another technical solution adopted by the present invention is: a detection method for the tacrolimus chemiluminescent immunoassay reagent, which is a chemiluminescent immunoassay based on magnetic particle separation technology. The detection method includes a sequential immunoreaction step, a step of washing the reaction solution of the immunoreaction using magnetic separation and washing equipment, and a step of adding a substrate solution to the washed reaction solution and detecting the luminescence intensity using a chemiluminescence detector. The immunoreaction step uses the reagent described above in the present invention and is specifically implemented as follows: the original solution or diluent of the sample to be tested is added to the test sample tube, then the first reagent and the second reagent are added sequentially, mixed, and incubated for the first time at 25-40°C, then the magnetic separation reagent is added, mixed, and incubated for the second time at 25-40°C.

[0023] The substrate solution is an alkaline phosphatase chemiluminescent substrate solution.

[0024] Furthermore, the first incubation time can be 10-20 minutes, usually 15 minutes; the second incubation time can be 3-15 minutes, usually 5 minutes.

[0025] Preferably, the alkaline phosphatase chemiluminescent substrate solution is a Tris buffer containing a certain concentration of dioxane compounds, such as a Tris buffer with a dioxane compound concentration of 0.6 mmol / L and a pH of 9.35.

[0026] The steps described above—washing the reaction solution of the immunoassay using magnetic separation and washing equipment, adding the substrate solution to the washed reaction solution, and detecting the luminescence intensity using a chemiluminescence detector—can all be performed using conventional methods, and the equipment and apparatus used are also conventional. The alkaline phosphatase chemiluminescent substrate solution is known to those skilled in the art and is commercially available.

[0027] Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0028] Accurate and precise detection of tacrolimus can be achieved using the reagents of this invention combined with traditional chemiluminescence immunoassay methods and equipment. The first and third reagents in this invention can be prepared using mature and stable processes, resulting in low production costs. Furthermore, due to the stability of the preparation process, batch-to-batch variability in reagent analysis is small, improving the inter-analytical precision of the detection. The preparation method of the second reagent in this invention effectively couples tacrolimus antigen to alkaline phosphatase with high coupling efficiency, further reducing reagent costs and ensuring detection effectiveness. The detection method of this invention offers good accuracy, high precision, high sensitivity, and a wide detection range. It allows for disordered sample pre-dilution and is simple and time-saving to operate. Compared with methods using imported reagents, the detection method of this invention has a significant cost advantage. Attached Figure Description

[0029] Figure 1 This is the standard curve of the chemiluminescent immunoassay for tacrolimus.

[0030] Figure 2 This is a linear analysis graph of the chemiluminescent immunoassay of tacrolimus. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0032] Example 1: Preparation of the first reagent

[0033] (1) Materials and instruments: Anti-tacrolimus monoclonal antibody (purity over 95 wt%, concentration 2 mg / mL) preserved in phosphate buffer; fluorescein isothiocyanate (FITC), sodium bicarbonate and other reagents should be chemically pure; G-25 gel purification column was purchased from GE.

[0034] (2) Preparation steps:

[0035] ① Prepare a 0.5 mg / mL FITC solution using 0.1–0.2 mol / L carbonate buffer solution with pH = 6.0–9.0;

[0036] ② Add the FITC solution prepared in step ① to the monoclonal antibody solution at a ratio of 1:20 of tacrolimus monoclonal antibody to FITC molecules, mix well, and let stand at room temperature for 12 hours to generate tacrolimus monoclonal antibody-FITC conjugate.

[0037] ③ Separate the reaction solution from step ② using a G-25 gel column to remove unreacted FITC and obtain a solution containing tacrolimus monoclonal antibody-FITC conjugate (i.e., FITC-labeled tacrolimus monoclonal antibody).

[0038] ④ Dilute the solution containing tacrolimus monoclonal antibody-FITC conjugate obtained in step ③ with 0.1 mol / L TRIS buffer containing 0.5% bovine serum albumin (BSA) at pH 8.0 to a concentration of 1.0–5.0 μg / mL and a pH of 6.0–9.0, which is the first reagent.

[0039] Example 2: Preparation of the second reagent

[0040] (1) Materials and instruments: Tacrolimus antigen (solid powder, purity over 95%); alkaline phosphatase (ALP solution, ALP purity of about 99%, specific activity of about 1500 U / mg, concentration of 10 mg / mL) preserved in phosphate buffer; DSS coupling agent purchased from THERMO; chemical reagents such as TRIS should be chemically pure; G-25 gel purification column is a product of GE.

[0041] (2) Preparation steps:

[0042] ① Take 1 mg of tacrolimus antigen, add DMSO to dissolve the antigen to a concentration of 20-50 mg / mL, add 0.5 mg of DSS, react at room temperature for 2 hours, dilute the reaction solution 1:10 with DMSO, and store at 2-8℃ for later use;

[0043] ② Take 1 mg of ALP solution and dilute it to 1 mg / mL with 0.1 M pH 9.5 NaHCO3 buffer. Add the tacrolimus-DMSO solution prepared in step ① to the diluted ALP buffer for ligation reaction. The volume of tacrolimus-DMSO solution added is 1 / 20 of the volume of ALP buffer. Let it stand at room temperature for 30 min, desalt it with a G-25 gel column, and store it at 2-8℃ for later use.

[0044] ③ Dilute the solution from step ② with 0.1 mol / L TRIS buffer containing 0.5% bovine serum albumin (BSA) at pH 8.0 to a concentration of 0.05–0.20 μg / mL and pH 6.0–9.0. This solution is the second reagent.

[0045] Example 3: Preparation of magnetic separation reagent

[0046] (1) Materials and Instruments:

[0047] The magnetic microparticle suspension should contain 5 wt% magnetic microparticles, each containing a carboxyl (COOH) active group. The carboxyl content per gram (g) of magnetic microparticles (dry weight) should be no less than 0.4 mmol. The microparticles should be superparamagnetic and have a diameter between 0.5 and 2 μm. The anti-FITC monoclonal antibody should be either a polyclonal or monoclonal antibody, with a purity of ≥90 wt% and a dilution titer exceeding 1:1,000,000. 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be chemically pure.

[0048] (2) Preparation steps:

[0049] ① Take a suspension of 100 mg of magnetic microparticles, magnetically separate the supernatant, and resuspend in 10 mL of 0.05 mol / L, pH 4.5-5 MES buffer;

[0050] ② Add 2-4 mg of anti-FITC monoclonal antibody and suspend at room temperature for 30-60 min;

[0051] ③ Add 0.5–1 mL of freshly prepared 10 mg / mL EDC aqueous solution and suspend at room temperature for 2–12 h;

[0052] ④ Magnetic separation: remove the supernatant and resuspend the sample in 0.1 mol / L TRIS buffer containing 0.5% bovine serum albumin (BSA) at pH 8.0 to a concentration of 1 mg / mL at pH 8.0. This is the magnetic separation reagent.

[0053] Example 4: Reagent for tacrolimus chemiluminescent immunoassay, comprising:

[0054] The first reagent (concentration of 0.75 μg / mL) prepared according to the method in Example 1, 50 mL;

[0055] The second reagent (concentration of 0.05 μg / mL) prepared according to the method in Example 2, 50 mL;

[0056] 50 mL of magnetic separation reagent prepared according to the method in Example 3.

[0057] Example 5: Reagent for quantitative detection of tacrolimus, comprising:

[0058] The first reagent (concentration of 0.5 μg / mL) prepared according to the method in Example 1, 5 mL;

[0059] The second reagent (concentration of 0.02 μg / mL) prepared according to the method of Comparative Example 2, 5 mL;

[0060] 5 mL of magnetic separation reagent prepared according to the method in Example 3.

[0061] Example 6: Chemiluminescent immunoassay of tacrolimus using the reagents from Example 4.

[0062] (1) Detection steps

[0063] ①Immune reaction: Add 30 μl of the original sample (serum or plasma) to the test tube, then add 50 μl of the original sample.

[0064] Mix the first reagent and 50 μl of the second reagent, and incubate at 37±1℃ for 15 min; add 50 μl of magnetic separation reagent, mix well, and incubate at 37±1℃ for 5 min.

[0065] ② Washing: Allow the magnetic particles to settle in a magnetic field, remove the supernatant, add 600 μl of washing solution, remove the magnetic field, shake to fully suspend the magnetic particles, then perform magnetic separation and remove the supernatant. Repeat this step 3 times;

[0066] ③ Add substrate solution to detect luminescence intensity: Add 150 μl of alkaline phosphatase chemiluminescence substrate solution (APCL-Ⅰ from Beijing Apis Biotechnology Co., Ltd., Tris buffer containing 0.6 mmol / L dioxane compounds, pH 9.35) to the detection tube, shake to fully suspend the magnetic particles, and detect the luminescence intensity per unit time.

[0067] (2) Set the reaction parameters for the fully automated chemiluminescence immunoassay analyzer (Table 1)

[0068] Table 1. Response parameters for tacrolimus detection using a fully automated chemiluminescence immunoassay analyzer.

[0069]

[0070] (3) Reagent performance evaluation experiment

[0071] The performance of the above-mentioned tacrolimus chemiluminescent immunoassay reagent was validated. The main performance indicators tested included appearance, fill volume, limit of detection, linearity, repeatability, batch-to-batch difference, accuracy, calibrator accuracy and homogeneity, and quality control accuracy and homogeneity. Experimental parameters were set according to the above method. The reagent performance validation results are as follows:

[0072] Appearance

[0073]

[0074] Appearance

[0075] Appearance Detection standard determination Calibrator Meets requirements The calibrator is a dark red liquid. conform to

[0076] Appearance

[0077] Appearance Detection standard determination Quality control products Meets requirements The quality control sample is a dark red liquid. conform to

[0078] Filling volume

[0079] Filling volume Detection standard determination Tacrolimus R1: 5.1mL, R2: 5.0mL, R3: 5.2mL R1≥5.0mL, R2≥5.0mL, R3≥5.0mL conform to

[0080] Filling volume

[0081]

[0082] Filling volume

[0083]

[0084] Unit: Limit of Detection (ng / mL)

[0085]

[0086] Linear range: 2.00–32.00 ng / mL

[0087]

[0088] Repeatability unit: ng / mL

[0089]

[0090]

[0091]

[0092] Inter-batch difference - second concentration

[0093]

[0094]

[0095] Inter-batch difference - third concentration

[0096]

[0097]

[0098]

[0099] Calibrator

[0100] Accuracy unit: ng / mL

[0101]

[0102]

[0103] Uniformity unit: ng / mL calibrator 1

[0104]

[0105] Uniformity

[0106] Calibrator 2 units: ng / mL

[0107]

[0108]

[0109] Uniformity

[0110] Calibrator 3 units: ng / mL

[0111]

[0112]

[0113] Quality control products

[0114] Expected result unit: ng / mL

[0115] # Actual test #1 Actual test #2 Actual test #3 standard determination Quality control product 1 4.98 5.02 5.01 4.49-5.55 conform to Quality control product 2 10.13 10.06 9.81 8.74-11.08 conform to Quality control product 3 19.96 20.03 19.91 18.26-23.02 conform to

[0116] Uniformity

[0117] Quality control sample 1 unit: ng / mL

[0118]

[0119] Quality control sample, 2 units: ng / mL

[0120]

[0121]

[0122] Quality control product 3

[0123]

[0124] The above-described embodiments are merely preferred embodiments for fully illustrating the present invention. The scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

Claims

1. A tacrolimus chemiluminescent immunoassay reagent, the reagent comprising an immunoreaction reagent for an immunoreaction step in chemiluminescent immunoassay, characterized in that: The immunoreaction reagent comprises a first reagent, a second reagent, and a magnetic separation reagent, wherein: The first reagent is a buffer solution containing a fluorescently labeled anti-tacrolimus monoclonal antibody and a pH of 6.0-9.0, wherein the concentration of the fluorescently labeled anti-tacrolimus monoclonal antibody is 1.0-5.0 μg / mL; The second reagent is a buffer solution containing tacrolimus-alkaline phosphatase conjugate with a pH of 6.0-9.0, wherein the concentration of the tacrolimus-alkaline phosphatase conjugate is 0.05-0.20 μg / mL; The magnetic separation reagent is a suspension of magnetic microparticles coated with anti-fluorescein monoclonal antibodies.

2. The tacrolimus chemiluminescent immunoassay reagent according to claim 1, characterized in that: The tacrolimus-alkaline phosphatase conjugate is composed of alkaline phosphatase and tacrolimus antigen linked by a cross-linking agent, succinimide octanoate.

3. The tacrolimus chemiluminescent immunoassay reagent according to claim 1, characterized in that: In the magnetic microparticles coated with anti-fluorescein monoclonal antibodies, there is a chemical coupling between the anti-fluorescein monoclonal antibodies and the magnetic microparticles.

4. A method for preparing a tacrolimus chemiluminescent immunoassay reagent as described in any one of claims 1 to 3, comprising the steps of preparing the first reagent, the second reagent, and the magnetic separation reagent respectively, characterized in that: The preparation process of the second reagent is as follows: ① Tacrolimus antigen and cross-linking agent disuccinimide octanoate are reacted in dimethyl sulfoxide solvent at room temperature to generate a conjugate of triiodothyronine antigen and disuccinimide octanoate, which is then stored at 2-8°C for later use. ② Mix the buffer solution containing alkaline phosphatase with the solution obtained in step ① at a molar ratio of alkaline phosphatase to the conjugate of triiodothyronine antigen and succinimide dioctanoate at 1:1.1~1.

3. React at room temperature to generate the tacrolimus-alkaline phosphatase conjugate. After the reaction is complete, desalt the reaction solution by passing it through a G-25 gel column. Adjust the concentration and pH value with an appropriate pH buffer to obtain the second reagent.

5. The preparation method according to claim 4, characterized in that: The purity of the tacrolimus antigen and the alkaline phosphatase is greater than or equal to 95 wt%, and the specific activity of the alkaline phosphatase exceeds 1000 u / mg. The concentration of the buffer solution for the alkaline phosphatase is 0.5~1.5 mg / mL.

6. The preparation method according to claim 4, characterized in that: The preparation method of the first reagent is as follows: Prepare a buffer solution containing fluorescein with a pH of 6.0-9.

0. Then, according to the molecular ratio of fluorescein to anti-tacrolimus monoclonal antibody of 20-200:1, mix the buffer solution containing fluorescein with a pH of 6.0-9.0 with the buffer solution containing anti-tacrolimus monoclonal antibody with a pH of 6.0-9.

0. After mixing, allow the mixture to stand at room temperature for reaction. Then, separate the reaction solution through a G-25 gel column to remove the free fluorescein, and obtain a solution containing fluorescein-labeled anti-tacrolimus monoclonal antibody. Then, adjust the concentration and pH with a buffer solution with an appropriate pH value to obtain the first reagent.

7. The preparation method according to claim 4, characterized in that: The magnetic separation reagent is prepared as follows: magnetic microparticles containing carboxyl active groups are reacted with antifluorescein monoclonal antibody at room temperature for 2-18 hours in the presence of a coupling agent. After the reaction is completed, magnetic separation is performed, the supernatant is removed, and the pH and concentration are adjusted with a buffer solution with an appropriate pH value to obtain the magnetic separation reagent.

8. The preparation method according to claim 7, characterized in that: The magnetic microparticles are superparamagnetic, with a diameter of 0.5~2μm, and the content of carboxyl active groups on each gram of magnetic microparticles is not less than 0.4mmol; the antifluorescein monoclonal antibody is a monoclonal antibody or a polyclonal monoclonal antibody with a purity greater than or equal to 90wt% and a dilution titer greater than 1:1,000,000; the coupling agent is carbodiimide.

9. A detection method for the tacrolimus chemiluminescent immunoassay reagent as described in any one of claims 1 to 3, the method being a chemiluminescent immunoassay based on magnetic particle separation technology, the detection method comprising the following steps: an immunoassay step, a step of washing the reaction solution of the immunoassay using a magnetic separation and washing device, and a step of adding a substrate solution to the washed reaction solution and detecting the luminescence intensity using a chemiluminescence detector, characterized in that: The specific implementation is as follows: Add the original solution or diluent of the sample to be tested to the test tube, then add the first reagent and the second reagent in sequence, mix well, and perform the first incubation at 25~40℃. Then add the magnetic separation reagent, mix well, and perform the second incubation at 25~40℃. The substrate solution is an alkaline phosphatase chemiluminescent substrate solution.

10. The detection method according to claim 9, characterized in that: The first incubation period is 10-20 minutes; the second incubation period is 3-15 minutes.

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