Detection reagent of delta9-tetrahydrocannabinol as well as preparation method and detection method of detection reagent

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a detection reagent of delta 9-tetrahydrocannabinol and a preparation method and a detection method thereof, the detection reagent comprises a first detection reagent and a second detection reagent, and the preparation method comprises the following steps: dissolving NaCl in a buffer solution to obtain the first detection reagent; and dissolving the bovine serum albumin-anti-delta9-tetrahydrocannabinol antibody immobilized latex microspheres in a buffer solution to obtain the second detection reagent. The detection accuracy is improved.

Description

Technical Field

[0001] The present application relates to the field of detection technology, and in particular to a detection reagent for Δ9-tetrahydrocannabinol and a preparation method and a detection method thereof. Background Art

[0002] Cannabis belongs to the Moraceae family and is an ancient cultivated crop. Because it contains Δ9-tetrahydrocannabinol (THC), which can strongly stimulate the central nervous system, it is called "the world's three major drugs" along with heroin and cocaine. Δ9-tetrahydrocannabinol, with a molecular weight of 314Da, is a small molecule hapten substance and is also a terpene compound in cannabis that has been studied in depth. THC has a good therapeutic effect in the treatment of glaucoma, malaria, constipation, rheumatic diseases, and pain, and recent studies have found that tetrahydrocannabinol has a wide range of cardiovascular activity. However, long-term marijuana users are prone to violence and suicidal tendencies due to behavioral toxicity caused by cumulative poisoning, so it is of great significance to carry out analytical research on marijuana drugs.

[0003] Common Δ9-THC detection methods:

[0004] (1) Enzyme-linked immunosorbent assay (ELISA) is a relatively simple method for detecting Δ9-tetrahydrocannabinol. Its basic principle is the competitive binding principle: Δ9-tetrahydrocannabinol is pre-bound to the surface of a solid phase carrier; during the assay, the sample to be tested and the enzyme-labeled antibody are reacted with the Δ9-tetrahydrocannabinol bound to the solid phase carrier according to a certain procedure to form an antigen-antibody complex. If Δ9-tetrahydrocannabinol exists in the sample, it will compete with the Δ9-tetrahydrocannabinol on the surface of the solid phase carrier for binding to the antibody. Wash away the unbound substances, remove other substances in the reaction solution by washing, and add a substrate for color development. The more Δ9-tetrahydrocannabinol in the sample, the fewer antibodies to the reference antigen will be bound to the bottom of the well, and the weaker the signal. The ELISA method has poor quantitative accuracy, long operation time, and low degree of automation, and is mostly used for qualitative detection;

[0005] (2) Radioimmunoassay (RIA) is a method in which radiolabeled Δ9-THC and unlabeled Δ9-THC to be detected in the sample are competitively bound to a limited amount of specific antibodies, and the content of Δ9-THC in the sample is estimated by measuring the free radiolabeled Δ9-THC. The RIA method takes a long time, the test results are unstable, the repeatability is worse than that of ELISA, and there is a risk of radioactive contamination.

[0006] (3) Gas chromatography-mass spectrometry, liquid chromatography and liquid chromatography-mass spectrometry are the gold standards for detecting Δ9-THC and can detect extremely small amounts of Δ9-THC in samples. However, the disadvantages are long cycles, high costs, and expensive instruments, which are not suitable for large-scale promotion and application. Gas chromatography technology is relatively mature and widely used, but it is limited by the fact that the mobile phase must be gas, and high temperatures often cause tetrahydrocannabinol to decarboxylate, resulting in high quantitative analysis results.

[0007] The detection process of Δ9-tetrahydrocannabinol in the prior art is complicated to operate and has low measurement accuracy. Summary of the invention

[0008] The purpose of the embodiments of the present application is to provide a detection reagent for Δ9-tetrahydrocannabinol and a preparation method and a detection method thereof to improve the accuracy of detection.

[0009] In order to solve the above technical problems, the present application provides a method for preparing a detection reagent for Δ9-tetrahydrocannabinol, which adopts the following technical solution:

[0010] A method for preparing a detection reagent for Δ9-tetrahydrocannabinol, wherein the detection reagent comprises a first detection reagent and a second detection reagent, comprising the following steps:

[0011] Dissolving NaCl in a buffer solution to obtain the first detection reagent;

[0012] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are dissolved in a buffer to obtain the second detection reagent.

[0013] Furthermore, the step of dissolving NaCl in a buffer to obtain the first detection reagent comprises:

[0014] Dissolve 7.0-9.0 g of NaCl in double distilled water and add buffer;

[0015] Double distilled water was added to make up to 1000 mL, and PEG 6000 and Tween 80 were added after shaking to obtain the first detection reagent.

[0016] Furthermore, the step of dissolving bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres in a buffer to obtain the second detection reagent comprises:

[0017] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are dissolved in a mixed solution of Tris buffer, bovine serum albumin, and Tween-20 to obtain the second detection reagent.

[0018] Furthermore, the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are prepared by the following steps:

[0019] preparing a bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate;

[0020] Performing an activation operation on the latex microspheres to obtain carboxyl-activated latex microspheres;

[0021] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate is coupled to the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

[0022] Furthermore, the step of preparing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate comprises:

[0023] The bovine serum albumin is sulfided using a 2-iminothiolane reagent in a NaH2PO3 solution, incubated, and dialyzed to obtain sulfided bovine serum albumin;

[0024] The anti-Δ9-tetrahydrocannabinol antibody was dissolved in a mixed solution containing sodium dihydrogen phosphate, sodium chloride and EDTA;

[0025] Add dimethyl sulfoxide dissolved with SMCC, mix well, and perform dialysis to obtain an anti-Δ9-tetrahydrocannabinol antibody conjugate;

[0026] The anti-Δ9-tetrahydrocannabinol antibody linker is mixed with the sulfurized bovine serum albumin to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate.

[0027] Furthermore, the molar ratio of the anti-Δ9-tetrahydrocannabinol antibody linker to the sulfated bovine serum albumin is 1:10.

[0028] Furthermore, the step of activating the latex microspheres to obtain carboxyl-activated latex microspheres comprises:

[0029] The latex microspheres, EDC, NHS and PBS buffer are uniformly mixed, centrifuged, and the supernatant is removed to obtain the carboxyl-activated latex microspheres.

[0030] Furthermore, the step of coupling the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody immobilized latex microspheres comprises:

[0031] In a tween-20 solution, mixing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres;

[0032] The mixture was incubated at room temperature, and the supernatant was removed after centrifugation to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

[0033] In order to solve the above technical problems, the present application also provides a detection reagent for Δ9-tetrahydrocannabinol (THC), which adopts the following technical solution:

[0034] A detection reagent for Δ9-tetrahydrocannabinol (THC), which is prepared by the above-mentioned preparation method of the detection reagent for Δ9-tetrahydrocannabinol.

[0035] In order to solve the above technical problems, the present application also provides a method for detecting Δ9-tetrahydrocannabinol, using the above detection reagent and adopting the following technical solution:

[0036] A method for detecting Δ9-tetrahydrocannabinol, comprising the following steps:

[0037] Adding the sample to be tested to the first detection reagent, measuring the absorbance after incubation to obtain an initial absorbance;

[0038] adding the second detection reagent, measuring the absorbance after incubation to obtain the reaction absorbance;

[0039] The concentration of Δ9-tetrahydrocannabinol is calculated according to the initial absorbance and the reaction absorbance.

[0040] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0041] The method used in the present application is latex immunoturbidimetry, which is simple to operate, rapid, highly sensitive, accurate, suitable for automation, and widely used. It can also rapidly measure both small and large molecules with high throughput using a fully automatic biochemical analyzer. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 is a flow chart of an embodiment of a method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to the present application;

[0044] Figure 2 is a flow chart of an embodiment of a method for detecting Δ9-tetrahydrocannabinol according to the present application;

[0045] Figure 3 This is a linear range result diagram of an embodiment of a detection reagent for Δ9-tetrahydrocannabinol (THC) according to the present application. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0047] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0048] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0049] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0050] The present application provides a method for preparing a detection reagent for Δ9-tetrahydrocannabinol, such as Figure 1 As shown, Figure 1 The present invention is a flow chart of an embodiment of a method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to the present application; the detection reagent includes a first detection reagent and a second detection reagent, and includes the following steps:

[0051] S101: dissolving NaCl in a buffer solution to obtain the first detection reagent;

[0052] S102: Dissolve the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres in a buffer solution to obtain the second detection reagent.

[0053] As an option of the present application, the step of dissolving NaCl in a buffer to obtain the first detection reagent includes:

[0054] Dissolve 7.0-9.0 g of NaCl in double distilled water and add buffer;

[0055] Double distilled water was added to make up to 1000 mL, and PEG 6000 and Tween 80 were added after shaking to obtain the first detection reagent.

[0056] As an option in the present application, the buffer includes at least one of Tris-HCl buffer, phosphate buffer, HEPES buffer, glycine buffer and barbiturate buffer.

[0057] As an option in the present application, when the buffer is Tris-HCl buffer, the final concentration of the Tris-HCl buffer is 0.05 mol / L.

[0058] The present application provides a specific embodiment of the preparation process of the first detection reagent (reagent R1): first dissolve NaCl (7.0-9.0 g) with double distilled water, then add Tris-HCl buffer, and finally add double distilled water to 1000 ml to make the final concentration of Tris 0.05 mol / L, shake well, then add a small amount of PEG 6000 and Tween80, and mix well.

[0059] As an optional step of the present application, the step of dissolving bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres in a buffer to obtain the second detection reagent comprises:

[0060] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are dissolved in a mixed solution of Tris buffer, bovine serum albumin, and Tween-20 to obtain the second detection reagent.

[0061] In this embodiment, the concentration of the Tris buffer is 50 mM, the concentration of bovine serum albumin is 0.1%, and the concentration of Tween-20 is 0.05% by mass.

[0062] As an option of the present application, the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody immobilized latex microspheres are prepared by the following steps:

[0063] preparing a bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate;

[0064] Performing an activation operation on the latex microspheres to obtain carboxyl-activated latex microspheres;

[0065] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate is coupled to the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

[0066] As an option of the present application, the step of preparing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate comprises:

[0067] The bovine serum albumin is sulfided using a 2-iminothiolane reagent in a NaH2PO3 solution, incubated, and dialyzed to obtain sulfided bovine serum albumin;

[0068] The anti-Δ9-tetrahydrocannabinol antibody was dissolved in a mixed solution containing sodium dihydrogen phosphate, sodium chloride and EDTA;

[0069] Add dimethyl sulfoxide dissolved with SMCC, mix well, and perform dialysis to obtain an anti-Δ9-tetrahydrocannabinol antibody conjugate;

[0070] The anti-Δ9-tetrahydrocannabinol antibody linker is mixed with the sulfurized bovine serum albumin to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate.

[0071] In this embodiment, the concentration of SMCC in the dimethyl sulfoxide is 5 mg / mL; in the mixed solution, the concentration of sodium dihydrogen phosphate is 100 mM, the concentration of sodium chloride is 150 mM, the concentration of EDTA is 1 mM, and the pH is 7.2; the concentration of the anti-Δ9-tetrahydrocannabinol antibody in the mixed solution is 2 mg / mL. The molar ratio of the anti-Δ9-tetrahydrocannabinol antibody to the SMCC is 1:3; the molar ratio of the anti-Δ9-tetrahydrocannabinol antibody linker to the sulfided bovine serum albumin is 1:10.

[0072] As an option of the present application, the step of activating the latex microspheres to obtain carboxyl-activated latex microspheres comprises:

[0073] The latex microspheres, EDC, NHS and PBS buffer are uniformly mixed, centrifuged, and the supernatant is removed to obtain the carboxyl-activated latex microspheres.

[0074] As an option of the present application, the step of coupling the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody immobilized latex microspheres comprises:

[0075] Mixing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres in a 0.2% tween-20 solution by mass fraction;

[0076] The mixture was incubated at room temperature, and the supernatant was removed after centrifugation to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

[0077] The present application provides a specific embodiment of the preparation process of the second detection reagent (reagent R2):

[0078] 1. Preparation of bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate

[0079] (1) bovine serum albumin (BSA) (1 mg) was sulfided using Traut (2-iminothiolane) reagent in 100 μL 14 mM NaH2PO3 to introduce sulfhydryl groups, wherein the molar ratio of Traut reagent to bovine serum albumin was 1:20-100;

[0080] After incubation at room temperature for 1 hour, excess Traut's reagent was removed by dialysis to obtain sulfated BSA.

[0081] (2) treating the anti-Δ9-tetrahydrocannabinol antibody with the cross-linking agent SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), specifically:

[0082] Dissolving the anti-Δ9-tetrahydrocannabinol antibody in 100 mM sodium dihydrogen phosphate, 150 mM sodium chloride and 1 mM EDTA (pH=7.2), wherein the concentration of the anti-Δ9-tetrahydrocannabinol antibody after dissolution is 2 mg / mL;

[0083] Add dimethyl sulfoxide (DMSO) dissolved with SMCC, the concentration of SMCC in DMSO is 5 mg / mL, and the molar ratio of anti-Δ9-tetrahydrocannabinol antibody to SMCC is 1:3;

[0084] After stirring at 25 °C for 2 h, the excess SMCC was removed by dialysis, and then the anti-Δ9-tetrahydrocannabinol antibody conjugate was obtained;

[0085] The anti-Δ9-tetrahydrocannabinol antibody linker was mixed with sulfated bovine serum albumin and reacted at room temperature for 1 hour to obtain anti-Δ9-tetrahydrocannabinol antibody-BSA, wherein the molar ratio of the anti-Δ9-tetrahydrocannabinol antibody linker to the sulfated bovine serum albumin was 1:10.

[0086] 2. Activation of latex microspheres

[0087] In a centrifuge tube, 100 μL of 5% (wt / vol) PS (polystyrene) latex microspheres and 100 μL of 2 mg / mL EDC / NHS, 10 mM PBS buffer (pH 7.0) were added. After stirring at 37°C for 15 min, the redundant EDC and NHS were removed by centrifugation to obtain carboxyl-activated PS latex microspheres.

[0088] 3. Coupling of activated latex microspheres with bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate

[0089] Finally, BSA-THC conjugates were immobilized on carboxyl-activated PS latex microspheres in 0.2% tween-20 solution by mass fraction.

[0090] After incubation at room temperature for 3 h, the monolithically coupled anti-THC antibody-BSA-latex microspheres were purified by centrifugation.

[0091] 4. Preparation of reagent R2

[0092] The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres were dissolved in a 50 mM Tris, 0.1% BSA and 0.05% Tween-20 buffer solution (pH=7.4) at a mass fraction of 1:10 to 1:50 to obtain a second detection reagent (reagent R2).

[0093] The present application also provides a Δ9-tetrahydrocannabinol (THC) detection reagent, which is prepared using the above-mentioned Δ9-tetrahydrocannabinol detection reagent preparation method.

[0094] The present application also provides a method for detecting Δ9-tetrahydrocannabinol, such as Figure 2 As shown, Figure 2 9-THC is a flow chart of an embodiment of a method for detecting Δ9-THC according to the present application. Using the above detection reagent, the following steps are included:

[0095] S201: adding a sample to be tested to the first detection reagent, measuring the absorbance after incubation, and obtaining an initial absorbance;

[0096] S202: adding the second detection reagent, measuring the absorbance after incubation, and obtaining the reaction absorbance;

[0097] S203: Calculating the concentration of Δ9-tetrahydrocannabinol according to the initial absorbance and the reaction absorbance.

[0098] In this embodiment, the present application detects Δ9-tetrahydrocannabinol in human urine; the method adopted in the present application is latex immunoturbidimetry, which is simple to operate, rapid, highly sensitive, accurate, suitable for automation, and widely used. It can also use a fully automatic biochemical analyzer to quickly and efficiently measure both small and large molecules.

[0099] The above-mentioned detection reagents of this application are suitable for fully automatic or semi-automatic biochemical analyzers of brands such as Beckman, Hitachi, Olympus, Toshiba, Roche, Abbott, Siemens, and Mindray. The determination method is as follows:

[0100] 1. Measurement conditions

[0101] The assay conditions of the kit described in this application

[0102] Temperature: 37℃

[0103] Main wavelength: 560nm

[0104] Sub-wavelength: 800nm

[0105] Sample volume: 5 μL

[0106] R1 dosage: 200 μL

[0107] R2 dosage: 40 μL

[0108] Analysis type: Two-point endpoint method

[0109] 2. The determination method is as follows:

[0110] Determine the blank absorbance, add 5 μL of the sample to be tested to 200 μL of the first detection reagent, pre-incubate for 5 minutes, measure the blank absorbance, add 40 μL of the second detection reagent, incubate for 15 minutes, and measure the reaction absorbance.

[0111] The amount of the sample to be tested, the amount of the first detection reagent and the amount of the second detection reagent can be adjusted according to the requirements of different types of biochemical analyzers and the ratio of sample and reagent amounts specified in the "determination conditions". If there is no specified wavelength in the instrument, you can select the value closest to the specified wavelength and enter it.

[0112] (3) Calibration and sample measurement

[0113] Use the calibrator in the kit to calibrate according to the calibration procedure in the manual of the analytical instrument used, the mode is multi-point calibration, and a working curve is established. After calibration, the urine sample is measured, and the antibody concentration in the sample can be calculated based on the working curve.

[0114] This application also provides experimental data:

[0115] Example 1: Analytical Performance Evaluation of Δ9-Tetrahydrocannabinol Detection Reagents

[0116] 1. Analytical sensitivity or minimum detection limit

[0117] Control reagent: a well-known foreign brand

[0118] Detection principle: latex enhanced immunoturbidimetry is used to detect turbidity changes at 570nm. The degree of change is linearly related to the Δ9-tetrahydrocannabinol content in the sample.

[0119] Sensitivity evaluation method: dilute the calibrator into a series of concentrations close to the sensitivity, and determine its concentrations as 0.05 mg / L, 0.10 mg / L, 0.15 mg / L, and 0.21 mg / L by ELISA method. Add zero standard as sample, use two kits, and repeat the measurement 10 times according to their respective measurement methods, and record the reaction absorbance values. The data are shown in the following table:

[0120] Table 1 Sensitivity evaluation

[0121]

[0122] The average value of the zero standard of the reagent of the present application, which was measured 10 times, was 0.019, the standard deviation SD was 0.008, and the sum of the average value and 3 times the standard deviation (0.019+3X0.008) 0.043 was taken as the blank limit. The CV of the kit at 0.05 mg / L was 9.1%, which was less than 10%. Therefore, the minimum detection sensitivity of this kit was 0.05 mg / L, and the minimum detection sensitivity of the control kit was 0.1 mg / L. The sensitivity of the kit of the present application was higher than that of the control kit.

[0123] 2. Accuracy and repeatability

[0124] Using a 4 mg / L calibrator and human urine with a labeled value of 0.42 mg / L as samples, the determination was repeated 20 times according to the method described in Example 2. As shown in the following table, the determination mean value X, standard deviation SD and coefficient of variation CV were calculated respectively.

[0125] Table 2 Accuracy and repeatability

[0126]

[0127]

[0128] The results showed that the relative deviation was used to examine the accuracy of the measurement, and the relative deviation was within the range of ±15%. The coefficient of variation was used to examine the intra-batch precision, and the coefficient of variation of the calibrator and urine samples were 2.11% and 2.45%, respectively.

[0129] 3. Batch-to-batch precision

[0130] Using 3 batches of the reagent described in the present application, the same urine sample was measured 5 times according to the method described in Example 2, and the coefficient of variation (CV) of the 5 measurement results was calculated to examine the batch precision. The results are shown in the following table.

[0131] Table 3 Inter-batch precision

[0132]

[0133] The results showed that the coefficient of variation between batches was 6.7%, indicating that the differences between reagent batches were good.

[0134] 4. Linear range

[0135] The high-value urine sample (concentration of 7.86 mg / L) close to the upper limit of the linear range was diluted with deionized water at 1:1, 1:3, 1:9, 1:16, 1:27, 1:54, and 1:99 to obtain 7 solutions with different concentrations. Each concentration was measured 3 times according to the method described in Example 2, and regression analysis was performed on the measured average value and the corresponding theoretical value. The results are shown in the following table and Figure 3 As shown, Figure 3 This is a linear range result diagram of an embodiment of a detection reagent for Δ9-tetrahydrocannabinol (THC) according to the present application.

[0136] Table 4 Linear range

[0137]

[0138] according to Figure 3 It can be seen that the results show that the calculated regression equation is y=0.9974x-0.0283, and the correlation coefficient r

[0139] =0.9986, indicating that the kit of the present application has a good correlation within the linear range of (0.05-8) mg / L.

[0140] 5. Stability

[0141] After the kit described in the present application was opened, it was stored at 2-8°C for 2 weeks. The calibrator with a labeled value of 8.12 mg / L was taken out and measured according to the method described in Example 2. The measurement was repeated 3 times, and the relative deviation between the detection average value and the labeled value was calculated. The results are shown in Table 5.

[0142] Table 5 Stability - 2 weeks after opening

[0143]

[0144] The experimental results show that the relative deviation between the average value of the test kit described in the present application and the labeled value is less than 3% after 2 weeks of opening the bottle, and the stability of the bottle after opening is good.

[0145] After the kit described in the present application was stored at 2-8°C for 16 months, the calibrator with a labeled value of 8.12 mg / L was taken out and measured according to the method described in Example 2. The measurement was repeated 3 times, and the relative deviation between the detection mean and the labeled value was calculated. The results are shown in Table 6.

[0146] Table 6 Stability - 16 months storage

[0147]

[0148] The results showed that the relative deviations between the detected values ​​and the labeled values ​​were less than 4%, indicating that the kit of the present application was relatively stable when stored at 2-8°C for 16 months.

[0149] It should be understood that, although the steps in the flowchart of the accompanying drawings are displayed in sequence as indicated by the arrows, these steps are not necessarily executed in sequence in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn or alternately with other steps or at least a part of the sub-steps or stages of other steps.

[0150] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for preparing a detection reagent for Δ9-tetrahydrocannabinol, characterized in that: The detection reagent comprises a first detection reagent and a second detection reagent, comprising the following steps: Dissolving NaCl in a buffer solution to obtain the first detection reagent; The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are dissolved in a buffer to obtain the second detection reagent.

2. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 1, characterized in that: The step of dissolving NaCl in a buffer to obtain the first detection reagent comprises: Dissolve 7.0-9.0 g of NaCl in double distilled water and add buffer; Double distilled water was added to make up to 1000 mL, and PEG 6000 and Tween 80 were added after shaking to obtain the first detection reagent.

3. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 1, characterized in that: The step of dissolving bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres in a buffer to obtain the second detection reagent comprises: The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are dissolved in a solution mixed with Tris buffer, bovine serum albumin, and Tween-20 to obtain the second detection reagent.

4. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 1, characterized in that: The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres are prepared by the following steps: preparing a bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate; Performing an activation operation on the latex microspheres to obtain carboxyl-activated latex microspheres; The bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate is coupled to the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

5. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 4, characterized in that: The steps of preparing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate include: The bovine serum albumin is sulfided using a 2-iminothiolane reagent in a NaH2PO3 solution, incubated, and dialyzed to obtain sulfided bovine serum albumin; The anti-Δ9-tetrahydrocannabinol antibody was dissolved in a mixed solution containing sodium dihydrogen phosphate, sodium chloride and EDTA; Add dimethyl sulfoxide dissolved with SMCC, mix well, and perform dialysis to obtain an anti-Δ9-tetrahydrocannabinol antibody conjugate; The anti-Δ9-tetrahydrocannabinol antibody linker is mixed with the sulfurized bovine serum albumin to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate.

6. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 5, characterized in that: The molar ratio of the anti-Δ9-tetrahydrocannabinol antibody linker to the sulfated bovine serum albumin is 1:

10.

7. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 4, characterized in that: The step of activating the latex microspheres to obtain carboxyl-activated latex microspheres comprises: The latex microspheres, EDC, NHS and PBS buffer are uniformly mixed, centrifuged, and the supernatant is removed to obtain the carboxyl-activated latex microspheres.

8. The method for preparing a detection reagent for Δ9-tetrahydrocannabinol according to claim 4, characterized in that: The step of coupling the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody immobilized latex microspheres comprises: In a tween-20 solution, mixing the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody conjugate with the carboxyl-activated latex microspheres; The mixture was incubated at room temperature, and the supernatant was removed after centrifugation to obtain the bovine serum albumin-anti-Δ9-tetrahydrocannabinol antibody-immobilized latex microspheres.

9. A detection reagent for Δ9-tetrahydrocannabinol (THC), characterized in that: The Δ9-tetrahydrocannabinol detection reagent is prepared by the preparation method of the Δ9-tetrahydrocannabinol detection reagent according to any one of claims 1 to 8.

10. A method for detecting Δ9-tetrahydrocannabinol, using the detection reagent according to claim 9, characterized in that: The steps include: Adding the sample to be tested to the first detection reagent, measuring the absorbance after incubation to obtain an initial absorbance; adding the second detection reagent, measuring the absorbance after incubation to obtain the reaction absorbance; The concentration of Δ9-tetrahydrocannabinol is calculated according to the initial absorbance and the reaction absorbance.

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