Enzyme linked immunosorbent assay kit for detecting plasticizer in edible oil and application of enzyme linked immunosorbent assay kit

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting a plasticizer in edible oil and application of the enzyme linked immunosorbent assay kit, and belongs to the technical field of enzyme linked immunosorbent assay. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a plasticizer coupling antigen, a plasticizer standard substance, an enzyme conjugate working solution, a plasticizer specific antibody working solution, a purification tube, a purification reagent, a standard substance diluent, a sample diluent, a substrate solution, a stop solution and a 10 * concentrated washing solution. The standard substance diluent provided by the invention is used for diluting a plasticizer standard substance at different temperatures to obtain a plasticizer standard solution, and the obtained plasticizer standard solution has basically consistent inhibition ratio, so that the stability of the kit is remarkably enhanced; meanwhile, the standard diluent and other components in the kit have a synergistic effect, so that the enzyme linked immunosorbent assay kit has the advantages of good specificity, high sensitivity, good accuracy and high stability when being used for detecting the plasticizer in the edible oil; therefore, the enzyme linked immunosorbent assay kit has a good application prospect in the detection of the plasticizer in the edible oil.

Description

Technical Field

[0001] This invention belongs to the field of enzyme-linked immunosorbent assay (ELISA) technology, specifically relating to an ELISA kit for detecting plasticizers in edible oils and its application. Background Technology

[0002] Oils and fats are an important part of people's diets, and "edible oil safety" is an important component of "food safety." The quality of edible oils directly affects consumers' health and safety. The quality and safety testing of edible oils has always been a difficult and hot issue in the field of food analysis. Phthalate esters (PAEs) are one of the most common exogenous contaminants in the oil industry. They are a large class of fat-soluble compounds. Oil crops are easily contaminated by exogenous pollutants in the environment during growth, causing plasticizers to accumulate in the plant and transfer out during the processing into edible oil. When processing oil comes into contact with plastic products containing plasticizers, the leaching process can also cause plasticizers to transfer into the edible oil. During storage, prolonged contact between edible oil and plastic containers can also lead to the transfer of plasticizers into the edible oil. The health administration department has set regulations on the maximum residue limits of plasticizers (Health Office Supervision Letter

[2011] No. 551): The maximum residue limits of di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), and dibutyl phthalate (DBP) in oils are 1.5 mg / kg, 9.0 mg / kg, and 0.3 mg / kg, respectively.

[0003] Currently, the detection methods for plasticizers in edible oils mainly rely on instrumental methods, including gas chromatography, high-performance liquid chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance. While instrumental methods offer advantages such as high sensitivity and good stability, they are expensive, involve complex and time-consuming sample preparation processes, and require large quantities of organic reagents. Therefore, they require well-equipped laboratories and specialized operators. Additionally, some enzyme-linked immunosorbent assay (ELISA) devices and reagents are available on the market, but their sensitivity is relatively low, they are easily affected by sample matrix interference, sample pretreatment steps are cumbersome, and the detection range is limited. Therefore, none of the above methods meet the requirements for rapid, low-cost, and efficient detection.

[0004] Given various factors, there is an urgent need to develop a detection method for plasticizers in various vegetable oils that offers advantages over instrumental methods, including faster detection speed, higher specificity, higher sensitivity, and lower cost. Immunoassay techniques based on antigen-antibody specific reactions are well-suited for this purpose. Currently, common immunoassay techniques for detecting plasticizers include colloidal gold immunochromatography, fluorescence immunoassay, and enzyme-linked immunosorbent assay (ELISA). Colloidal gold immunochromatography is low-cost and convenient, but it can only detect qualitatively, not quantitatively. While fluorescence immunoassay can detect quantitatively, the problem of non-specific staining remains unresolved, resulting in insufficient objectivity in result interpretation and a relatively complex procedure. Traditional ELISA kits require the creation of standard curves for 5-6 concentration points per experiment. Errors in the operation of individual standard concentration points or issues related to plate viscosity (CV), particularly edge effects, can lead to a deterioration in the linearity of the standard curve, affecting the accuracy of the detection results. Furthermore, creating complete standard curves for each experiment increases testing costs and leads to waste when testing scattered samples. Therefore, traditional enzyme-linked immunosorbent assay (ELISA) kits suffer from problems such as high operational requirements and poor reproducibility; in addition, the complex matrix of edible oils and their susceptibility to contamination by plasticizer migration make them unsuitable for grassroots testing needs. Therefore, developing an ELISA kit suitable for the rapid, efficient, stable, and accurate detection of plasticizers in edible oils has significant economic and social benefits. Summary of the Invention

[0005] The purpose of this invention is to provide an enzyme-linked immunosorbent assay (ELISA) kit for detecting plasticizers in edible oils and its application. This kit addresses the problems of existing ELISA kits, such as high operational requirements, poor repeatability, and high cost when testing scattered samples.

[0006] In a first aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting plasticizers in edible oils, comprising: an ELISA plate coated with plasticizer-conjugated antigen, plasticizer standards, enzyme conjugate working solution, plasticizer-specific antibody working solution, purification tubes, purification reagents, standard diluent, sample diluent, substrate solution, stop solution, and 10× concentrated wash buffer; wherein the standard diluent comprises: NaCl with a mass concentration of 0.5-1.5% (m / v), dimethyl sulfoxide with a volume percentage of 3-7% (v / v), mannitol with a mass concentration of 0.1-1% (m / v), PVP-40 with a mass concentration of 0.1-0.3% (m / v), EDTA with a mass concentration of 0.1-1% (m / v), trehalose with a mass concentration of 1-5% (m / v), and 0.05-2M citrate buffer, with a pH value of 5.8-6.5.

[0007] In this invention, by optimizing the components of the standard diluent and controlling the content of each specific component within a specific range, the plasticizer standard solutions obtained by diluting the plasticizer standard with the standard diluent at different temperatures have a basically consistent inhibition rate, thereby significantly enhancing the stability of the kit. At the same time, the standard diluent works synergistically with other components in the kit, thus giving the enzyme-linked immunosorbent assay kit advantages such as high specificity, high sensitivity, high accuracy, and high stability when detecting plasticizers in edible oils.

[0008] In some implementations, the coating solution used in the preparation of ELISA plates coated with plasticizer-conjugated antigens includes: 0.05-0.2 mol / L citrate buffer with a pH of 5.8-6.5.

[0009] In some embodiments, the preparation of the enzyme conjugate working solution includes mixing the enzyme conjugate and the enzyme conjugate / antibody diluent at a ratio of 1:200 to 1:1000; wherein the enzyme conjugate includes horseradish peroxidase-conjugated goat anti-mouse IgG.

[0010] In some embodiments, the preparation of the plasticizer-specific antibody working solution includes: mixing the plasticizer-specific antibody and the enzyme conjugate / antibody dilution solution at a ratio of 1:5000 to 1:100000; wherein the plasticizer-specific antibody includes a plasticizer monoclonal antibody.

[0011] In some embodiments, the enzyme conjugate / antibody diluent comprises: 0.03-0.1% casein, 0.1-0.5% (m / v) BSA, 0.1-0.5% (m / v) skim milk powder, 1-3% (m / v) sucrose, 0.1-0.5% (m / v) SG09, 0.05-0.2% (v / v) proclin950, 0.005-0.2% (v / v) β-mercaptoethanol, 0.01-0.2% (v / v) Tween-20, and 0.01-0.2 M phosphate buffer at a pH of 6.5-7.5.

[0012] In some embodiments, the purification tube is equipped with solid-phase extraction packing material; and / or, the purification reagent includes a weakly basic reagent; and / or, the 10× concentrated washing solution includes: 0.5-2% (v / v) Tween-20, 0.1-1% (m / v) sodium azide, and 0.1-1M phosphate buffer with a pH of 7-7.8.

[0013] In some implementations, the sample diluent includes: 1.5-2.5% (m / v) NaCl, 0.1-1% (m / v) mannitol, 0.1-0.3% (m / v) PVP-40, 0.1-1% (m / v) EDTA, 1-5% (m / v) trehalose, and 0.05-2M citrate buffer with a pH of 5.8-6.5.

[0014] In a second aspect, the present invention provides the application of any of the above-described enzyme-linked immunosorbent assay kits in the detection of plasticizers in edible oils.

[0015] In a third aspect, the present invention provides a method for detecting plasticizers in edible oils using any of the above-mentioned enzyme-linked immunosorbent assay (ELISA) kits, comprising the following steps: extracting the sample to be tested using an organic solvent, adding the resulting subnaphthalene to a purification tube, adding purification reagent and performing extraction, adding sample diluent to the resulting supernatant to obtain the test solution; adding standard diluent and the test solution to the microwells of an ELISA plate coated with plasticizer-conjugated antigen, then adding enzyme conjugate working solution and plasticizer-specific antibody working solution, mixing and incubating at room temperature, washing with 1× washing buffer, adding substrate solution, and incubating at room temperature in the dark, finally adding stop solution, and measuring the absorbance of each well at wavelengths of 450 nm / 630 nm; fitting a real-time standard curve with the absorbance values ​​of the standard diluent and the absorbance values ​​of standard solutions containing different concentrations of plasticizers preset in the kit, thereby determining the content of plasticizers in the test solution.

[0016] In some implementations, the determination of absorbance values ​​of pre-set reagent kits containing different concentrations of phthalic acid standard solutions includes: diluting phthalic acid standards to different concentrations using standard diluent; adding different concentrations of phthalic acid standard solutions to the wells of an ELISA plate coated with phthalic acid-conjugated antigen; adding enzyme conjugate working solution and phthalic acid-specific antibody working solution; mixing and incubating at room temperature; washing with 1× washing buffer; adding substrate solution and incubating at room temperature in the dark; finally adding stop solution; and measuring the absorbance of each well at wavelengths of 450 nm and 630 nm.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention optimizes the components of the standard diluent and controls the content of each specific component within a specific range. This ensures that the phthalate standard solutions obtained by diluting the phthalate standard at different temperatures using this standard diluent have a basically consistent inhibition rate, thereby significantly enhancing the stability of the kit. Simultaneously, the standard diluent works synergistically with other components in the kit, giving the enzyme-linked immunosorbent assay (ELISA) kit advantages in detecting phthalates in edible oils, including high specificity, high sensitivity, high accuracy, and high stability. Therefore, this ELISA kit has promising application prospects in the detection of phthalates in edible oils. Attached Figure Description

[0018] Figure 1 This is the standard curve obtained in Embodiment 1 of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0021] Currently, existing enzyme-linked immunosorbent assay (ELISA) kits suffer from problems such as high operational requirements, poor reproducibility, and high costs when testing scattered samples.

[0022] To address the problems of existing enzyme-linked immunosorbent assay (ELISA) kits, such as high operational requirements, poor repeatability, and high cost when testing scattered samples, this invention provides an ELISA kit for detecting plasticizers in edible oils and its application.

[0023] In a first aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting plasticizers in edible oils, comprising: an ELISA plate coated with plasticizer-conjugated antigen, plasticizer standards, enzyme conjugate working solution, plasticizer-specific antibody working solution, purification tubes, purification reagents, standard diluent, sample diluent, substrate solution, stop solution, and 10× concentrated wash buffer; wherein the standard diluent comprises: NaCl with a mass concentration of 0.5-1.5% (m / v), dimethyl sulfoxide with a volume percentage of 3-7% (v / v), mannitol with a mass concentration of 0.1-1% (m / v), PVP-40 with a mass concentration of 0.1-0.3% (m / v), EDTA with a mass concentration of 0.1-1% (m / v), trehalose with a mass concentration of 1-5% (m / v), and 0.05-2M citrate buffer, with a pH value of 5.8-6.5.

[0024] In the standard diluent provided by this invention, the mass concentration of NaCl can be, for example, 0.5%, 1%, 1.5% (m / v) or other values ​​within this range; the volume percentage of dimethyl sulfoxide can be, for example, 3%, 5%, 7% (v / v) or other values ​​within this range; the mass concentration of mannitol can be, for example, 0.1%, 0.5%, 1% (m / v) or other values ​​within this range; the mass concentration of PVP-40 can be, for example, 0.1%, 0.2%, 0.3% (m / v) or other values ​​within this range; the mass concentration of EDTA can be, for example, 0.1%, 0.5%, 1% (m / v) or other values ​​within this range; the mass concentration of trehalose can be, for example, 1%, 3%, 5% (m / v) or other values ​​within this range; the concentration of citrate buffer can be, for example, 0.05M, 0.1M, 2M or other values ​​within this range; and the pH value can be, for example, 5.8, 6, 6.5 or other values ​​within this range.

[0025] The enzyme-linked immunosorbent assay (ELISA) kit provided by this invention optimizes the components of the standard diluent and controls the content of each specific component within a specific range. This ensures that the phthalate standard solutions obtained by diluting the phthalate standard at different temperatures using the standard diluent have a basically consistent inhibition rate, thereby significantly enhancing the stability of the kit. At the same time, the standard diluent works synergistically with other components in the kit, giving the ELISA kit advantages such as high specificity, high sensitivity, high accuracy, and high stability when detecting phthalates in edible oils.

[0026] In some implementations, the coating solution used in the preparation of ELISA plates coated with plasticizer-conjugated antigens includes: 0.05-0.2 mol / L citrate buffer with a pH of 5.8-6.5.

[0027] In the coating solution provided by the present invention, the concentration of citrate buffer can be, for example, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L or other values ​​within this range; the pH value can be, for example, 5.8, 6, 6.5 or other values ​​within this range.

[0028] In this invention, by optimizing the composition and content of the coating solution, the differences between the wells of the ELISA plate can be reduced, thereby ensuring the stability of subsequent detection results.

[0029] In some embodiments, the preparation of the enzyme conjugate working solution includes mixing the enzyme conjugate and the enzyme conjugate / antibody diluent at a ratio of 1:200 to 1:1000 (e.g., 1:200, 1:500, 1:1000 or other values ​​within this range); wherein the enzyme conjugate comprises horseradish peroxidase-conjugated goat anti-mouse IgG.

[0030] In this invention, by optimizing the concentration of the enzyme conjugate working solution, the stability of the enzyme conjugate working solution can be improved, thereby enhancing the detection performance of the kit.

[0031] In some embodiments, the preparation of the plasticizer-specific antibody working solution includes: mixing the plasticizer-specific antibody and the enzyme conjugate / antibody diluent at a ratio of 1:5000-1:100000 (e.g., 1:5000, 1:20000, 1:100000 or other values ​​within this range); wherein the plasticizer-specific antibody includes a plasticizer monoclonal antibody.

[0032] In this invention, by optimizing the concentration of the plasticizer-specific antibody working solution, the stability of the plasticizer-specific antibody working solution can be improved, thereby enhancing the detection performance of the kit.

[0033] In some embodiments, the enzyme conjugate / antibody diluent comprises: 0.03-0.1% (m / v) casein, 0.1-0.5% (m / v) BSA, 0.1-0.5% (m / v) skim milk powder, 1-3% (m / v) sucrose, 0.1-0.5% (m / v) SG09, 0.05-0.2% (v / v) proclin 950, 0.005-0.2% (v / v) β-mercaptoethanol, 0.01-0.2% (v / v) Tween-20, and 0.01-0.2 M phosphate buffer at a pH of 6.5-7.5.

[0034] In the enzyme conjugate / antibody diluent provided by this invention, the mass concentration of casein can be, for example, 0.03%, 0.05%, 0.1% (m / v) or other values ​​within this range; the mass concentration of BSA can be, for example, 0.1%, 0.24%, 0.5% (m / v) or other values ​​within this range; the mass concentration of skim milk powder can be, for example, 0.1%, 0.24%, 0.5% (m / v) or other values ​​within this range; the mass concentration of sucrose can be, for example, 1%, 2%, 3% (m / v) or other values ​​within this range; the mass concentration of SG09 can be, for example, 0.1%, 0.2%, 0.5% (m / v) or other values ​​within this range; proclin The volume percentage of 950 can be, for example, 0.05%, 0.1%, 0.2% (v / v) or other values ​​within this range; the volume percentage of β-mercaptoethanol can be, for example, 0.005%, 0.01%, 0.2% (v / v) or other values ​​within this range; the volume percentage of Tween-20 can be, for example, 0.01%, 0.05%, 0.2% (v / v) or other values ​​within this range; the concentration of phosphate buffer can be, for example, 0.01M, 0.1M, 0.2M or other values ​​within this range; the pH value can be, for example, 6.5, 7, 7.5 or other values ​​within this range.

[0035] In this invention, by optimizing the components and content of the enzyme conjugate / antibody diluent, the stability of the enzyme conjugate working solution and the plasticizer-specific antibody working solution can be significantly improved, thereby enhancing the detection performance of the kit.

[0036] In some embodiments, the purification tube is equipped with solid-phase extraction packing material; and / or, the purification reagent includes a weakly basic reagent (preferably borate buffer); and / or, the 10× concentrated washing solution includes: 0.5-2% (v / v) Tween-20, 0.1-1% (m / v) sodium azide, and 0.1-1M phosphate buffer with a pH of 7-7.8.

[0037] In the 10× concentrated washing solution provided by this invention, the volume percentage of Tween-20 can be, for example, 0.5%, 1%, 2% (v / v) or other values ​​within this range; the mass concentration of sodium azide can be, for example, 0.1%, 0.4%, 1% (m / v) or other values ​​within this range; the concentration of phosphate buffer can be, for example, 0.1M, 0.2M, 1M or other values ​​within this range; and the pH value can be, for example, 7, 7.4, 7.8 or other values ​​within this range.

[0038] Understandably, the solid-phase extraction packing material and purification reagents in the purification tube can be selected according to actual needs, as long as they can efficiently purify the sample.

[0039] In some implementations, the sample diluent includes: 1.5-2.5% (m / v) NaCl, 0.1-1% (m / v) mannitol, 0.1-0.3% (m / v) PVP-40, 0.1-1% (m / v) EDTA, 1-5% (m / v) trehalose, and 0.05-2M citrate buffer with a pH of 5.8-6.5.

[0040] In the sample diluent provided by this invention, the mass concentration of NaCl can be, for example, 1.5%, 2%, 2.5% (m / v) or other values ​​within this range; the mass concentration of mannitol can be, for example, 0.1%, 0.5%, 1% (m / v) or other values ​​within this range; the mass concentration of PVP-40 can be, for example, 0.1%, 0.2%, 0.3% (m / v) or other values ​​within this range; the mass concentration of EDTA can be, for example, 0.1%, 0.5%, 1% (m / v) or other values ​​within this range; the mass concentration of trehalose can be, for example, 1%, 3%, 5% (m / v) or other values ​​within this range; the concentration of citrate buffer can be, for example, 0.05M, 0.1M, 2M or other values ​​within this range; and the pH value can be, for example, 5.8, 6, 6.5 or other values ​​within this range.

[0041] In this invention, by optimizing the composition and content of the sample diluent, the sample recovery rate can be improved, the matrix effect can be eliminated, and the generation of false positive and false negative results can be significantly reduced.

[0042] In a second aspect, the present invention provides the application of any of the above-described enzyme-linked immunosorbent assay kits in the detection of plasticizers in edible oils.

[0043] The detection principle of the enzyme-linked immunosorbent assay (ELISA) kit provided by this invention is as follows: When the phthalate-conjugated antigen is pre-coated on the ELISA plate, after adding the sample solution or standard solution, the phthalate-specific antibody working solution is added. The phthalate in the sample competes with the phthalate-conjugated antigen coated on the ELISA plate for the phthalate-specific antibody. Enzyme-labeled anti-antibody is added for amplification. TMB is used for color development. The absorbance value of the sample is negatively correlated with the phthalate content. Finally, a real-time standard curve is fitted by the absorbance value of the standard dilution and the absorbance values ​​of the standard solutions containing different concentrations of phthalate preset in the kit, so that the phthalate content in the sample can be calculated.

[0044] In a third aspect, the present invention provides a method for detecting plasticizers in edible oils using any of the above-mentioned enzyme-linked immunosorbent assay (ELISA) kits, comprising the following steps: extracting the sample to be tested using an organic solvent, adding the resulting subnaphthalene to a purification tube, adding purification reagent and performing extraction, adding sample diluent to the resulting supernatant to obtain the test solution; adding standard diluent and the test solution to the microwells of an ELISA plate coated with plasticizer-conjugated antigen, then adding enzyme conjugate working solution and plasticizer-specific antibody working solution, mixing and incubating at room temperature, washing with 1× washing buffer, adding substrate solution, and incubating at room temperature in the dark, finally adding stop solution, and measuring the absorbance of each well at wavelengths of 450 nm / 630 nm; fitting a real-time standard curve with the absorbance values ​​of the standard diluent and the absorbance values ​​of standard solutions containing different concentrations of plasticizers preset in the kit, thereby determining the content of plasticizers in the test solution.

[0045] The detection method provided by this invention uses a pre-set absorbance value of a standard solution containing different concentrations of plasticizer in the kit. This absorbance value is then used to fit a real-time standard curve with the absorbance value of the standard dilution. This eliminates the need to test five standard concentrations for each sample test to obtain a standard curve; instead, only the absorbance value of a single standard dilution needs to be tested to determine the plasticizer content in the sample. This invention's single standard point setting is suitable for high-throughput sample detection and saves costs when dealing with small sample volumes. Furthermore, it avoids the accuracy of plasticizer content in samples being affected by differences in the standard curve caused by human operation, environmental temperature fluctuations, etc. In addition, the sample pretreatment steps are simple and time-saving, and the enzyme-linked immunosorbent assay (ELISA) kit has advantages such as high specificity, high sensitivity, and high accuracy.

[0046] In some implementations, the determination of absorbance values ​​of pre-set reagent kits containing different concentrations of phthalic acid standard solutions includes: diluting phthalic acid standards to different concentrations using standard diluent; adding different concentrations of phthalic acid standard solutions to the wells of an ELISA plate coated with phthalic acid-conjugated antigen; adding enzyme conjugate working solution and phthalic acid-specific antibody working solution; mixing and incubating at room temperature; washing with 1× washing buffer; adding substrate solution and incubating at room temperature in the dark; finally adding stop solution; and measuring the absorbance of each well at wavelengths of 450 nm and 630 nm.

[0047] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0048] Example 1: Component Optimization of Enzyme-Linked Immunosorbent Assay Kit 1.1 Optimization of coating solution components in the preparation of enzyme-linked immunosorbent assay (ELISA) plates coated with plasticizer-conjugated antigens 1) Prepare coating solution 1: 0.1 mol / L carbonate buffer, pH 9.6; coating solution 2: 0.1 mol / L PB buffer, pH 7.4; coating solution 3: 0.1 mol / L citrate buffer, pH 6. 2) Dilute the plasticizer-conjugated antigen to 0.1 μg / mL with the coating solution from step 1), add 100 μL to each well, incubate overnight at 4°C, discard the coating solution, wash 3 times with 1× washing solution for 15 s each time, discard the liquid in the well, absorb the residual water with absorbent paper, add 200 μL of blocking solution (0.02 mol / L phosphate buffer containing 3% (w / v) skim milk powder, pH 7.4) to each well, incubate at 37°C for 2 h, discard the liquid in the well, dry, vacuum seal with aluminum foil, and store at 4°C for later use; 3) The working solution of the enzyme conjugate was determined by matrix titration experiment, with an absorbance value of 0.8-1.2 as the reference standard. The enzyme conjugate was mixed with 1× washing buffer at a ratio of 1:1000-1:100000 to obtain the working solution of the enzyme conjugate. 50 μL of deionized water and 50 μL of enzyme conjugate working solution were added to the wells of the above different microplates, mixed well and incubated at room temperature for 20 min. After washing 5 times and drying, 100 μL of substrate solution was added to each well and incubated at room temperature in the dark for 10 min. 100 μL of stop solution was added and the absorbance value was measured at wavelengths of 450 nm and 630 nm. The coefficient of variation of each well in the microplate was calculated. The results are shown in Table 1 below.

[0049] Table 1. Coefficient of variation results for each well of the ELISA plate.

[0050] As can be seen from Table 1, using coating solution 3 to prepare the ELISA plate can result in a smaller coefficient of variation for each well, thus ensuring the stability of the subsequent kit detection results.

[0051] 1.2 Optimization of components in enzyme conjugate working solution and plasticizer-specific antibody working solution 1) Preparation of enzyme conjugate / antibody dilution buffer: 0.05% (m / v) casein, 0.24% (m / v) BSA, 0.24% (m / v) skim milk powder, 2% (m / v) sucrose, 0.2% (m / v) SG09, 0.1% (v / v) proclin 950, 0.01% (v / v) β-mercaptoethanol, 0.05% (v / v) Tween-20, 0.02M phosphate buffer, pH 7; 2) Using a matrix titration experiment and with an absorbance value of 1.8-2.2 as the reference standard, the phthalic acid-specific antibody working solution was prepared by mixing the enzyme conjugate / antibody diluent from step 1) at a ratio of 1:5000-1:100000. The horseradish peroxidase-conjugated goat anti-mouse IgG was mixed with the enzyme conjugate / antibody diluent from step 1) at a ratio of 1:200-1:1000 to prepare the enzyme conjugate working solution. 50 μL of standard diluent, 50 μL of enzyme conjugate working solution, and 50 μL of phthalic acid-specific antibody were added to the wells of the ELISA plate as determined above. After mixing the working solution, incubate at room temperature for 20 min, wash 5 times, pat dry, add 100 μL of substrate solution to each well, incubate at room temperature in the dark for 10 min, add 100 μL of stop solution, and measure the absorbance at 450 nm / 630 nm wavelengths to determine that the dilution factor of the plasticizer-specific antibody working solution is 20,000 times and the dilution factor of the enzyme conjugate working solution is 500 times. Divide them into two portions, one portion is refrigerated at 4℃ and the other portion is placed in a constant temperature incubator at 37℃. Accelerated comparison experiments are performed at different intervals to detect the stability of the plasticizer-specific antibody and enzyme conjugate working solutions. The results are shown in Table 2 below.

[0052] Table 2. Results of accelerated comparison experiments using plasticizer-specific antibody and enzyme conjugate working solutions.

[0053] As can be seen from Table 2, using the optimized enzyme conjugate / antibody diluent can improve the stability of the plasticizer-specific antibody and enzyme conjugate working solution prepared with the enzyme conjugate / antibody diluent, thereby improving the detection performance of the kit.

[0054] 1.3 Optimization of Standard Diluent Components 1) Determination of the components of the standard diluent Based on the standard diluent with 1% (m / v) NaCl, 5% (v / v) dimethyl sulfoxide, 0.1M citrate buffer, and pH 6 as the main components, the effects of adding mannitol, PVP-40, EDTA, and trehalose on the performance of the standard diluent were further investigated. Specifically, the following experiments were conducted: Mannitol was added to the main components of the standard diluent to make their mass concentrations 0.1%, 1%, and 5% (m / v), respectively. PVP-40 was added to the main components of the standard diluent to achieve mass concentrations of 0.1%, 0.3%, and 1% (m / v). EDTA was added to the main components of the standard diluent to achieve mass concentrations of 0.1%, 0.3%, and 1% (m / v). Trehalose was added to the main components of the standard diluent to achieve mass concentrations of 1%, 5%, and 10% (m / v). A total of 12 standard diluents and their main components were used as control groups. The above 13 reagents were used as standard diluents. The plasticizer standard (100 ppm, dissolved in dimethyl sulfoxide) was diluted to 0 μg / kg and 10 μg / kg. The plasticizer-specific antibody was diluted to 20,000 times and the enzyme conjugate to 500 times using enzyme conjugate / antibody diluent. 50 μL of standard was added to each well of the enzyme-labeled plate, followed by 50 μL of enzyme conjugate working solution and 50 μL of antibody working solution. After mixing, the plate was incubated at room temperature for 20 min, washed 5 times, and patted dry. 100 μL of substrate solution was added to each well, and the plate was incubated at room temperature in the dark for 10 min. 100 μL of stop solution was added, and the absorbance was measured at wavelengths of 450 nm and 630 nm. The results are shown in Table 3 below.

[0055] Table 3 Results of the standard diluent composition optimization experiment

[0056] As shown in Table 3, adding mannitol at a mass concentration of 0.1% or 1% (m / v), PVP-40 at a mass concentration of 0.1% or 0.3% (m / v), EDTA at a mass concentration of 0.1%, 0.3%, or 1% (m / v), or trehalose at a mass concentration of 1% or 5% (m / v) to the main components of the standard diluent had no significant effect on the inhibition of the standard or the OD value of the zero standard. The results indicate that adding specific amounts of mannitol, PVP-40, EDTA, and trehalose can enhance the stability of the standard diluent.

[0057] 2) Standard Curve Stability Experiment Preparation of standard diluent: 1% (m / v) NaCl, 5% (v / v) dimethyl sulfoxide, 1% (m / v) mannitol, 0.3% (m / v) PVP-40, 1% (m / v) EDTA, 5% (m / v) trehalose, and 0.1M citrate buffer, pH 6. Using this standard diluent, plasticizer standards were prepared into standard solutions of 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg. Using the main components of the standard diluent as a control group, plasticizer standards were prepared into standard solutions of 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg. ELISA detection was performed at 35℃, 25℃, and 15℃, respectively. The results are shown in Table 4 below.

[0058] Table 4. Experimental results of standard curve stability

[0059] As can be seen from Table 4, by adding specific amounts of mannitol, PVP-40, EDTA and trehalose to the standard diluent, the standard solutions of different concentrations obtained by using this standard diluent can have basically consistent inhibition rates at different ambient temperatures, which significantly improves the detection performance of the kit at different ambient temperatures.

[0060] 1.4 Optimization of Sample Diluent Components 1) Preparation of sample diluent To maintain consistency with the standard solution, the sample diluent can be adjusted based on the standard diluent to counteract matrix interference. The specific procedure is as follows: Remove dimethyl sulfoxide from the standard diluent, and then adjust the amount of NaCl added to achieve a concentration between 1-5% (m / v). Prepare the sample diluent as follows: Sample dilution 1: 1% (m / v) NaCl, 1% (m / v) mannitol, 0.3% (m / v) PVP-40, 1% (m / v) EDTA, 5% (m / v) trehalose, 0.1M citrate buffer, pH 6; Sample diluent 2: The composition is basically the same as that of sample diluent 1, except that the mass concentration of NaCl is 2% (m / v). Sample diluent 3: The composition is basically the same as that of sample diluent 1, except that the mass concentration of NaCl is 3% (m / v). Sample diluent 4: The composition is basically the same as that of sample diluent 1, except that the mass concentration of NaCl is 4% (m / v). Sample diluent 5: The composition is basically the same as that of sample diluent 1, except that the mass concentration of NaCl is 5% (m / v).

[0061] 2) Sample pretreatment Weigh 100g of edible oil sample that was negative for plasticizers, add plasticizer standard to it, theoretically so that the final plasticizer content in the sample is 500μg / kg, mix thoroughly and use an instrument to confirm the plasticizer content in the sample, and obtain a plasticizer positive sample with a total plasticizer content of 496μg / kg. Five groups of samples were weighed, each containing one tube of negative sample and one tube of positive sample, with 1.5g of sample in each tube. 1.5mL of dimethyl sulfoxide was added, and the mixture was extracted for 3min. Then, the mixture was centrifuged at 4000r / min for 1min to obtain the lower supernatant. 1mL of the lower supernatant was taken and added to a purification tube (equipped with solid-phase extraction packing material) and mixed. 0.2mL of purification reagent (borate buffer) was added, and the mixture was vortexed for 1min. After centrifugation for 1min, 0.1mL of the upper supernatant was taken and 0.9mL of sample diluent was added. The mixture was vortexed for 1min to obtain the test solution.

[0062] Using the standard diluent from step 1.3, the plasticizer standard (100 ppm, dissolved in dimethyl sulfoxide) was diluted to 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg. The plasticizer-specific antibody was diluted to 20,000 times and the enzyme conjugate to 500 times using the enzyme conjugate / antibody diluent. 50 μL of the standard was added to each well of the microplate, followed by 50 μL of enzyme conjugate working solution and 50 μL of plasticizer-specific antibody working solution. After mixing, the plate was incubated at room temperature for 20 min, washed 5 times, and patted dry. 100 μL of substrate solution was added to each well, and the plate was incubated at room temperature in the dark for 10 min. 100 μL of stop solution was added, and the absorbance was measured at wavelengths of 450 nm and 630 nm. The results are shown in Table 5 below.

[0063] Table 5 Optimization results of sample diluent components

[0064] As can be seen from Table 5, optimizing the composition and content of the sample diluent can improve the sample recovery rate, eliminate the influence of the matrix, and significantly reduce the occurrence of false positive and false negative results.

[0065] 1.5 Determination of absorbance values ​​of standard solutions containing different concentrations of plasticizers pre-set in the kit Using the optimized standard diluent described above, the plasticizer standard was diluted to standard solutions of 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg. The plasticizer-specific antibody was diluted 20,000 times and the enzyme conjugate was diluted 500 times using the enzyme conjugate / antibody diluent described above. 50 μL of each solution was added to wells containing a coating concentration of 0.1 μg / mL. Five standards at concentrations of 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg were added, along with 50 μL of enzyme conjugate working solution and 50 μL of antibody working solution. After mixing, the mixture was incubated at room temperature for 20 min, washed five times, and patted dry. 100 μL of substrate solution was added to each well, and the mixture was incubated at room temperature in the dark for 10 min. 100 μL of stop solution was added, and the absorbance was measured at wavelengths of 450 nm and 630 nm. The percentage absorbance of the standards was calculated using the following formula: Percentage absorbance (%) = B / B0 × 100%; where B is the average absorbance of the standard solution, and B0 is the average absorbance of the 0 μg / kg standard solution. The percentage absorbance of the standards was measured repeatedly and recorded. The average value was taken, and the results are shown in Table 6 below.

[0066] Table 6. Percentage absorbance values ​​of standard samples

[0067] Using the four concentrations of 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg in Table 6 as the x-axis and the logit value of their corresponding percentage absorbance as the y-axis, a linear fit was performed to obtain the pre-set standard curves containing standard solutions of different concentrations of plasticizer. Figure 1 ).

[0068] Furthermore, the phthalate content in the sample was tested using the kit via single-standard measurement (the test method of this invention) and full-standard curve measurement (the conventional method).

[0069] Specifically: 10 groups of samples were weighed, each group containing one tube of negative sample (ND) and one tube of positive sample (496 μg / kg), with 1.5 g of sample per tube. 1.5 mL of dimethyl sulfoxide was added, and the mixture was extracted for 3 min. Then, it was centrifuged for 1 min to obtain the lower supernatant. 1 mL of the lower supernatant was taken and added to a purification tube (equipped with solid-phase extraction packing material) and mixed. 0.2 mL of purification reagent (borate buffer) was added, and the mixture was extracted for 1 min. After centrifugation for 1 min, the upper supernatant was taken and 0.9 mL of sample diluent was added. The mixture was vortexed for 1 min to obtain the test solution. These 10 groups of test sample solutions were distributed to 10 different personnel for testing.

[0070] Using the optimized standard diluent described above, the plasticizer standard (100 ppm, dissolved in dimethyl sulfoxide) was diluted to 0 μg / kg, 3 μg / kg, 9 μg / kg, 27 μg / kg, and 81 μg / kg. The plasticizer-specific antibody was diluted 20,000-fold with the enzyme conjugate / antibody diluent, and the enzyme conjugate was diluted 500-fold. 50 μL of the standard was added to each well of the microplate, followed by 50 μL of enzyme conjugate working solution and 50 μL of antibody working solution. After mixing, the plate was incubated at room temperature for 20 min, washed 5 times, patted dry, and then the contents of each well were added... 100 μL of substrate solution was incubated at room temperature in the dark for 10 min. 100 μL of stop solution was added, and the absorbance was measured at wavelengths of 450 nm and 630 nm. A standard curve was plotted based on the concentration and absorbance of the plasticizer standard solution to determine the plasticizer content in the test solution (i.e., full standard curve measurement, conventional method). At the same time, the plasticizer content was calculated using the standard curve provided with the kit of this invention by comparing the absorbance values ​​of the standard dilution solution (0 μg / kg standard) and the test sample solution (i.e., single standard measurement, method of this invention). The results of the comparison are shown in Table 7 below.

[0071] Table 7 Comparison of full standard curve measurements and single standard curve measurements

[0072] As can be seen from Table 7, using the kit of the present invention for single-label detection results in more stable single-label values, which to some extent reduces detection errors caused by differences in personnel operation and environmental factors.

[0073] Example 2 Composition of the enzyme-linked immunosorbent assay kit This embodiment provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting plasticizers in edible oils, comprising the following components: 1) ELISA plates coated with plasticizer-conjugated antigens; 2) Plasticizer standard; 3) Enzyme conjugate working solution (obtained by mixing horseradish peroxidase-conjugated goat anti-mouse IgG with enzyme conjugate / antibody dilution at a ratio of 1:200-1:1000); 4) Plasticizer-specific antibody working solution (obtained by mixing plasticizer monoclonal antibody with enzyme conjugate / antibody dilution solution at a ratio of 1:5000-1:100000); 5) Purification tube (equipped with solid-phase extraction packing); 6) Purification reagent (weakly alkaline reagent, preferably borate buffer); 7) Standard dilution buffer (including: 0.5-1.5% (m / v) NaCl, 3-7% (v / v) dimethyl sulfoxide, 0.1-1% (m / v) mannitol, 0.1-0.3% (m / v) PVP-40, 0.1-1% (m / v) EDTA, 1-5% (m / v) trehalose, 0.05-2M citrate buffer, pH 5.8-6.5); 8) Sample diluent (including: 1.5-2.5% (m / v) NaCl, 0.1-1% (m / v) mannitol, 0.1-0.3% (m / v) PVP-40, 0.1-1% (m / v) EDTA, 1-5% (m / v) trehalose, 0.05-2M citrate buffer, pH 5.8-6.5); 9) Substrate solution (TMB solution); 10) Termination solution (2 mol / L hydrochloric acid solution); 11) 10× concentrated washing solution (0.5-2% (v / v) Tween-20, 0.1-1% (m / v) sodium azide, 0.1-1M phosphate buffer, pH 7-7.8). 12) Preset absorbance values ​​of standard solutions containing different concentrations of plasticizer; Among them, the enzyme conjugate / antibody diluents in 3) and 4) include: casein with a mass concentration of 0.03-0.1% (m / v), BSA with a mass concentration of 0.1-0.5% (m / v), skim milk powder with a mass concentration of 0.1-0.5% (m / v), sucrose with a mass concentration of 1-3% (m / v), SG09 with a mass concentration of 0.1-0.5% (m / v), proclin 950 with a volume percentage of 0.05-0.2% (v / v), β-mercaptoethanol with a volume percentage of 0.005-0.2% (v / v), Tween-20 with a volume percentage of 0.01-0.2% (v / v), and phosphate buffer with a pH of 6.5-7.5.

[0074] Example 3: Detection of plasticizers in actual edible oils 1) Sample pretreatment Weigh 1.5g of edible oil sample into each tube, add 1.5mL of dimethyl sulfoxide, extract for 3min, then centrifuge for 1min to obtain the lower supernatant. Take 1mL of the lower supernatant and add it to the purification tube, mix well, then add 0.2mL of purification reagent, extract for 1min, centrifuge for 1min, take the upper supernatant, add 0.9mL of sample diluent, vortex for 1min to obtain the test solution.

[0075] 2) Detection using a kit Add 50 μL of standard dilution solution (0 μg / kg phthalic acid) to the microplate wells coated with phthalic acid conjugated antigen, followed by 50 μL of enzyme conjugate working solution, and then 50 μL of phthalic acid-specific antibody working solution. Mix well and incubate at room temperature for 20 min. Wash 5 times, pat dry, add 100 μL of substrate solution to each well, incubate at room temperature in the dark for 10 min, add 100 μL of stop solution, and measure the absorbance at 450 nm / 630 nm using an automated microplate reader.

[0076] By fitting the absorbance values ​​of the standard dilution solution and the absorbance values ​​of the standard solutions containing different concentrations of plasticizers preset in the kit to generate a real-time standard curve, the content of plasticizers in the sample can be calculated. Based on the detection results, it can be determined whether there is plasticizer migration in the edible oil. The results are shown in Table 8 below.

[0077] Table 8 Actual Sample Detection Results

[0078] As can be seen from Table 8, the detection data of the kit of the present invention are similar to the data detected by the instrument, indicating that the kit of the present invention has the advantage of high accuracy.

[0079] Example 4: Performance Testing of the Reagent Kit 4.1 Sample Precision Test Edible oils with a plasticizer concentration of 370 μg / kg were tested using the above method. Three different batches of the kit were used, and the test was repeated 5 times for each concentration. The coefficient of variation was calculated, and the results are shown in Table 9 below.

[0080] Table 9 Precision test results of edible oil samples

[0081] As can be seen from Table 9, the coefficient of variation is small when using different batches of the kit of the present invention for detection, indicating that the kit of the present invention has good batch stability and precision.

[0082] 4.2 Sample Accuracy Test Two edible oil samples with plasticizer contents of 300 μg / kg and 500 μg / kg were taken and tested according to the method described above in the kit of this invention. Four replicates were performed for each sample, and the accuracy was calculated. The results are shown in Table 10 below.

[0083] Table 10. Accuracy results of the reagent kit

[0084] As can be seen from Table 10, the kit of the present invention has an accuracy of 80-115% in detecting edible oil samples, and the coefficient of variation is within 15%. Therefore, the kit of the present invention has good accuracy.

[0085] 4.3 Reagent kit stability test The kit was stored at 2-8℃. After 12 months of testing, the maximum absorbance (zero standard), 50% inhibition concentration, and actual measured values ​​of plasticizer addition were all within the normal range. Considering that abnormal storage conditions may occur during transportation and use, the kit was placed at 37℃ for 15 days for accelerated aging experiments. The results are shown in Table 11.

[0086] Table 11 Results of accelerated aging test of the reagent kit

[0087] As can be seen from Table 11, all indicators of the kit fully meet the requirements. From the above results, it can be concluded that the kit of this invention can be stored at 2-8℃ for more than 12 months.

[0088] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0089] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for detecting plasticizers in edible oils, characterized in that, include: ELISA plates coated with plasticizer-conjugated antigens, plasticizer standards, enzyme conjugate working solutions, plasticizer-specific antibody working solutions, purification tubes, purification reagents, standard diluents, sample diluents, substrate solutions, stop solutions, and 10× concentrated wash solutions; The standard diluent comprises: 0.5-1.5% NaCl (w / v), 3-7% dimethyl sulfoxide (v / v), 0.1-1% mannitol (w / v), 0.1-0.3% PVP-40 (w / v), 0.1-1% EDTA (w / v), 1-5% trehalose (w / v), and 0.05-2M citrate buffer with a pH of 5.8-6.

5.

2. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The coating solution used in the preparation of the enzyme-linked immunosorbent assay (ELISA) plate coated with plasticizer-conjugated antigen includes: 0.05-0.2 mol / L citrate buffer with a pH of 5.8-6.

5.

3. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The preparation of the enzyme conjugate working solution includes: mixing the enzyme conjugate and the enzyme conjugate / antibody diluent at a ratio of 1:200-1:1000; The enzyme conjugate includes horseradish peroxidase-conjugated goat anti-mouse IgG.

4. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The preparation of the plasticizer-specific antibody working solution includes: mixing the plasticizer-specific antibody and the enzyme conjugate / antibody dilution solution at a ratio of 1:5000-1:100000; The plasticizer-specific antibodies include plasticizer monoclonal antibodies.

5. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 3 or 4, characterized in that, The enzyme conjugate / antibody diluent comprises: 0.03-0.1% casein, 0.1-0.5% BSA, 0.1-0.5% skim milk powder, 1-3% sucrose, 0.1-0.5% SG09, 0.05-0.2% proclin 950, 0.005-0.2% β-mercaptoethanol, 0.01-0.2% Tween-20, and 0.01-0.2M phosphate buffer with a pH of 6.5-7.

5.

6. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The purification tube is equipped with solid-phase extraction packing material; and / or The purification reagent includes a weakly alkaline reagent; and / or, The 10× concentrated washing solution comprises: 0.5-2% Tween-20 by volume, 0.1-1% sodium azide by mass, and 0.1-1M phosphate buffer with a pH of 7-7.

8.

7. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The sample diluent comprises: 1.5-2.5% NaCl, 0.1-1% mannitol, 0.1-0.3% PVP-40, 0.1-1% EDTA, 1-5% trehalose, and 0.05-2M citrate buffer with a pH of 5.8-6.

5.

8. The application of the enzyme-linked immunosorbent assay kit as described in any one of claims 1-7 in the detection of plasticizers in edible oils.

9. A method for detecting plasticizers in edible oils using the enzyme-linked immunosorbent assay kit according to any one of claims 1-7, characterized in that, Includes the following steps: The sample to be tested was extracted using an organic solvent. The resulting subnaphthalene was added to a purification tube, and purification reagent was added again for further extraction. Sample diluent was added to the resulting supernatant to obtain the test solution. Add standard dilution and the test solution to the microwells of the ELISA plate coated with plasticizer-conjugated antigen, respectively. Then add enzyme conjugate working solution and plasticizer-specific antibody working solution, mix well and incubate at room temperature. After washing with 1× washing solution, add substrate solution and incubate at room temperature in the dark. Finally, add stop solution and measure the absorbance of each well at wavelengths of 450 nm / 630 nm. A real-time standard curve is generated by fitting the absorbance values ​​of the standard dilution solution and the absorbance values ​​of standard solutions containing different concentrations of plasticizers preset in the kit, thereby determining the content of plasticizers in the test solution.

10. The method according to claim 9, characterized in that, The assay of absorbance values ​​of the pre-set kit containing different concentrations of phthalic acid standard solutions includes: diluting the phthalic acid standard into different concentrations using standard diluent; adding the different concentrations of phthalic acid standard solutions to the microwells of an ELISA plate coated with phthalic acid conjugate antigen; then adding enzyme conjugate working solution and phthalic acid-specific antibody working solution; mixing and incubating at room temperature; washing with 1× washing buffer; adding substrate solution and incubating at room temperature in the dark; finally adding stop solution; and measuring the absorbance of each well at wavelengths of 450 nm and 630 nm.

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