A mycophenolic acid hapten and its antigen preparation

Abstract

The application discloses a kind of rice mycotic acid hapten and antigen preparation application, it is related to antigen preparation technical field, introduce sulfydryl in the 2 of rice mycotic acid, and obtain rice mycotic acid antigen by coupling with protein through connecting arm, rice mycotic acid hapten and antigen preparation application include the following specific flow: S1, the synthesis of rice mycotic acid hapten;S2, the preparation of rice mycotic acid immunogen;S3, the preparation of rice mycotic acid coating original;S4, the preparation of rice mycotic acid monoclonal antibody;S5, the preparation of rice mycotic acid immunofluorescence detection card;S6, rice mycotic acid detection in food;S7, detection limit experiment;S8, false positive rate, false negative rate experiment.The rice mycotic acid hapten retains the structural characteristics of rice mycotic acid, better exposes rice mycotic acid structural characteristics, improves the immunity and immune recognition ability of antigen, lays foundation for preparing specific, high sensitivity antibody, and improves its rapid detection ability.

Description

Technical Field

[0001] This invention relates to the field of antigen preparation technology, specifically to a bongkrekic acid hapten and its antigen preparation and application. Background Technology

[0002] Bongkrekic acid (BA), chemically known as 3-carboxymethyl-17-methoxy-6,18,21-trimethyl-eicosano-2,4,8,12,14,18,20-heptaenoic acid, is a food poisoning toxin produced by *Pseudomonas cocovenenans* subsp. *farinofermentans*. Ingestion of food contaminated with this toxin can cause poisoning in humans or animals, and in severe cases, death. Bongkrekic acid is a major cause of poisoning in fermented cornmeal products, spoiled fresh white fungus, and other spoiled starchy products. With the increasing diversity of these food products and their preparation methods, the demand for detection capabilities for bongkrekic acid, as well as rapid detection capabilities adapted to the fast pace of the market, is growing daily.

[0003] In existing technologies, the main analytical methods for detecting bongkrekic acid are high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, both of these methods have drawbacks such as being time-consuming, having complex pretreatment processes, and requiring high equipment investment, making them unsuitable for current market demands. Immunoassay can effectively compensate for the shortcomings of instrumental methods, making the development of an immunoassay method for bongkrekic acid particularly important.

[0004] The key to a reliable immunoassay lies in obtaining antibodies with high specificity and sensitivity. To achieve this, a well-designed hapten structure is crucial. Bongkrekic acid has three carboxyl groups in its chemical structure, all of which can couple with amino groups in proteins. However, this coupling increases steric hindrance interference in the protein structure, affecting immune recognition, resulting in complex antigen structures, low effective antigen yield, and even protein cross-linking.

[0005] Based on the above objectives, we propose a bongkrekic acid hapten and its antigen preparation and application. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a bongkrekic acid hapten and its antigen preparation and application, thereby solving the problems mentioned in the background section.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a bongkrekic acid hapten, wherein a thiol group is introduced at the 2-position of bongkrekic acid, and the bongkrekic acid antigen is obtained by coupling with a protein through a linker arm. The structure of the bongkrekic acid hapten is shown below:

[0008]

[0009] To further optimize this technical solution, the synthesis of the bongkrekic acid hapten includes the following specific steps:

[0010] Weigh 3 mg of bongkrekic acid and add it to 100 mg of bis-2-dimethylaminoethyl ether and bis-p-toluenesulfonylamine ionic liquid, and heat to 60°C;

[0011] Add 10 mg of sodium sulfide to the liquid and keep it at a warm temperature for 18 hours;

[0012] Cool the liquid to room temperature, add 3 mL of deionized water for extraction, let stand for 20 min, take the aqueous phase, repeat the extraction step three times and combine the aqueous phases;

[0013] The combined aqueous phases were extracted three times with 30 mL x 3 ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate overnight, and the ethyl acetate was removed under reduced pressure to obtain bongkrekic acid hapten.

[0014] A bongkrekic acid hapten and its antigen preparation and application, wherein the antigen preparation and application are based on the above-mentioned bongkrekic acid hapten, and the antigen preparation and application include the following specific process:

[0015] S1, Synthesis of bongkrekic acid hapten;

[0016] S2, Preparation of bongkrekic acid immunogen;

[0017] S3, Preparation of bongkrekic acid coating agent;

[0018] S4. Preparation of bongkrekic acid monoclonal antibody;

[0019] S5. Preparation of bongkrekic acid immunofluorescence detection card;

[0020] S6. Detection of bongkrekic acid in food;

[0021] S7, Detection limit test;

[0022] S8, False Positive Rate and False Negative Rate Experiment.

[0023] To further optimize this technical solution, the preparation of bongkrekic acid immunogen in step S2 includes the following specific operational procedures:

[0024] Weigh 8 mg of hemocyanin and 2 mg of sodium 4-N-maleimide methylcyclohexane-1-carboxylic acid sulfonate succinimide ester and add them to 1 mL of BB buffer at pH 8.5. Activate at room temperature for 4 h to form an activation solution.

[0025] Dissolve 0.8 mg of bongkrekic acid hapten in 0.2 mL of DMF, add dropwise to the activation solution, and react at room temperature for 8 h;

[0026] Ultrafiltration with a 3KD ultrafiltration tube removes unbound low molecular weight substances to obtain bongkrekic acid immunogen.

[0027] To further optimize this technical solution, step S3, the preparation of the bongkrekic acid coating agent includes the following specific operational procedures:

[0028] Weigh 8 mg of bovine serum albumin and 2 mg of sodium 4-N-maleimide methylcyclohexane-1-carboxylic acid sulfonate succinimide ester and add them to 1 mL of BB buffer at pH 8.5. Activate at room temperature for 4 h to form an activation solution.

[0029] Dissolve 0.8 mg of bongkrekic acid hapten in 0.2 mL of DMF, add dropwise to the activation solution, and react at room temperature for 8 h;

[0030] Ultrafiltration with a 3KD ultrafiltration tube removes unbound low molecular weight substances to obtain bongkrekic acid-coated antigen.

[0031] To further optimize this technical solution, the preparation method of bongkrekic acid monoclonal antibody in step S4 includes the following specific operational procedures:

[0032] Immunization: 0.1 mg of bongkrekic acid immunogen was injected into the peritoneal cavity of 6-week-old BALB / c mice. The mice were immunized once every three weeks for a total of three immunizations. Tail blood was collected one week after the third immunization to measure the titer and inhibition rate.

[0033] Cell fusion and cloning: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. Positive wells were screened by indirect ELISA. Positive wells were further screened by indirect competitive ELISA. Hybridoma cell lines that could stably secrete monoclonal antibodies were cloned by limiting dilution method. The obtained hybridoma cell lines were cryopreserved in liquid nitrogen.

[0034] Monoclonal antibody preparation: Frozen cell lines were revived, cultured, and injected into the peritoneal cavity of BALB / c mice. Ascites fluid was collected 7 days later. The purified ascites fluid was bongkrekic acid monoclonal antibody. The antibody titer of bongkrekic acid monoclonal antibody was 64000, and the IC10 was 2ug / kg.

[0035] To further optimize this technical solution, the preparation of the kombucigenin immunofluorescence detection card in step S5 includes the following specific process:

[0036] S501, activation of fluorescent microspheres;

[0037] S502, antibody labeling;

[0038] S503, preparation of coupling pad;

[0039] S504, Sample pad preparation;

[0040] S505, preparation of the reaction membrane;

[0041] S506, Assembly.

[0042] To further optimize this technical solution, step S6, the detection of bongkrekic acid in food, includes the following specific procedures:

[0043] Sample pretreatment: Weigh 2g of sample into a 20mL centrifuge tube, add 10mL of acetonitrile, vortex for 2min, centrifuge at 4000rpm for 5min at room temperature, take 5mL of the upper organic phase, blow dry under nitrogen in a 50℃ water bath, and reconstitute with 200uL of 0.01M PBS.

[0044] Detection: Add the sample to be tested onto the sample pad and perform an immunofluorescence reaction. Place the immunofluorescence detection card for measuring bongkrekic acid on the fluorescence card reader and read the intensity of the fluorescence signal.

[0045] Result interpretation: When both the test line and the control line show orange fluorescence under 345nm excitation light, and the test line is darker than the control line or the two lines are the same or similar in color, the test result is negative. When the test line and the control line show orange fluorescence under 345nm excitation light, while the test line does not show fluorescence or is lighter in color than the control line, the test result is positive.

[0046] To further optimize this technical solution, step S7 includes the following experimental contents for the detection limit experiment:

[0047] Take blank samples and add bongkrekic acid to final concentrations of 2ug / kg, 4ug / kg, and 8ug / kg. Detect the samples according to step S6, repeating three times.

[0048] To further optimize this technical solution, step S8 includes the following experiments for false positive rate and false negative rate:

[0049] Take a blank sample, add bongkrekic acid to a final concentration of 4 ug / kg, and test according to step S6. Repeat three times. 50 samples of each type, and test three batches of test strips separately.

[0050] Compared with the prior art, the present invention provides a bongkrekic acid hapten and its antigen preparation and application, which has the following beneficial effects:

[0051] This bongkrekic acid hapten and its antigen preparation and application involve introducing a thiol group at the 2-position of bongkrekic acid and coupling it with a protein via a linker arm to obtain a hapten, while completely preserving the structural characteristics of bongkrekic acid. This better exposes the structural features of bongkrekic acid, improves the immunogenicity and immune recognition ability of the antigen, lays the foundation for the preparation of antibodies with high specificity and sensitivity, and enhances its rapid detection capability. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the synthetic route for a bongkrekic acid hapten proposed in this invention.

[0053] Figure 2 This is a schematic diagram of the structure of a bongkrekic acid hapten proposed in this invention;

[0054] Figure 3 This is a schematic diagram of the process for preparing and applying a bongkrekic acid hapten and its antigen, as proposed in this invention. Detailed Implementation

[0055] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Example:

[0057] A bongkrekic acid hapten was obtained by analyzing the chemical structure of bongkrekic acid, which revealed that the double bond at position 2 was relatively reactive. A thiol group was introduced at position 2 of the bongkrekic acid, and the hapten was coupled to a protein via a linker arm. The structure of this bongkrekic acid hapten is as follows: Figure 1 As shown.

[0058] like Figure 2 As shown, in this embodiment, the synthesis of the bongkrekic acid hapten includes the following specific steps:

[0059] Weigh 3 mg of bongkrekic acid and add it to 100 mg of bis(2-dimethylaminoethyl) ether and bis(p-toluenesulfonyl)amine ionic liquid, and heat to 60°C;

[0060] Add 10 mg of sodium sulfide to the liquid and keep it at a warm temperature for 18 hours;

[0061] Cool the liquid to room temperature, add 3 mL of deionized water for extraction, let stand for 20 min, take the aqueous phase, repeat the extraction step three times and combine the aqueous phases;

[0062] The combined aqueous phases were extracted three times with 30 mL x 3 ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate overnight, and the ethyl acetate was removed under reduced pressure to obtain bongkrekic acid hapten.

[0063] like Figure 3 As shown, a bongkrekic acid hapten and its antigen preparation and application are described. The antigen preparation and application based on the aforementioned bongkrekic acid hapten includes the following specific steps:

[0064] S1, Synthesis of bongkrekic acid hapten.

[0065] S2. Preparation of bongkrekic acid immunogen.

[0066] In this embodiment, 8 mg of hemocyanin (KLH) and 2 mg of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC) were weighed and added to 1 mL of BB buffer at pH 8.5, and activated at room temperature for 4 h. 0.8 mg of bongkrekic acid hapten was dissolved in 0.2 mL of DMF and added dropwise to the above activation solution, and reacted at room temperature for 8 h. Unbound low molecular weight substances were removed by ultrafiltration using a 3 KD ultrafiltration tube to obtain the bongkrekic acid immunogen.

[0067] S3. Preparation of bongkrekic acid coating agent.

[0068] In this embodiment, 8 mg of bovine serum albumin (BSA) and 2 mg of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (Sulfo-SMCC) were weighed and added to 1 mL of BB buffer at pH 8.5, and activated at room temperature for 4 h. 0.8 mg of bongkrekic acid hapten was dissolved in 0.2 mL of DMF and added dropwise to the above activation solution, and reacted at room temperature for 8 h. Unbound low molecular weight substances were removed by ultrafiltration using a 3 kDa ultrafiltration tube to obtain the bongkrekic acid coated antigen.

[0069] S4. Preparation of bongkrekic acid monoclonal antibody.

[0070] In this embodiment:

[0071] Immunization: 0.1 mg of bongkrekic acid immunogen was injected intraperitoneally into 6-week-old BALB / c mice, followed by three immunizations at three-week intervals. Tail blood was collected one week after the third immunization to measure the titer and inhibition rate.

[0072] Cell fusion and cloning: Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. Positive wells were screened using indirect ELISA, and further screened using indirect competitive ELISA. Hybridoma cell lines that stably secrete monoclonal antibodies were cloned using limiting dilution. The obtained hybridoma cell lines were cryopreserved in liquid nitrogen.

[0073] Monoclonal antibody preparation: Frozen cell lines were revived, cultured, and injected into the peritoneal cavity of BALB / c mice. Ascites fluid was collected 7 days later, and the purified ascites fluid yielded bongkrekic acid monoclonal antibody. The antibody titer of this bongkrekic acid monoclonal antibody was 64000, with an IC10 of 2 μg / kg.

[0074] S5. Preparation of bongkrekic acid immunofluorescence detection card.

[0075] In this embodiment;

[0076] Activation of fluorescent microspheres: Add the fluorescent microspheres to be activated to a buffer solution, wherein the buffer solution is a 2-(N-morpholino)ethanesulfonic acid solution with a pH of 6.0. Wash the microspheres with sonication for several minutes, centrifuge at high speed for 15 minutes, discard the supernatant, and repeat the washing step. Collect the solid material and add it to a buffer solution of 2-(N-morpholino)ethanesulfonic acid solution with a pH of 6.0. Add N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to the buffer solution in sequence, with a mass ratio of NHS to EDC of 4:1. Shake the reaction solution at room temperature for 30 minutes, centrifuge the above reaction solution for 15 minutes, discard the supernatant, collect the solid material and dissolve it in PB buffer (0.05 mol / L, pH 7.4) to obtain the fluorescent microsphere solution.

[0077] Antibody labeling: Dissolve bongkrekic acid antibody in PBS buffer (pH 7.4), dilute the antibody concentration to 1 mg / mL, and add it to the fluorescent microsphere solution. Incubate at room temperature with shaking for 60 min. Add 10 μL of ethanolamine and incubate at room temperature with shaking for 60 min. Then add bovine serum albumin solution (BSA solution) and block for 60 min. Centrifuge the above reaction system for 15 min, discard the supernatant, collect the solid material, and resuspend it in PB buffer (0.05 mol / L, pH 7.4) to obtain fluorescent microsphere-labeled bongkrekic acid antibody. Store at 4℃.

[0078] Preparation of the conjugate pad: Cut a 20mm×4mm glass fiber membrane, take an appropriate amount of fluorescent microsphere-labeled bongkrekic acid antibody and spray it onto the glass fiber membrane at 4ul / cm using a BIODOT sprayer, and dry at 37℃ for 1h to obtain the conjugate pad.

[0079] Sample pad preparation: Sample pad 2: Cut a 1.1cm×10cm glass cellulose membrane, immerse it evenly in the sealing solution for 10min, and vacuum dry it under clean conditions at room temperature to obtain the sample pad for later use.

[0080] Preparation of reaction membrane: Spray bongkrekic acid semi-antibody-BSA conjugate solution onto T line, and spray secondary antibody solution that recognizes bongkrekic acid antibody onto C line.

[0081] Assembly: The sample pad, coupling pad, reaction membrane, and absorbent pad are pasted onto the PVC board in sequence.

[0082] S6. Detection of bongkrekic acid in food.

[0083] In this embodiment:

[0084] Sample pretreatment: Weigh 2g of sample into a 20mL centrifuge tube, add 10mL of acetonitrile, vortex for 2min, centrifuge at 4000rpm for 5min at room temperature, take 5mL of the upper organic phase, blow dry with nitrogen in a 50℃ water bath, and reconstitute with 200uL of 0.01M PBS.

[0085] Detection: The method for detecting bongkrekic acid using the above-mentioned immunofluorescence detection card includes the following steps:

[0086] The sample to be tested is added to the sample pad for an immune reaction.

[0087] Place the immunofluorescence detection card for measuring bongkrekic acid on the fluorescence card reader and read the intensity of the fluorescence signal.

[0088] Result Interpretation: When both the detection line and the control line show orange fluorescence under 345nm excitation light, and the detection line is darker than the control line or the two lines are the same or similar in color, the result is negative. When the control line shows orange fluorescence under 345nm excitation light, while the detection line does not show fluorescence or is lighter in color than the control line, the result is positive.

[0089] S7, Detection Limit Experiment.

[0090] In this embodiment, blank samples were added with bongkrekic acid to final concentrations of 2ug / kg, 4ug / kg, and 8ug / kg, and the test was performed according to step S6, repeated three times. The result showed that the detection limit of this test strip was 4ug / kg.

[0091] S8, False Positive Rate and False Negative Rate Experiment.

[0092] In this embodiment, blank samples were added with bongkrekic acid to a final concentration of 4 μg / kg and tested according to step S6, repeated three times. Fifty samples of each type were used, and three batches of test strips were tested separately. The results showed that the false positive rate and false negative rate were both 0.

[0093] The beneficial effects of this invention are:

[0094] This bongkrekic acid hapten and its antigen preparation and application involve introducing a thiol group at the 2-position of bongkrekic acid and coupling it with a protein via a linker arm to obtain a hapten, while completely preserving the structural characteristics of bongkrekic acid. This better exposes the structural features of bongkrekic acid, improves the immunogenicity and immune recognition ability of the antigen, lays the foundation for the preparation of antibodies with high specificity and sensitivity, and enhances its rapid detection capability.

[0095] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0096] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mycophenolic acid hapten, characterized in that, The structure of the bongkrekic acid hapten is as follows: 。 2. The mycophenolic acid hapten according to claim 1, characterized in that, The synthesis of the bongkrekic acid hapten comprises the following specific steps: Take 3 mg of bongkrekic acid and add to 100 mg of bis 2-dimethylaminoethyl ether and bis p-toluenesulfonyl amine ionic liquid, heat to 60 DEG C; Add 10 mg of sodium sulfide to the liquid, and incubate for 18 h; Cool the liquid to room temperature, add 3 mL of deionized water for extraction, and take the water phase after standing for 20 min, repeat the extraction step three times and combine the water phases; Add 30 mL x 3 ethyl acetate to the combined water phase for extraction three times, combine the organic phases, dry with anhydrous sodium sulfate overnight, and evaporate the ethyl acetate under reduced pressure to obtain the bongkrekic acid hapten.

3. A diagnostic application of mycophenolic acid hapten, based on the diagnostic application of mycophenolic acid hapten according to any one of claims 1 to 2, characterized in that, The antigen preparation application comprises the following specific process: S1, synthesis of the bongkrekic acid hapten; In the step S1, the synthesis of the bongkrekic acid hapten comprises the following specific steps: Take 3 mg of bongkrekic acid and add to 100 mg of bis 2-dimethylaminoethyl ether and bis p-toluenesulfonyl amine ionic liquid, heat to 60 DEG C; Add 10 mg of sodium sulfide to the liquid, and incubate for 18 h; Cool the liquid to room temperature, add 3 mL of deionized water for extraction, and take the water phase after standing for 20 min, repeat the extraction step three times and combine the water phases; Add 30 mL x 3 ethyl acetate to the combined water phase for extraction three times, combine the organic phases, dry with anhydrous sodium sulfate overnight, and evaporate the ethyl acetate under reduced pressure to obtain the bongkrekic acid hapten; S2, preparation of the bongkrekic acid immunogen; In the step S2, the preparation of the bongkrekic acid immunogen comprises the following specific operation process: Take 8 mg of hemocyanin and 2 mg of 4-N-maleimidomethyl cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt and add to 1 mL of BBS buffer at pH 8.5, activate at room temperature for 4 h to form an activation solution; Dissolve 0.8 mg of the bongkrekic acid hapten in 0.2 mL of DMF, and dropwise add to the activation solution, and react at room temperature for 8 h; Remove the unbound low molecular weight substances by ultrafiltration with a 3KD ultrafiltration tube to obtain the bongkrekic acid immunogen; S3, preparation of the bongkrekic acid coating agent; In the step S3, the preparation of the bongkrekic acid coating agent comprises the following specific operation process: Take 8 mg of bovine serum albumin and 2 mg of 4-N-maleimidomethyl cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt and add to 1 mL of BBS buffer at pH 8.5, activate at room temperature for 4 h to form an activation solution; Dissolve 0.8 mg of the bongkrekic acid hapten in 0.2 mL of DMF, and dropwise add to the activation solution, and react at room temperature for 8 h; Remove the unbound low molecular weight substances by ultrafiltration with a 3KD ultrafiltration tube to obtain the bongkrekic acid coating agent; S4, preparation of the bongkrekic acid monoclonal antibody; In the step S4, the preparation method of the bongkrekic acid monoclonal antibody comprises the following specific operation process: Immunization: take 0.1 mg of the bongkrekic acid immunogen and inject into the abdominal cavity of a 6-week-old BALB / C mouse, immunize once every three weeks, a total of three times, and take tail blood one week after the third immunization to measure the titer and inhibition rate; Cell fusion and cloning: the immune mouse spleen cells were fused with SP2 / 0 myeloma cells, and the positive wells were screened by indirect ELISA, and the positive wells were further identified and screened by indirect competitive ELISA, and the hybridoma cell strain capable of stably secreting monoclonal antibody was cloned by limited dilution method, and the obtained hybridoma cell strain was stored in liquid nitrogen; Monoclonal antibody preparation: the frozen cell strain was recovered, cultured, and injected into the abdominal cavity of a BALB / C mouse, and the ascites was collected after 7 days, and the ascites was purified to obtain the mycophenolic acid monoclonal antibody, and the antibody titer of the mycophenolic acid monoclonal antibody was 64000, and the IC10 was 2ug / kg; S5, preparation of mycophenolic acid immunofluorescence detection card; In the step S5, the preparation of the mycophenolic acid immunofluorescence detection card includes the following specific process: S501, fluorescence microsphere activation; S502, antibody labeling; S503, conjugate pad preparation; S504, sample pad preparation; S505, reaction membrane preparation; S506, assembly; S6, detection of mycophenolic acid in food; In the step S6, the detection of mycophenolic acid in food includes the following specific process: Sample pretreatment: weigh 2g sample in a 20mL centrifuge tube, add 10mL acetonitrile, vortex for 2min, centrifuge at 4000rpm for 5min at room temperature, take 5mL of the upper organic phase, dry under nitrogen at 50℃ water bath, and redissolve in 200uL 0.01M PBS; Detection: add the sample to be determined to the sample pad, perform immunoreaction, place the immunofluorescence detection card for detecting mycophenolic acid on the fluorescence card reader, and read the intensity of the fluorescence signal; Result judgment: when the detection line and the quality control line show orange fluorescence under 345nm excitation light irradiation, and the detection line is darker than the quality control line or the colors are the same or similar, it means that the detection result is negative, and when the detection line and the quality control line are under 345nm excitation light irradiation, the quality control line shows orange fluorescence, and the detection line does not show fluorescence or shows lighter color than the quality control line, which means that the detection result is positive; S7, detection limit experiment; In the step S7, the detection limit experiment includes the following experimental contents: Take the blank sample, add mycophenolic acid to a final concentration of 2ug / kg, 4ug / kg, and 8ug / kg, and detect according to the process of step S6, and repeat three times; S8, false positive rate and false negative rate experiment; In the step S8, the false positive rate and false negative rate experiment includes the following experimental contents: Take the blank sample, add mycophenolic acid to a final concentration of 4ug / kg, and detect according to the process of step S6, and repeat three times; 50 samples of each sample, three batches of test strips are detected respectively.

Images