Cyclosporine detection kit and application thereof

Abstract

The invention discloses a cyclosporine detection kit and application thereof, the cyclosporine detection kit comprises a reagent R1 comprising a magnetic bead coated cyclosporine antibody and a Tris buffer solution I, a reagent R2 comprising an alkaline phosphatase labeled cyclosporine antigen and an AP stabilizer, a reagent R3 comprising an MES buffer solution, and a reagent R4 comprising an MES buffer solution. The sample treating agent comprises a Tris buffer solution III containing copper sulfate pentahydrate, sodium sulfite, a preservative, methanol and ethylene glycol. According to the cyclosporin detection kit and the application thereof, the R1, R2 and R3 reagents are combined and applied to the to-be-detected sample treated by the specific Tris buffer solution I sample treating agent, the specific detection of cyclosporin is improved through the synergistic effect of the specific Tris buffer solution I, the AP stabilizer and the specific MES buffer solution, the detection accuracy and stability are good, the repeatability is good, and the kit is suitable for large-scale popularization and application. The problems of cross reaction and insufficient stability during detection of a to-be-detected sample are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to a cyclosporine detection kit and its application, and belongs to the technical field of biological detection. BACKGROUND

[0002] Cyclosporine is a potent immunosuppressant and is widely used for anti-rejection treatment after organ transplantation. Due to its narrow therapeutic window, large individual differences, and close relationship between blood concentration and efficacy and toxicity, blood concentration monitoring is required. Current detection methods include: 1) high performance liquid chromatography, which is highly accurate but complex to operate and time-consuming, and is not suitable for rapid clinical detection; 2) enzyme-linked immunosorbent assay, which is simple to operate but has low sensitivity and is easily disturbed; 3) fluorescence immunoassay, which has high sensitivity but is greatly affected by sample matrix; and 4) chemiluminescent immunoassay, which has the advantages of high sensitivity, wide linear range, and high degree of automation, but the currently available kits still have problems such as high cross-reactivity and insufficient stability. SUMMARY

[0003] To at least solve the above-mentioned problems of the prior art, the present application provides a cyclosporine detection kit and its application, which has high stability, specificity, accuracy and sensitivity. The detection kit can accurately test cyclosporine by using chemiluminescent immunoassay.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solution: a cyclosporine detection kit, comprising R1 reagent, R2 reagent, R3 reagent and sample processing agent.

[0005] The R1 reagent comprises magnetic bead-coated cyclosporine antibody and Tris buffer solution I.

[0006] The R2 reagent comprises alkaline phosphatase-labeled cyclosporine antigen and AP stabilizer.

[0007] The R3 reagent comprises MES buffer.

[0008] The sample processing agent comprises Tris buffer solution II, wherein the Tris buffer solution II contains CuSO4·5H2O, Na2SO3, Proclin300, methanol and ethylene glycol.

[0009] Preferably, in the sample processing agent, the concentration of the Tris buffer solution II is 40-100mM, the pH is 7.5-8, and it contains 50-120mM CuSO4·5H2O, 10-40mM Na2SO3, 0.01-0.04w / w% Proclin300, 32-45v / v% methanol and 25-33v / v% ethylene glycol.

[0010] Preferably, in the R1 reagent, the concentration of the Tris buffer solution I is 40-100 mM, the pH is 7-7.5, and the Tris buffer solution I contains BSA, trehalose, mannitol, Tween-20 and Proclin 300.

[0011] Preferably, in the Tris buffer solution I, 0.5-3 w / w% of BSA, 0.5-2.5 w / w% of trehalose, 0.2-2 w / w% of mannitol, 0.1-1.2 w / w% of Tween-20 and 0.05-0.2 w / w% of Proclin 300 are contained.

[0012] Preferably, in the R3 reagent, the concentration of the MES buffer solution is 40-100 mM, the pH is 6.0-6.5, and the MES buffer solution further contains NaCl and Proclin 300.

[0013] Preferably, in the MES buffer solution, 0.5-1.2 w / w% of NaCl and 0.05-0.2 w / w% of Proclin 300 are contained.

[0014] Preferably, in the R1 reagent, the concentration of the magnetic bead-coated cyclosporine antibody magnetic beads is 0.05-0.5 mg / mL.

[0015] Preferably, in the magnetic bead-coated cyclosporine antibody, the mass ratio of magnetic beads to cyclosporine antibody is 100-10:1.

[0016] Preferably, in the R2 reagent, the concentration of the alkaline phosphatase-labeled cyclosporine antigen cyclosporine antigen is 0.05-0.2 μg / mL.

[0017] Preferably, in the alkaline phosphatase-labeled cyclosporine antigen, the mass ratio of alkaline phosphatase to cyclosporine antigen is 1-10:1.

[0018] The application also provides a method for using the cyclosporine detection kit, comprising the following steps:

[0019] (1) adding a sample processing agent to the sample to be tested, vortexing, centrifuging, and taking the centrifugal liquid to a sample tube as a sample;

[0020] (2) placing the sample, R1 reagent, R2 reagent and R3 reagent in a reaction tube, shaking and mixing, incubating, and then testing by chemiluminescence immunoassay to determine the luminescence value corresponding to the sample concentration, and calculating the cyclosporine concentration in the sample according to the standard curve.

[0021] Preferably, the volume ratio of the sample to be tested to the sample processing agent is 1:1.

[0022] Preferably, the ratio of the sample, the R1 reagent, the R2 reagent and the R3 reagent is 1:2:5:10.

[0023] Preferably, the standard curve is prepared by dissolving cyclosporine-containing standard samples into different concentrations of standard samples, adding sample treatment agent to the standard samples, vortexing, centrifuging, taking the centrifugal liquid, and loading the centrifugal liquid into empty standard sample tubes as standard sample liquid.

[0024] The cyclosporine detection kit of the present application uses Tris buffer solution II containing CuSO4·5H2O, Na2SO3, Proclin300, methanol and ethylene glycol as a sample treatment agent to treat the sample to be tested, which not only avoids cross-reaction during detection of the sample to be tested, but also improves the detection accuracy and stability of the detection target in the sample; the cyclosporine detection kit of the present application uses the combination of the R1 reagent including the magnetic bead-coated cyclosporine antibody and the Tris buffer solution I, the R2 reagent including the alkaline phosphatase-labeled cyclosporine antigen and the AP stabilizer, and the R3 reagent including the protective MES buffer, which makes the detection value of cyclosporine in the sample fall within the acceptable error range of the actual value, has good accuracy, improves the specificity and sensitivity of cyclosporine detection, further reduces the error, and improves the stability of the detection; at the same time, the Tris buffer solution I of the present application contains trehalose, mannitol and Tween-20, which greatly improves the storage stability of the magnetic bead-coated cyclosporine antibody; the cyclosporine detection kit of the present application uses chemiluminescence immunoassay for testing, which can meet the needs of rapid and accurate detection in the clinic, has high accuracy, high sensitivity, high specificity, and good stability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The standard curve of Example 1 of the present application is shown in the following figure. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the implementation of the present application. The described embodiments are only a part of the embodiments of the present application, not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application. If no specific conditions are specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments, components used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0027] The application provides a cyclosporine detection kit, which comprises an R1 reagent, an R2 reagent, an R3 reagent and a sample processing agent.

[0028] The R1 reagent comprises a magnetic bead coated cyclosporine antibody and a Tris buffer solution I, wherein the Tris buffer solution I contains BSA, trehalose, mannitol, Tween-20 and Proclin 300.

[0029] The R2 reagent comprises an alkaline phosphatase labeled cyclosporine antigen and an AP stabilizer, wherein the AP stabilizer used in the application is selected from Shanghai Aladdin Biochem Technology Co., Ltd., item number: A615749-MOPF-250ml, specification: based on MOPS buffer, pH 6.3±0.4, no protein.

[0030] The R3 reagent comprises a MES buffer solution, wherein the MES buffer solution contains NaCl and Proclin 300.

[0031] The sample processing agent comprises a Tris buffer solution II, wherein the Tris buffer solution II contains CuSO4·5H2O, Na2SO3, Proclin 300, methanol and ethylene glycol.

[0032] In the sample processing agent, the concentration of the Tris buffer solution II is 50-100 mM, the pH is 7.5-8, and the Tris buffer solution II contains 50-120 mM CuSO4·5H2O, 10-40 mM Na2SO3, 0.01-0.04 w / w % Proclin 300, 32-45 v / v % methanol and 25-33 v / v % ethylene glycol.

[0033] In the R1 reagent, the concentration of the Tris buffer solution I is 40-100 mM, the pH is 7-7.5, and the Tris buffer solution I contains 0.5-3 w / w % BSA, 0.5-2.5 w / w % trehalose, 0.2-2 w / w % mannitol, 0.1-1.2 w / w % Tween-20 and 0.05-0.2 w / w % Proclin 300.

[0034] In the R3 reagent, the concentration of the MES buffer solution is 50-100 mM, the pH is 6.0-6.5, and the MES buffer solution contains 0.5-1.2 w / w % NaCl and 0.05-0.2 w / w % Proclin 300.

[0035] In the R1 reagent, the magnetic bead concentration of the magnetic bead coated cyclosporine antibody is 0.05-0.5 mg / mL.

[0036] The mass ratio of the magnetic beads to the cyclosporine antibody in the magnetic bead coated cyclosporine antibody used in the application is 100-10:1; the preparation process of the specific magnetic bead coated cyclosporine antibody is as follows: first, the magnetic beads are mixed with EDC and NHS in proportion, 1 mg of the magnetic beads corresponds to 5 μg of EDC and 2.5 μg of NHS, then the mixture is placed in a PBS buffer solution with a pH of 5-6.5 and activated at 20-30°C for 15-30 min, after the activation is completed, the buffer solution is removed by centrifugation, then the activated magnetic beads are resuspended by adding a PBS buffer solution, the cyclosporine monoclonal antibody is added and mixed, the mass ratio of the magnetic beads to the cyclosporine monoclonal antibody is 100-10:1, then cross-linking is performed at 20-30°C for 3 h, after the cross-linking is completed, the buffer solution is removed by centrifugation, and the magnetic bead coated cyclosporine antibody with a particle size of 1-3 μm is obtained, then the magnetic bead coated cyclosporine antibody is blocked by using a Tris buffer solution I containing BSA, trehalose, mannitol, Tween-20 and Proclin 300 for 2 h, after the blocking is completed, the buffer solution is removed by centrifugation, and the carboxyl magnetic bead coated with the valproic acid antibody is obtained.

[0037] The cyclosporine antigen concentration of the alkaline phosphatase labeled cyclosporine antigen used in the application is 0.05-0.2 μg / mL.

[0038] The mass ratio of the alkaline phosphatase to the cyclosporine antigen in the alkaline phosphatase labeled cyclosporine antigen used in the application is 1-10:1; the preparation process of the specific alkaline phosphatase labeled cyclosporine antigen is as follows: the cyclosporine antigen, the alkaline phosphatase and the TBS buffer solution are mixed, the mass ratio of the alkaline phosphatase to the cyclosporine antigen is 1-10:1, then dialysis is performed overnight at 2-8°C, then glutaraldehyde is added and the reaction is performed in the dark for 2-3 h, the glutaraldehyde accounts for 0.5% of the total mass of the cyclosporine antigen and the alkaline phosphatase, after the reaction is completed, the TBS buffer solution is added, then dialysis is performed overnight at 2-8°C, and the alkaline phosphatase labeled cyclosporine antigen is obtained after the dialysis is completed.

[0039] The above-mentioned cyclosporine detection kit of the application tests the cyclosporine in a sample by using a chemiluminescence immunoassay, and the specific application method steps are as follows:

[0040] (1) The sample processing agent is added to the sample to be tested, the volume ratio of the sample to be tested to the sample processing agent is 1:1, vortex oscillation is performed for 30 s, centrifugation is performed at 12000 rpm for 10 min, the centrifugal liquid is taken and loaded into a sample tube as a sample;

[0041] (2) The sample, the R1 reagent, the R2 reagent and the R3 reagent are placed in a reaction tube, the volume ratio of the sample to the R1 reagent, the R2 reagent and the R3 reagent is 1:2:5:10, the mixture is shaken and mixed, incubation is performed at 37°C for 15 min, then the chemiluminescence immunoassay is used for testing, the luminescence value corresponding to the sample concentration is determined, and the cyclosporine concentration in the sample is calculated according to the standard curve.

[0042] For the standard curve: dissolve the cyclosporine-containing standard sample, and prepare standard samples of different concentrations. Add sample treatment agent to the standard samples, vortex mix, centrifuge, and take the centrifugal liquid to fill the empty standard sample tube as the standard sample liquid. The standard sample liquid is used to replace the sample in step (2) for testing. The luminescence value corresponding to the standard sample concentration is determined, and the standard curve of the standard sample concentration and the luminescence value is prepared.

[0043] Example 1

[0044] A cyclosporine detection kit, comprising R1 reagent, R2 reagent, R3 reagent, and sample treatment agent; wherein,

[0045] I. The R1 reagent comprises magnetic bead-coated cyclosporine antibody and Tris buffer solution I:

[0046] 1) In the magnetic bead-coated cyclosporine antibody, the magnetic bead particle size is 1 μm, and the mass ratio of magnetic bead to cyclosporine antibody is 50:1;

[0047] 2) The Tris buffer solution I is prepared by using Tris, HCl, BSA, trehalose, mannitol, Tween-20, Proclin 300, and pure water, with a concentration of 50 mM Tris buffer solution I, pH 7-7.5, and containing 2 w / w% BSA, 1.5 w / w% trehalose, 1 w / w% mannitol, 0.8 w / w% Tween-20, and 0.1 w / w% Proclin 300;

[0048] 3) The magnetic bead-coated cyclosporine antibody is diluted with the Tris buffer solution I of 2) above, so that the magnetic bead concentration is 0.2 mg / mL, i.e. the R1 reagent;

[0049] II. The R2 reagent comprises alkaline phosphatase-labeled cyclosporine antigen and AP stabilizer:

[0050] 4) In the alkaline phosphatase-labeled cyclosporine antigen, the mass ratio of alkaline phosphatase to cyclosporine antigen is 5:1;

[0051] 5) The AP stabilizer is based on MOPS buffer, pH 6.5; no protein;

[0052] 6) The alkaline phosphatase-labeled cyclosporine antigen is diluted with the AP stabilizer of 5) above, so that the cyclosporine antigen concentration is 0.1 μg / mL, i.e. the R2 reagent;

[0053] III. The R3 reagent comprises MES buffer:

[0054] 7) MES buffer: 50 mM MES buffer, pH 6.0-6.5, containing 0.9 w / w% NaCl and 0.1 w / w% Proclin 300, prepared using MES, NaCl, Proclin 300, and purified water, i.e. R3 reagent;

[0055] IV. The sample treatment agent comprises Tris buffer solution II:

[0056] 8) Tris buffer solution II: 50 mM Tris buffer solution II, pH 7.5-8, containing 100 mM CuSO4-5H2O, 30 mM Na2SO3, 0.02 w / w% Proclin 300, 40 v / v% methanol, and 30 v / v% ethylene glycol, prepared using Tris, HCl, CuSO4-5H2O, Na2SO3, methanol, ethylene glycol, Proclin 300, and purified water, i.e. sample treatment agent;

[0057] V. The test kit composed of R1 reagent, R2 reagent, R3 reagent, and sample treatment agent is used to test cyclosporine in a sample by chemiluminescent immunoassay. The specific application method steps are as follows:

[0058] (1) Add the sample treatment agent to the sample to be tested, and vortex for 30 s at a volume ratio of sample to be tested to sample treatment agent of 1:1. Centrifuge at 12000 rpm for 10 min, and take the centrifugal liquid to fill into a sample tube as a sample;

[0059] (2) Place 10 μL of the sample, 20 μL of R1 reagent, 50 μL of R2 reagent, and 100 μL of R3 reagent in a reaction tube, shake to mix, incubate at 37°C for 15 min, and then test by chemiluminescent immunoassay to determine the luminescence value corresponding to the sample concentration. The cyclosporine concentration in the sample is calculated according to the standard curve.

[0060] For the standard curve: dissolve the standard sample containing cyclosporine to prepare standard samples of different concentrations. The cyclosporine concentrations are 57.508 ng / mL, 123.984 ng / mL, 250.921 ng / mL, 420.153 ng / mL, 598 ng / mL, 773.944 ng / mL, 915.562 ng / mL, 1123.221 ng / mL, and 1490.566 ng / mL. Add the sample treatment agent to the standard sample, vortex to mix, centrifuge, take the centrifugal liquid to fill into an empty standard sample tube as a standard sample liquid, and test the standard sample liquid instead of the sample in step (2) to determine the luminescence value corresponding to the standard sample concentration. As shown in Table 1, a standard curve (y) of the standard sample concentration (x) and the luminescence value is prepared according to the data in Table 1, as shown in Figure 1The standard curve formula is shown as y=(a-d) / (1+(x / c) b )+d, a=49061623017, b=1.12326364, c=0.00439662, d=59082.41498.

[0061] Table 1

[0062] Serial number Standard sample concentration (ng / ml) Luminous value 1 57.508 1255296 2 123.984 511982.5 3 250.921 312180.5 4 420.153 183086 5 598.389 148042 6 773.944 117119 7 915.562 106703 8 1123.221 98452 9 1490.566 94272

[0063] In addition, cyclosporine is added to the negative sample to prepare a blood sample with a concentration of 60 ng / mL to 1500 ng / mL. The detection reagent kit and application method of the embodiment are used for detection of the luminescence value, and the corresponding cyclosporine concentration value is calculated according to the standard curve. At the same time, the cyclosporine concentration is detected by liquid chromatography-tandem mass spectrometry, and the difference and deviation between the cyclosporine concentration detected by liquid chromatography-tandem mass spectrometry and the cyclosporine chemiluminescence immunoassay kit are calculated, as shown in Table 2. It should be noted that the difference between the concentration detected by liquid chromatography-tandem mass spectrometry and the concentration detected by chemiluminescence immunoassay is within the range of ±5 ng / ml, and the deviation is within the range of ±8%, which indicates that the cyclosporine detection kit of the embodiment has good accuracy.

[0064] Table 2

[0065]

[0066] As can be seen from Table 2, the difference between the concentration detected by liquid chromatography-tandem mass spectrometry and the concentration detected by chemiluminescence immunoassay is within the range of ±5 ng / ml, and the deviation is within the range of ±3%, which indicates that the cyclosporine detection kit of the embodiment has high accuracy in detecting cyclosporine, and the detection result has high reliability, and the application of the cyclosporine detection kit of the embodiment can be promoted.

[0067] Example 2

[0068] A cyclosporine detection kit, comprising R1 reagent, R2 reagent, R3 reagent and sample processing agent; wherein,

[0069] I. The R1 reagent comprises magnetic bead coated cyclosporine antibody and Tris buffer solution I:

[0070] 1) In the magnetic bead coated cyclosporine antibody, the magnetic bead particle size is 1 μm, and the mass ratio of magnetic bead to cyclosporine antibody is 50:1;

[0071] 2) Tris buffer solution I: 50 mM Tris buffer solution I, pH 7-7.5, containing 0.5 w / w% BSA, 0.5 w / w% trehalose, 0.2 w / w% mannitol, 0.1 w / w% Tween-20 and 0.05 w / w% Proclin 300, prepared using Tris, HCl, BSA, trehalose, mannitol, Tween-20, Proclin 300 and pure water;

[0072] 3) Dilute the magnetic bead-coated cyclosporine antibody using the Tris buffer solution I of 2) above, so that the magnetic bead concentration is 0.2 mg / mL, i.e. R1 reagent;

[0073] II. R2 reagent includes alkaline phosphatase-labeled cyclosporine antigen and AP stabilizer:

[0074] 4) In the alkaline phosphatase-labeled cyclosporine antigen, the mass ratio of alkaline phosphatase to cyclosporine antigen is 5:1;

[0075] 5) AP stabilizer, based on MOPS buffer, pH 6.5; protein-free;

[0076] 6) Dilute the alkaline phosphatase-labeled cyclosporine antigen using the AP stabilizer of 5) above, so that the cyclosporine antigen concentration is 0.1 μg / mL, i.e. R2 reagent;

[0077] III. R3 reagent includes MES buffer:

[0078] 7) MES buffer: 50 mM MES buffer, pH 6.0-6.5, containing 0.5 w / w% NaCl and 0.05 w / w% Proclin 300, prepared using MES, NaCl, Proclin 300 and pure water, i.e. R3 reagent;

[0079] IV. Sample treatment reagent includes Tris buffer solution II:

[0080] 8) Tris buffer solution II: 50 mM Tris buffer solution II, pH 7.5-8, containing 50 mM CuSO4-5H2O, 10 mM Na2SO3, 0.01 w / w% Proclin 300, 32 v / v% methanol and 25 v / v% ethylene glycol, prepared using Tris, HCl, CuSO4-5H2O, Na2SO3, methanol, ethylene glycol, Proclin 300 and pure water, i.e. sample treatment reagent;

[0081] V. The test kit composed of R1 reagent, R2 reagent, R3 reagent and sample treatment reagent tests cyclosporine in a sample by chemiluminescent immunoassay, and the specific application method steps are as follows:

[0082] (1) Add a sample processing agent to the sample to be tested, the volume ratio of the sample to be tested to the sample processing agent is 1:1, vortex for 30s, centrifuge at 12000 rpm for 10 min, take the centrifugal liquid and load it into a sample tube as a sample;

[0083] (2) Place 10 μL of the sample, 20 μL of R1 reagent, 50 μL of R2 reagent, and 100 μL of R3 reagent in a reaction tube, shake and mix, incubate at 37°C for 15 min, then test using a chemiluminescence immunoassay to determine the luminescence value corresponding to the sample concentration, and calculate the cyclosporin concentration in the sample according to the standard curve; the standard curve is prepared in the same manner as in Example 1.

[0084] Example 3

[0085] A cyclosporin detection kit, comprising R1 reagent, R2 reagent, R3 reagent, and a sample processing agent; wherein,

[0086] I. The R1 reagent comprises magnetic bead-coated cyclosporin antibodies and Tris buffer solution I:

[0087] 1) In the magnetic bead-coated cyclosporin antibodies, the magnetic bead particle size is 1 μm, and the mass ratio of magnetic beads to cyclosporin antibodies is 50:1;

[0088] 2) Tris buffer solution I: using Tris, HCl, BSA, trehalose, mannitol, Tween-20, Proclin 300, and pure water to prepare a 50 mM Tris buffer solution I with a pH of 7-7.5, and containing 3 w / w% BSA, 2.5 w / w% trehalose, 2 w / w% mannitol, 1.2 w / w% Tween-20, and 0.2 w / w% Proclin 300;

[0089] 3) Dilute the magnetic bead-coated cyclosporin antibodies with the Tris buffer solution I of 2) above, so that the magnetic bead concentration is 0.2 mg / mL, i.e. the R1 reagent;

[0090] II. The R2 reagent comprises alkaline phosphatase-labeled cyclosporin antigen and AP stabilizer:

[0091] 4) In the alkaline phosphatase-labeled cyclosporin antigen, the mass ratio of alkaline phosphatase to cyclosporin antigen is 5:1;

[0092] 5) AP stabilizer, based on MOPS buffer, pH 6.5; no protein;

[0093] 6) Dilute the alkaline phosphatase-labeled cyclosporin antigen with the AP stabilizer of 5) above, so that the cyclosporin antigen concentration is 0.1 μg / mL, i.e. the R2 reagent;

[0094] Three, R3 reagent includes MES buffer solution:

[0095] 7) MES buffer solution: using MES, NaCl, Proclin 300, pure water to prepare 50mM MES buffer solution, pH is 6.0-6.5, and contains 0.5w / w% NaCl and 0.2w / w% Proclin 300, namely R3 reagent;

[0096] Four, sample processing agent includes Tris buffer solution II:

[0097] 8) Tris buffer solution II: using Tris, HCl, CuSO4·5H2O, Na2SO3, methanol, ethylene glycol, Proclin 300, pure water to prepare 50mM Tris buffer solution II, pH is 7.5-8, and contains 120mM CuSO4·5H2O, 30mM Na2SO3, 0.05w / w% Proclin 300, 45v / v% methanol and 33v / v% ethylene glycol, namely sample processing agent;

[0098] Five, the detection kit composed of R1 reagent, R2 reagent, R3 reagent and sample processing agent tests cyclosporine in sample by chemiluminescence immunoassay, and the specific application method steps are as follows:

[0099] (1) adding sample processing agent to the sample to be tested, the volume ratio of sample to be tested to sample processing agent is 1:1, vortexing for 30s, centrifuging at 12000rpm for 10min, and taking the centrifugal liquid to load into the sample tube as the sample;

[0100] (2) placing 10μL sample, 20μL R1 reagent, 50μL R2 reagent and 100μL R3 reagent in the reaction tube, shaking and mixing, incubating at 37℃ for 15min, and then testing by chemiluminescence immunoassay to determine the luminescence value corresponding to the sample concentration, and calculating the cyclosporine concentration in the sample according to the standard curve. The standard curve is prepared in the same way as in Example 1.

[0101] Sensitivity, repeatability and accuracy evaluation: the cyclosporine detection kits and application methods of Examples 1-3 are used to detect the sample to be tested with cyclosporine concentration of 60ng / ml, 510ng / ml and 1250ng / ml respectively, the detection number of each sample is 8 times, and the mean value, deviation, standard deviation and coefficient of variation are calculated, as shown in Table 1. The CV value is less than 10% and the deviation value is less than 10%, which meets the standard.

[0102] Table 3

[0103]

[0104] From Table 3, it can be seen that the detection kits of Examples 1-3 have low bias and CV values for cyclosporine detection, good detection effect, excellent performance, and excellent sensitivity, repeatability and accuracy.

[0105] Precision evaluation: the detection kits of Examples 1-3 and the application methods thereof were used to detect samples to be tested with cyclosporine concentrations of 60 ng / ml and 120 ng / ml, each sample was detected at 1 day, 3 days, 5 days, 7 days, 11 days and 14 days, and each sample was detected twice a day, and the mean value, standard deviation and coefficient of variation were calculated, as shown in Table 4. The CV value less than 10% meets the standard.

[0106] Table 4

[0107]

[0108]

[0109] From Table 4, it can be seen that the detection kits of Examples 1-3 have low CV values for cyclosporine detection at 1 day, 3 days, 5 days, 7 days, 11 days and 14 days, each sample is detected twice a day, and the detection kits of Examples 1-3 have excellent precision performance.

[0110] From Tables 3 and 4, the detection kit of the present application has high specificity for cyclosporine and can accurately test the concentration of cyclosporine.

[0111] Stability evaluation: the detection kits of Examples 1-3 were stored at 2-8°C for 2 months, 4 months, 6 months, 8 months, 10 months, 12 months and 14 months, and samples to be tested with cyclosporine concentrations of 120 ng / ml, 250 ng / ml and 895 ng / ml were detected, and the decrease rate in 14 months was calculated, as shown in Table 5.

[0112] Table 5

[0113]

[0114] From Table 5, it can be seen that the detection kits of Examples 1-3 have good stability in 14 months and have long-term stability.

[0115] Comparative Example 1

[0116] A detection kit for cyclosporine, which is different from Example 1 in that the detection kit only includes R1 reagent, R2 reagent and R3 reagent.

[0117] The step (1) in the application method is: adding Tris buffer solution to the sample to be tested, the volume ratio of the sample to be tested to the Tris buffer solution is 1:1, vortexing for 30s, centrifuging at 12000rpm for 10min, and taking the centrifugal liquid into a sample tube as a sample; wherein the Tris buffer solution is a 50mM Tris buffer solution containing 0.02w / w% Proclin300, and the pH is 7.5-8;

[0118] The rest is exactly the same.

[0119] Comparative Example 2

[0120] A cyclosporin detection kit, different from Example 1 is that the sample treatment agent comprises Tris buffer solution II, the Tris buffer solution II is prepared by using Tris, HCl, Na2SO3, methanol, ethylene glycol, Proclin300, pure water to prepare a 50mM Tris buffer solution II, the pH is 7.5-8, and contains 30mM Na2SO3, 0.02w / w% Proclin300, 40v / v% methanol and 30v / v% ethylene glycol;

[0121] The rest is exactly the same.

[0122] Comparative Example 3

[0123] A cyclosporin detection kit, different from Example 1 is that the sample treatment agent comprises Tris buffer solution II, the Tris buffer solution II is prepared by using Tris, HCl, CuSO4·5H2O, Na2SO3, ethylene glycol, Proclin300, pure water to prepare a 50mM Tris buffer solution II, the pH is 7.5-8, and contains 100mM CuSO4·5H2O, 30mM Na2SO3, 0.02w / w% Proclin300 and 30v / v% ethylene glycol;

[0124] The rest is exactly the same.

[0125] Comparative Example 4

[0126] A cyclosporin detection kit, different from Example 1 is that the sample treatment agent comprises Tris buffer solution II, the Tris buffer solution II is prepared by using Tris, HCl, CuSO4·5H2O, Na2SO3, methanol, Proclin300, pure water to prepare a 50mM Tris buffer solution II, the pH is 7.5-8, and contains 100mM CuSO4·5H2O, 30mM Na2SO3, 0.02w / w% Proclin300 and 40v / v% methanol;

[0127] The rest is exactly the same.

[0128] Comparative Example 5

[0129] A cyclosporine detection kit differs from Example 1 in that: Tris buffer solution II: a 50 mM Tris buffer solution II is prepared using Tris, HCl, CuSO4·5H2O, methanol, ethylene glycol, Proclin300, and pure water, with a pH of 7.5-8 and containing 100 mM CuSO4·5H2O, 0.02 w / w% Proclin300, 40 v / v% methanol, and 30 v / v% ethylene glycol;

[0130] The rest is exactly the same.

[0131] Comparative Example 6

[0132] A cyclosporine detection kit differs from Example 1 in that: Tris buffer solution II: 50 mM Tris buffer solution II is prepared using Tris, HCl, CuSO4·5H2O, Na2SO3, methanol, ethylene glycol, Proclin300, and pure water, with a pH of 7.5-8 and containing 100 mM CuSO4·5H2O, 30 mM Na2SO3, 0.1 w / w% Proclin300, 40 v / v% methanol, and 30 v / v% ethylene glycol;

[0133] The rest is exactly the same.

[0134] The cyclosporine detection kits and application methods of Example 1 and Comparative Examples 1 to 6 were used to test samples with a cyclosporine concentration of 60 ng / ml and 120 ng / ml, respectively. Each sample was tested 8 times, and the mean, deviation, standard deviation, and coefficient of variation were calculated, as shown in Table 6. At the same time, the cyclosporine detection kits of Example 1 and Comparative Examples 1 to 6 were subjected to accelerated stability tests on test samples with a cyclosporine concentration of 120 ng / ml for 1 day, 3 days, 7 days, 11 days, and 14 days, as shown in Table 7.

[0135] Table 6

[0136]

[0137]

[0138] Table 7

[0139]

[0140] From Table 6, compared with Example 1, the CV value and bias value of Comparative Example 1, which is only the Tris buffer solution containing Proclin 300, are greatly increased, and the detection effect is poor. The non-specific reaction of the detection kit of Comparative Example 1 to cyclosporine is high, and the sensitivity, repeatability, stability and accuracy are low. Compared with Example 1, the Tris buffer solution II used in Comparative Examples 2-5 lacks any one of CuSO4·5H2O, Na2SO3, methanol and ethylene glycol, which significantly increases the CV value and bias value, reduces the detection effect, leads to the increase of the non-specific reaction of the detection kit to cyclosporine, and reduces the sensitivity, repeatability, stability and accuracy of the detection kit of cyclosporine. In addition, it is found from Table 7 that, compared with Example 1, the Tris buffer solution II of Comparative Example 1 containing only Proclin 300, the Tris buffer solution II of Comparative Example 5 lacking Na2SO3 and the Tris buffer solution II of Comparative Example 6 with excessive Proclin 300 all have a greater impact on the stability of the detection kit of cyclosporine.

[0141] Comparative Example 7

[0142] A detection kit of cyclosporine, which is different from Example 1 in that: the Tris buffer solution I is prepared by using Tris, HCl, BSA, trehalose, mannitol, Proclin 300 and pure water to prepare a 50mM Tris buffer solution I with a pH of 7-7.5 and containing 2w / w% BSA, 1.5w / w% trehalose, 1w / w% mannitol and 0.1w / w% Proclin 300;

[0143] The rest is exactly the same.

[0144] Comparative Example 8

[0145] A detection kit of cyclosporine, which is different from Example 1 in that: the Tris buffer solution I is prepared by using Tris, HCl, BSA, trehalose, Proclin 300 and pure water to prepare a 50mM Tris buffer solution I with a pH of 7-7.5 and containing 2w / w% BSA, 1.5w / w% trehalose and 0.1w / w% Proclin 300;

[0146] The rest is exactly the same.

[0147] Comparative Example 9

[0148] A cyclosporin detection kit, different from Example 1 in that Tris buffer solution I: 50 mM Tris buffer solution I is prepared using Tris, HCl, BSA, mannitol, Tween-20, Proclin 300, pure water, pH 7-7.5, and containing 2 w / w% BSA, 1 w / w% mannitol, 0.8 w / w% Tween-20 and 0.1 w / w% Proclin 300;

[0149] The rest is exactly the same.

[0150] Comparative Example 10

[0151] A cyclosporin detection kit, different from Example 1 in that Tris buffer solution I: 50 mM Tris buffer solution I is prepared using Tris, HCl, BSA, mannitol, Tween-20, Proclin 300, pure water, pH 7-7.5, and containing 2 w / w% BSA, 1 w / w% mannitol, 0.8 w / w% Tween-20 and 0.1 w / w% Proclin 300;

[0152] The rest is exactly the same.

[0153] The cyclosporin detection kits of Example 1 and Comparative Examples 7-10 and the application method were used to detect samples to be tested with cyclosporin concentrations of 60 ng / ml and 120 ng / ml, each sample was detected 8 times, and the mean, deviation, standard deviation, and coefficient of variation were calculated, as shown in Table 8; at the same time, the cyclosporin detection kits of Example 1 and Comparative Examples 1-6 were used to test the accelerated stability of samples to be tested with cyclosporin concentrations of 120 ng / ml at 1 day, 3 days, 7 days, 11 days, and 14 days, as shown in Table 9.

[0154] Table 8

[0155]

[0156] Table 9

[0157]

[0158] As can be seen from Table 8 and Table 9, compared with Example 1, the Tris buffer solution I of Comparative Example 7 lacks Tween-20, which makes the CV value and the bias value significantly increase, the detection effect is poor, and the reagent kit storage effect is poor, resulting in insufficient stability of the cyclosporine detection reagent kit, affecting the repeatability and accuracy of the kit detection, and Comparative Examples 8-10 further lack one of BSA, trehalose, and mannitol, which makes the reagent kit storage effect further deteriorate, the stability of the cyclosporine detection reagent kit is further reduced, thereby significantly reducing the repeatability and accuracy of the kit detection.

[0159] Comparative Example 11

[0160] A cyclosporine detection reagent kit, which is different from Example 1 in that: the MES buffer solution is prepared by using MES, Proclin 300, and pure water to prepare a 50mM MES buffer solution with a pH of 6.0-6.5 and containing 0.1w / w% Proclin 300;

[0161] The rest is exactly the same.

[0162] Comparative Example 12

[0163] A cyclosporine detection reagent kit, which is different from Example 1 in that: the detection reagent kit only includes R1 reagent, R2 reagent, and sample processing agent;

[0164] Step (2) in the application method: the reaction tube contains 10μL sample, 20μL R1 reagent, 50μL R2 reagent, and 100μL pure water;

[0165] The rest is exactly the same.

[0166] Comparative Example 13

[0167] A cyclosporine detection reagent kit, which is different from Example 1 in that: three, R3 reagent includes HEPES buffer solution;

[0168] 7) HEPES buffer solution: using HEPES, Proclin 300, and pure water to prepare a 50mM HEPES buffer solution with a pH of 6.8-7.2 and containing 0.1w / w% Proclin 300, i.e. R3 reagent;

[0169] The rest is exactly the same.

[0170] The cyclosporine detection reagent kits of Example 1 and Comparative Examples 11-13 and the application method are used to detect samples with cyclosporine concentrations of 60ng / ml and 120ng / ml, each sample is detected 8 times, and the mean, bias, standard deviation, and coefficient of variation are calculated, as shown in Table 10.

[0171] Table 10

[0172]

[0173] From Table 10, compared with Comparative Examples 11-13, the R3 reagent of Example 1 uses MES buffer containing NaCl, which synergizes with the R1 reagent and the R2 reagent, is more conducive to reducing the CV value and the bias value, improves the detection effect, and can more improve the repeatability and accuracy of the cyclosporine detection kit for cyclosporine detection.

[0174] It is apparent for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit and essential characteristics of the present application. Therefore, the embodiments should be considered in all aspects as illustrative and not restrictive, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended to encompass all changes falling within the meaning and range of equivalents of the claims.

[0175] Furthermore, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the present specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A cyclosporine detection kit, characterized in that: Including R1 reagent, R2 reagent, R3 reagent and sample processing agent; The R1 reagent includes magnetic bead-coated cyclosporine antibody and Tris buffer solution I; The R2 reagent includes alkaline phosphatase-labeled cyclosporine antigen and AP stabilizer; The R3 reagent includes MES buffer; The sample treatment agent includes Tris buffer solution II, wherein the Tris buffer solution II contains CuSO4·5H2O, Na2SO3, Proclin300, methanol and ethylene glycol.

2. A cyclosporine detection kit according to claim 1, characterized in that: In the sample treatment agent, the Tris buffer solution II has a concentration of 40-100 mM, a pH of 7.5-8, and contains 50-120 mM CuSO4·5H2O, 10-40 mM Na2SO3, 0.01-0.04 w / w% Proclin300, 32-45 v / v% methanol, and 25-33 v / v% ethylene glycol.

3. A cyclosporine detection kit according to claim 1, characterized in that: In the R1 reagent, the concentration of the Tris buffer solution I is 40-100 mM, the pH is 7-7.5, and the Tris buffer solution I contains BSA, trehalose, mannitol, Tween-20 and Proclin 300.

4. A cyclosporine detection kit according to claim 3, characterized in that: The Tris buffer solution I contains 0.5-3w / w% BSA, 0.5-2.5w / w% trehalose, 0.2-2w / w% mannitol, 0.1-1.2w / w% Tween-20 and 0.05-0.2w / w% Proclin300.

5. A cyclosporine detection kit according to claim 1, characterized in that: In the R3 reagent, the concentration of the MES buffer is 40-100 mM, and the pH is 6.0-6.5; and the MES buffer also contains NaCl and Proclin 300.

6. A cyclosporine detection kit according to claim 5, characterized in that: The MES buffer contains 0.5-1.2 w / w% NaCl and 0.05-0.2 w / w% Proclin 300.

7. A cyclosporine detection kit according to claim 1, characterized in that: In the R1 reagent, the concentration of the cyclosporine antibody-coated magnetic beads is 0.05-0.5 mg / mL.

8. A cyclosporine detection kit according to claim 1, characterized in that: In the R2 reagent, the concentration of the alkaline phosphatase-labeled cyclosporine antigen is 0.05 to 0.2 μg / mL.

9. A method for using the cyclosporine detection kit according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) Adding a sample treatment agent to the sample to be tested, vortexing, centrifuging, and taking the centrifuge liquid into a sample tube as the sample; (2) The sample, R1 reagent, R2 reagent, and R3 reagent are placed in a reaction tube, shaken to mix, incubated, and then tested using a chemiluminescence immunoassay to measure the luminescence value corresponding to the sample concentration, and the cyclosporine concentration in the sample is calculated based on the standard curve.

10. The application method of the cyclosporine detection kit according to claim 9, characterized in that: The volume ratio of the sample to be tested to the sample treatment agent is 1:1; the volume ratio of the sample, R1 reagent, R2 reagent and R3 reagent is 1:2:5:10.

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