N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten and preparation method and application thereof

Abstract

The present invention discloses a N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten and a preparation method and application thereof, and belongs to the technical field of environmental pollutant detection. The present invention first provides a N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten, and obtains an artificial antigen by coupling a carrier protein, obtains a 6PPD-quinone antibody from an immunized animal, and uses the antibody to prepare an enzyme-linked immunosorbent assay kit to detect and analyze 6PPD-quinone in a sample. The kit provided by the present invention has strong specificity, high sensitivity, simple operation, and fast analysis speed, which makes up for the shortcomings of precision instrument analysis and realizes rapid analysis of trace 6PPD-quinone residues in complex environmental media.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental pollutant detection, and specifically relates to an N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten and a preparation method and application thereof. Background Art

[0002] P-phenylenediamine quinone compounds (PPDQs) are derivatives of p-phenylenediamine antioxidants (PPD) and have a certain distribution and potential toxic effects in the environment. This type of compound is mainly released from rubber products containing p-phenylenediamine antioxidants, such as tires. Due to wear and aging during use, these antioxidant components will penetrate into the environment, especially in areas with busy traffic roads. Studies have shown that p-phenylenediamine quinones are ubiquitous in urban water environments, air, and soil. In addition, p-phenylenediamine quinone compounds have also been detected in human urine, indicating that long-term exposure to this type of pollutant may pose a great risk to human health, and its widespread existence and potential hazards have attracted people's attention. Therefore, strengthening the monitoring and detection of PPDQs in the environment is a necessary means to control this type of pollution.

[0003] 6PPD-quinone (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone) is a common phenylenediamine quinone compound. The precursor of 6PPD-quinone is an antioxidant 6PDD (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) widely used in rubber tires. It is oxidized and converted into 6PPD-quinone in an ozone environment. 6PPD-quinone is widely present in environmental media such as surface water, dust, and atmospheric particulate matter. Exposure to 6PPD-quinone can cause acute death of silver salmon in urban streams, and can also cause death of farmed fish such as brook trout and rainbow trout. Sublethal concentrations can also affect the behavior of zebrafish embryos and can accumulate in the liver, kidneys, and lungs of mice. Therefore, it is of great significance to establish a method or detection product that can quickly, sensitively, and efficiently detect 6PPD-quinone.

[0004] At present, the main analytical detection methods for 6PPD-quinone are high-performance liquid chromatography or high-performance liquid chromatography-mass spectrometry. Although these methods have high accuracy, they have the disadvantages of complex sample pretreatment procedures, long analysis cycles, large solvent consumption, and high costs. It is difficult to achieve rapid and efficient analysis and is not suitable for screening and detection of large quantities of samples. For example, patent document CN 118777481 A discloses a method for detecting rubber additives and oxidation products in surface water by liquid chromatography-tandem mass spectrometry. The method specifically includes adding salt in an appropriate amount to the water sample, enriching the object to be detected by solid phase extraction technology, and then using HPLC-MS / MS analysis to quantitatively detect 17 rubber additives and oxidation products including 6PPD-quinone in surface water. The quantitative limit of this method for 6PPD-quinone is 0.1ng / L, and the detection limit is 0.32ng / L. For example, patent document CN 118549510 A discloses a rapid analysis method for p-phenylenediamine quinone compounds, which uses nanospray laser induced plasma mass spectrometry to detect and analyze trace p-phenylenediamine quinone pollutants, achieving a detection limit of 0.005 ng / mL for 6PPD-quinone. Although the method is highly sensitive, it requires precision instruments and professional analysts to operate, is costly, and cannot be used for rapid on-site detection.

[0005] In order to make up for the defects of the prior art, the present invention provides a rapid and efficient 6PPD-quinone detection method. The method is based on the unique advantages of antigen-antibody in qualitative and quantitative analysis in immunoassay methods, and can quickly and sensitively identify trace 6PPD-quinone in environmental samples, which has important scientific significance and application value for the detection of 6PPD-quinone residues. Summary of the invention

[0006] The present invention overcomes the shortcomings of the prior art and provides a rapid analysis method for 6PPD-quinone. The method detects and analyzes 6PPD-quinone in a sample based on enzyme-linked immunosorbent assay technology. The method has strong specificity, high sensitivity, simple operation, and fast analysis speed, which makes up for the shortcomings of precision instrument analysis and realizes rapid and sensitive analysis of trace 6PPD-quinone residues in complex environmental media.

[0007] The present invention includes the following technical solutions:

[0008] In the first aspect of the present invention, the present invention provides an N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten, characterized in that the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten has a structure shown in Formula I, or a pharmaceutically acceptable salt thereof:

[0009] (I).

[0010] The N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten provided by the present invention is obtained by introducing a short spacer arm modification on the basis of the 6PPD-quinone structure. The hapten itself contains a carboxyl end and can be directly coupled with a carrier through a cross-linking agent, and can be used as a hapten without further structural changes. At the same time, the introduced short spacer arm makes it easier to expose the specific binding site after connecting it with the carrier protein, thereby improving the immunogenicity of the antigen.

[0011] In a second aspect of the present invention, the present invention provides a method for preparing the above-mentioned N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten, characterized in that the method comprises the following steps:

[0012] The synthetic route is as follows:

[0013] ;

[0014] (1) mixing 6PPD-quinone and tert-butyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, reacting at 70-90° C. for 10-14 hours, separating and purifying to obtain an intermediate product;

[0015] (2) The intermediate product is dissolved in dichloromethane, trifluoroacetic acid is added, and the mixture is stirred at room temperature for 2-4 hours, and then separated and purified to obtain octyl salicylate hapten.

[0016] Preferably, in step (1), octyl salicylate and tert-butyl 4-bromobutyrate are mixed in a molar ratio of 1.2:1 and reacted at 80° C. for 12 hours.

[0017] The inventors replaced the tert-butyl 4-bromobutyrate in the above step (1) with methyl 4-bromobutyrate, ethyl 4-bromobutyrate and 4-bromobutyric acid of the same molar mass, and found that the target hapten can be obtained in high yield only by using tert-butyl 4-bromobutyrate as the raw material. This is because the intermediate product obtained by reacting tert-butyl 4-bromobutyrate with 6PPD-quinone has a higher hydrolysis efficiency in the subsequent hydrolysis reaction due to the presence of a tert-butyl ester group, and the by-products formed in the hydrolysis reaction are less, so the yield of the target hapten is higher.

[0018] The separation and purification operation in step (1) is as follows: evaporate to dryness after the reaction is completed, add dichloromethane to dissolve the residue, add silica gel for adsorption, spin dry for column chromatography, elute with a mixture of petroleum ether and ethyl acetate, collect the eluate, and concentrate under reduced pressure to obtain an intermediate product. The volume ratio of petroleum ether to ethyl acetate is (5-15):1, preferably 10:1.

[0019] The separation and purification operation in step (2) is as follows: after the reaction is completed, the mixture is concentrated under reduced pressure, dichloromethane is added to dissolve the residue, silica gel is added for adsorption, the mixture is spin-dried for column chromatography, and a mixture of petroleum ether and ethyl acetate is used for elution, the eluate is collected, and the mixture is concentrated under reduced pressure to obtain 6PPD-quinone hapten. The volume ratio of petroleum ether to ethyl acetate is (3-7):1, preferably 5:1.

[0020] In a specific embodiment of the present invention, the method for preparing the 6PPD-quinone hapten comprises the following steps:

[0021] S1. Rinse the 10 ml round-bottom flask, blow dry it with ethanol, fix it on the stirrer, and add a stir bar;

[0022] S2, weigh 100 mg of 6PPD-quinone raw material and add it to the container prepared in step S1, add 5 ml of N,N-dimethylformamide (DMF) to dissolve, stir, add 92.6 mg of anhydrous potassium carbonate, and react at room temperature for 30 min;

[0023] S3. After the reaction is completed, add 74.5 μl of tert-butyl 4-bromobutyrate and react at 80° C. for 12 hours;

[0024] S4, weigh after evaporation, add 5 mL of dichloromethane to dissolve the residue, add 2 times the weight of silica gel to mix the sample, evaporate the sample to dryness, perform column chromatography, elute with a mixture of petroleum ether: ethyl acetate = 10:1, collect the main product point, and concentrate under reduced pressure to obtain a red solid intermediate product;

[0025] S5. Add 5 ml of dichloromethane to the red solid intermediate product to dissolve it, then add 1 ml of trifluoroacetic acid, stir at room temperature to react for 3 hours, concentrate under reduced pressure and weigh, add 5 mL of dichloromethane to dissolve the residue, add 2 times the weight of silica gel to mix the sample, evaporate the sample to dryness and perform column chromatography, elute with petroleum ether: ethyl acetate = 5:1, collect the main product spots, and concentrate under reduced pressure to obtain the hapten shown in formula I.

[0026] 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 6PPD-quinone hapten and full antigen compounds can be synthesized, they should all fall within the protection scope of the present invention.

[0027] In a third aspect, the present invention provides a 6PPD-quinone whole antigen, characterized in that the whole antigen is obtained by coupling the 6PPD-quinone hapten described in the first aspect of the present invention with a carrier protein.

[0028] 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.

[0029] 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.

[0030] In a specific embodiment of the present invention, the carrier protein is selected from bovine serum albumin, and the 6PPD-quinone hapten is coupled with the bovine serum albumin to obtain the 6PPD-quinone immunogen.

[0031] In a specific embodiment of the present invention, the carrier protein is selected from ovalbumin, and the 6PPD-quinone hapten is coupled with the ovalbumin to obtain the 6PPD-quinone coating.

[0032] In a fourth aspect, the present invention provides a 6PPD-quinone antibody, characterized in that the antibody is obtained by immunizing an animal with the 6PPD-quinone immunogen described in the third aspect of the present invention.

[0033] Those skilled in the art can obtain the antibody by immunizing animals with the 6PPD-quinone 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.

[0034] In a fifth aspect, the present invention provides a use of the 6PPD-quinone hapten described in the first aspect of the present invention, the 6PPD-quinone full antigen described in the third aspect of the present invention, and the 6PPD-quinone antibody described in the fourth aspect of the present invention in at least one of the following:

[0035] (1) Application in the detection of 6PPD-quinone;

[0036] (2) Application in the preparation of products for detecting 6PPD-quinone.

[0037] The products include but are not limited to test strips, membrane strips, test kits, chips, models or detection platforms.

[0038] In some embodiments of the present invention, the product is an enzyme-linked immunosorbent assay (ELISA) kit.

[0039] In a sixth aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting 6PPD-quinone, the kit comprising an antibody working solution, and the antibody working solution is prepared by diluting the 6PPD-quinone antibody described in the fourth aspect of the present invention.

[0040] Furthermore, the kit also includes an ELISA plate coated with the 6PPD-quinone 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).

[0041] Furthermore, the kit also includes a 6PPD-quinone standard working solution, which is a PBS solution with 6PPD-quinone 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.

[0042] In the seventh aspect, the present invention provides a method for detecting 6PPD-quinone, characterized in that the method comprises: utilizing an indirect competitive ELISA method, using the 6PPD-quinone coated original ELISA plate described in the third aspect of the present invention, adding a sample solution to be tested, and simultaneously adding the 6PPD-quinone 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 6PPD-quinone concentration in the sample.

[0043] The technical solution provided by the present invention has the following technical advantages:

[0044] (1) Compared with the haptens in the prior art, the 6PPD-quinone hapten provided by the present invention has a carboxyl end in its structure and can be directly coupled to a carrier through a cross-linking agent. After being connected to a 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 6PPD-quinone hapten can specifically bind to B lymphocytes and produce a good immune response effect.

[0045] (2) The hapten synthesis method of the present invention is simple, and the purity of the prepared product reaches 99% and the yield is as high as 94%. It can be directly coupled with protein to immunize animals to produce specific antibodies against 6PPD-quinone.

[0046] (3) The antibodies provided by the present invention can be used to detect 6PPD-quinone, especially the polyclonal antibodies have high specific recognition performance for 6PPD-quinone; the antiserum titer is 1:9000, and the best mouse polyclonal antiserum IC50 is 0.209 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a diagram showing the mass spectrometry detection result of the 6PPD-quinone hapten in Example 1;

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

[0049] Figure 3is the MALDI-TOF-MAS image of the 6PPD-quinone hapten-BSA complex in Example 2;

[0050] Figure 4 is the MALDI-TOF-MAS image of the 6PPD-quinone hapten-BSA complex in Comparative Example 2;

[0051] Figure 5 It is the MALDI-TOF-MAS image of the 6PPD-quinone-BSA coupling product in Comparative Example 3;

[0052] Figure 6 This is the ELISA standard curve obtained in Example 3. DETAILED DESCRIPTION

[0053] 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.

[0054] Example 1 Preparation and structural identification of 6PPD-quinone hapten

[0055] 1) Preparation of 6PPD-quinone hapten

[0056] The synthetic route is as follows:

[0057] ;

[0058] S1. Rinse the 10 ml round-bottom flask, blow dry it with ethanol, fix it on the stirrer, and add a stir bar;

[0059] S2, weigh 100 mg of 6PPD-quinone raw material and add it to the container prepared in step S1, add 5 ml of N,N-dimethylformamide (DMF) to dissolve, stir, add 92.6 mg of anhydrous potassium carbonate, and react at room temperature for 30 min;

[0060] S3. After the reaction is completed, add 74.5 μl of tert-butyl 4-bromobutyrate and react at 80° C. for 12 hours;

[0061] S4, weigh after evaporation, add 5 mL of dichloromethane to dissolve the residue, add 2 times the weight of silica gel to mix the sample, evaporate the sample to dryness, perform column chromatography, elute with a mixture of petroleum ether: ethyl acetate = 10:1, collect the main product point, and concentrate under reduced pressure to obtain a red solid intermediate product;

[0062] S5. Add 5 ml of dichloromethane to the red solid intermediate product to dissolve it, then add 1 ml of trifluoroacetic acid, stir at room temperature for 3 hours, weigh after vacuum concentration, add 5 ml of dichloromethane to dissolve the residue, add 2 times the weight of silica gel to mix the sample, rotary evaporate the sample to perform column chromatography, elute with petroleum ether: ethyl acetate = 5:1, collect the main product spot, and vacuum concentrate to obtain the hapten shown in formula I. The purity of the product reaches 99% and the yield reaches 94%.

[0063] 2) Structural identification of 6PPD-quinone hapten

[0064] The obtained product was identified by mass spectrometry, and the identification results were as follows: Figure 1 As shown, the peak of M / Z 385 (M+H+) can be clearly seen, which is consistent with the target molecular weight (384.48), indicating that 6PPD-quinone hapten was successfully synthesized.

[0065] Example 2 Preparation and structural identification of 6PPD-quinone artificial antigen

[0066] 1. Preparation of 6PPD-quinone artificial antigen (active ester method)

[0067] 1. Synthesis of immunogen (6PPD-quinone hapten-BSA coupling)

[0068] (1) Dissolve 17.2 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), and activate by stirring at room temperature for 2-3 hours to obtain Solution I.

[0069] (2) 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 until fully dissolved to obtain Solution II.

[0070] 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;

[0071] (3) Take the above solution I and add it dropwise into the above solution II while stirring. React at room temperature (20-25°C) with magnetic stirring (500 rpm) for 12 hours to obtain solution III.

[0072] (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.

[0073] 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.

[0074] 2. Synthesis of coating source (6PPD-quinone hapten-OVA coupling)

[0075] Replace 50 mg BSA in step 1 with 33.6 mg OVA to obtain the coating stock solution.

[0076] 2. Identification of 6PPD-quinone artificial antigen

[0077] The identification method is as follows: the conjugate of 6PPD-quinone hapten and carrier protein BSA is detected by MALDI-TOF-MS. 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 6PPD-quinone hapten and carrier protein BSA is desalted and mixed with matrix-assisted solution, spotted on the MALDI target, and placed in the mass spectrometer for detection. The binding ratio of 6PPD-quinone hapten and carrier protein BSA is calculated according to the following formula: coupling ratio = (MC-MB) / MA, where MA, MB and MC represent the relative molecular masses of hapten, protein and conjugate, respectively.

[0078] The immunogen MALDI-TOF-MS identification results showed that the coupling ratio was: R = (74537.55-66302.94) / 384.48 = 21.418 ( Figure 2 and Figure 3 ). That is, in the immunogen, the molar ratio of the coupling of the 6PPD-quinone hapten (Formula I) and bovine serum albumin (BSA) is 21.418:1

[0079] Example 3 Preparation and detection of 6PPD-quinone antibody

[0080] 1. Immunization of Balb / c mice

[0081] The protein concentration of 6PPD-quinone 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 6PPD-quinone hapten-BSA.

[0082] For the first immunization of 6-week-old female Balb / c mice, the 6PPD-quinone hapten-BSA immunogen was diluted to 1 mg / mL (diluted with 0.01 mol / L PBS), mixed with an equal volume of Freund's complete adjuvant, and fully emulsified. Five mice were inoculated subcutaneously at the back of the neck at multiple points. The antigen dose was 100 μg / mouse, 0.2 ml per mouse. The second immunization was performed 14 days later, and 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.

[0083] 2. Detection of 6PPD-quinone mouse antiserum

[0084] 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.

[0085] 1. Determination of antiserum titer, the steps are as follows:

[0086] (1) Coating: Dilute the coating antigen (6PPD-quinone 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 h, pour off the liquid in the wells, wash the plate three times with PBST buffer (pH 7.2), and spin dry the washing solution;

[0087] (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.

[0088] (3) Add antiserum: add 50 μL 0.01 M PBS (pH 7.4) to each column well, then add 50 μL diluted 6PPD-quinone antiserum, dilute the antibody starting from 1:4000 with 0.01 M PBS in a gradient of 2, for a total of 4 dilutions, add 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;

[0089] At the same time, unimmunized Balb / c mouse serum was set as a negative control;

[0090] (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;

[0091] (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;

[0092] (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.

[0093] The results showed that the antibody titer in the serum was 1:9000.

[0094] 2. Determination of antiserum sensitivity, the steps are as follows:

[0095] (1) The coating and blocking process is the same as the coating and blocking process in “1. Determination of antiserum titer” above;

[0096] (2) Adding standards and antibodies: Add 50 μL of 6PPD-quinone standard solution and 50 μL of mouse serum diluted solution after three immunizations for one week 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 is PBS buffer, and the concentrations of the standards are 0, 0.003, 0.01, 0.03, 0.09, 0.27, 0.81 and 2.43 ng / mL, respectively, with three replicates for each concentration;

[0097] (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;

[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] With -log10 (competitor) value as the horizontal axis and OD450nm value as the vertical axis, the four-parameter equation of Origin 8.0 was used for fitting to establish a standard curve to obtain the IC50 value, such as Figure 6 shown.

[0102] The results showed that the IC50 of mouse polyclonal antibody serum with the best immune effect was 0.209 ng / mL.

[0103] 3. Determination of minimum detection limit

[0104] The detection method for 6PPD-quinone provided by the present invention has a minimum detection limit (LOD) of 0.058 ng / mL.

[0105] Comparative Example 1

[0106] The difference between this comparative example and Example 1 is that the reaction temperature of 6PPD-quinone and tert-butyl 4-bromobutyrate is changed to room temperature. The purity of the obtained product is 10% and the yield is 15%.

[0107] Comparative Example 2

[0108] The difference between this comparative example and Example 2 is that the 6PPD-quinone complete antigen is prepared by a mixed anhydride method, and the specific method is as follows:

[0109] 1. Synthesis of Immunogen (6PPD-quinone Hapten-BSA Conjugation)

[0110] (1) Dissolve 17.2 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, and dropwise add 6 μL of isobutyl chloroformate (0-4°C) and stir for 30 min to obtain Solution I.

[0111] (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.

[0112] (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;

[0113] (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.

[0114] 2. Synthesis of Coating Material (6PPD-Quinone Hapten-OVA Coupling)

[0115] Prepare the coating stock solution by replacing 50 mg BSA in step 1 with 33.6 mg OVA.

[0116] 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 4 , the coupling ratio is calculated to be: R=18.192. That is, in the immunogen, the molar ratio of the coupling of the 6PPD-quinone hapten to bovine serum albumin (BSA) is 18.192:1. The coupling ratio of the immunogen prepared by the active ester method is 21.418. In comparison, the coupling ratio of the coupling of 6PPD-quinone hapten to BSA by the active ester method is higher.

[0117] Referring to the method provided in Example 3, the immunogen prepared in Comparative Example 2 was used to immunize Balb / c mice to obtain polyclonal 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 3.603 ng / mL. The antibody titer obtained by immunizing mice with the immunogen prepared by the active ester method was 1:9000, and the IC50 of the mouse polyantibody serum was 0.209 ng / mL. Therefore, by comparing the antibody titer and IC50 data obtained by the immunogen obtained by the ester coupling 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 coupling method has better immunogenicity.

[0118] Comparative Example 3

[0119] 6PPD-quinone was used as a hapten and coupled with BSA and OVA to obtain an immunogen and a coating agent. The structure of the 6PPD-quinone is shown in Formula II:

[0120] (Formula II)

[0121] 1. Synthesis of Immunogen (6PPD-quinone-BSA Conjugation)

[0122] (1) Dissolve 13.3 mg of 6PPD-quinone (Formula II) in 1 mL of N,N-dimethylformamide (DMF). After complete dissolution, cool to (0-4°C) in an ice bath and add 18.2 mg of N,N'-carbonyldiimidazole. Stir for 30 min, then return to room temperature (20-25°C) and stir for 2 hours to activate the mixture, to obtain Solution I.

[0123] (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.

[0124] (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;

[0125] (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.

[0126] 2. Synthesis of Coating Agent (6PPD-quinone-OVA Coupling)

[0127] Prepare the coating stock solution by replacing 50 mg BSA in step 1 with 33.6 mg OVA.

[0128] The prepared immunogen was identified using the method provided in Example 2. The immunogen MALDI-TOF-MS identification result is as follows: Figure 5 The coupling ratio is calculated to be: R=11.901. That is, in the immunogen, the molar ratio of the coupling of 6PPD-quinone and bovine serum albumin (BSA) is 11.901:1.

[0129] The immunogen prepared in Comparative Example 3 was used to immunize Balb / c mice to obtain antibodies. The antibody was tested for antiserum titer and sensitivity by the method provided in Reference Example 3. 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 80 ng / mL.

[0130] The experimental results of comparative example 3 show that the immunogen coupling efficiency obtained by directly coupling BSA with 6PPD-quinone as a hapten is extremely low, while the present application uses tert-butyl 4-bromobutyrate to modify and hydrolyze 6PPD-quinone to obtain the hapten shown in formula I, which is very beneficial for coupling BSA and significantly improves the coupling efficiency. In addition, the whole antigen obtained by directly coupling 6PPD-quinone with a carrier protein through its own amino group has poor immunogenicity, low antibody titer, and low antibody activity. The present invention introduces a short spacer arm based on the 6PPD-quinone structure to fully expose the specific binding site, so that the prepared antigen has better immunogenicity, and the antibody obtained through the antigen has a high titer and good activity.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten, characterized in that: It has the structure shown in formula (I): （Ⅰ）。 2. The method for preparing the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten according to claim 1, characterized in that: The steps include: (1) mixing 6PPD-quinone and tert-butyl 4-bromobutyrate in a molar ratio of (0.8-1.6):1, reacting at 70-90° C. for 10-14 hours, separating and purifying to obtain an intermediate product; (2) The intermediate product is dissolved in dichloromethane, trifluoroacetic acid is added, and the reaction is stirred at room temperature for 2-4 hours. The product is separated and purified to obtain N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten.

3. The preparation method according to claim 2, characterized in that: In the step (1), 6PPD-quinone and tert-butyl 4-bromobutyrate are mixed in a molar ratio of 1.2:1 and reacted at 80° C. for 12 hours.

4. A N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone complete antigen, characterized in that: The whole antigen is obtained by coupling the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten described in 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 6PPD-quinone hapten is coupled with bovine serum albumin to obtain 6PPD-quinone immunogen; the carrier protein is selected from ovalbumin, and 6PPD-quinone hapten is coupled with ovalbumin to obtain 6PPD-quinone coating.

5. An N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone antibody, characterized in that: The antibody is obtained by immunizing an animal with the 6PPD-quinone immunogen according to claim 4.

6. Use of the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone hapten according to claim 1, or the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone full antigen according to any one of claim 4, or the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone antibody according to claim 5 in at least one of the following: (1) Application in the detection of 6PPD-quinone, which does not include a method for diagnosing a disease; (2) Application in the preparation of products for detecting 6PPD-quinone; The product is selected from a test strip, a membrane strip, a test kit, a chip, a model or a detection platform.

7. An enzyme-linked immunosorbent assay kit, characterized in that: The kit is used for detecting 6PPD-quinone, and comprises an antibody working solution, wherein the antibody working solution is prepared by diluting the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone antibody according to claim 5.

8. The kit according to claim 7, characterized in that The kit comprises an ELISA plate obtained by coating the 6PPD-quinone coating source as claimed in claim 4, a goat anti-mouse antibody labeled with horseradish peroxidase and a 6PPD-quinone standard working solution.

9. A method for detecting 6PPD-quinone, characterized in that: The method comprises: using the indirect competitive ELISA method, using the 6PPD-quinone coated original coated ELISA plate as claimed in claim 4, adding the sample solution to be tested, and simultaneously adding the N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone antibody as claimed in claim 5, incubating, washing and patting dry, adding enzyme-labeled sheep anti-mouse antibody, coloring and terminating with the substrate, and measuring the absorbance value with an ELISA instrument; drawing an ELISA standard curve, substituting the absorbance value into the standard curve to obtain the 6PPD-quinone concentration in the sample; the detection method does not include a method for diagnosing a disease.

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