A methylphenylhydrazine hapten, its preparation method and application

Abstract

The application discloses a cresotanazole hapten, a preparation method and application thereof, and belongs to the technical field of cresotanazole content determination. The cresotanazole hapten is obtained by modifying cresotanazole by using 4-bromobutyric acid ethyl ester, and the obtained hapten structure itself contains a carboxyl end, can be directly coupled with a carrier protein through a crosslinking agent, has a high coupling ratio, and is easier to expose a specific binding site after being connected with the carrier protein, and has good immunogenicity. The cresotanazole antibody prepared according to the hapten has high specific recognition performance and high sensitivity, can be further prepared into an immunological analysis kit for rapid detection, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of drometrizole content determination, and particularly relates to a drometrizole hapten and a preparation method and application thereof. Background Art

[0002] Ultraviolet rays in sunlight are a major cause of skin damage. Prolonged exposure to UV rays can cause erythema, itching, hyperpigmentation, and even skin cancer. Therefore, to inhibit or mitigate UV damage to human skin, sunscreens are often added to cosmetics. Sunscreens are the core of sunscreen cosmetic formulas, and their effectiveness is directly related to the amount of sunscreen added. Because most UV absorbers are relatively unstable, some manufacturers incorporate high doses of sunscreens or even banned sunscreens into their product formulas to achieve higher sun protection. Excessive amounts of sunscreen can pose certain health risks, such as roughening the skin, causing skin and eye irritation, photosensitivity reactions, and even, in severe cases, cancer. In recent years, quality issues have frequently occurred during random inspections of sunscreen cosmetics, with discrepancies between the measured sunscreen content and type and the label information, resulting in high failure rates. Therefore, it is crucial to develop specific, accurate, and user-friendly sunscreen analysis methods to improve the quantitative detection of UV absorbers in sunscreen products.

[0003] Benzotriazoles are often added to cosmetics for sun protection due to their excellent absorption of short-wave and medium-wave ultraviolet rays. Therefore, my country's 2015 edition of the "Safety Technical Specifications for Cosmetics" specifies permitted sunscreens, stipulating that only two UV absorbers—drometrizole trisiloxane (DTTSO) and methylene bis-benzotriazolyl tetramethylbutylphenol (UV-360)—may be used in sunscreen cosmetics, with maximum usage limits set. All other benzotriazole UV absorbers are prohibited. However, investigations have revealed that banned benzotriazole UV absorbers, such as drometrizole, are still being misused in sunscreen cosmetics on the market.

[0004] Dromtriazole (molecular formula C 13 H 11 N₃O (relative molecular mass 255.3) is a UV absorber that absorbs the ultraviolet radiation from sunlight and fluorescent light sources. In cosmetics and personal care products, drometrizole can be used both as a sunscreen ingredient and as a UV absorber to prevent deterioration. It is also commonly used as a UV absorber and stabilizer in the synthesis of plastics, polyesters, dyes, and rubber, and in the polymerization and manufacturing of polymers to prevent degradation caused by excessive UV radiation. Therefore, testing for drometrizole can provide a basis for quality control of products containing drometrizole.

[0005] Currently, detection methods for drometrizole primarily include liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. For example, patent document CN 119125375 A discloses a method for determining drometrizole in textiles. The method comprises extracting drometrizole from the textile to be tested with an extraction solvent, followed by detection and analysis using a high-performance liquid chromatography-diode array detector (LC-DAD). This method exhibits good linearity within the drometrizole mass concentration range of 10-50 μg / mL, with a detection limit of 21.2 mg / kg, a recovery rate of 94.1-97.4%, and a relative standard deviation of 3.4-5.9%. For example, the non-patent document "Determination of drometrizole and daptriazole trisiloxane in sunscreen cosmetics by high-performance liquid chromatography" discloses a method for analyzing drometrizole using reversed-phase high-performance liquid chromatography. The method first extracts drometrizole from the cosmetics using a water-tetrahydrofuran-methanol solvent, separates it using a C18 column, and uses HPLC-DAD for qualitative and quantitative analysis. The method showed good linearity in the mass concentration range of 5.0-500 mg / L of drometrizole, with a limit of quantification of 5.0 mg / kg and a recovery of 90-110%.

[0006] The above existing methods all have the disadvantages of expensive instruments and complicated operations. They can only be performed in the laboratory, are inconvenient to use, and cannot achieve rapid on-site detection. Based on this, the present invention provides a method for detecting drometrizole based on enzyme-linked immunosorbent assay technology, which can quickly determine the drometrizole content in the sample to be tested and is suitable for rapid on-site detection. Summary of the Invention

[0007] The fundamental factors affecting the quality of immunoassays are the specificity and sensitivity of antibodies. The properties of antibodies depend on the molecular structure of the immune hapten. The design and synthesis of the immune hapten is the basis and key step for producing specific antibodies and establishing rapid detection technology. Based on this, the first object of the present invention is to provide a hapten for drometrizole, which is obtained by structurally modifying drometrizole. The hapten structure itself contains a carboxyl terminus and can be directly coupled to a carrier protein via a cross-linking agent, resulting in a high coupling ratio. After connection with the carrier protein, the specific binding site is more easily exposed, resulting in better immunogenicity. The second object of the present invention is to provide a complete antigen and antibody obtained based on the hapten. Another object of the present invention is to provide a kit and a detection method for detecting drometrizole.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a drometrizole hapten, characterized in that the drometrizole hapten has a structure shown in Formula I, or a pharmaceutically acceptable salt thereof:

[0010] (Ⅰ).

[0011] In a second aspect, the present invention provides a method for preparing a hapten of drometrizole, characterized in that the preparation method comprises the following steps:

[0012] (1) Mix drometrizole and ethyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, and react at 30-50°C for 3-5 hours to obtain an intermediate product;

[0013] (2) Dissolve the intermediate product in methanol, then add 2M NaOH solution and allow to hydrolyze at 30-50°C for 1-3 hours;

[0014] (3) After the hydrolysis is completed, 2M HCl is added to adjust the pH to 3.5-4.5, and a white solid is precipitated. The solid is filtered, washed, and dried to obtain the drometrizole hapten shown in Formula I.

[0015] Preferably, in step (1), the molar ratio of drometrizole to ethyl 4-bromobutyrate is 1.2:1, and the reaction is carried out at 40° C. for 4 hours to obtain an intermediate product.

[0016] Preferably, the hydrolysis condition in step (2) is 40° C. for 2 hours.

[0017] Preferably, in step (3), 2M HCl is used to adjust the pH to 4.0.

[0018] In a specific embodiment of the present invention, the method for preparing the drometrizole hapten comprises the following steps:

[0019] S1: Rinse a 50 ml round-bottom flask, blow dry it with ethanol, fix it on a stirrer, and add a stir bar;

[0020] S2: Weigh 225 mg of drometrizole raw material and add it to the container prepared in step S1. Dissolve it in 2.5 ml of N,N-dimethylformamide (DMF). Stir. Add 48 mg of sodium hydride and react at room temperature for 30 min.

[0021] S3: After the reaction is completed, add 172 μl of ethyl 4-bromobutyrate and react at 40°C for 4 hours. Evaporate to dryness and weigh. Add 5 mL of methanol to dissolve the residue. Add 1000 μl of 2 M NaOH solution and heat at 40°C for 2 hours for hydrolysis.

[0022] S4: After the hydrolysis reaction is complete, the pH is adjusted to 4 with 2M HCl to precipitate a white solid, which is filtered, and the filter cake is washed with distilled water and dried in a forced air drying oven at 40°C to obtain the hapten represented by Formula I.

[0023] In the above preparation process, the inventors replaced the above-mentioned ethyl 4-bromobutyrate with methyl 4-bromobutyrate, tert-butyl 4-bromobutyrate, and 4-bromobutyric acid of the same molar mass. It was found that the target hapten could be obtained using ethyl 4-bromobutyrate as a raw material, and the yield was high. Although the intermediate product prepared using tert-butyl 4-bromobutyrate as a raw material was more efficient in the subsequent hydrolysis step, the yield and purity data showed that there was no significant difference between the target hapten prepared using ethyl 4-bromobutyrate as a raw material, and those skilled in the art know that the price of ethyl 4-bromobutyrate is much lower than that of tert-butyl 4-bromobutyrate, so the present invention preferably uses ethyl 4-bromobutyrate as a raw material for preparation.

[0024] The fixed values ​​described in each synthesis step of the preparation method provided by the present invention are only optimized values, but the protection scope of the present invention is not limited thereto. As long as the corresponding drometrizole hapten and full antigen compounds can be synthesized, they should all fall within the protection scope of the present invention.

[0025] In a third aspect, the present invention provides a whole drometrizole antigen, characterized in that the whole antigen is obtained by coupling the drometrizole hapten described in the first aspect of the present invention with a carrier protein.

[0026] The coupling method is selected from the active ester method (i.e., biocoupling method) or the mixed anhydride method; the active ester method is preferred.

[0027] The carrier protein is selected from any one or a combination of two or more of thyroglobulin (BTG), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), diphtheria toxoid, a non-toxic mutant of diphtheria toxin, tetanus toxoid and bacterially expressed proteins.

[0028] In a specific embodiment of the present invention, the carrier protein is bovine serum albumin, and the drometrizole hapten is coupled to the bovine serum albumin to obtain the drometrizole immunogen.

[0029] In a specific embodiment of the present invention, the carrier protein is ovalbumin, and the drometrizole hapten is coupled to the ovalbumin to obtain the drometrizole coating.

[0030] In a fourth aspect, the present invention provides a drometrizole antibody, characterized in that the antibody is obtained by immunizing an animal with the drometrizole immunogen described in the third aspect of the present invention.

[0031] Those skilled in the art can obtain the antibody by immunizing an animal with the drometrizole immunogen described in the third aspect of the present invention through conventional techniques. In a specific embodiment of the present invention, the animal is a mouse.

[0032] In a fifth aspect, the present invention provides a use of the drometrizole hapten described in the first aspect of the present invention, the drometrizole full antigen described in the third aspect of the present invention, and the drometrizole antibody described in the fourth aspect of the present invention in at least one of the following:

[0033] (1) Application in the detection of drometrizole;

[0034] (2) Application in the preparation of products for detecting drometrizole.

[0035] The products include but are not limited to test strips, membrane strips, test kits, chips, models or detection platforms.

[0036] In some embodiments of the present invention, the product is an enzyme-linked immunosorbent assay (ELISA) kit.

[0037] In a sixth aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting drometrizole. The kit comprises an antibody working solution, which is prepared by diluting the drometrizole antibody described in the fourth aspect of the present invention.

[0038] Furthermore, the kit also includes an ELISA plate coated with the drometrizole coating agent described in the third aspect of the present invention and a goat anti-mouse antibody labeled with horseradish peroxidase (HRP) (enzyme-labeled secondary antibody).

[0039] Furthermore, the kit also includes a drometrizole standard working solution, which is a PBS solution with drometrizole concentrations of 0 ng / mL, 0.003 ng / mL, 0.01 ng / mL, 0.03 ng / mL, 0.09 ng / mL, 0.27 ng / mL, 0.81 ng / mL and 2.43 ng / mL.

[0040] In a seventh aspect, the present invention provides a method for detecting drometrizole, characterized in that the method comprises: using an indirect competitive ELISA method, using the drometrizole-coated original coated ELISA plate described in the third aspect of the present invention, adding a sample solution to be tested, and at the same time adding the drometrizole antibody described in the fourth aspect of the present invention, incubating, washing and patting dry, adding an enzyme-labeled goat anti-mouse antibody, developing color with a substrate, terminating, and measuring the absorbance value with a microplate reader; drawing an ELISA standard curve, substituting the absorbance value into the standard curve to obtain the drometrizole concentration in the sample.

[0041] The technical solution provided by the present invention has the following technical advantages:

[0042] (1) The sample pre-treatment steps of the existing detection methods for drometrizole are cumbersome and costly, and are not suitable for screening and testing of large quantities of samples. The drometrizole detection method based on enzyme-linked immunosorbent assay provided by the present invention has unique advantages in the qualitative and quantitative analysis of antigens and antibodies, and its advantages of simple and rapid operation, low cost, high sensitivity, and large sample size make up for the shortcomings of instrumental analysis, laying a research and development foundation for the rapid detection of drometrizole residues.

[0043] (2) Compared with the haptens in the prior art, the drometrizole hapten of the present invention contains a carboxyl end and can be directly coupled to a carrier through a cross-linking agent. After being connected to the carrier protein, it is easier to expose the specific binding site and has higher sensitivity. When preparing specific antibodies, the hapten and protein are coupled so that the drometrizole hapten can specifically bind to B lymphocytes and produce a good immune response effect.

[0044] (3) The hapten synthesis method of the present invention is simple, and the purity of the prepared hapten product reaches 99% and the yield is as high as 90%. It can be directly coupled with a protein to immunize animals to produce specific antibodies against drometrizole.

[0045] (4) The antibodies provided by the present invention can be used to detect drometrizole, especially the polyclonal antibodies have high specific recognition performance for drometrizole; the antiserum titer is 1:12000, and the best mouse polyclonal antiserum IC50 is 0.216 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram showing the mass spectrometry identification results of the drometrizole hapten in Example 1;

[0047] Figure 2 This is a diagram showing the nuclear magnetic resonance identification results of the drometrizole hapten in Example 1;

[0048] Figure 3 is the MALDI-TOF-MAS image of BSA in Example 2;

[0049] Figure 4 This is a MALDI-TOF-MAS image of the drometrizole hapten-BSA complex in Example 2;

[0050] Figure 5 This is a MALDI-TOF-MAS image of the drometrizole hapten-BSA complex in Comparative Example 2;

[0051] Figure 6 This is the MALDI-TOF-MAS image of the drometrizole-BSA coupling product in Comparative Example 3;

[0052] Figure 7This is the ELISA standard curve obtained in Example 3. DETAILED DESCRIPTION

[0053] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0054] Example 1 Preparation and structural identification of drometrizole hapten

[0055] 1) Preparation of drometrizole hapten

[0056] The synthetic route is as follows:

[0057] ;

[0058] S1: Rinse a 50 ml round-bottom flask, blow dry it with ethanol, fix it on a stirrer, and add a stir bar;

[0059] S2: Weigh 225 mg of drometrizole raw material and add it to the container prepared in step S1. Dissolve it in 2.5 ml of N,N-dimethylformamide (DMF). Stir. Add 48 mg of sodium hydride and react at room temperature for 30 min.

[0060] S3: After the reaction is complete, add 172 μl of ethyl 4-bromobutyrate and react at 40°C for 4 hours. Evaporate to dryness and weigh the residue. Dissolve the residue in 5 mL of methanol and add 1000 μl of 2 M NaOH solution. Heat at 40°C for 2 hours for hydrolysis.

[0061] S4: After the hydrolysis reaction is complete, the pH is adjusted to 4 with 2M HCl to precipitate a white solid, which is filtered. The filter cake is washed with distilled water and dried in a 40°C forced air oven to obtain the hapten represented by Formula I. The product has a purity of 99% and a yield of 90%.

[0062] 2) Structural identification of the drometrizole hapten

[0063] The obtained product was identified by mass spectrometry and nuclear magnetic resonance. Figure 1 As shown in the figure, it is clear that the peak of M / Z 312 (M+H+) and the peak of M / Z 334 (M+Na+) are consistent with the target molecular weight (311.34). Figure 2 As shown, it contains the main structure of the target molecule. The above results indicate that the synthesis of drometrizole hapten is successful.

[0064] Example 2 Preparation and Structural Identification of Dromtrizole Artificial Antigen

[0065] 1. Preparation of artificial antigen of drometrizole (active ester method)

[0066] 1. Synthesis of Immunogen (Dromtrizole Hapten-BSA Conjugate)

[0067] S1: Dissolve 17.4 mg of the hapten (Formula I) prepared in Example 1 in 1 mL of N,N-dimethylformamide (DMF). After complete dissolution, add 10.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.2 mg of N-hydroxysuccinimide (NHS). Stir and activate at room temperature for 2-3 hours to obtain Solution I.

[0068] S2: Weigh 50 mg of bovine serum albumin (BSA) and dissolve it in 3.5 mL of 0.1 M carbonate buffer. Stir at 200 rpm for 10 min to fully dissolve the BSA to obtain Solution II.

[0069] The pH of the 0.1 M carbonate buffer solution is 9.6, the solvent is water, and the solutes and their concentrations are as follows: Na2CO3 1.59 g / L, NaHCO3 2.94 g / L;

[0070] S3: Take the above solution I and add it dropwise to the above solution II while stirring. React with magnetic stirring (500 rpm) at room temperature (20-25°C) for 12 hours to obtain solution III.

[0071] S4: Place the solution III into a dialysis bag (10 cm) rinsed with distilled water, and dialyze against 1 L of 0.01 M PBS (pH = 7.4) for 3 days at 4°C with stirring. Change the dialysate three times a day. Centrifuge the dialyzed product at 5000 rpm for 6 min, aliquot into 1.5 ml / tubes, number the antigens, and store at -20°C for later use.

[0072] The solvent of the 0.01 M PBS (pH = 7.4) is water, and the solutes and their concentrations are as follows: 0.27 g / L potassium dihydrogen phosphate, 1.42 g / L sodium hydrogen phosphate, 8 g / L sodium chloride, and 0.2 g / L potassium chloride.

[0073] 2. Synthesis of Coating Material (Dromtrizole Hapten-OVA Conjugate)

[0074] Replace 50 mg of BSA in step 1 with 33.6 mg of OVA to obtain the original coating solution.

[0075] 2. Identification of artificial antigens of drometrizole

[0076] The identification method is as follows: The conjugate of drometrizole hapten and carrier protein BSA is detected by MALDI-TOF-MS. Changes in molecular mass data indicate the successful conjugation of the hapten to the protein and can be used to calculate the protein-to-hapten conjugation ratio. The conjugate of drometrizole hapten and carrier protein BSA is desalted and mixed with matrix-assisted solution. The mixture is spotted onto a MALDI target and detected in a mass spectrometer. The binding ratio of drometrizole hapten to carrier protein BSA is calculated according to the following formula: Conjugation ratio = (MC-MB) / MA, where MA, MB, and MC represent the relative molecular masses of the hapten, protein, and conjugate, respectively.

[0077] The immunogen MALDI-TOF-MS identification results showed that the coupling ratio was: R = (73905.18-66302.94) / 311.34 = 24.418 ( Figure 3 and Figure 4 ). That is, in the immunogen, the molar ratio of the drometrizole hapten (Formula I) to bovine serum albumin (BSA) is 24.418:1.

[0078] Example 3 Preparation and detection of drometrizole antibodies

[0079] 1. Immunization of Balb / c mice

[0080] The protein concentration of drometrizole hapten-BSA is calculated based on the total protein amount, that is, the total protein concentration multiplied by the added volume is the added amount of drometrizole hapten-BSA.

[0081] For the first immunization of 6-week-old female Balb / c mice, dilute the drometrizole hapten-BSA immunogen to 1 mg / mL (diluted with 0.01 mol / L PBS), mix with an equal volume of Freund's complete adjuvant, and fully emulsify. Inoculate subcutaneously at multiple points on the back of the neck of 5 mice at a dose of 100 μg / mouse, 0.2 ml per mouse. For the second immunization 14 days later, emulsify the immunogen with an equal volume of Freund's incomplete adjuvant at the same dose as the first immunization, and boost immunizations are performed three times.

[0082] 2. Detection of mouse antiserum against drometrizole

[0083] One week after the three immunizations, 20 μL of blood was collected from the tail of the mice, centrifuged, and the serum was collected. The titer was measured using the classic checkerboard method and the sensitivity was measured using the indirect competitive ELISA method.

[0084] 1. Determination of antiserum titer, the steps are as follows:

[0085] (1) Coating: Dilute the coating antigen (Dromtrizole hapten-OVA) to 0.2 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6), add 100 μL / well to the ELISA plate, incubate in a 37°C incubator for 2 hours, pour off the liquid in the well, wash the plate three times with PBST buffer (pH 7.2), and spin dry the washing solution;

[0086] (2) Blocking: Add 150 μL of blocking solution (2% skim milk) to each well and block at 37°C for 1 h. Shake dry the liquid in the wells, wash the plate three times with PBST buffer (pH 7.2), and pat dry the washing solution.

[0087] (3) Add antiserum: add 50 μL of 0.01 M PBS (pH 7.4) to each column well, and then add 50 μL of diluted drometrizole antiserum. The antibody is diluted with 0.01 M PBS starting from 1:4000, with a gradient of 2, for a total of 4 dilution gradients. The sample volume is 50 μL per well. Incubate at 37°C for 30 min, wash three times with PBST buffer (pH 7.2), pat dry, incubate for 30 min, wash three times with PBST buffer (pH 7.2), and pat dry;

[0088] At the same time, serum from unimmunized Balb / c mice was used as a negative control;

[0089] (4) Add enzyme-labeled secondary antibody: add 100 μL HRP-goat anti-mouse IgG (5000-fold diluted in PBS) to each well, incubate at 37°C for 30 min, wash three times with PBST buffer (pH 7.2), and pat dry;

[0090] (5) Color development: Mix the horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide at a volume ratio of 1:1, add 100 μL to each well, incubate at 37°C for 15 min, and then add 50 μL of stop solution (2 mol / L H2SO4) to each well;

[0091] (6) Reading determination: Read the absorbance (OD) using a microplate reader at a wavelength of 450 nm. The titer of the antiserum is determined by the antibody dilution corresponding to a negative OD value of no more than 0.15 and a maximum OD450nm value between 1.5 and 1.8.

[0092] The results showed that the antibody titer in the serum was 1:12000.

[0093] 2. Determination of antiserum sensitivity, the steps are as follows:

[0094] (1) The coating and blocking process is the same as that in “1. Determination of Antiserum Titer” above;

[0095] (2) Adding standards and antibodies: Add 50 μL of drometrizole standard solution and 50 μL of mouse serum diluted one week after three immunizations to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry;

[0096] The solvent of the standard solution was PBS buffer, and the concentrations of the standard were 0, 0.003, 0.01, 0.03, 0.09, 0.27, 0.81, and 2.43 ng / mL, with three replicates for each concentration;

[0097] (3) Add enzyme-labeled secondary antibody: Add 100 μL of enzyme-labeled secondary antibody dilution solution to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry;

[0098] (4) Color development: Add 100 μL of color development solution to each well and incubate at 37°C for 15 min;

[0099] (5) Termination: Add 50 μL of 2 mol / L concentrated sulfuric acid to each well;

[0100] (6) Reading: Measure the OD value of each well at a wavelength of OD450nm.

[0101] The standard curve was established by fitting the four-parameter equation of Origin 8.0 with -log10 (competitor) value as the horizontal axis and OD450nm value as the vertical axis ( Figure 7 ) to obtain IC50 values.

[0102] The results showed that the IC50 of the mouse polyclonal antibody serum with the best immune effect was 0.216 ng / mL.

[0103] 3. Minimum detection limit determination

[0104] The method for detecting drometrizole provided by the present invention has a minimum detection limit (LOD) of 0.037 ng / mL.

[0105] Comparative Example 1

[0106] The difference between this comparative example and Example 1 is that the reaction time of drometrizole and ethyl 4-bromobutyrate is changed to 1 hour.

[0107] The purity of the obtained product was 50% and the yield was 50%.

[0108] Comparative Example 2

[0109] The difference between this comparative example and Example 2 is that the mixed anhydride method is used to prepare the drometrizole complete antigen. The specific method is as follows:

[0110] 1. Synthesis of Immunogen (Dromtrizole Hapten-BSA Conjugate)

[0111] (1) Dissolve 17.4 mg of the hapten (Formula I) prepared in Example 1 in 1 mL of N,N-dimethylformamide (DMF). After complete dissolution, cool the mixture to 0-4°C in an ice bath, add 32 μL of tri-n-butylamine, stir for 15 minutes, and then add 6 μL of isobutyl chloroformate dropwise at 0-4°C for 30 minutes of stirring to activate the mixture, thereby obtaining Solution I.

[0112] (2) Weigh 50 mg of bovine serum albumin (BSA) and dissolve it in 3.5 mL of 0.01 M PBS (pH = 7.4). Stir at 200 rpm for 10 min to fully dissolve the BSA to obtain Solution II.

[0113] (3) Add the above solution I dropwise to the above solution II while stirring, and react at room temperature (20-25°C) with magnetic stirring (500 rpm) for 12 h to obtain solution III;

[0114] (4) Solution III was placed in a dialysis bag (10 cm) rinsed with distilled water, and dialyzed against 1 L of 0.01 M PBS (pH = 7.4) for 3 days at 4°C with stirring. The dialysate was changed 3 times a day. The dialyzed product was centrifuged at 5000 rpm for 6 min, and the aliquots were divided into 1.5 ml / tubes. The antigens were numbered and stored at -20°C for future use.

[0115] 2. Synthesis of Coating Source (Dromtrizole Hapten-OVA Conjugate)

[0116] Prepare the coating stock solution by replacing 50 mg BSA in step 1 with 33.6 mg OVA.

[0117] The immunogen prepared by the mixed anhydride method was identified using the method provided in Example 2. The MALDI-TOF-MS identification results of the immunogen are as follows: Figure 5 The calculated coupling ratio is R = 24.086. This means that the molar ratio of the drometrizole hapten to bovine serum albumin (BSA) in the immunogen is 24.086:1. Comparing the coupling ratio data for BSA coupling using the active ester method, it can be seen that the active ester method achieves a higher coupling ratio for drometrizole hapten and BSA.

[0118] Using the method provided in Example 3, the immunogen prepared in Comparative Example 2 was used to immunize Balb / c mice to obtain antibodies. The antibodies were subjected to antiserum titer determination and sensitivity determination. The results showed that the antibody titer in the serum was 1:4000, and the IC50 of the mouse polyantibody serum with the best immune effect was 5.55 ng / mL. Comparing the antibody titer and IC50 data obtained by coupling the immunogen obtained by the active ester method, it can be found that the coupling method of the hapten and the carrier protein significantly affects the immunogenicity of the prepared full antigen, and the full antigen obtained by coupling by the active ester method has better immunogenicity.

[0119] Comparative Example 3

[0120] Using cresol red as a hapten, coupling with BSA and OVA respectively to obtain immunogen and coating antigen. The structure of cresol red is shown as formula II:

[0121] (Formula II)

[0122] 1. Synthesis of immunogen (cresol red-BSA coupling)

[0123] (1) Dissolve 10.07 mg of cresol red (formula II) in 1 mL of N,N-dimethylformamide (DMF), after complete dissolution, cool to (0-4°C) in an ice bath, add N,N'-carbonyldiimidazole 18.5 mg, stir for 30 min, then restore to room temperature (20-25°C) and stir for 2 hours to obtain solution I;

[0124] (2) Weigh 50 mg of bovine serum albumin (BSA) and dissolve it in 3.5 mL of 0.01 M PBS (pH=7.4), stir at 200 rpm for 10 min, and fully dissolve to obtain solution II;

[0125] (3) Take the above solution I and add it dropwise to the above solution II while stirring, and stir at room temperature (20-25°C) at 500 rpm for 12 h to obtain solution III;

[0126] (4) Put the solution III into a dialysis bag (10 cm) washed with distilled water, dialyze against 1 L of 0.01 M PBS (pH=7.4) for 3 days, stir at 4°C, change the dialysis solution 3 times a day, centrifuge the dialysis product at 5000 rpm for 6 min, and store at -20°C for future use.

[0127] 2. Synthesis of coating antigen (cresol red-OVA coupling)

[0128] Use 33.6 mg of OVA instead of 50 mg of BSA in step 1 to prepare the coating antigen solution.

[0129] Use the method provided in Example 2 to identify the prepared immunogen, and the MALDI-TOF-MS identification result of the immunogen is as follows: Figure 6 The coupling ratio is calculated to be R=14.040, i.e., the molar ratio of cresol red to bovine serum albumin (BSA) in the immunogen is 14.040:1.

[0130] The antibody was obtained by immunizing Balb / c mice with the immunogen prepared according to the method provided in Reference Example 3. The antiserum titer and sensitivity determination of the antibody showed that the antibody titer in the serum was 1:3000, and the immune effect of the best mouse multiple antiserum IC50 was 53 ng / mL.

[0131] The above results show that, without modification, directly coupling BSA with cresol red azole as a hapten results in very low coupling efficiency, while the use of 4-bromobutyric acid ethyl ester to modify cresol red azole and then hydrolyze it to obtain the hapten shown in Formula I is very advantageous for coupling BSA, and the coupling efficiency is significantly improved. Moreover, the whole antigen immunogenicity of cresol red azole directly coupled with a carrier protein through the phenolic hydroxyl group thereof is poor, and the specific binding site is not fully exposed, resulting in low antibody titer.

[0132] The above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the technical solutions recorded in the above examples can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A hapten of drometrizole, characterized in that The drometrizole hapten has the structure shown in Formula I, or a pharmaceutically acceptable salt thereof: （Ⅰ） The drometrizole hapten is prepared by the following method: (1) Mix drometrizole and ethyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, and react at 30-50°C for 3-5 hours to obtain an intermediate product; (2) Dissolve the intermediate product in methanol, then add 2 M NaOH solution and allow to hydrolyze at 30-50°C for 1-3 hours; (3) After the hydrolysis is completed, 2M HCl is added to adjust the pH to 3.5-4.5, and a white solid is precipitated. The solid is filtered, washed, and dried to obtain the drometrizole hapten shown in Formula I.

2. The method for preparing the drometrizole hapten according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Mix drometrizole and ethyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, and react at 30-50°C for 3-5 hours to obtain an intermediate product; (2) Dissolve the intermediate product in methanol, then add 2 M NaOH solution and allow to hydrolyze at 30-50°C for 1-3 hours; (3) After the hydrolysis is completed, 2M HCl is added to adjust the pH to 3.5-4.5, and a white solid is precipitated. The solid is filtered, washed, and dried to obtain the drometrizole hapten shown in Formula I.

3. A complete antigen of drometrizole, characterized in that: The complete antigen is obtained by coupling the drometrizole hapten according to claim 1 with a carrier protein, and the coupling method is selected from the active ester method; the carrier protein is selected from bovine serum albumin, and the drometrizole hapten is coupled with bovine serum albumin to obtain the drometrizole immunogen; the carrier protein is selected from ovalbumin, and the drometrizole hapten is coupled with ovalbumin to obtain the drometrizole coating.

4. A drometrizole antibody, characterized in that The antibody is obtained by immunizing an animal with the drometrizole immunogen according to claim 3.

5. Use of the drometrizole hapten according to claim 1, the drometrizole full antigen according to claim 3, and the drometrizole antibody according to claim 4 in at least one of the following: (1) Application in the detection of drometrizole; (2) Application in the preparation of products for detecting drometrizole.

6. An enzyme-linked immunosorbent assay kit for detecting drometrizole, comprising an antibody working solution, wherein the antibody working solution is prepared by diluting the drometrizole antibody according to claim 4.

7. The kit according to claim 6, characterized in that The kit further comprises an ELISA plate coated with the drometrizole coating agent according to claim 3 and a goat anti-mouse antibody labeled with horseradish peroxidase.

8. The kit according to claim 6, characterized in that The kit also includes a drometrizole standard working solution.

9. A method for detecting drometrizole, characterized in that: The method comprises: using an indirect competitive ELISA method, coating an ELISA plate with the drometrizole coating agent according to claim 3, adding a sample solution to be tested, and simultaneously adding the drometrizole antibody according to claim 4, incubating, washing, and patting dry, adding an enzyme-labeled goat anti-mouse antibody, developing color with a substrate, terminating, and measuring the absorbance value with an enzyme reader; drawing an ELISA standard curve, and substituting the absorbance value into the standard curve to obtain the drometrizole concentration in the sample.

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