Enzyme-linked immunoassay kit for benzopyrene in edible oil, kitchen fume and automobile exhaust and method of using the same

Abstract

This invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting benzo[a]pyrene in edible oil, kitchen fumes, and vehicle exhaust. The kit includes: a sample reconstitution solution, an enzyme conjugate / antibody diluent, a standard / sample diluent, a benzo[a]pyrene standard, a benzo[a]pyrene-specific mouse monoclonal antibody, an enzyme conjugate, an ELISA plate coated with benzo[a]pyrene antigen, a chromogenic solution, a stop solution, and a washing solution. This invention also discloses a method for detecting benzo[a]pyrene in edible oil, kitchen fumes, and vehicle exhaust using the kit, including sample pretreatment, ELISA detection, and result processing and analysis. The sample reconstitution solution, enzyme conjugate / antibody diluent, and standard / sample diluent provided by this invention overcome the solubility problem of benzo[a]pyrene in edible oil, kitchen fumes, and vehicle exhaust while maintaining antibody sensitivity, and can also significantly improve antibody titer, thus possessing significant value for widespread application.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay, specifically relating to an enzyme-linked immunosorbent assay kit for detecting benzo[a]pyrene in oils and fats, which is particularly suitable for detecting benzo[a]pyrene in samples containing oil components such as edible oils, kitchen fumes, and automobile exhaust. Background Technology

[0002] Benzo[a]pyrene, a polycyclic aromatic hydrocarbon containing a benzene ring, is a potent carcinogen. Depending on the position of the fused benzene ring, benzo[a]pyrene has several isomers, the two most common being benzo[a]pyrene, which is carcinogenic, and benzo[e]pyrene, which is also highly carcinogenic. It is generally produced during production and daily life through the incomplete combustion of fuels such as petroleum, coal, and natural gas, or the high-temperature decomposition of organic matter. It is widely present in the environment along with other polycyclic aromatic hydrocarbons, including in air, water, soil and sediments, smoked foods, grilled and fried foods, and is also emitted into the air by automobile exhaust. Currently, as one of the most toxic carcinogens among polycyclic aromatic hydrocarbons, the state has clear regulations on the limits of benzo[a]pyrene in air, food, cooking oil, fumes, and emissions, and strictly regulates and controls its use. Therefore, the detection of benzo[a]pyrene content in air, food, cooking oil, and fumes is extremely important for human health.

[0003] Currently, the detection of benzo[a]pyrene in edible oils primarily relies on instrumental methods: gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and liquid chromatography-fluorescence detection. These methods require large and expensive equipment, are relatively complex and time-consuming, and consume large amounts of toxic organic reagents, such as dichloromethane, easily generating significant amounts of toxic waste. Therefore, well-equipped laboratories are necessary for benzo[a]pyrene detection in oils. Enzyme-linked immunosorbent assay (ELISA) is another method for determining benzo[a]pyrene. This comprehensive technique combines antigen-antibody immune reactions with highly efficient enzyme catalysis, offering advantages such as high specificity, high sensitivity, and ease of operation. However, commercially available rapid benzo[a]pyrene detection products are scarce. Most products have limited applicability, suitable only for simple samples like water, and have long detection times (over 1.5 hours, excluding pretreatment), making them unsuitable for rapid analysis and detection of large volumes of samples. For complex samples like edible oils, there are currently no stable, rapid, and reliable rapid detection products. This is because benzo[a]pyrene has a planar rigid structure with five coplanar benzene rings. It is a nonpolar molecule, insoluble in water, but highly soluble in edible oils. Therefore, the detection of benzo[a]pyrene in edible oils faces the following challenges: extraction and purification are difficult; immunoassay methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay all require antigen-antibody reactions, which must be carried out in an aqueous phase. The poor water solubility of benzo[a]pyrene leads to poor reaction sensitivity. To solve this problem, organic reagents with relatively high concentrations need to be introduced into the system; the introduction of organic reagents affects the stability of rapid detection reagents; the kit must include a standard solution, but benzo[a]pyrene is insoluble in water. Therefore, pure organic reagents must be used as solvents, and the solution must be diluted just before use. Otherwise, benzo[a]pyrene will precipitate from the standard solution and adsorb onto the container walls.

[0004] In the prior art, CN105738611A discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting benzo[a]pyrene residues in soil and water. The matrix composition of water samples is relatively simple, making it relatively easy to extract benzo[a]pyrene. Another example is CN115028733A, which provides an ELISA method and kit for detecting pyrene and benzo[a]pyrene residues in aquatic products such as fish, shrimp, and crab. However, these ELISA kits face the following challenges when detecting benzo[a]pyrene in oils: 1) The main component of oils is triglycerides, which can hinder the reaction between antibodies and antigens (forming a hydrophobic layer on the surface of micropores or proteins, hindering the binding of antigens and antibodies, or the insertion of hydrophobic groups into the protein, leading to conformational changes); 2) The influence of pigments, vitamins, terpenoids, etc., in oils on antigen-antibody reactions; 3) Recycled kitchen oils and oils repeatedly fried or boiled tend to have more complex matrix compositions due to the introduction of other food-derived components; 4) Organic reagents with good benzo[a]pyrene solubility are often miscible with oils. Meanwhile, the aforementioned kits are all designed for the detection of benzo[a]pyrene content in water samples, soil, or aquatic products, and research on enzyme-linked immunosorbent assay (ELISA) for benzo[a]pyrene in oils is lacking. Benzo[a]pyrene is highly soluble in oils, making it difficult to detect in samples such as edible oils, kitchen fumes, and vehicle exhaust, which is a major challenge in detecting benzo[a]pyrene in oils. Therefore, effectively separating and purifying benzo[a]pyrene from oils while considering efficiency, cost, sensitivity, and environmental friendliness, and eliminating the influence of these impurities on ELISA, has become a major obstacle for rapid immunoassay methods. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem that benzo[a]pyrene is easily soluble in oils but poorly soluble in water. A stable, rapid, simple, and highly sensitive enzyme-linked immunosorbent assay (ELISA) kit has been developed for detecting residual benzo[a]pyrene in edible oils. This invention is not limited by laboratory equipment conditions and can enable rapid detection of benzo[a]pyrene in oils anytime and anywhere.

[0006] Another object of the present invention is to provide a rapid quantitative method for detecting benzo[a]pyrene in edible oils.

[0007] To achieve the above objectives, this invention employs the following measurement principle: the antigen in the wells of the ELISA plate competes with the analyte in the sample for binding to the added benzo[a]pyrene antibody. After washing, unbound benzo[a]pyrene antibody is removed, leaving the antigen-benzo[a]pyrene antibody conjugate. This conjugate undergoes an enzyme reaction, followed by the addition of a substrate for color development. Since the amounts of antigen and antibody are constant, a higher concentration of the analyte in the sample results in more antibody binding and less antibody binding to the antigen on the ELISA plate, leading to a weaker color reaction. The benzo[a]pyrene content in the standard / sample is negatively correlated with its absorbance. A log-logit standard curve of concentration versus absorbance is established using a series of known concentrations of benzo[a]pyrene standards and their absorbances. By substituting the absorbance of the sample into the curve, the concentration of benzo[a]pyrene in the sample can be determined. Furthermore, since the reaction needs to be carried out in the aqueous phase, in order to solve the solubility problem of benzo[a]pyrene while maintaining high sensitivity, this invention uses a methanol reconstitution solution for reconstitution, and adds glycerol, trehalose, and sorbitol to the enzyme conjugate / antibody diluent, and adds Mg to the standard / sample diluent. 2+ Sodium citrate effectively maintains the stability of core components such as antibodies and enzyme conjugates, increases the solubility of benzo[a]pyrene and maintains its stability in aqueous solution, while simultaneously altering and maintaining the dominant conformation of the antibody (the antibody preferentially reacts with free benzo[a]pyrene in solution; and maintains the binding performance of the antibody to the antigen on the microwells when the analyte is absent). To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The enzyme-linked immunosorbent assay (ELISA) kit provided by this invention comprises the following components:

[0009] (1) Enzyme conjugate / antibody dilution;

[0010] (2) Standard diluent and sample diluent;

[0011] (3) Sample reconstitution solution

[0012] The components of the enzyme-linked immunosorbent assay kit also include benzo[a]pyrene standard, benzo[a]pyrene-specific mouse monoclonal antibody, enzyme conjugate, ELISA plate coated with benzo[a]pyrene antigen, chromogenic solution, stop solution, and washing solution.

[0013] The enzyme conjugate / antibody diluent is formulated as follows: 20 mmol / L phosphate buffer, 5% v glycerol, 1 wt% trehalose, 2 wt% sorbitol, 0.1 wt% preservative, and also contains 0.1 wt% SG09 or 0.1 wt% BSA, with a pH of 7.4.

[0014] The standard diluent formulation is: 50 v% methanol aqueous solution, 40-332.5 nmol / L mg. 2+(Generally, magnesium sulfate can be used, and the concentration of magnesium sulfate in the standard diluent is calculated to be 0.05-0.4 wt%), and sodium citrate 0.05-2 wt%; the sample diluent formulation is 40-665 nmol / L mg. 2+ (Generally, magnesium sulfate can be used, and the concentration of magnesium sulfate in the standard dilution solution is 0.05-0.8wt%), and sodium citrate is 0.05-4wt%.

[0015] In each of the formulations, v% represents volume percentage and wt% represents mass percentage.

[0016] Preferably, the preservative is proclin 950.

[0017] Preferably, the standard diluent is formulated as a 50% v% methanol aqueous solution, further containing 166.2 nmol / L g. 2+ (Generally, magnesium sulfate can be used; the concentration of magnesium sulfate in the standard diluent is calculated to be 0.2 wt% MgSO4), 0.5 wt% sodium citrate; the sample diluent formulation is 332.4 nmol / L Mg. 2+ (Generally, magnesium sulfate can be used. The concentration of magnesium sulfate in the standard dilution solution is 0.4wt% MgSO4, and 1wt% sodium citrate can be used.)

[0018] The benzo[a]pyrene standard solution has several gradient concentrations. Generally, the gradient concentrations (i.e., dilution factors) of the standard solution can be optimized and adjusted according to the actual situation, which is a conventional technique in this field.

[0019] The enzyme-labeled plate coated with benzo[a]pyrene antigen used BaP-BSA (11.5 mg / mL) as the antigen, and the coating solution was 0.05 mol / L carbonate buffer at pH 9.6.

[0020] The benzo[a]pyrene specific antibody is 2.5 mg / mL BaP-ab, and its working solution is a mixture of the antibody and the above-mentioned enzyme conjugate / antibody dilution at a ratio of 1:200000.

[0021] The enzyme conjugate is Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG,FcγFragment Specific (1.5 mL), and its working solution is a mixture of the enzyme conjugate and the above enzyme conjugate dilution at a ratio of 1:1000.

[0022] The colorimetric solution is a single-component colorimetric solution VII (Biopanda, TMB-S-004), and the stop solution is 1 mol / L HCl. The selection of the colorimetric solution and the stop solution is a conventional technique in the art, and other commonly used colorimetric solutions and stop solutions that can achieve the same effect can also be used.

[0023] In step 1 of this invention, steps (6) and (7) involve adding pure methanol solution for reconstitution to maximize the extraction of benzo[a]pyrene from the sample. Specifically, benzo[a]pyrene is slightly soluble in methanol, and methanol is miscible with water; therefore, methanol acts as a solubilizer. While existing patent CN105738611A also uses 30% methanol, its function is to alter the conformation of the antibody, thereby changing the binding performance between the antibody and the analyte. This is significantly different from the technical effect and mechanism of action of this invention, and is fundamentally different. The applicant of this invention, by using methanol, acetonitrile, acetone, etc., as reconstitution control solutions, prepared solutions containing 10 ppb of benzo[a]pyrene by mixing these solvents with water in different proportions and comparing them with a blank control, found that only the methanol of this invention has the best solubilizing effect.

[0024] The enzyme conjugate / antibody diluent in this invention's kit exhibits a synergistic effect among SG09, glycerol, trehalose, and sorbitol. The mixture formed by these components helps maintain the stability of the antibody and enzyme conjugate without affecting the kit's sensitivity. This is because glycerol, trehalose, and sorbitol can replace water in forming hydrogen bonds with proteins, preventing protein denaturation caused by phase changes in the solution (such as freezing and thawing) under extreme conditions, which could lead to the inactivation of core components like the enzyme conjugate, antibody, and antigen, thus affecting the product's shelf life. Extensive experiments have shown that only by using SG09, glycerol, trehalose, and sorbitol synergistically according to the above-described formulation can the long-term stability of the antibody and enzyme conjugate be effectively guaranteed. The absence of one or more of these components significantly reduces the effectiveness.

[0025] The Mg content of the standard / sample diluent in the kit of this invention 2+ It can disrupt the hydration layer of proteins, that is, change the conformation of antibodies, thereby increasing the antibody's affinity for benzo[a]pyrene, similar to "in Mg2+". 2+ Under the influence of sodium citrate, the antibody forms an optimal conformation, which is highly conducive to binding with benzo[a]pyrene. Sodium citrate provides a slightly alkaline solution environment, which is beneficial for the binding of antibodies to free benzo[a]pyrene, resulting in improved sensitivity. In addition, citrate has antioxidant properties, which can also prevent benzo[a]pyrene from being oxidized, thus benefiting the long-term preservation of the kit. It is also a chelating agent that can bind with Mg2+. 2+ Chelation enhances its long-term stability in water; it can effectively prevent Mg from forming. 2+ It reacts with carbon dioxide in the air to form magnesium carbonate. Extensive experiments have shown that only by adding Mg to the standard / sample diluent according to this invention...2+ Sodium citrate is needed to significantly improve antibody titer while maintaining sensitivity.

[0026] The present invention also provides a method for using the benzo[a]pyrene enzyme-linked immunosorbent assay kit, comprising the following steps:

[0027] 1. Sample pretreatment:

[0028] 1.1 Saponification: Add 4 mL of 2 mol / L NaOH ethanol solution to 0.2 g of oil sample and mix well. Let it stand at room temperature (20-25℃) for 5 min to saponify. Add 4 mL of double-distilled water to dissolve the saponified product, then add 2 mL of n-hexane and shake vigorously. Centrifuge at 4000 rpm for 5 min. Take 1 mL of the upper n-hexane layer, which is the solution to be purified.

[0029] 1.2 Purification:

[0030] 1) Activate the BaP SPE column sequentially with 1 mL of dichloromethane and 1 mL of n-hexane.

[0031] 2) Load the solution to be purified from step 1.1 into the BaP SPE column;

[0032] 3) Rinse with 1 mL of n-hexane, repeat once;

[0033] 4) Add 1 mL of dichloromethane to elute, and collect all the eluent;

[0034] 5) Dry under nitrogen / air at 40℃, then reconstitute with 0.2 mL of sample reconstitution solution;

[0035] 6) Take 100 μL of the above sample reconstitution solution, add 100 μL of sample diluent and mix well to obtain the test solution;

[0036] 2. Incubation: Add 50 μL of standard working solution or 50 μL of test solution from step 1 to the enzyme-labeled plate coated with benzo[a]pyrene antigen, then add 50 μL of enzyme conjugate and 50 μL of antibody working solution, mix well and incubate at room temperature in the dark for 20 min.

[0037] 3. Washing and color development: Pour out the reaction solution in the microwell, repeat twice filling 360μL of washing solution and soaking for 15s before pouring out, add 100μL of color development solution, and incubate at room temperature in the dark for 10min.

[0038] 4. Termination and reading: Add 100 μL of stop solution to each well and read the value at a wavelength of 450 nm. The reading should be completed within 15 min.

[0039] 5. Results Analysis: Substitute the above absorbance values ​​into the data software to calculate the concentration of benzo[a]pyrene in the sample. Calculate the binding rate (B / B0) using the absorbance OD of the obtained standard. Plot a standard curve with logit(B / B0) as the ordinate and log(benzo[a]pyrene concentration) as the abscissa to obtain the linear equation. Calculate the B / B0 of the sample solution using the same method, and determine the corresponding benzo[a]pyrene concentration based on the equation. The formula for calculating B / B0 is:

[0040]

[0041] Wherein, B is expressed as the absorbance of the standard solution or sample solution, and B0 is expressed as the absorbance of the zero-concentration standard solution.

[0042] During incubation in step 2, ensure that all reagents have been brought back to 20-25°C.

[0043] The standard working solution in step 2 is to dilute the benzo[a]pyrene standard with the standard diluent to concentrations of 0, 0.2, 0.6, 1.8, and 5.4 μg / L (ppb), respectively.

[0044] Preferably, the washing solution in step 3 is distilled water or deionized water.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The kit provided by this invention employs a competitive ELISA detection method. It involves pretreating the sample, diluting the enzyme conjugate and antibody with a specifically formulated enzyme conjugate / antibody diluent, and diluting the standard and sample with specific standard and sample diluents. Compared to existing technologies, the kit and its usage method provided by this invention offer high specificity and strong affinity, improving detection sensitivity and accuracy, with a minimum sensitivity of 0.2 μg / L (ppb). More importantly, the technique provided by this invention significantly improves antibody titer while maintaining antibody sensitivity, and reduces organic reagent consumption by more than 70%. Using a coating antigen to coat the ELISA plate provides better coating and longer shelf life compared to antibody coating, thereby improving the precision and stability of the kit. This invention also addresses the limitation of current benzo[a]pyrene detection kits, which are mostly suitable for simple samples such as water and aquatic products. It overcomes the difficulties of complex matrix components, significant sample matrix interference, and difficult extraction and purification of samples such as edible oil, kitchen fumes, and automobile exhaust, demonstrating significant value for widespread application. Attached Figure Description

[0047] Figure 1This is the standard curve of benzo[a]pyrene in Experiment Example 4, where the X-axis is the logarithm of the concentration of the benzo[a]pyrene standard solution, and the Y-axis is logit{the optical density value of the benzo[a]pyrene standard solution divided by the optical density value of the "zero" pore (B / B0)}.

[0048] Figure 2 This is the standard curve of benzo[a]pyrene under the optimal sensitivity of Experiment Example 4, where the X-axis is the logarithm of the concentration of the benzo[a]pyrene standard solution, and the Y-axis is logit{the optical density value of the benzo[a]pyrene standard solution divided by the optical density value of the "zero" pore (B / B0)}.

[0049] Figure 3 This is the liquid chromatography standard curve of benzo[a]pyrene in Experiment Example 4, where the X-axis represents the concentration of the benzo[a]pyrene standard solution and the Y-axis represents the peak area of ​​the benzo[a]pyrene standard solution determined by liquid chromatography. The equation relating Y and X is Y = 4258.9524X - 362.2412.

[0050] Figure 4 The liquid chromatogram of the benzo[a]pyrene standard solution in Experimental Example 4 is shown. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The detection methods described in the following schemes all use benzo[a]pyrene (i.e., Bap) as the detection target. Unless otherwise specified, they are all conventional methods. Unless otherwise specified, the reagents and materials can be purchased from commercial channels. The specific procurement sources are shown in Table 1 below.

[0053] Table 1. Types and sources of reagents and materials

[0054]

[0055]

[0056] In the following experimental examples, the enzyme conjugate / antibody diluent was formulated with 20 mmol / L phosphate buffer, 0.1 wt% preservative, and 0.1 wt% SG09 or 0.1 wt% BSA. The concentrations of glycerol, trehalose, and sorbitol were determined according to the specific experimental requirements.

[0057] The basic formulation of the standard / sample diluent is a 50 v% methanol aqueous solution.

[0058] The initial formulation of the sample reconstitution solution was pure methanol.

[0059] The standard working solution is prepared by diluting the benzo[a]pyrene standard with the aforementioned standard / sample diluent to a concentration of 10 μg / L (ppb).

[0060] In the following experimental examples, unless otherwise specified, the kits used consist of enzyme conjugate / antibody diluent; standard / sample diluent; sample reconstitution solution; benzo[a]pyrene standard; benzo[a]pyrene specific antibody; enzyme conjugate; ELISA plate coated with benzo[a]pyrene antigen; chromogenic solution; stop solution; and washing solution. The kits are used to perform ELISA detection of benzo[a]pyrene using the following method:

[0061] 1. Sample pretreatment:

[0062] (1) Saponification: Weigh 0.2g of edible oil (rapeseed oil, Luhua brand, purchased from the supermarket), add 4mL of 2mol / L NaOH ethanol solution, mix well and let stand for 5min to saponify, then add 4mL of double-distilled water to dissolve the saponified product, then add 2mL of n-hexane and shake vigorously for 2min, centrifuge at 4000rpm for 5min, and take 1mL of the upper n-hexane layer, which is the liquid to be purified;

[0063] (2) Activation: First add 1 mL of dichloromethane to the column, and when the liquid level reaches the column bed, add 1 mL of n-hexane to the column, and wait for all the liquid to flow out;

[0064] (3) Sample loading: Add all the solution to be purified in (1) into the activated column at a flow rate of 1-2 mL / min, and discard the effluent;

[0065] (4) Eluting: Add 1 mL of n-hexane to elute the column, let it drip naturally, discard the eluent, and repeat once;

[0066] (5) Elution: Add 1 mL of dichloromethane to elute the column at a flow rate of 1-2 mL / min, and collect all the eluent;

[0067] (6) Nitrogen blowing and reconstitution: The collected liquid in (5) was dried under nitrogen / air at 40°C and reconstituted with 0.2 mL of sample reconstitution solution;

[0068] (7) Dilution: Take 100 μL of the sample reconstituted solution from (6), add 100 μL of sample diluent and mix well to obtain the test solution.

[0069] 2. Incubation: Add 50 μL of standard working solution or 50 μL of test solution from step 1 to the enzyme-labeled plate coated with benzo[a]pyrene antigen, then add 50 μL of enzyme conjugate and 50 μL of antibody working solution, mix well and incubate at room temperature in the dark for 20 min.

[0070] 3. Washing: Pour out the reaction solution from the micropores, fill with 360μL of washing solution, soak for 15 seconds, pour out, and repeat the process twice more, adding 360μL of washing solution and soaking for 15 seconds each time, then spin dry.

[0071] 4. Color development: Add 100 μL of substrate, cover with the plate, and incubate at room temperature in the dark for 10 min;

[0072] 5. Termination and reading: Add 100 μL of stop solution to each well and read the value at a wavelength of 450 nm. The reading should be completed within 15 min.

[0073] 6. Results Analysis: Substitute the above absorbance values ​​into the data software to calculate the concentration of benzo[a]pyrene in the sample. Calculate the binding rate (B / B0) using the average absorbance of the obtained standards. Plot a standard curve with logit (binding rate) as the ordinate and the logarithm of the benzo[a]pyrene concentration as the abscissa to obtain the linear equation. Calculate the binding rate of the sample solution using the same method, and determine the corresponding benzo[a]pyrene concentration of the sample based on the equation. The formula for calculating the binding rate is:

[0074]

[0075] Where B is the average absorbance of the standard solution or sample solution, and B0 is the average absorbance of the 0-concentration standard solution.

[0076] Experimental Example 1: Selection of Sample Reconstitution Solution

[0077] Based on the characteristic that benzo[a]pyrene is insoluble in water but readily soluble in oil, methanol, acetonitrile, DMSO, DMF, acetone, and ethanol, which are miscible with water and can dissolve benzo[a]pyrene, were selected as experimental subjects. Solutions obtained by mixing these solvents with water in different proportions were designed as new solvents (see Tables 2-1 to 2-6 for details). These were used to prepare benzo[a]pyrene solutions with a concentration of 10 ppb—the experimental group—and benzo[a]pyrene solutions with a concentration of 0 ppb—the blank control group. The 10 ppb benzo[a]pyrene was obtained by diluting 10,000 ppb benzo[a]pyrene standard (dissolved in acetone) with the above-mentioned new solvents. The blank control was the solvent itself. For example, if methanol:water = 1:9, i.e., a 10% methanol aqueous solution, was used as the solvent, the experimental group was a 10% methanol aqueous solution containing 10 ppb benzo[a]pyrene, and the blank control group was a 10% methanol aqueous solution, and so on for the other reagents. The antibody working solution had a concentration of 1:20000 and the enzyme conjugate working solution had a concentration of 1:1000. The enzyme conjugate / antibody diluent was diluted to the corresponding concentrations, and enzyme-linked immunosorbent assay (ELISA) was performed according to the above method. The optimal co-solvent was selected by analyzing the blank absorbance and the binding rate (B / B0, hereinafter referred to as B / B0) of 10 ppb benzo[a]pyrene in the experimental group. The concentrations of each component and the detection results are shown in Tables 2-1 to 2-6 below.

[0078] Table 2-1 Selection of Methanol

[0079] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) Methanol:water = 1:9 0.545 0.56 Methanol:water = 1:1 1.6305 1.245 pure methanol 2.777 3.314 pure water 0.1011 0.1208

[0080] Table 2-2 Selection of Acetonitrile

[0081] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) Acetonitrile:water = 1:9 1.345 1.289 Acetonitrile:water = 1:1 1.156 1.125 Pure acetonitrile 0.045 0.035 pure water 0.1011 0.1208

[0082] Table 2-3 Selection of DMSO

[0083] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) DMSO:water=5:95 1.565 1.578 DMSO:water=1:9 1.212 1.238 Pure DMSO 0.6534 0.6467 pure water 0.1011 0.1208

[0084] Table 2-4 Selection of DMF

[0085] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) DMF:water=5:95 1.652 1.6037 DMF:water=1:9 1.3135 1.3471 Pure DMF 0.7524 0.7902 pure water 0.1011 0.1208

[0086] Table 2-5 Selection of Acetone

[0087] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) Acetone:water = 1:9 0.4894 0.4029 Acetone:water = 1:1 1.5428 1.1412 Pure acetone 3.031 3.256 pure water 0.1011 0.1208

[0088] Table 2-6 Selection of Ethanol

[0089] solvent Blank control group (BaP 0ppb) Experimental group (BaP 10ppb) Ethanol:water = 1:9 0.3585 0.3982 Ethanol:water = 1:1 1.178 1.2307 pure ethanol 2.426 2.2568 pure water 0.1011 0.1208

[0090] Results Analysis: Firstly, for a commercially available kit for detecting benzo[a]pyrene in oils, the two most fundamental metrics are sensitivity and cost. The sensitivity of an ELISA kit can be indirectly reflected by the B / B0 ratio of a specific concentration of standard. The amount of antibody used, the key raw material, is the core of the product cost. The amount of antibody used can be characterized by its titer, which can be indirectly reflected by the absorbance of the zero standard. Therefore, by calculating the B / B0 values ​​of different solvent experimental groups (benzo[a]pyrene 10 ppb), we can determine the effect of the concentration change of the studied reagent in water on the antibody sensitivity, i.e., ΔB / B0. 10 ppb (μg / L) was chosen because the limit standard for benzo[a]pyrene in vegetable oils is 10 ppb. Simultaneously, we need to consider the impact of error on the experimental results; therefore, we need to combine the CV detection data from the coated plate for judgment.

[0091] Using the absorbance of the double-distilled water blank control group as a complete control, the relative change in absorbance between the blank control group and the complete control with different solvents, i.e., ΔOD0, was calculated. However, we need to consider the influence of error on the experimental results, therefore, it is necessary to combine the CV detection data of the coated plate for judgment.

[0092] The results obtained from the above formula analysis are shown in Table 2-7.

[0093] Table 2-7 Results Analysis

[0094] solvent <![CDATA[B / B0]]> absorbance of blank control <![CDATA[△B / B0]]> <![CDATA[△OD0]]> water 119.5％* 0.1011 19.5％ 0 10% methanol water 102.8％* 0.545 2.8％ 391.2％ 50% methanol water 76.4％ 1.6305 -24.6％ 1369.6％ methanol 119.3％* 2.777 19.3％ 2402.9％ 10% acetonitrile 95.8％ 1.345 -4.2％ 1112.3％ 50% acetonitrile 97.3％ 1.156 -2.7％ 941.9％ Acetonitrile 77.8％ 0.045 -22.2％ -59.4％ 5% DMSO 100.8％ 1.565 0.8％ 1310.5％ 10% DMSO 102.1％* 1.212 2.1％ 992.4％ DMSO 99.0％ 0.6534 -1％ 488.9％ 5% DMF 97.1％ 1.652 -2.9％ 1389.0％ 10% DMF 102.6％* 1.3135 2.6％ 1083.9％ DMF 105.0％* 0.7524 5.0％ 578.1％ 10% acetone 82.3％ 0.4894 -17.7％ 341.1％ 50% acetone 74.0％ 1.5428 -26％ 1290.5％ acetone 107.4％* 3.031 7.4％ 2631.9％ 10% ethanol 111.1％* 0.3585 11.1％ 223.1％ 50% ethanol 104.5％* 1.178 4.5％ 961.7％ ethanol 93.0％ 2.426 -7％ 2086.6％

[0095] Data Explanation: The plate absorbance CV is 5%, and the blank absorbance is 0.025. With a confidence interval of 99%, the absorbance fluctuation of the complete control is ±45.1%. In the absence of a standard, the theoretical value of B / B0 is 100%. According to the plate absorbance CV, with a confidence interval of 99% (in the absence of a standard), the fluctuation range of B / B0 is 90%-110%. Therefore, we can see that as long as B / B0 ≥ 90%, i.e., ΔB / B0 ≥ -10%, the standard and antibody in the solution have not reacted or there is no standard.

[0096] Note: Data marked with "*" are based on actual conditions. The theoretical maximum value of B / B0 is 100%, indicating that no standard product exists.

[0097] Based on the above analysis, methanol was chosen as the primary solvent because a 50% methanol-water solution balances sensitivity and cost. Acetone was chosen as an alternative primarily because it is not commonly used and is classified as a hazardous chemical.

[0098] Experimental Example 2: Formulation Selection of Enzyme Conjugate / Antibody Diluent

[0099] The basic formulation of the enzyme conjugate / antibody diluent is 20 mmol / L phosphate buffer. Proteins, carbohydrates (disaccharides, such as sucrose and trehalose), reducing agents, hydrophilic polymers, and salts are then introduced one by one. Enzyme conjugate / antibody working solutions are prepared using corresponding enzyme conjugate / antibody diluents, with a concentration of 1:1000 for the enzyme conjugate working solution and 1:6000 for the antibody working solution. All other operating steps remain unchanged. The standard diluent uses 50% methanol. The effectiveness of the components is determined by titer and sensitivity. Sensitivity is indirectly determined by analyzing the binding rate of standard working solutions at different concentrations, using the following formula:

[0100]

[0101] Changes in potency are determined by the change in absorbance of blank standards with different formulations, using the following formula: The detection results of each component are shown in Tables 3-1 to 3-6 below.

[0102] Table 3-1 Selection of Casein

[0103]

[0104] Table 3-2 Selection of FBS (Fetal Bovine Serum)

[0105]

[0106] Table 3-3 Selection of Glycerin

[0107]

[0108] Table 3-4 Selection of Sorbitol

[0109]

[0110] Table 3-5 Selection of Sucrose

[0111]

[0112]

[0113] Table 3-6 Selection of Trehalose

[0114]

[0115] Table 3-7 Selection of PEG8000

[0116]

[0117] Table 3-8 Selection of Citric Acid

[0118]

[0119] Table 3-9 Selection of Sodium Citrate

[0120]

[0121] Table 3-10 Tween 20 Selection

[0122]

[0123] Table 3-11 Selection of EDTA (Sodium ethylenediaminetetraacetate)

[0124]

[0125]

[0126] Based on the above experimental results, we can see that when the reagent components contain proteins and hydrophilic polymers, the antibody titer remains basically unchanged. However, as the content of these components increases, the sensitivity decreases rapidly, causing the final product's detection limit to fail to meet the national standard limit, thus limiting its practical application. Disaccharide compounds and glycerol content less than 10% have virtually no significant impact on antibody titer and sensitivity. Commercially available kits have shelf-life requirements. Since the working concentrations of their antibodies and enzyme conjugates are below μg / mL, the protein content in the reagents is generally increased to above 1 mg / mL by adding extraneous proteins, thereby extending the shelf life of the antibodies and enzyme conjugates. However, based on the above experiments, we found that proteins and hydrophilic polymers should generally not be used in the enzyme conjugate working solution and antibody working solution of the benzo[a]pyrene ELISA kit. In view of this situation, the working solution formulation for the enzyme conjugate / antibody dilution was selected as follows: 20 mmol / L phosphate buffer, 5% glycerol, 1% trehalose, 2% sorbitol, 0.25% sodium citrate and 0.1% preservative, and also contains 0.1% SG09 or 0.1% BSA, with a pH of 7.4.

[0127] Experimental Example 3: Formulation Selection of Standard / Sample Diluent

[0128] Based on Experiment 2, it was found that the sensitivity of antibodies decreases with the introduction of proteins and hydrophilic polymers into reagent components and with increasing concentrations. Small hydrophilic compounds such as disaccharides and glycerol have virtually no significant effect on antibody titer and sensitivity. Metal ions are often destabilizing factors for proteins. Introducing metal ions into the standard / sample diluent weakens the influence of proteins in the reagent components on antibody sensitivity during the antigen-antibody reaction. This achieves the goal of improving sensitivity without affecting the stability of the enzyme conjugate and antibody working solution. Therefore, we screened the optimal cation through single-factor experiments, adding corresponding concentrations of CaCl2, ZnSO4, MgSO4, FeCl3, NaCl, MgCl2, and Mg(CH3COO)2 to a 50% methanol aqueous solution. The optimal formulation of the sample / standard diluent was determined by the change in absorbance (ΔOD) of the zero-standard solution and the change in the binding ratio B / B0 (ΔB / B0) of the standard solution containing a known concentration of benzo[a]pyrene. △OD represents the change in absorbance of the zero standard solution; △B / B0 represents the change in binding rate of the standard solution containing a known concentration of benzo[a]pyrene, where B / B 0新标稀 The binding rate of known concentrations of benzo[a]pyrene prepared by diluting the new formulation standard, B / B 0 50％甲醇水 The binding rates of benzo[a]pyrene at the same concentration prepared with 50% methanol and water; the concentrations of each component and the detection results are shown in Tables 4-1 to 4-7:

[0129] Table 4-1 Selection of CaCl2

[0130]

[0131]

[0132] Table 4-2 Selection of ZnSO4

[0133] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 0.050％ 1.9941 1.4563 73.0％ 0.005％ 2.2402 1.5836 70.7％ Comparison 1.935 1.105 57.1％

[0134] Table 4-3 Selection of MgSO4

[0135] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 0.050％ 2.3647 1.6603 70.2％ 0.005％ 2.1041 1.3716 65.2％ Comparison 1.935 1.105 57.1％

[0136] Table 4-4 Selection of FeCl3

[0137] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 0.050％ 1.1684 0.708 60.6％ 0.005％ 2.2595 1.4948 66.2％ Comparison 1.935 1.105 57.1％

[0138] Table 4-5 Selection of NaCl

[0139] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 2％ 0.405 0.1478 36.5％ 0.5％ 0.6212 0.3000 48.3％ Comparison 1.935 1.105 57.1％

[0140] Table 4-6 Selection of MgCl2

[0141] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 0.1％ 2.5539 1.7682 69.2％ 0.01％ 2.2724 1.4676 64.6％ Comparison 1.935 1.105 57.1％

[0142] Table 4-7 Selection of Mg(CH3COO)2

[0143] concentration Absorbance (0 ppb) Absorbance (1 ppb) B / B0(1ppb) 0.10％ 2.5184 1.7599 69.9％ 0.01％ 2.2409 1.4525 64.8％ Comparison 1.935 1.105 57.1％

[0144] Table 4-8 Results of changes in absorbance of zero standard and known concentration of benzo[a]pyrene binding rate of dilute standard solutions with different formulations

[0145]

[0146]

[0147] Data Explanation: The plate absorbance CV is 5%, and the blank absorbance is 0.025. At a 99% confidence interval, the control absorbance fluctuation is ±5.2%. When ΔOD > 5.2%, it indicates that the new formulation can improve antibody titer; when ΔOD < -5.2%, it indicates that the new formulation decreases antibody titer. With 1 ppb standard, the B / B0 of 50% methanol is 57.1%. Based on the plate absorbance CV, at a 99% confidence interval, the B / B0 fluctuation range for 1 ppb standard is 57.1% ± 5.7%. Therefore, when ΔB / B0 > 5.7%, it indicates that the new formulation decreases antibody sensitivity; when ΔB / B0 < -5.7%, it indicates that the new formulation improves antibody sensitivity.

[0148] Based on the above results, we found that as Mg 2+ Increasing the concentration of Mg can significantly improve the antibody titer, due to the high concentration of Mg. 2+ Increased concentration leads to excessively high OD values, thus distorting the 1ppb binding rate data in Mg. 2+ In the presence of [a specific substance], by optimizing the antibody working concentration to its optimal level, and then testing with a 1 ppb standard, it was found that its binding rate was essentially the same as that of a 50% methanol standard at the same concentration. Further experiments revealed the optimal Mg [concentration]. 2+ At a concentration of 166.2 nmol / L, the antibody titer reaches 1:200,000, which translates to an antibody concentration of 12.5 ng / mL. Furthermore, the absorbance of the product meets the factory standards for commercially available kits. This is because Mg was introduced into the standard working solution. 2+ In order to maintain Mg 2+ To improve stability, sodium citrate was removed from the enzyme / antibody working solution and then added to the standard working solution, along with Mg. 2+ Formation of complexes.

[0149] The final reagent formula is as follows:

[0150] Enzyme conjugate / antibody diluent formulation: 20 mmol / L phosphate buffer at pH 7.4, 5% glycerol, 1% trehalose, 2% sorbitol, 0.1% proclin 950, and also contains 0.1% SG09 or 0.1% BSA;

[0151] Standard diluent formulation: 50% methanol aqueous solution, 166.2 nmol / L MgSO4, 0.5% sodium citrate;

[0152] Sample diluent formulation: 332.4 nmol / L MgSO4, 1% sodium citrate.

[0153] In summary, the final concentration of the antibody working solution decreased from 500 ng / mL to 12.5 ng / mL; the product sensitivity increased from 10 μg / kg (ppb) to 0.2 μg / kg (ppb). Furthermore, compared to traditional liquid chromatography, the consumption of organic reagents in the sample pretreatment process in this experimental example was reduced by more than 70%.

[0154] Experimental Example 4: Detection of Benzo[a]pyrene in Edible Oils

[0155] 1. Blind Sample Comparison: Taking various edible oils on the market as examples, the above-mentioned enzyme-linked immunosorbent assay (ELISA) steps and liquid chromatography (GB5009.27-2016 National Food Safety Standard for the Determination of Benzo[a]pyrene in Food and GB5009.265-2021 National Food Safety Standard for the Determination of Polycyclic Aromatic Hydrocarbons in Food) were used for detection to obtain accuracy experimental data. The original data of the benzo[a]pyrene ELISA kit standard curve are shown in Table 4-9, and the benzo[a]pyrene ELISA standard curve is shown in Table 4-9. Figure 1 ;

[0156] Table 4-9 Raw data of the standard curve for the benzo[a]pyrene ELISA kit

[0157]

[0158] The accuracy of ELISA is expressed as recovery rate. A recovery rate of 100% or higher, and a recovery rate of ≥80% and ≤120% indicates a satisfactory ELISA test result (e.g., recovery rate = 100%). Figure 2 The results (as shown) are consistent with the HPLC detection results (the HPLC detection method is the reference method disclosed in GB5009.27-2016, and the standard curve is shown in the figure). Figure 3 As shown, the liquid chromatogram is as follows: Figure 4 (As shown in Table 5). If a result of "not detected" is displayed at their respective detection limits, it indicates that the two conclusions are consistent, only the detection limits are different.

[0159] Table 5 Accuracy Experiment Data

[0160]

[0161]

[0162] Based on the accuracy experimental data in Table 5 above, compared with the HPLC results, the overall compliance of the ELISA method is 41 / 42 = 97.62%.

[0163] One sample each from the high, medium, and low concentration points within the linear range was selected to study the precision of product detection within the linear range. The low concentration point was chosen to be as close as possible to the lower limit of detection (lower critical point of the linear range), and the medium concentration point was chosen to be as close as possible to the midpoint of the standard curve (IC50). 50 The high concentration points should be located as close as possible to the upper boundary of the linear interval to comprehensively assess the detection precision within the linear interval. Using samples 7, 10, and 20 from Table 5 as experimental subjects, six different parallel groups were set up, and the mean and standard deviation of the ELISA detection results between parallel groups were calculated. The precision results obtained using the coefficient of variation (CV) are shown in Table 6.

[0164] Table 6 Precision Experiment Data

[0165]

[0166] Using No. 15 blended oil as a blank sample, 20 parallel samples were prepared and detected using the enzyme-linked immunosorbent assay (ELISA) method described above. The results of the detection limit data are shown in Table 7.

[0167] Table 7 Detection Limit Data

[0168]

[0169] In summary, the sample pretreatment and detection methods provided by this invention significantly improve antibody titer against benzo[a]pyrene in edible oils while maintaining the same sensitivity. The sensitivity is reduced to a minimum of 0.075 μg / L, and the titer reaches 1:200,000 (12.5 ng / mL antibody dosage). The accuracy of this enzyme-linked immunosorbent assay (ELISA) reaches 97.6% (blind sample comparison results), the precision within the linear range is 7.6%–11%, the detection limit is 0.8 μg / kg, and the linear range is 0.8–21.6 μg / kg, meeting the requirements of the national standard GB2762-2022 for the limits of contaminants in food. This ELISA method can be applied to the detection of benzo[a]pyrene in edible oils. Based on the properties of edible oils, kitchen fumes, and vehicle exhaust, it can be inferred that the kit and detection method provided by this invention can also be used for kitchen fumes and vehicle exhaust.

[0170] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An enzyme-linked immunosorbent assay (ELISA) kit for detecting benzo[a]pyrene in edible oils, kitchen fumes, and automobile exhaust, characterized in that, The kit includes sample reconstitution solution, enzyme conjugate / antibody diluent, standard diluent, and sample diluent; The sample reconstitution solution was a 50% methanol aqueous solution; The enzyme conjugate / antibody diluent is formulated as follows: 5-200 mmol / L phosphate buffer, 5 v / v% glycerol, 1 wt% trehalose, 2 wt% sorbitol and 0.01-0.5 v / v% preservative, and also contains 0.1 wt% SG09 or 0.1 wt% BSA, with a pH of 7.

4. The standard diluent is: 50 v% methanol aqueous solution, 166.2 nmol / L mg. 2+ and 0.5 wt% sodium citrate; the sample diluent had a concentration of 332.4 nmol / L mg. 2+ And 1 wt% sodium citrate.

2. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 1, characterized in that, The components of the enzyme-linked immunosorbent assay kit also include benzo[a]pyrene standard, benzo[a]pyrene-specific mouse monoclonal antibody, enzyme conjugate, ELISA plate coated with benzo[a]pyrene antigen, chromogenic solution, stop solution and washing solution.

3. The method of using the benzo[a]pyrene enzyme-linked immunosorbent assay kit according to claim 1 or 2, characterized in that, Includes the following steps: S1. Sample pretreatment: The sample to be tested is first saponified, then subjected to liquid-liquid extraction, activation, purification, washing and elution, and drying; then reconstituted with the sample reconstitution solution; finally, the reconstituted sample solution is diluted with the sample diluent to obtain the test solution. S2. The standard is serially diluted with the standard diluent to obtain the standard working solution; the test solution from step S1 and the standard working solutions of different concentrations are added to the microwells of the enzyme-labeled plate coated with benzo[a]pyrene antigen, and then the enzyme conjugate and antibody working solution are added, mixed well and incubated at room temperature in the dark. S3. Pour out the reaction solution from the microwell, wash and spin dry, add substrate, and incubate at room temperature in the dark. S4. Add stop solution to stop color development, and take readings within 15 minutes at a wavelength of 450nm. S5. Result Analysis: Plot a standard curve based on the standard solution and relative absorbance value, calculate the sample concentration in the sample well, and finally determine the concentration of benzo[a]pyrene in the sample to be tested.

4. The method of use according to claim 3, characterized in that, In step S2, the standard is diluted with the standard diluent to concentrations of 0, 0.2, 0.6, 1.8, and 5.4 μg / L, respectively.

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