Enzyme linked immunosorbent assay kit for detecting diisononyl phthalate and application of enzyme linked immunosorbent assay kit

Abstract

The invention relates to the technical field of biochemical detection, in particular to an enzyme linked immunosorbent assay kit for detecting diisononyl phthalate and application of the enzyme linked immunosorbent assay kit. The invention relates to an enzyme linked immunosorbent assay kit for detecting diisononyl phthalate. The enzyme linked immunosorbent assay kit comprises the following components: an elisa plate coated with a coating antigen, a diisononyl phthalate specific antibody working solution, an enzyme marker working solution, a diisononyl phthalate standard substance solution, a substrate developing solution, a stopping solution, a washing solution, a sample diluting solution and a purifying agent. According to the invention, the content of diisononyl phthalate in an edible oil sample is qualitatively or quantitatively detected by mainly adopting an indirect competitive ELISA method; the sample pretreatment requirement is low, the sample pretreatment process is simple, and a large batch of edible oil samples can be rapidly detected at the same time; the main reagent is provided in the form of a working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, and particularly relates to an enzyme-linked immunosorbent assay kit for detecting diisononyl phthalate and its application. Background Art

[0002] Diisononyl phthalate (DINP) belongs to phthalate esters and is a plasticizer (also known as a plasticizer) commonly used in industrial production at present. It widely exists in food packaging, cosmetics, medical devices, and environmental water bodies. Such plasticizers are not food or food additives, but can enter food through environmental migration, food packaging migration, and illegal addition, etc., and have a toxic effect on human health. For example, it can cause endocrine disorders, affect normal fertility, and long-term accumulation in the body can lead to deformities, canceration, and mutations, and also cause great harm to the cardiovascular, digestive, and urinary systems. Therefore, relevant documents stipulate that it is strictly prohibited to artificially add phthalate esters to food and food additives. The maximum residue limits of diisononyl phthalate (DINP), bis(2-ethylhexyl) phthalate (DEHP), and dibutyl phthalate (DBP) in food and food additives are 9.0 mg / kg, 1.5 mg / kg, and 0.3 mg / kg respectively.

[0003] Currently, the detection methods for phthalate esters reported in the literature include gas chromatography, ultra-high performance liquid chromatography, high performance liquid chromatography-mass spectrometry, gas chromatography-ion trap mass spectrometry, etc. For the detection of diisononyl phthalate in food, "GB 5009.271-2016 Determination of Phthalate Esters in Foods" is used, and for the detection of phthalate esters in food packaging, "GB 31604.30-2016 Determination and Migration Amount Determination of Phthalate Esters in Food Contact Materials and Articles" is used. Both detection methods use gas chromatography-mass spectrometry (GC-MS) for determination. This method is sensitive, accurate, has strong specificity and good resolution, and can simultaneously determine multiple drugs. However, it requires expensive instruments, the sample pretreatment is complex, time-consuming and laborious, the detection cost is relatively high, it cannot be operated on-site, and it requires professional personnel to operate, so its application is limited.

[0004] Therefore, how to provide a detection method for diisononyl phthalate with simple operation, low cost, and fast detection speed is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] The object of the present invention is to provide an enzyme-linked immunosorbent assay kit for detecting diisononyl phthalate and its application. The present invention provides a diisononyl phthalate (DINP) detection kit with simple operation, low cost, fast detection speed, no need for large-scale instrument equipment, and low detection limit, so as to better meet the detection work carried out by food enterprises, relevant government departments, etc.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a preparation method of a diisononyl phthalate hapten, comprising the following steps:

[0008] (1) Take 4-nitrophthalic acid and isononyl alcohol and mix them, and reflux and react under the catalysis of a strong acid to obtain diisononyl 4-nitrophthalate;

[0009] (2) Take diisononyl 4-nitrophthalate, absolute ethanol and ammonium formate and mix them, and react under the catalysis of a palladium / carbon catalyst to obtain diisononyl 4-aminophthalate, which is the diisononyl phthalate hapten.

[0010] Preferably, in step (1), the mass-volume ratio of 4-nitrophthalic acid to isononyl alcohol is 8-12 g: 35-45 ml; the temperature of the reflux reaction is 100-140 °C, and the time is 5-7 h; the strong acid is 98% concentrated sulfuric acid.

[0011] Preferably, in step (2), the mass-volume ratio of diisononyl 4-nitrophthalate, absolute ethanol and ammonium formate is 3-5 g: 20-30 ml: 2-3 g; the temperature of the reaction is 23-27 °C, and the time is 1.5-2 h.

[0012] The present invention also provides the diisononyl phthalate hapten prepared by the above method.

[0013] The present invention also provides an enzyme-linked immunosorbent assay kit for detecting diisononyl phthalate, comprising the following components: an enzyme-labeled plate coated with a coating antigen, a working solution of a diisononyl phthalate-specific antibody, a working solution of an enzyme-labeled substance, a standard solution of diisononyl phthalate, a substrate chromogenic solution, a termination solution, a washing solution, a sample diluent, a purifying agent;

[0014] The coating antigen is a diisononyl phthalate conjugate antigen; the diisononyl phthalate conjugate antigen is obtained by conjugating the diisononyl phthalate hapten with a carrier protein;

[0015] The working solution of the diisononyl phthalate-specific antibody is a specific antibody prepared using the diisononyl phthalate conjugate antigen as an immunogen.

[0016] Preferably, the carrier protein is one of bovine serum albumin, chicken ovalbumin, keyhole limpet hemocyanin, mouse serum protein, thyroglobulin, rabbit serum protein, human serum protein or fibrinogen; preferably bovine serum albumin.

[0017] Preferably, the specific antibody is a monoclonal antibody or a polyclonal antibody; preferably a polyclonal antibody.

[0018] Preferably, the enzyme-labeled working solution is HRP-goat anti-mouse antibody or HRP-goat anti-rabbit antibody; preferably HRP-goat anti-mouse antibody.

[0019] The buffer solution of the enzyme-labeled working solution is 0.5 - 2 w / V% BSA, 0.01 - 0.1 w / V% Tween-20, 0.5 - 2 w / V% glycerol, 0.03 - 0.1 w / V% Proclin-300, 0.05 - 0.2 mol / L phosphate buffer, with a pH value of 7.0 - 7.4;

[0020] The concentrations of the dinonyl phthalate standard solutions are 0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, and 8 mg / L respectively;

[0021] The buffer solution of the dinonyl phthalate standard solution is: 0.01% - 0.1% (v / v) β-hydroxypropyl cyclodextrin, 0.5% - 2% (v / v) glycerol, 10% - 40% (v / v) methanol aqueous solution, 0.1 - 0.3 mol / L phosphate buffer, with a pH value of 7.2 - 7.8.

[0022] Preferably, when the labeled enzyme is horseradish peroxidase, the substrate chromogenic solution is composed of substrate solution A and substrate solution B, wherein solution A is hydrogen peroxide or urea peroxide, and solution B is o-phenylenediamine or tetramethylbenzidine; preferably, solution A is urea peroxide and solution B is tetramethylbenzidine.

[0023] Preferably, the termination solution is 1 - 2 mol / L sulfuric acid or phosphate buffer; preferably a sulfuric acid solution.

[0024] The washing solution is: 0.5% - 1.0% (v / v) Tween-20, 0.03% - 0.1% (v / v) Proclin-300, 0.05 - 0.2 mol / L phosphate buffer, with a pH of 7.0 - 7.5;

[0025] The sample diluent is: 0.01% - 0.1% (v / v) β-hydroxypropyl cyclodextrin, 0.5% - 2% (v / v) glycerol, 0.03% - 0.1% (v / v) Proclin-300, 0.1 - 0.3 mol / L phosphate buffer, with a pH of 7.2 - 7.8;

[0026] The purifying agent is one or more of C18 packing material, Florisil, polystyrene, and neutral alumina.

[0027] The present invention also provides the application of the described enzyme-linked immunosorbent assay kit in detecting diisononyl phthalate in edible oil.

[0028] The present invention also provides a method for detecting diisononyl phthalate in edible oil by using the described enzyme-linked immunosorbent assay kit, which comprises the following steps:

[0029] (a) Pretreatment of the edible oil sample;

[0030] Take an edible oil sample, mix it with water and methanol, centrifuge and take the supernatant. Add a purifying agent and water to the supernatant, then centrifuge again and take the supernatant. Mix the supernatant with a sample diluent to obtain a test solution;

[0031] (b) Detecting the test solution obtained in step a;

[0032] (c) Analyzing the detection result.

[0033] The detection principle of the present invention is as follows: Diisononyl phthalate coating antigen is pre-coated on a microplate strip. After adding a sample solution or a standard product solution, then add a working solution of a diisononyl phthalate-specific antibody. Diisononyl phthalate in the sample competes with the coating antigen coated on the enzyme-labeled plate for the diisononyl phthalate-specific antibody. Add an enzyme label for amplification, and use a chromogenic solution for color development. The absorbance value of the sample is negatively correlated with the content of diisononyl phthalate. By comparing with a standard curve, the residual amount of diisononyl phthalate in the sample can be obtained; at the same time, according to the depth of the color in the enzyme-labeled plate wells and by comparing with the colors of standard product solutions with a series of concentrations, the concentration range of the residual amount of diisononyl phthalate in the sample can be roughly judged.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Existing detection methods for edible oil include large-scale instrument methods such as liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry. When detecting diisononyl phthalate in edible oil, these methods usually require the use of a purification column, and the price of the purification column ranges from 70 to 280 yuan. It not only has high costs, long time, but also complex operations. However, the present invention does not require a purification column and only needs a kit and an enzyme-labeled instrument to perform quantitative detection.

[0036] (2) Since diisononyl phthalate is similar to oils and fats in physical and chemical properties and is very difficult to extract from edible oils, there is no kit for directly detecting diisononyl phthalate in edible oils in the prior art. There is no such product in this field at home and abroad. The present invention fills the blank in this research field. At present, except for this kit, there is no enzyme-linked immunosorbent assay kit that can quantitatively detect diisononyl phthalate in edible oils.

[0037] (3) The present invention mainly uses the indirect competitive ELISA method to qualitatively or quantitatively detect the content of diisononyl phthalate in edible oil samples; it has low requirements for sample pretreatment, the sample pretreatment process is simple, and it can quickly detect a large number of edible oil samples at the same time; the main reagents are provided in the form of working solutions, and the test method is convenient and feasible, with the characteristics of high specificity, high sensitivity, high precision, high accuracy, etc.

[0038] (4) The detection equipment of the present invention is simple. Only a small oscillator, a centrifuge and an enzyme-labeled instrument are needed to complete the quantitative detection. However, the existing large-scale instrument methods such as gas chromatography not only require an oscillator and a centrifuge, but also a nitrogen blower and expensive, bulky and complex gas chromatography instruments, mass spectrometers, etc.

[0039] (5) The pretreatment method of the edible oil samples of the present invention is simple, without nitrogen blowing and without using a purification column. It can be directly extracted and diluted, with low cost. The existing instrument detection methods not only need to use a solid-phase extraction column, but also need to be dried with nitrogen, which is not only complex in operation, but also high in cost.

[0040] (6) The detection results are accurate, and the coincidence rate with liquid chromatography is more than 95%.

[0041] (7) The detection limit is low. The detection limit of diisononyl phthalate in edible oils reaches 6 mg / kg, which can meet the existing monitoring standards.

[0042] (8) It can be industrially produced, and is suitable for the large-scale and rapid screening of samples by food and drug supervision departments, enterprises, supermarkets, etc., and has better commercial value and application value. Description of the Drawings

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0044] Figure 1 It is a synthetic route diagram of the diisononyl phthalate hapten.

[0045] Figure 2It is the infrared spectrum of the diisononyl phthalate hapten.

[0046] Figure 3 It is the standard curve of the kit. Detailed implementation

[0047] The present invention provides a method for preparing a diisononyl phthalate hapten, which includes the following steps:

[0048] (1) Take 4-nitrophthalic acid and isononyl alcohol and mix them, and reflux and react under the catalysis of a strong acid to obtain diisononyl 4-nitrophthalate;

[0049] (2) Take diisononyl 4-nitrophthalate, absolute ethanol and ammonium formate and mix them, and react under the catalysis of a palladium / carbon catalyst to obtain diisononyl 4-aminophthalate, which is the diisononyl phthalate hapten.

[0050] In the present invention, the mass-volume ratio of the 4-nitrophthalic acid and isononyl alcohol in step (1) is 8-12 g: 35-45 ml; preferably 9-11 g: 37-43 ml; more preferably 10 g: 40 ml.

[0051] In the present invention, the temperature of the reflux reaction is 100-140 °C, and the time is 5-7 h; preferably the temperature of the reflux reaction is 110-130 °C, and the time is 6 h; more preferably the temperature of the reflux reaction is 120 °C, and the time is 6 h.

[0052] In the present invention, the strong acid is 98% concentrated sulfuric acid.

[0053] In the present invention, the mass-volume ratio of the diisononyl 4-nitrophthalate, absolute ethanol and ammonium formate in step (2) is 3-5 g: 20-30 ml: 2-3 g; preferably 4 g: 22-28 ml: 2.5 g; more preferably 4 g: 25 ml: 2.5 g

[0054] In the present invention, the temperature of the reaction in step (2) is 23-27 °C, and the time is 1.5-2 h; preferably the temperature of the reaction is 23-27 °C, and the time is 1.5-2 h; more preferably the temperature of the reaction is 25 °C, and the time is 2 h.

[0055] The present invention also provides the diisononyl phthalate hapten prepared by the above method.

[0056] The present invention also provides an enzyme-linked immunosorbent assay kit for the detection of diisononyl phthalate, comprising the following components: an enzyme-labeled plate coated with a coating antigen, a working solution of a diisononyl phthalate-specific antibody, a working solution of an enzyme-labeled substance, a diisononyl phthalate standard solution, a substrate chromogenic solution, a termination solution, a washing solution, a sample diluent, and a purifying agent;

[0057] The coating antigen is a diisononyl phthalate-conjugated antigen; the diisononyl phthalate-conjugated antigen is obtained by conjugating the diisononyl phthalate hapten with a carrier protein;

[0058] The working solution of the diisononyl phthalate-specific antibody is a specific antibody prepared using the diisononyl phthalate-conjugated antigen as an immunogen.

[0059] In the present invention, the carrier protein is one of bovine serum albumin, chicken ovalbumin, keyhole limpet hemocyanin, mouse serum protein, thyroid protein, rabbit serum protein, human serum protein, or fibrinogen; preferably bovine serum albumin.

[0060] In the present invention, the specific antibody is a monoclonal antibody or a polyclonal antibody; preferably a polyclonal antibody.

[0061] In the present invention, the working solution of the enzyme-labeled substance is an HRP-goat anti-mouse antibody or an HRP-goat anti-rabbit antibody; preferably an HRP-goat anti-mouse antibody.

[0062] In the present invention, the buffer solution of the working solution of the enzyme-labeled substance is 0.5 - 2 w / V% BSA, 0.01 - 0.1 w / V% Tween-20, 0.5 - 2 w / V% glycerol, 0.03 - 0.1 w / V% Proclin-300, 0.05 - 0.2 mol / L phosphate buffer solution, with a pH value of 7.0 - 7.4; preferably, the buffer solution of the working solution of the enzyme-labeled substance is 2 w / V% BSA, 0.1 w / V% Tween-20, 2 w / V% glycerol, 0.1 w / V% Proclin-300, 0.2 mol / L phosphate buffer solution, with a pH value of 7.4.

[0063] The concentrations of the diisononyl phthalate standard solutions are 0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, and 8 mg / L respectively;

[0064] The buffer solution of the dinonyl phthalate standard solution is: β-hydroxypropyl cyclodextrin with a volume concentration of 0.01% - 0.1%, glycerol with a volume concentration of 0.5% - 2%, methanol aqueous solution with a volume concentration of 10% - 40%, phosphate buffer solution with a volume concentration of 0.1 - 0.3 mol / L, and the pH value is 7.2 - 7.8; preferably, it is β-hydroxypropyl cyclodextrin with a volume concentration of 0.1%, glycerol with a volume concentration of 2%, methanol aqueous solution with a volume concentration of 40%, phosphate buffer solution with a volume concentration of 0.3 mol / L, and the pH value is 7.8.

[0065] In the present invention, when the labeled enzyme is horseradish peroxidase, the substrate chromogenic solution is composed of substrate solution A and substrate solution B. Among them, solution A is hydrogen peroxide or urea peroxide, and solution B is o-phenylenediamine or tetramethylbenzidine; preferably, solution A is urea peroxide and solution B is tetramethylbenzidine.

[0066] In the present invention, the termination solution is sulfuric acid or phosphate buffer solution with a concentration of 1 - 2 mol / L; preferably, it is 1 mol / L sulfuric acid solution.

[0067] In the present invention, the washing solution is: Tween-20 with a volume concentration of 1.0%, Proclin-300 with a volume concentration of 0.1%, 0.2 mol / L phosphate buffer solution, and pH 7.5; preferably, it is Tween-20 with a volume concentration of 1.0%, Proclin-300 with a volume concentration of 0.1%, 0.2 mol / L phosphate buffer solution, and pH 7.5.

[0068] In the present invention, the sample diluent is: β-hydroxypropyl cyclodextrin with a volume concentration of 0.01% - 0.1%, glycerol with a volume concentration of 0.5% - 2%, Proclin-300 with a volume concentration of 0.03% - 0.1%, phosphate buffer solution with a volume concentration of 0.1 - 0.3 mol / L, and the pH value is 7.2 - 7.8; preferably, it is β-hydroxypropyl cyclodextrin with a volume concentration of 0.1%, glycerol with a volume concentration of 2%, Proclin-300 with a volume concentration of 0.1%, phosphate buffer solution with a volume concentration of 0.3 mol / L, and the pH value is 7.8.

[0069] In the present invention, the purifying agent is one or more of C18 filler, florisil, polystyrene, and neutral alumina; preferably, it is C18 filler.

[0070] The present invention also provides the application of the described enzyme-linked immunosorbent assay kit in detecting dinonyl phthalate in edible oil.

[0071] The present invention also provides a method for detecting dinonyl phthalate in edible oil by using the described enzyme-linked immunosorbent assay kit, which includes the following steps:

[0072] (a) Pretreatment of the edible oil sample;

[0073] Take the edible oil sample, mix it with water and methanol, centrifuge, and take the supernatant. Add a purifying agent and water to the supernatant, centrifuge again, and take the supernatant. Mix the supernatant with a sample diluent to obtain the test solution to be detected;

[0074] (b) Detect the test solution obtained in step a;

[0075] (c) Analyze the test results.

[0076] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0077] Sources of the following experimental materials:

[0078] Horseradish peroxidase: Jackson Immuno Research, USA, 115 - 035 - 003. β - Hydroxypropyl cyclodextrin: Beijing Vokai Biotechnology Co., Ltd., A49081. Surfactant S9: Seebio Technology (Shanghai) Co., Ltd., ACH0313E. Tween - 20: Sinopharm Chemical Reagent Co., Ltd., 30189328. C18 packing material: Sinopharm Chemical Reagent Co., Ltd., F80020430100. Florisil: Sinopharm Chemical Reagent Co., Ltd., C205455000. Neutral alumina: Sinopharm Chemical Reagent Co., Ltd., 20002360. Polystyrene: Sinopharm Chemical Reagent Co., Ltd., C178895000. Proclin - 300: Sigma - Aldrich, USA, 48912 - U.

[0079] Bovine serum albumin (BSA): MP Biomedicals, USA, 02FC007780. Keyhole limpet hemocyanin (KLH): Sigma - Aldrich, USA, H7017. Sodium caseinate: Sigma - Aldrich, USA, C8654. Trehalose: Shanghai Aladdin Biochemical Technology Co., Ltd., T100010. Anhydrous methanol (chromatographic grade): Shanghai Macklin Biochemical Co., Ltd., M813904. Glycerol: Sinopharm Chemical Reagent Co., Ltd., C327255000. Peanut oil: Luhua 5S pressed first - grade peanut oil. DBP: National Standard Material Center of Tanmo Quality Inspection, BW900928 - 100 - A.

[0080] DEHP: Standard Substance Center of Tanmo Quality Inspection, BW900947-500-A. DINP: Standard Substance Center of Tanmo Quality Inspection, 70145. DIBP: Standard Substance Center of Tanmo Quality Inspection, GBW(E)100964. DIOP: Standard Substance Center of Tanmo Quality Inspection, 71741. BBP: Standard Substance Center of Tanmo Quality Inspection, BW900519-1000-A. DMP: Standard Substance Center of Tanmo Quality Inspection, BW901199-1000-A. Tetranitrophthalic acid: Sinopharm Chemical Reagent Co., Ltd., XW06102752. Isooctyl alcohol: Sinopharm Chemical Reagent Co., Ltd., TRCM325655250MG. Concentrated sulfuric acid: Sinopharm Chemical Reagent Co., Ltd., 10021608.

[0081] Palladium / carbon catalyst (Pd / C): Sinopharm Chemical Reagent Co., Ltd., 20050665. Ammonium formate: Sinopharm Chemical Reagent Co., Ltd., 30011618. Potassium dihydrogen phosphate: Sinopharm Chemical Reagent Co., Ltd., 10017618. Disodium hydrogen phosphate: Sinopharm Chemical Reagent Co., Ltd., 20040618. Sodium carbonate: Sinopharm Chemical Reagent Co., Ltd., 10019260. Sodium bicarbonate: Sinopharm Chemical Reagent Co., Ltd., 10018960. Sodium chloride: Sinopharm Chemical Reagent Co., Ltd., 10019308. Potassium chloride: Sinopharm Chemical Reagent Co., Ltd., 10016308.

[0082] Preparation of the components of the kit in Example 1

[0083] 1. Preparation of the dinonyl phthalate hapten

[0084] Take a clean 100 mL three-necked round-bottom flask, place 10 g of 4-nitrophthalic acid at the bottom of the flask, then slowly add 37.2 mL of isononyl alcohol, add 1.55 mL of concentrated H2SO4 as a catalyst while stirring, stir and reflux at 120 °C, and drop the reaction mixture onto a thin-layer chromatography silica gel plate to check whether the reaction is complete; until the raw material spots disappear by thin-layer chromatography (TLC) detection (developer: a small amount of potassium permanganate dissolved in acetone solvent; developing agent, n-hexane:ethyl acetate = 5:1); the reaction is complete after stirring and refluxing for 6 h. Distill off the unreacted isononyl alcohol and the generated water; at this time, pour the liquid while it is hot into ice water, stir magnetically for 5 min and then let it stand for a period of time to separate layers, wash the lower yellow oily liquid with 10% Na2CO3 solution until the water layer is colorless (pH 7.5); finally, dehydrate and dry the oily crude product with anhydrous sodium sulfate to obtain a yellow oily liquid, dinonyl 4-nitrophthalate, which is the DINP hapten intermediate.

[0085] Take a clean round-bottom flask with a volume of 200 mL and add a magnetic stir bar. Sequentially add 4 g of diisononyl 4-nitrophthalate, 25 mL of absolute ethanol, 150 mg of palladium / carbon catalyst (Pd / C), and 2.3 g of ammonium formate, then seal the flask. React at room temperature in the dark for 2 hours. Filter to remove the palladium / carbon catalyst (Pd / C), filter twice to obtain a golden-yellow liquid. Wash the filter residue, wash each time with an equal volume, and combine the washing solutions. Stir magnetically and evaporate under reduced pressure to remove the solvent ethanol to obtain an orange-yellow oily liquid. Wash with ethanol 3 times and recrystallize to obtain the solid diisononyl 4-aminophthalate, which is the DINP hapten.

[0086] Use a Fourier transform infrared spectrometer (FTIR) to measure the infrared spectrum of diisononyl 4-aminophthalate to confirm the successful synthesis of the DINP hapten. Turn on and preheat: Turn on the main spectrometer and computer, and start the operating software. Preheat for at least 30 minutes (if the laboratory humidity is high, extend to 1 hour). Parameter settings: Scanning range: 4000 - 400 cm-1 (covering the characteristic region of organic functional groups). Resolution: 4 cm-1. Number of scans: 32 times. Detector selection: DTGS. Install a blank salt plate (without sample) and ensure the surface of the salt plate is clean. Click "Collect Background", and the software automatically subtracts the interference of CO2 and H2O in the optical path. Insert the clamped salt plate sample holder into the optical path of the sample chamber. Click "Scan" and observe the real-time spectrum: Check if the baseline is straight. If the baseline is tilted, it may be due to a too thick liquid film or the salt plate not being tightly pressed. Obtain the infrared spectrum of the diisononyl phthalate hapten, as Figure 2 shown.

[0087] 2. Preparation of diisononyl phthalate immunogen

[0088] Couple the diisononyl phthalate hapten with keyhole limpet hemocyanin (KLH) by the diazotization method to obtain the immunogen.

[0089] Weigh 50 mg of keyhole limpet hemocyanin (KLH) and dissolve the KLH in 10 mL of 0.1 mol / L carbonate buffer solution (CB solution), and place it in the refrigerator at 4 °C for later use. Weigh 5 mg of the diisononyl 4-aminophthalate hapten prepared in step 1 into a test tube, add 2 mL of dimethylformamide (DMF), and completely dissolve the diisononyl 4-aminophthalate hapten under ice bath conditions. Weigh 3.4 mg of NaNO2 and dissolve it fully in 1 mL of water. Add the NaNO2 solution to the diisononyl 4-aminophthalate hapten solution under ice bath conditions and react for 3 h under ice bath conditions. Slowly drip the activated diisononyl 4-aminophthalate hapten solution into the KLH solution and react overnight under ice bath conditions. Dialyze with 0.01 mol / L PBS at 4 °C for 3 days, changing the dialysis solution 3 times a day to remove unreacted small molecules; aliquot and store at -20 °C for later use.

[0090] 3. Preparation of the coating antigen of diisononyl phthalate

[0091] The coating antigen was obtained by coupling the hapten of diisononyl phthalate with bovine serum albumin (BSA) using the diazotization method.

[0092] Weigh 30 mg of bovine serum albumin (BSA), dissolve the BSA in 10 mL of 0.1 mol / L carbonate buffer solution (CB solution), and place it in a refrigerator at 4 °C for later use. Weigh 5 mg of the hapten of diisononyl phthalate prepared in step 1 into a test tube, add 2 mL of dimethylformamide (DMF), and completely dissolve the hapten of diisononyl phthalate under ice bath conditions. Weigh 3.4 mg of NaNO₂ and dissolve it fully in 1 mL of water. Add the NaNO₂ solution to the hapten solution of diisononyl phthalate under ice bath conditions and react for 3 h under ice bath conditions. Slowly drip the activated hapten solution of diisononyl phthalate into the BSA solution and react overnight under ice bath conditions. Dialyze with 0.01 mol / L PBS at 4 °C for 3 days, changing the dialysis fluid 3 times a day to remove unreacted small molecule substances; aliquot and store at -20 °C for later use.

[0093] 4. Preparation of monoclonal antibody of diisononyl phthalate

[0094] Animal immunization: Inject the immunogen of diisononyl phthalate into Balb / c mice at an immunization dose of 150 μg / mouse to produce antiserum.

[0095] Cell fusion and cloning: After the mouse serum test results are relatively high, take its spleen cells and fuse them with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). Use competitive ELISA to measure the cell supernatant and screen positive wells. Clone the positive wells using the limiting dilution method until a hybridoma cell line secreting monoclonal antibody of diisononyl phthalate is obtained.

[0096] Cell cryopreservation and resuscitation: Prepare a cell suspension of 1×10 6 cells / mL with the monoclonal hybridoma cell line using the cryopreservation solution and store it in liquid nitrogen for a long time. When resuscitating, take out the cryopreservation tube, immediately place it in a water bath at 37 °C for rapid thawing. After centrifuging to remove the cryopreservation solution, transfer it to a culture flask for cultivation.

[0097] Production and purification of monoclonal antibody: Inject 0.5 mL / mouse of sterilized paraffin oil into the abdominal cavity of Balb / c mice. After 7 days, inject 5×10 5 cells / mouse of the stable monoclonal hybridoma cell line into the abdominal cavity. After 7 days, collect the ascites. Purify the ascites using the caprylic acid - saturated ammonium sulfate method and store it at -20 °C.

[0098] 5. Preparation of the enzyme - labeled plate of diisononyl phthalate

[0099] Dilute the diisononyl phthalate coating source to 1 μg / mL with coating buffer, add 100 μL to each well, incubate at 4°C in the dark for 16 h, pour off the liquid in the wells, wash once with washing buffer for 30 s each time, pat dry, then add 150 μL of blocking solution to each well, incubate at 37°C in the dark for 2 h, pour off the liquid in the wells, pat dry, dry at 37°C for 0.5 h, and store in a vacuum sealed aluminum film; the coating buffer is a carbonate buffer with a pH value of 9.3 and 0.1 mol / L.

[0100] 6. Preparation of enzyme marker working solution.

[0101] The HRP-goat anti-mouse antibody was diluted to 2 μg / mL with a buffer; the enzyme marker buffer was 2 w / V% BSA, 0.1 w / V% surfactant Tween-20, 2 w / V% glycerol, 0.1 w / V% Proclin-300, and 0.2 mol / L phosphate buffer, all of which were volume concentrations, with a pH value of 7.4.

[0102] Example 2 Construction of diisononyl phthalate ELISA kit

[0103] Using the raw materials prepared in Example 1, a diisononyl phthalate enzyme-linked immunosorbent assay kit was constructed to include the following components:

[0104] (1) ELISA plate coated with diisononyl phthalate;

[0105] (2) diisononyl phthalate specific antibody working solution;

[0106] (3) Enzyme marker working solution;

[0107] (4) 6 bottles of diisononyl phthalate standard solution, with concentrations of 0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, and 8 mg / L respectively; the buffer solution of the diisononyl phthalate standard solution is: 0.1% β-hydroxypropyl cyclodextrin by volume, 2% glycerol by volume, 40% methanol aqueous solution by volume, and 0.3 mol / L phosphate buffer by volume, with a pH value of 7.5.

[0108] (5) The substrate color developing solution consists of solution A and solution B, wherein solution A is urea peroxide and solution B is tetramethylbenzidine;

[0109] (6) The stop solution is 2 mol / L sulfuric acid solution;

[0110] (7) the washing solution is a phosphate buffer having a pH value of 7.5 and containing 1.0% Tween-20, 0.1% Proclin-300, and 0.2 mol / L;

[0111] (8) The sample diluent is a phosphate buffer solution with a pH value of 7.5, containing 0.1% β-hydroxypropyl cyclodextrin by volume concentration, 2% glycerol by volume concentration, 0.1% Proclin-300 by volume concentration, and 0.3 mol / L by volume concentration.

[0112] (9) The sample purifying agent is C18 packing powder.

[0113] Example 3 Detection of Diisononyl Phthalate in Edible Oil Samples

[0114] 1. Sample Pretreatment

[0115] (1) Weigh 1 g of the sample into a 10 mL glass centrifuge tube, sequentially add 1 mL of water (Wahaha purified water), 4 mL of methanol (chromatographic grade), and vortex for 1 min; centrifuge at 4000 rpm for 5 min;

[0116] (2) Take 2 mL of the supernatant into a glass centrifuge tube, add 1 g of the purifying agent, then add 0.8 mL of water (Wahaha purified water), and shake for 1 min; centrifuge at 4000 rpm for 5 min;

[0117] (3) Take a clean glass test tube, sequentially add 0.5 mL of the sample diluent and 0.7 mL of the sample supernatant, and vortex for 1 min;

[0118] (4) Take 50 μL of the mixed solution for analysis.

[0119] 2. Detection Using the Kit Prepared in Example 2

[0120] Sequentially add 50 μL / well of the diisononyl phthalate standard solution or the pretreated sample solution, 50 μL / well of the enzyme-labeled working solution, and 50 μL / well of the diisononyl phthalate-specific antibody working solution to the micro wells of the enzyme-linked immunosorbent assay (ELISA) plate; gently shake and mix well, cover with a cover plate membrane, and react at 25 °C in the dark for 30 min; pour out the liquid in the wells, add 260 μL of the washing solution to each well and wash thoroughly 5 times, with an interval of 10 s each time, and pat dry with absorbent paper; add 50 μL of the substrate chromogenic solution A urea peroxide and 50 μL of the substrate chromogenic solution B 3,3',5,5'-tetramethylbenzidine (TMB) to each well, gently shake and mix well, cover with a cover plate membrane, and develop color at 25 °C in the dark for 10 min. Then add 50 μL of the termination solution 2 mol / L sulfuric acid to each well, gently shake and mix well, and measure the absorbance value (OD value) of each well with an ELISA reader at a wavelength of 450 nm.

[0121] 3. Analysis of Detection Results

[0122] The absorbance average value (B) of the standard solution at each obtained concentration is divided by the absorbance value (B0) of the first standard solution (0 standard), and then multiplied by 100% to obtain the percentage absorbance value. Using the logarithm of the concentration (mg / L) of the diisononyl phthalate standard as the X-axis and the percentage absorbance value as the Y-axis, a standard curve is plotted, as Figure 3 shown. Calculate the percentage absorbance value of the sample solution in the same way. The diisononyl phthalate content corresponding to each sample can be calculated from the standard curve, and multiplying by its corresponding dilution factor gives the actual content of diisononyl phthalate in the sample.

[0123] Example 4 Determination Test of Technical Parameters of Diisononyl Phthalate Enzyme-Linked Immunosorbent Assay Kit

[0124] 1. Sensitivity and Detection Limit of the Kit

[0125] The sensitivity of the kit was determined by the conventional method. The lowest point of the standard curve of the kit was 0.5 mg / L, the range of the standard curve was 0.5 - 8 mg / L, and the floating range of IC50 (50% inhibition concentration) was 1.2 - 1.6 mg / L; 20 blank edible oil samples were detected, and the concentrations corresponding to each percentage absorbance value were found from the standard curve. The detection limit was expressed as the average value of the concentrations of 20 samples plus 3 times the standard deviation. The result showed that the detection limit of this method for edible oil samples was 6 mg / kg.

[0126] Table 1 Experiment for Determining Sample Detection Limit

[0127]

[0128]

[0129] 2. Sample Precision and Accuracy Tests

[0130] Taking the recovery rate as the evaluation index for accuracy and the relative standard deviation (RSD%) of the detection results of repeatedly measuring a sample at a certain concentration as the evaluation index for precision. The calculation formulas are: Recovery rate (%) = actual measured value / theoretical value × 100%, where the theoretical value is the added concentration of the sample; Relative standard deviation RSD% = SD / X × 100%, where SD is the standard deviation and X is the average value of the measured data.

[0131] The addition recovery determination of edible oil samples was carried out with diisononyl phthalate at three concentrations of 6 mg / kg, 9 mg / kg, and 12 mg / kg. Each sample was made into 4 parallels and measured with three batches of different reagents. The average recovery rate and precision results of the samples are shown in the following table.

[0132] Table 2 Precision and Accuracy Tests

[0133]

[0134]

[0135]

[0136] The peanut oil samples were spiked with diisononyl phthalate (DINP) at three concentrations of 6 mg / kg, 9 mg / kg, and 12 mg / kg, and the average recoveries were between 81.67% and 110%; the relative standard deviations within and between batches were both less than 15%.

[0137] 3. Cross-reactivity test

[0138] Drugs with similar structures and functions to diisononyl phthalate were selected for ELISA determination, and their 50% inhibitory concentrations (IC 50 ) were obtained from the standard curves of various drugs, and the cross-reactivity of the kit to other drugs was calculated using the following formula.

[0139] Cross-reactivity (CR) = IC 50 (DINP) / IC 50 (analogue) × 100%

[0140] Table 3 Cross-reactivity test

[0141] structural analog crossing rate (%) diisononyl phthalate (DINP) 100 di(2-ethylhexyl) phthalate (DEHP) <1 dibutyl phthalate (DBP) <1 diisobutyl phthalate (DIBP) <1 benzyl butyl phthalate (BBP) <1 diisooctyl phthalate (DIOP) <1 dimethyl phthalate (DMP) <1 parabens <1 butyl formate <0.1

[0142] It can be seen from Table 3 that the cross-reactivities of the monoclonal antibody against diisononyl phthalate (DINP) with other structural analogues such as DEHP, DBP, DIBP, BBP, DIOP, and DMP are all less than 1%, indicating that the monoclonal antibody against diisononyl phthalate (DINP) has high specificity.

[0143] 4. Kit stability test

[0144] It can be seen from Table 4 that the storage condition of the kit is 2 - 8°C. After 12 months of determination, the maximum absorbance value (zero standard), IC 50 value, and the actual measured value of the added diisononyl phthalate are all within the normal range. Considering that there may be abnormal storage conditions during transportation and use, the kit was placed under the storage conditions of 37°C, -20°C, and shaking for 15 days for accelerated aging experiments, and the IC 50 of the standard curves under each condition was measured, and the relevant variation was calculated. The results are shown in Tables 5, 6, and 7. Under the conditions of 37°C, -20°C, and shaking environment, the kit has good stability, and the IC 50 and related variations are guaranteed within the normal fluctuation range. From the above results, it can be concluded that the kit can be stored at 2 - 8°C for at least 12 months or more.

[0145] Table 4 Storage Test at 44°C

[0146]

[0147] Table 5 Thermal Acceleration Test at 37°C

[0148]

[0149] Table 6 Storage Test at -20°C

[0150]

[0151] Table 7 Storage Test with Shaking

[0152]

[0153] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a diisononyl phthalate hapten, characterized in that: The steps include: (1) mixing 4-nitrophthalic acid and isononyl alcohol, and subjecting the mixture to a reflux reaction under the catalysis of a strong acid to obtain diisononyl 4-nitrophthalate; (2) 4-nitrodiisononylphthalate, anhydrous ethanol and ammonium formate are mixed and reacted under the catalysis of palladium / carbon catalyst to obtain 4-aminodiisononylphthalate, which is diisononylphthalate hapten.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass volume ratio of 4-nitrophthalic acid and isononanol is 8-12 g:35-45 ml; the temperature of the reflux reaction is 100-140° C., and the time is 5-7 h; and the strong acid is 98% concentrated sulfuric acid.

3. The preparation method according to claim 1, characterized in that: In step (2), the mass volume ratio of 4-nitrophthalic acid diisononyl ester, anhydrous ethanol and ammonium formate is 3-5g:20-30ml:2-3g; the reaction temperature is 23-27°C and the reaction time is 1.5-2h.

4. The diisononyl phthalate hapten prepared by the method according to any one of claims 1 to 3.

5. An enzyme-linked immunosorbent assay kit for detecting diisononyl phthalate, characterized in that: The invention comprises the following components: an ELISA plate coated with a coating source, a diisononyl phthalate specific antibody working solution, an enzyme marker working solution, a diisononyl phthalate standard solution, a substrate color developing solution, a stop solution, a washing solution, a sample diluent, and a purifying agent; The coating is originally a diisononyl phthalate-coupled antigen; the diisononyl phthalate-coupled antigen is obtained by coupling the diisononyl phthalate hapten described in claim 4 with a carrier protein; The diisononyl phthalate-specific antibody working solution is a specific antibody prepared by using diisononyl phthalate-coupled antigen as an immunogen.

6. The kit according to claim 5, characterized in that The carrier protein is one of bovine serum albumin, chicken ovalbumin, keyhole limpet hemocyanin, mouse serum protein, thyroid protein, rabbit serum protein, human serum protein or fibrinogen; the specific antibody is a monoclonal antibody or a polyclonal antibody.

7. The kit according to claim 5, characterized in that The enzyme marker working solution is HRP-goat anti-mouse antibody or HRP-goat anti-rabbit antibody; The buffer of the enzyme marker working solution is 0.5-2w / V% BSA, 0.01-0.1w / V% Tween-20, 0.5-2w / V% glycerol, 0.03-0.1w / V% Proclin-300, 0.05-0.2mol / L phosphate buffer, and the pH value is 7.0-7.4; The concentrations of the diisononyl phthalate standard solutions are 0 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, and 8 mg / L, respectively; The buffer solution of the diisononyl phthalate standard solution is: 0.01% to 0.1% beta-hydroxypropyl cyclodextrin by volume, 0.5% to 2% propylene glycol by volume, 10% to 40% methanol aqueous solution by volume, 0.1 to 0.3 mol / L phosphate buffer by volume, and the pH value is 7.2 to 7.

8.

8. The kit according to claim 5, characterized in that When the labeling enzyme is horseradish peroxidase, the substrate color developing solution is composed of substrate solution A and substrate solution B, wherein solution A is hydrogen peroxide or urea peroxide, and solution B is o-phenylenediamine or tetramethylbenzidine; the stop solution is 1-2 mol / L sulfuric acid or phosphate buffer; The washing solution is: 0.5% to 1.0% Tween-20 by volume, 0.03% to 0.1% Proclin-300 by volume, 0.05 to 0.2 mol / L phosphate buffer, pH 7.0 to 7.5; The sample diluent comprises: β-hydroxypropyl cyclodextrin with a volume concentration of 0.01% to 0.1%, glycerol with a volume concentration of 0.5% to 2%, Proclin-300 with a volume concentration of 0.03% to 0.1%, and phosphate buffer with a volume concentration of 0.1 to 0.3 mol / L, pH 7.2 to 7.8; The purifier is one or more of C18 filler, Florisil, polystyrene, and neutral alumina.

9. Use of the enzyme-linked immunosorbent assay kit according to any one of claims 5 to 8 in detecting diisononyl phthalate in edible oil.

10. A method for detecting diisononyl phthalate in edible oil using the enzyme-linked immunosorbent assay kit according to any one of claims 5 to 8, characterized in that: The steps include: (a) Edible oil sample pretreatment; Take the edible oil sample, water and methanol, mix them and centrifuge to obtain the supernatant, add the purifier and water to the supernatant and centrifuge again to obtain the supernatant; add the sample diluent to the supernatant and mix to obtain the test solution; (b) testing the test solution obtained in step a; (c) Analysis of test results.

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