Cresol trozole hapten as well as preparation method and application thereof

Abstract

The invention discloses a cresol trozole hapten as well as a preparation method and application thereof, and belongs to the technical field of cresol trozole content determination. The cresol trozole hapten is obtained by carrying out structural modification on cresol trozole by using ethyl 4-bromobutyrate, the obtained hapten structure contains a carboxyl terminal, can be directly coupled with a carrier protein through a cross-linking agent, has a high coupling ratio, is easier to expose a specific binding site after being connected with the carrier protein, and has good immunogenicity. The cresol trozole antibody prepared according to the hapten has high specific recognition performance and high sensitivity, can be further prepared into an immunoassay 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 factor in causing damage to human skin. Long-term ultraviolet radiation can cause skin problems such as erythema, itching, pigmentation and skin cancer. Therefore, in order to inhibit or reduce the damage of ultraviolet rays to human skin, some sunscreens are often added to cosmetics. Sunscreens are the core of sunscreen cosmetics formulas, and the sunscreen effect of sunscreen cosmetics is related to the content of sunscreens added in the formula. Since most ultraviolet absorbers have poor stability, in order to obtain cosmetics with higher sunscreen performance, some manufacturers add high-dose sunscreens or prohibited sunscreens to product formulas. When sunscreens are added in excess, they will cause certain harm to human health, such as making the skin rough, causing skin and eye irritation, and producing photosensitivity reactions. In severe cases, they may even cause cancer. In recent years, in the supervision and random inspection of sunscreen cosmetics, quality problems such as the actual measured content and type of sunscreens not being consistent with the label identification have occurred from time to time, and the unqualified rate is high. Therefore, it is very necessary to establish a sunscreen analysis method with strong specificity, high accuracy and simple operation to improve the quantitative detection of ultraviolet absorbers in sunscreen products.

[0003] Benzotriazole substances are often added to cosmetics for sun protection because they have a good absorption effect on short-wave or medium-wave ultraviolet rays. For this reason, my country's 2015 version of the "Safety Technical Specifications for Cosmetics" explains the sunscreens that are allowed to be used, stipulating that only two ultraviolet absorbers, drometrizole trisiloxane (DTTSO) and methylene bis-benzotriazolyl tetramethylbutylphenol (UV-360), can be used in sunscreen cosmetics, and their maximum usage is limited. The rest of the benzotriazole ultraviolet absorbers are prohibited. However, the investigation found that there is still abuse of banned benzotriazole ultraviolet absorbers in sunscreen cosmetics on the market, such as drometrizole.

[0004] Dromtriazole (molecular formula C 13 H 11 N3O, relative molecular mass 255.3) is a UV absorber that can absorb the ultraviolet part of sunlight and fluorescent light sources. In the field of cosmetics and personal care products, drometrizole can be used as a sunscreen ingredient and a UV absorber to prevent cosmetics and personal care products from deteriorating; it is also often used as a UV absorber and stabilizer in the synthesis of plastics, polyesters, dyes, and rubbers, and is used in the polymerization and production of polymer materials to avoid aging of polymer materials caused by excessive ultraviolet radiation. Therefore, the detection of drometrizole can provide a basis for the quality control of products containing drometrizole.

[0005] At present, the detection methods for drometrizole mainly 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, which includes adding an extraction solvent to extract drometrizole in the textile to be tested, and using a high-performance liquid chromatograph-diode array detector LC-DAD for detection and analysis. The method has a good linear relationship in the mass concentration of drometrizole in the range of 10-50 μg / mL, 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 fentrizole trisiloxane in sunscreen cosmetics by high-performance liquid chromatography" discloses a method for analyzing drometrizole using reversed-phase high-performance liquid chromatography, first using a water-tetrahydrofuran-methanol solvent to extract drometrizole in cosmetics, separating with a C18 column, and performing qualitative and quantitative analysis with HPLC-DAD. The method has a good linear relationship in the range of 5.0-500 mg / L of drometrizole mass concentration, with a limit of quantification of 5.0 mg / kg and a recovery rate of 90-110%.

[0006] The above existing methods all have the defects of expensive instruments and complicated operations, and can only be performed in the laboratory, which is 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 object to be detected and is suitable for rapid on-site detection. Summary of the invention

[0007] The fundamental factors affecting the quality of immunoassay are the specificity and sensitivity of antibodies. The properties of antibodies depend on the molecular structure of immune haptens. The design and synthesis of immune haptens are the basis and key steps for producing specific antibodies and establishing rapid detection technology. Based on this, the first object of the present invention is to provide a hapten of drometrizole, wherein the hapten is obtained by structurally modifying drometrizole. The hapten structure itself contains a carboxyl end, which can be directly coupled to a carrier protein through a cross-linking agent, has a high coupling ratio, and is more likely to expose specific binding sites after being connected to the carrier protein, and has better immunogenicity. The second object of the present invention is to provide a full 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: 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: (I).

[0009] In a second aspect, the present invention provides a method for preparing a drometrizole hapten, characterized in that the preparation method comprises the following steps: (1) mixing drometrizole and ethyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, reacting at 30-50° C. for 3-5 hours to obtain an intermediate product; (2) Dissolve the intermediate product in methanol, add 2M NaOH solution and carry out hydrolysis reaction 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.

[0010] 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.

[0011] Preferably, the hydrolysis condition in step (2) is a hydrolysis reaction at 40° C. for 2 hours.

[0012] Preferably, in step (3), 2M HCl is used to adjust the pH to 4.0.

[0013] In a specific embodiment of the present invention, the method for preparing the drometrizole hapten comprises the following steps: S1: Rinse a 50 ml round-bottom flask, blow dry it with ethanol, fix it on a stirrer, and add a stir bar; S2: Weigh 225 mg of drometrizole raw material and add it to the container prepared in step S1, add 2.5 ml of N,N-dimethylformamide (DMF) to dissolve, stir, add 48 mg of sodium hydride, and react at room temperature for 30 min; S3: After the reaction is completed, add 172 μl of ethyl 4-bromobutyrate, 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; 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 forced air drying oven at 40°C to obtain the hapten represented by formula I.

[0014] In the above-mentioned preparation process, the inventor replaced the above-mentioned 4-bromobutyric acid ethyl ester with 4-bromobutyric acid methyl ester, 4-bromobutyric acid tert-butyl ester and 4-bromobutyric acid of the same molar mass, and found that the target hapten can be obtained with 4-bromobutyric acid ethyl ester as raw material, and the yield is higher. Although the intermediate product prepared with 4-bromobutyric acid tert-butyl ester as raw material has a higher hydrolysis efficiency in the subsequent hydrolysis step, the yield and purity data show that there is no significant difference between it and the target hapten prepared with 4-bromobutyric acid ethyl ester as raw material, and those skilled in the art know that the price of 4-bromobutyric acid ethyl ester is much lower than 4-bromobutyric acid tert-butyl ester, so the present invention is preferably prepared with 4-bromobutyric acid ethyl ester as raw material.

[0015] 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.

[0016] 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.

[0017] The coupling method is selected from the active ester method (i.e., the biocoupling method) or the mixed anhydride method; the active ester method is preferred.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Those skilled in the art can obtain the antibody by immunizing animals 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.

[0023] 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: (1) Application in the detection of drometrizole; (2) Application in the preparation of products for detecting drometrizole.

[0024] The products include but are not limited to test strips, membrane strips, test kits, chips, models or detection platforms.

[0025] In some embodiments of the present invention, the product is an enzyme-linked immunosorbent assay (ELISA) kit.

[0026] In a sixth aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting drometrizole, the kit comprising an antibody working solution, and the antibody working solution is prepared by diluting the drometrizole antibody described in the fourth aspect of the present invention.

[0027] Furthermore, the kit also includes an ELISA plate coated with the drometrizole coating source described in the third aspect of the present invention and a goat anti-mouse antibody (enzyme-labeled secondary antibody) labeled with horseradish peroxidase (HRP).

[0028] 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.

[0029] 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 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 enzyme-labeled sheep anti-mouse antibody, coloring and terminating with a substrate, and determining the absorbance value with an ELISA instrument; drawing an ELISA standard curve, substituting the absorbance value into the standard curve to obtain the drometrizole concentration in the sample.

[0030] The technical solution provided by the present invention has the following technical advantages: (1) The sample pretreatment steps of the detection method for drometrizole in the prior art are complicated 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 has the advantages of simple and rapid operation, low cost, high sensitivity, and large sample size. It makes up for the shortcomings of instrumental analysis and lays a research and development foundation for the rapid detection of drometrizole residues.

[0031] (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, after the hapten and protein are coupled, the drometrizole hapten can specifically bind to B lymphocytes and produce a good immune response effect.

[0032] (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.

[0033] (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

[0034] Figure 1 This is a diagram showing the mass spectrometry identification result of the drometrizole hapten in Example 1; Figure 2 This is a diagram showing the nuclear magnetic resonance identification result of the drometrizole hapten in Example 1; Figure 3 is the MALDI-TOF-MAS image of BSA in Example 2; Figure 4 is a MALDI-TOF-MAS image of the drometrizole hapten-BSA complex in Example 2; Figure 5 It is the MALDI-TOF-MAS image of the drometrizole hapten-BSA complex in Comparative Example 2; Figure 6 It is the MALDI-TOF-MAS image of the drometrizole-BSA coupling product in Comparative Example 3; Figure 7 This is the ELISA standard curve obtained in Example 3. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1 Preparation and structural identification of drometrizole hapten 1) Preparation of drometrizole hapten The synthetic route is as follows: ; S1: Rinse a 50 ml round-bottom flask, blow dry it with ethanol, fix it on a stirrer, and add a stir bar; S2: Weigh 225 mg of drometrizole raw material and add it to the container prepared in step S1, add 2.5 ml of N,N-dimethylformamide (DMF) to dissolve, stir, add 48 mg of sodium hydride, and react at room temperature for 30 min; S3: After the reaction is completed, add 172 μl of ethyl 4-bromobutyrate and react at 40°C for 4 hours. After evaporation to dryness, 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; 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 drying oven to obtain the hapten represented by formula I. The purity of the product reaches 99% and the yield reaches 90%.

[0037] 2) Structural identification of drometrizole hapten The obtained product was identified by mass spectrometry and nuclear magnetic resonance. Figure 1 As shown in the figure, it can be clearly seen 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 the drometrizole hapten was successful.

[0038] Example 2 Preparation and structural identification of drometrizole artificial antigen 1. Preparation of artificial antigen of drometrizole (active ester method) 1. Synthesis of Immunogen (Dromtrizole Hapten-BSA Conjugate) S1: 17.4 mg of the hapten (Formula I) prepared in Example 1 was dissolved in 1 mL of N,N-dimethylformamide (DMF). After complete dissolution, 10.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 6.2 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred and activated at room temperature for 2-3 hours to obtain Solution I. S2: Weigh 50 mg of bovine serum albumin (BSA), dissolve it in 3.5 mL of 0.1 M carbonate buffer, stir at 200 rpm for 10 min to fully dissolve, and obtain solution II; The pH of the 0.1M 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; S3: Take the above solution I, add it dropwise into the above solution II, stir while adding, and react at room temperature (20-25°C) with magnetic stirring (500 rpm) for 12 hours to obtain solution III; S4: Place the solution III into a dialysis bag (10 cm) rinsed with distilled water, and dialyze against 1L 0.01M PBS (pH=7.4) for 3 days at 4°C with stirring. Change the dialysate 3 times a day. Centrifuge the dialyzed product at 5000 rpm for 6 min, dispense into 1.5 ml / tubes, number the antigens, and store at -20°C for later use.

[0039] 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.

[0040] 2. Synthesis of coating material (Dromtrizole hapten-OVA conjugate) Substitute 33.6 mg OVA for 50 mg BSA in step 1 to obtain the original coating solution.

[0041] 2. Identification of artificial antigen of drometrizole The identification method is as follows: the conjugate of drometrizole hapten and carrier protein BSA is detected by MALDI-TOF-MS, and the change of molecular mass data can indicate whether the conjugation of hapten and protein is successful or not, and the conjugation ratio of protein to hapten can be calculated. The conjugate of drometrizole hapten and carrier protein BSA is desalted and mixed with matrix-assisted solution, spotted on the MALDI target, and placed in a mass spectrometer for detection. The conjugation ratio of drometrizole hapten and 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 hapten, protein and conjugate, respectively.

[0042] 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) coupled to bovine serum albumin (BSA) is 24.418:1.

[0043] Example 3 Preparation and detection of drometrizole antibodies 1. Immunization of Balb / c mice 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.

[0044] 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 5 mice subcutaneously at multiple points on the back of the neck. The antigen dose is 100 μg / mouse, 0.2 ml per mouse. The second immunization was performed 14 days later. The immunogen was emulsified with an equal volume of Freund's incomplete adjuvant. The immunization dose was the same as the first immunization dose, and the number of booster immunizations was 3 times.

[0045] 2. Detection of mouse antiserum against drometrizole One week after the three immunizations, 20 μL of blood was collected from the tail of the mice and centrifuged to collect serum. The titer was measured by the classic checkerboard method and the sensitivity was measured by the indirect competitive ELISA method.

[0046] 1. Determination of antiserum titer, the steps are as follows: (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 wells, wash the plate three times with PBST buffer (pH 7.2), and spin dry the washing solution; (2) Blocking: Add 150 μL of blocking solution (2% skim milk) to each well and block at 37°C for 1 h. Shake off the liquid in the wells, wash the plate three times with PBST buffer (pH 7.2), and pat dry the washing solution. (3) Add antiserum: add 50 μL 0.01 M PBS (pH 7.4) to each well, then add 50 μL diluted drometrizole antiserum, starting from 1:4000, the antibody is diluted with 0.01 M PBS in a gradient of 2, a total of 4 dilution gradients, the sample volume is 50 μL per well, incubate at 37°C for 30 min, wash 3 times with PBST buffer (pH 7.2), pat dry, incubate for 30 min, wash 3 times with PBST buffer (pH 7.2), pat dry; At the same time, unimmunized Balb / c mouse serum was set as a negative control; (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; (5) Color development: Mix the horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine solution and 30% hydrogen peroxide in 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; (6) Reading determination: Read the absorbance (OD) using a microplate reader at a wavelength of 450 nm. The titer of the antiserum is 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.

[0047] The results showed that the antibody titer in the serum was 1:12000.

[0048] 2. Determination of antiserum sensitivity, the steps are as follows: (1) The coating and blocking process is the same as the coating and blocking process in “1. Determination of antiserum titer” above; (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; The solvent of the standard solution was PBS buffer, and the concentrations of the standard solution 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; (3) Add enzyme-labeled secondary antibody: Add 100 μL of enzyme-labeled secondary antibody dilution to each well, incubate at 37°C for 30 min, then wash three times with PBST solution and pat dry; (4) Color development: Add 100 μL of color development solution to each well and incubate at 37°C for 15 min. (5) Termination: Add 50 μL of 2 mol / L concentrated sulfuric acid to each well; (6) Reading: Measure the OD value of each well at a wavelength of OD450nm.

[0049] 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 the IC50 value.

[0050] The results showed that the IC50 of mouse polyclonal antibody serum with the best immune effect was 0.216 ng / mL.

[0051] 3. Determination of minimum detection limit The method for detecting drometrizole provided by the present invention has a minimum detection limit (LOD) of 0.037 ng / mL.

[0052] Comparative Example 1 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.

[0053] The purity of the obtained product was 50% and the yield was 50%.

[0054] Comparative Example 2 The difference between this comparative example and Example 2 is that the mixed anhydride method is used to prepare the drometrizole whole antigen, and the specific method is as follows: 1. Synthesis of Immunogen (Dromtrizole Hapten-BSA Conjugation) (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 to (0-4°C) in an ice bath, add 32 μL of tri-n-butylamine, stir for 15 min, add 6 μL of isobutyl chloroformate dropwise (0-4°C), and activate by stirring for 30 min to obtain Solution I. (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 until fully dissolved to obtain Solution II. (3) Add the above solution I dropwise into the above solution II, stirring while adding, and react at room temperature (20-25°C) with magnetic stirring (500 rpm) for 12 h to obtain solution III; (4) Place the solution III into a dialysis bag (10 cm) rinsed with distilled water, and dialyze against 1L 0.01M PBS (pH = 7.4) for 3 days at 4°C with stirring. Change the dialysate 3 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. 2. Synthesis of coating material (Dromtrizole hapten-OVA coupling) Prepare the coating stock solution by replacing 50 mg BSA in step 1 with 33.6 mg OVA.

[0055] The immunogen prepared by the mixed anhydride method was identified using the method provided in Example 2. The MALDI-TOF-MS identification result of the immunogen is as follows: Figure 5 , the calculated coupling ratio is: R=24.086. That is, in the immunogen, the molar ratio of the drometrizole hapten to bovine serum albumin (BSA) is 24.086:1. By comparing the coupling ratio data of BSA coupling by the active ester method, it can be found that the coupling ratio of drometrizole hapten and BSA coupling by the active ester method is higher.

[0056] 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, and 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. By comparing the antibody titer and IC50 data obtained by 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 the active ester method has better immunogenicity.

[0057] Comparative Example 3 Using drometrizole as a hapten, it was coupled with BSA and OVA to obtain an immunogen and a coating agent. The structure of drometrizole is shown in Formula II: (Formula II) 1. Synthesis of Immunogen (Dromtrizole-BSA Conjugate) (1) Dissolve 10.07 mg of drometrizole (Formula II) in 1 mL of N,N-dimethylformamide (DMF). After complete dissolution, cool to (0-4°C) in an ice bath, add 18.5 mg of N,N'-carbonyldiimidazole, stir for 30 minutes, return to room temperature (20-25°C), and stir and activate for 2 hours to obtain Solution I; (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 until fully dissolved to obtain Solution II. (3) Add the above solution I dropwise into the above solution II, stirring while adding, and react at room temperature (20-25°C) with magnetic stirring (500 rpm) for 12 h to obtain solution III; (4) Place the solution III into a dialysis bag (10 cm) rinsed with distilled water and dialyzed against 1 L 0.01 M PBS (pH = 7.4) for 3 days at 4 °C with stirring. Change the dialysate 3 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.

[0058] 2. Synthesis of coating material (Dromtrizole-OVA coupling) Prepare the coating stock solution by replacing 50 mg BSA in step 1 with 33.6 mg OVA.

[0059] The prepared immunogen was identified using the method provided in Example 2. The immunogen MALDI-TOF-MS identification result is as follows: Figure 6 The coupling ratio is calculated to be: R=14.040. That is, in the immunogen, the molar ratio of the coupling of drometrizole to bovine serum albumin (BSA) is 14.040:1.

[0060] Referring to the method provided in Example 3, the immunogen prepared in Comparative Example 3 was used to immunize Balb / c mice to obtain antibodies. The antibodies were subjected to antiserum titer and sensitivity tests, and the results showed that the antibody titer in the serum was 1:3000, and the IC50 of the mouse polyantibody serum with the best immune effect was 53 ng / mL.

[0061] The above results show that, in the case of no modification, the coupling efficiency of BSA is extremely low when drometrizole is directly used as a hapten. However, the hapten shown in Formula I obtained by modifying drometrizole with ethyl 4-bromobutyrate and then hydrolyzing it is very beneficial for coupling with BSA, and the coupling efficiency is significantly improved. In addition, the whole antigen obtained by directly coupling drometrizole to the carrier protein through its own phenolic hydroxyl group has poor immunogenicity and fails to fully expose the specific binding site, resulting in low titer of the obtained antibody.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hapten of drometrizole, characterized in that: The drometrizole hapten has a structure shown in Formula I, or a pharmaceutically acceptable salt thereof: （Ⅰ）。 2. The method for preparing the drometrizole hapten according to claim 1, characterized in that: The preparation method comprises the following steps: (1) mixing drometrizole and ethyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, reacting at 30-50° C. for 3-5 hours to obtain an intermediate product; (2) Dissolve the intermediate product in methanol, add 2M NaOH solution and carry out hydrolysis reaction 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 drometrizole whole antigen, characterized in that: The whole 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.

4. The whole drometrizole antigen according to claim 3, characterized in that: The carrier protein is selected from bovine serum albumin, and the drometrizole hapten is coupled with the bovine serum albumin to obtain the drometrizole immunogen; the carrier protein is selected from ovalbumin, and the drometrizole hapten is coupled with the ovalbumin to obtain the drometrizole coating source.

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

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

7. An enzyme-linked immunosorbent assay kit for detecting drometrizole, the kit comprising an antibody working solution, wherein the antibody working solution is prepared by diluting the drometrizole antibody according to claim 5.

8. The kit according to claim 7, characterized in that The kit also includes an ELISA plate coated with the drometrizole coating source as claimed in claim 4 and a goat anti-mouse antibody labeled with horseradish peroxidase.

9. The kit according to claim 7, characterized in that The kit also includes a drometrizole standard working solution.

10. A method for detecting drometrizole, characterized in that: The method comprises: using an indirect competitive ELISA method, using the drometrizole coating source as claimed in claim 4 to coat an ELISA plate, adding a sample solution to be tested, and simultaneously adding the drometrizole antibody as claimed in claim 5, incubating, washing and patting dry, adding an enzyme-labeled goat anti-mouse antibody, developing color through a substrate, terminating, and measuring the absorbance value with an ELISA reader; drawing an ELISA standard curve, substituting the absorbance value into the standard curve to obtain the drometrizole concentration in the sample.

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