Rapid detection test strip for main hazard factors in edible vegetable oil and its application

Abstract

The present invention relates to a rapid detection test strip for major hazard factors in edible vegetable oils and its application. It includes a base plate, one side of the base plate is sequentially pasted with a water-absorbing pad, a detection pad, a gold-labeled pad, and a sample pad from top to bottom, and adjacent pads are overlapped and connected at the connection point. The detection pad is based on a nitrocellulose membrane, and a quality control line and a detection line are horizontally arranged on the nitrocellulose membrane. The detection lines are located below the quality control lines, and the number is two or three, which are spaced apart. Each detection line is coated with a protein conjugate to be detected, and the detected objects are benzo[a]pyrene, aflatoxin B1, and / or capsaicinoids. The quality control line is coated with a rabbit anti-mouse polyclonal antibody; the gold-labeled pad is horizontally sprayed with a gold-labeled monoclonal antibody to be detected corresponding to the detected objects. Synchronous and rapid detection of one or more target objects among benzo[a]pyrene, aflatoxin B1, and capsaicinoids can be achieved on a test strip.

Description

Technical Field

[0001] The invention provides a test strip for quickly detecting main harmful factors in edible vegetable oil and application thereof. Background Art

[0002] Cooking oil safety is one of the most pressing food safety issues, affecting every household, every day, and every meal. In the new era, people's yearning for a better life has led to higher and stricter demands for cooking oil quality and safety. The greatest risks to cooking oil safety are aflatoxins, benzo[a]pyrene, and waste cooking oil. Aflatoxins and benzo[a]pyrene are both strong carcinogens. For example, AFB1 is 10 times more toxic than potassium cyanide, 68 times more toxic than arsenic, and 10,000 times more toxic than the pesticide 666, making it a Class I carcinogen. Benzo[a]pyrene (BaP) is a strong carcinogen, teratogen, and mutagen, and can induce lung, respiratory, stomach, digestive tract, bladder, and skin cancers. Natural capsaicin, dihydrocapsaicin, and synthetic capsaicin have long been used as characteristic indicators for identifying waste cooking oil. Therefore, combining benzo[a]pyrene with aflatoxins and capsaicinoids to test edible oil quality and safety can significantly improve the accuracy of edible oil safety assessments. Currently, the primary methods for detecting benzo[a]pyrene, aflatoxins, and capsaicinoids include instrumental analysis methods such as liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. These instrumental methods offer excellent stability, high sensitivity, and accuracy, but they suffer from complex pretreatment steps and high sample testing costs. Immunochromatography, based on antigen-antibody specific reactions, utilizes an immune competitive model to analyze the target compound content in a sample. Summary of the Invention

[0003] The problem to be solved by the present invention is to provide a rapid detection test strip for major hazard factors in edible vegetable oils and its application, which can be used to evaluate the major hazard factors in edible vegetable oils by synchronously and rapidly detecting the content of benzo[a]pyrene, aflatoxin B1 and capsaicinoids.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] A rapid test strip for major hazard factors in edible vegetable oils comprises a base plate, one side of which is adhered with a water-absorbing pad, a test pad, a gold-labeled pad, and a sample pad in order from top to bottom, with adjacent pads overlapping and connected at the joints. The test pad is based on a nitrocellulose membrane, and a quality control line and a test line are transversely arranged on the nitrocellulose membrane. The test lines are located below the quality control line, and there are two or three test lines at intervals. Each test line is coated with a test substance-protein conjugate, wherein the test substances are benzo[a]pyrene, aflatoxin B1, and capsaicinoids, and the test substance-protein conjugates are benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) and aflatoxin B1-ovalbumin conjugate (AFB1-OVA). , capsaicinoid-ovalbumin conjugate (CAP-OVA), the quality control line is coated with rabbit anti-mouse polyclonal antibody; the gold label pad is horizontally sprayed with gold-labeled monoclonal antibody corresponding to the object to be tested, and the monoclonal colloid of the object to be tested is anti-benzo[a]pyrene monoclonal antibody, anti-aflatoxin B1 monoclonal antibody, and anti-capsaicinoid monoclonal antibody; the anti-benzo[a]pyrene monoclonal antibody is secreted by the hybridoma cell line BBBE1H1 with the deposit number of CCTCC NO: C201882.

[0006] According to the above scheme, the absorbent pad is 16-18 mm long and 3-4 mm wide; the detection pad is 18-30 mm long and 3-4 mm wide; the gold label pad is 10-12 mm long and 3-4 mm wide; the sample pad is 12-15 mm long and 3-4 mm wide, and the overlapping length of adjacent pads is 1-3 mm.

[0007] According to the above solution, the absorbent pad is absorbent paper.

[0008] According to the above solution, the distance between each two adjacent detection lines on the detection pad is 2-3 mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7 mm.

[0009] According to the above scheme, the coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter on the detection line of the detection pad coated with benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) is 100-300 ng; the coating amount of aflatoxin B1-ovalbumin conjugate (AFB1-OVA) required per centimeter on the detection line coated with aflatoxin B1-ovalbumin conjugate is 100-300 ng; the coating amount of capsaicinoid-ovalbumin conjugate (CAP-OVA) required per centimeter on the detection line coated with capsaicinoid-ovalbumin conjugate (CAP-OVA) is 100-300 ng; and the coating amount of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 50-200 ng.

[0010] According to the above scheme, the particle size of the colloidal gold used in the gold label pad is 15 to 20 nm; the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length on the gold label pad is 100 to 200 ng, the amount of colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody required is 100 to 200 ng, and the amount of colloidal gold-labeled anti-capsaicinoid monoclonal antibody required is 100 to 600 ng.

[0011] According to the above scheme, preferably, the IC50 of the anti-aflatoxin B1 monoclonal antibody is less than or equal to 1.6 ppb, such as the monoclonal antibody secreted by the hybridoma cell line 3G1 with the deposit number CCTCC NO. C201014 disclosed in Chinese Patent No. 201210117614.9.

[0012] According to the above scheme, the IC50 of the anti-capsaicinoid monoclonal antibody is less than or equal to 8.5 ppb, such as the monoclonal antibody secreted by the hybridoma cell line YQQD8 with the deposit number CCTCC NO: C201534 disclosed in Chinese Patent No. 201610079095X.

[0013] The method for preparing the immunochromatographic test strip for the simultaneous detection of benzo[a]pyrene, aflatoxin B1, and capsaicinoids as described above comprises the following steps:

[0014] (1) Preparation of absorbent pad

[0015] Cut the absorbent paper to make an absorbent pad;

[0016] (2) Preparation of detection pad

[0017] Test line coating:

[0018] Benzo[a]pyrene-ovalbumin conjugate (BAP-OVA), aflatoxin B1-ovalbumin conjugate (AFB1-OVA) and capsaicinoid-ovalbumin conjugate (CAP-OVA) were prepared into 0.25-0.5 mg / mL coating solutions with coating buffer, and coated on nitrocellulose membranes by spraying. Two or three test lines were obtained according to the analyte to be measured, and then dried at 37-40°C for 30-60 minutes. The test lines coated with benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) were The coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter is 100-300 ng; the coating amount of aflatoxin B1-ovalbumin conjugate (AFB1-OVA) required per centimeter on the test line coated with aflatoxin B1-ovalbumin conjugate is 100-300 ng; the coating amount of capsaicinoid-ovalbumin conjugate (CAP-OVA) required per centimeter on the test line coated with capsaicinoid-ovalbumin conjugate is 100-300 ng, and the spacing between each two adjacent test lines is 2-3 mm;

[0019] Coating of quality control line:

[0020] Prepare a 0.2-0.4 mg / mL coating solution of rabbit anti-mouse polyclonal antibody using coating buffer, and coat it horizontally on a nitrocellulose membrane by dot spraying at a position 3-7 mm away from the test line close to the quality control line to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the quality control line is 50-200 ng, and then dry at 37-40°C for 1-2 hours.

[0021] (3) Preparation of sample pad

[0022] Soak the glass fiber membrane in the blocking solution, take it out, dry it at 37-40℃ for 6-10 hours to obtain the sample pad, and then store it in a desiccator at room temperature;

[0023] (4) Preparation of gold label pad

[0024] The glass fiber membrane is soaked in a blocking solution, taken out, and dried at 37-40° C. for 6-10 hours. A mixed solution of a monoclonal antibody to be detected labeled with colloidal gold is sprayed horizontally on the dried glass fiber membrane in a dot spraying manner according to the object to be detected. The monoclonal antibody to be detected labeled with colloidal gold is a colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution, a colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody solution, or a colloidal gold-labeled anti-capsaicinoid substance. A monoclonal antibody solution is prepared, wherein the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length is 100-200 ng, the amount of colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody required is 100-200 ng, and the amount of anti-capsaicinoid monoclonal antibody required is 100-600 ng, followed by vacuum freeze-drying for 2-4 hours and storage in a desiccator at room temperature; the anti-benzo[a]pyrene monoclonal antibody is secreted and produced by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882;

[0025] (5) Assembly of test strips

[0026] On one side of the cardboard, a water-absorbing pad, a detection pad, a gold label pad and a sample pad are pasted in sequence from top to bottom, and adjacent pads are overlapped at the connection points with an overlapping length of 1 to 3 mm, thereby obtaining a colloidal gold immunochromatographic test strip for simultaneously detecting mixed contamination of benzo[a]pyrene, aflatoxin B1 and capsaicinoids.

[0027] According to the above scheme, the coating buffer contains per 10 mL: 0.1-0.2 g of bovine serum albumin, 0.08 g of sodium chloride, 0.029 g of disodium hydrogen phosphate dodecahydrate, 0.002 g of potassium chloride, and 0.002 g of potassium dihydrogen phosphate;

[0028] According to the above scheme, the blocking solution in step (3) and step (4) contains the following per 100 mL: 1-2 g of ovalbumin, 2-5 g of sucrose, 0.02-0.05 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate.

[0029] According to the above scheme, the colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution was prepared by an unsaturated labeling method, which is as follows: 50.0 mL of a commercially available colloidal gold solution with a mass concentration of 0.01% was taken, the pH value was adjusted with 0.4 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2 mL of A 0.1 mg / mL aqueous solution of anti-benzo[a]pyrene monoclonal antibody was stirred for 30 minutes. A 10% bovine serum albumin aqueous solution was added to a final bovine serum albumin concentration of 1%, and stirring was continued for 30 minutes. After incubation at 4°C for 2 hours, the mixture was centrifuged at 1500 rpm for 15 minutes, the supernatant was collected, and the precipitate was discarded. The supernatant was centrifuged at 12000 rpm for 30 minutes, the supernatant was discarded, and 40.0 mL of labeled wash buffer was added. The mixture was centrifuged again at 12000 rpm for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in labeled wash buffer to obtain 5.0 mL of concentrate, which was refrigerated at 4°C until use.

[0030] The colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody is prepared by an unsaturated labeling method, which specifically comprises the following steps: taking 50.0 mL of a commercially available colloidal gold solution having a mass concentration of 0.01%, adjusting the pH value with 0.4 mL of a 0.1 mol / L potassium carbonate aqueous solution, slowly adding 1.5 mL of a 0.1 mg / mL anti-aflatoxin B1 monoclonal antibody aqueous solution while stirring, and continuing to stir for 30 minutes; adding a 10% bovine serum albumin aqueous solution to a final bovine serum albumin mass concentration of 1%, and continuing to stir for 30 minutes; placing the solution at 4° C. for 2 hours, centrifuging at 1500 rpm for 15 minutes, taking the supernatant, and discarding the precipitate; centrifuging the supernatant at 12000 rpm for 30 minutes, discarding the supernatant, adding 40.0 mL of a labeled washing storage solution; centrifuging the solution at 12000 rpm for 30 minutes, discarding the supernatant, and resuspending the precipitate in the labeled washing storage solution to obtain 5.0 mL of a concentrate, which is then placed in a 4° C. refrigerator for standby use;

[0031] The colloidal gold-labeled anti-capsaicinoid monoclonal antibody solution is prepared by an unsaturated labeling method, which specifically comprises the following steps: taking 50.0 mL of a commercially available colloidal gold solution having a mass concentration of 0.01%, adjusting the pH value with 0.425 mL of a 0.1 mol / L potassium carbonate aqueous solution, slowly adding 2.5 mL of a 0.1 mg / mL anti-capsaicinoid monoclonal antibody aqueous solution while stirring, and continuing to stir for 30 minutes; adding a 10% bovine serum albumin aqueous solution to a final serum albumin mass concentration of 1%, and continuing to stir for 30 minutes; placing the solution at 4° C. for 2 hours, centrifuging at 1500 rpm for 15 minutes, taking the supernatant, and discarding the precipitate; centrifuging the supernatant at 12000 rpm for 30 minutes, discarding the supernatant, adding 40.0 mL of a labeled washing and preservative solution; centrifuging the solution at 12000 rpm for 30 minutes, discarding the supernatant, and resuspending the precipitate in the labeled washing and preservative solution to obtain 5.0 mL of a concentrate, which is then placed in a 4° C. refrigerator for later use;

[0032] The 0.1 mol / L potassium carbonate aqueous solution is prepared by dissolving 13.8 g potassium carbonate in pure water to a volume of 1000 mL and filtering through a 0.22 μm filter membrane. The labeled washing and preservation solution is prepared by dissolving 2.0 g polyethylene glycol-20000, 0.2 g sodium azide, and 0.1235 g boric acid in pure water to a volume of 1000 mL and filtering through a 0.22 μm filter membrane.

[0033] The immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids as described above is used as follows: the sample extract is diluted with a sample sustained-release solution and then added to the sample pad of an immunochromatographic test strip for detecting mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids for detection, which serves as the test strip; an equal volume of the sample sustained-release solution is taken as a negative control solution and added dropwise to the sample pad of another immunochromatographic test strip for detecting mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids, which serves as a control test strip; after 15-20 minutes, the test strip and the control test strip are subjected to color development comparison:

[0034] When the color of the test line coated with the benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the benzo[a]pyrene content in the test sample solution is less than 5 ng / mL; when the color is lighter than the corresponding test line, it indicates that the benzo[a]pyrene content in the test sample solution is equal to or higher than 5 ng / mL and lower than 50 ng / mL; when no color is developed, it indicates that the benzo[a]pyrene content in the test sample solution is equal to or higher than 50 ng / mL;

[0035] When the color of the test line coated with aflatoxin B1-ovalbumin conjugate (AFB1-OVA) on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the aflatoxin B1 content in the test sample solution is lower than 1 ng / mL; when the color is lighter than the corresponding test line, it indicates that the aflatoxin B1 content in the test sample solution is equal to or higher than 1 ng / mL and lower than 20 ng / mL; when no color is developed, it indicates that the aflatoxin B1 content in the test sample solution is equal to or higher than 20 ng / mL;

[0036] When the color of the test line coated with capsaicinoid-ovalbumin conjugate (CAP-OVA) on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the content of capsaicinoids in the test sample solution is less than 5 ng / mL; when the color is lighter than the corresponding test line, it indicates that the content of capsaicinoids in the test sample solution is equal to or higher than 5 ng / mL and lower than 80 ng / mL; when no color is developed, it indicates that the content of capsaicinoids in the test sample solution is equal to or higher than 80 ng / mL;

[0037] When the quality control line does not show color, the test strip is considered invalid regardless of whether the test line of the test strip shows color;

[0038] Finally, the contents of benzo[a]pyrene, aflatoxin B1 and capsaicinoids in the test sample are obtained through conversion.

[0039] According to the above scheme, the sample is edible vegetable oil, and the pretreatment steps are: ultrasonic extraction of edible vegetable oil with n-hexane, taking the supernatant, diluting it 8-12 times with water, then adding immunomagnetic beads, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with sample diluent to obtain the supernatant, which is the test solution, wherein:

[0040] When detecting benzo[a]pyrene and aflatoxin B1, the immunomagnetic beads are magnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies and anti-aflatoxin monoclonal antibodies;

[0041] When detecting benzo[a]pyrene and capsaicinoids, the immunomagnetic beads are magnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies and anti-capsaicinoid monoclonal antibodies;

[0042] When detecting benzo[a]pyrene, aflatoxin B1, and capsaicinoids, the immunomagnetic beads are magnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin monoclonal antibodies, and / or anti-capsaicinoid monoclonal antibodies;

[0043] The immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin monoclonal antibodies and / or anti-capsaicinoid monoclonal antibodies include magnetic beads and anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin B1 monoclonal antibodies and / or anti-capsaicinoid monoclonal antibodies coupled based on the COOH groups on the magnetic beads and the amino groups of the antibodies.

[0044] According to the above scheme, the sample sustained-release solution is PBS containing 0.5% Tween 20, pH 7.4.

[0045] According to the above scheme, the mass ratio of anti-benzo[a]pyrene monoclonal antibody to magnetic beads in the immunomagnetic beads is 2:1~1:5; the mass ratio of anti-aflatoxin B1 monoclonal antibody to magnetic beads is 2:1~1:5; and the mass ratio of anti-capsaicinoid monoclonal antibody to magnetic beads is 2:1~1:5.

[0046] The preparation method of the magnetic beads coupled with the benzo[a]pyrene monoclonal antibody and the aflatoxin and / or capsaicinoid monoclonal antibody comprises the following steps: washing the magnetic beads to remove impurities; adding anti-benzo[a]pyrene monoclonal antibody and anti-aflatoxin B1 monoclonal antibody and / or anti-capsaicinoid monoclonal antibody for coupling, and magnetic separation; blocking the coupled magnetic beads with a blocking solution; after the blocking reaction is completed, performing magnetic separation, discarding the supernatant, obtaining the immunomagnetic beads, and storing them for later use.

[0047] The working principle of the rapid detection test strip for major hazard factors in edible vegetable oils is explained as follows, taking the simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids as an example: When the sample solution to be tested is added to the sample pad at the lower end of the test strip, the sample solution to be tested moves along the test strip toward the absorbent pad by capillary action. When it moves to the gold-labeled pad, the colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody, the colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody, and the colloidal gold-labeled anti-capsaicinoid monoclonal antibody are dissolved. When the sample contains benzo[a]pyrene, benzo[a]pyrene will bind to the anti-benzo[a]pyrene monoclonal antibody labeled with colloidal gold on the gold label pad and migrate upward together. When it reaches the detection line I where the benzo[a]pyrene-ovalbumin conjugate antigen is fixed, the antigen will compete with benzo[a]pyrene for binding to the limited antigen binding sites on the anti-benzo[a]pyrene monoclonal antibody labeled with colloidal gold. The higher the benzo[a]pyrene content in the sample, the more the antigen on the detection line can bind to the benzo[a]pyrene monoclonal antibody. The less colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibodies that can be bound, the lighter the color of the color band formed on the test line. When the sample contains aflatoxin B1, aflatoxin B1 will bind to the colloidal gold-labeled anti-aflatoxin B1 monoclonal antibodies on the gold pad and migrate upward together. When it reaches the test line II where the aflatoxin B1-ovalbumin conjugate (AFB1-OVA) antigen is fixed, the antigen will bind to aflatoxin B1. 1. Compete for binding with the limited antigen binding sites on the colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody. The higher the aflatoxin B1 content in the sample, the less colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody the antigen on the test line can bind to, and the lighter the color band formed on the test line. When the sample contains capsaicinoids, the capsaicinoids will bind to the colloidal gold-labeled anti-capsaicinoid monoclonal antibody on the gold label pad and migrate upward together. When they reach the test line III where the capsaicinoid-ovalbumin conjugate (CAP-OVA) antigen is fixed, the antigen will compete with the capsaicinoids for binding to the limited antigen binding sites on the colloidal gold-labeled anti-capsaicinoid monoclonal antibody. The higher the capsaicinoid content in the sample, the less colloidal gold-labeled anti-capsaicinoid monoclonal antibody the antigen on the test line can bind to, and the lighter the color band formed on the test line.

[0048] When the number of colloidal gold-labeled antibodies bound to the antigens on the three test lines is less than a certain threshold, no red lines will appear on the three test lines. Regardless of whether the sample contains any of the three targets, colloidal gold-labeled antibodies to the targets, or the conjugates of the colloidal gold-labeled anti-target antibodies and targets, that are not captured by the antigens on the test lines will continue to migrate to the quality control line and bind to the rabbit anti-mouse polyclonal antibody on the quality control line, resulting in enrichment and color development. Therefore, by comparing the color development of the test lines coated with benzo[a]pyrene-ovalbumin conjugate, aflatoxin B1-ovalbumin conjugate (AFB1-OVA), and capsaicinoid-ovalbumin conjugate (CAP-OVA) on the test strip with the corresponding test lines on the control strip, the mixed contamination of the three targets (benzo[a]pyrene, aflatoxin B1, and capsaicinoids) in the sample can be determined.

[0049] Beneficial effects of the present invention:

[0050] (1) Rapid and simultaneous detection of benzo[a]pyrene, aflatoxin B1, and capsaicinoids. The colloidal gold immunochromatographic test strip provided by the present invention can achieve simultaneous and rapid detection of benzo[a]pyrene, aflatoxin B1, or one or two of the target substances capsaicinoids on a single test strip. The antibodies used are all monoclonal antibodies. The benzo[a]pyrene monoclonal antibody used has good specificity and has a cross-reactivity with other structural analogs such as benz[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene, all less than 15%, and has high sensitivity. There is no interference between the detection of the various targets, and the method is simple and rapid.

[0051] (2) High sensitivity. The colloidal gold immunochromatographic test strip provided by the present invention has a minimum detection limit of 5 ng / mL for benzo[a]pyrene in the test solution, a minimum detection limit of 1 ng / mL for aflatoxin B1, and a minimum detection limit of 5 ng / mL for CAP toxin. These detection limits can meet the EU's limit requirements for these three target substances in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a front view of the colloidal gold immunochromatographic test strip for simultaneously detecting mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids of the present invention;

[0053] Figure 2 The result judgment diagram of the samples (numbered 001, 002, and 003) in Example 2;

[0054] In the figure: 1 cardboard, 2 absorbent pad, 3 test pad, 4 gold label pad, 5 sample pad, 6 quality control line, 7 test line III, 8 test line II, 9 test line I, 10 control test strip, 11 test strip; DETAILED DESCRIPTION

[0055] Example 1: Preparation of anti-benzo[a]pyrene monoclonal antibodies

[0056] The anti-benzo[a]pyrene monoclonal antibody is secreted by the BBBE1H1 hybridoma cell line with a deposit number of CCTCC NO: C201882, and is prepared by:

[0057] The anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1 was injected into BALB / c mice that had been treated with Freund's incomplete adjuvant. The ascites of the mice was collected and the antibodies were purified using the caprylic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered with double-layer filter paper, 4 o C, centrifuge at 12000 r / min for more than 15 min, aspirate the supernatant, mix the obtained ascites supernatant with 4 volumes of acetate buffer, slowly add octanoic acid under stirring, the volume of octanoic acid required for each milliliter of ascites is 30-35 μL, mix at room temperature for 30-60 min, 4 o C for more than 2 h. 12000 r / min, 4 o The mixture was centrifuged at 4°C for more than 30 min, and the precipitate was discarded. The supernatant was filtered with double-layer filter paper, and 1 / 10 of the filtrate volume was added with a phosphate buffer with a molar concentration of 0.1 mol / L and a pH of 7.4. The pH of the mixture was adjusted to 7.4 with a 2 mol / L sodium hydroxide solution. Ammonium sulfate was slowly added in an ice bath to a final concentration of 0.277 g / mL. o C for more than 2 h, then 12000 r / min, 4 o C centrifugation for more than 30 min, discard the supernatant, resuspend the precipitate in phosphate buffer with a molar concentration of 0.01 mol / L and a pH of 7.4, which is 1 / 10 of the original ascites volume, put it into a dialysis bag, dialyze it with 0.01 mol / L PBS for two days, and then dialyze it with PB for two days. Take out the protein solution in the dialysis bag, centrifuge it, collect the supernatant, discard the precipitate, and put it into -70 o After pre-freezing C, place it in a freeze dryer and freeze-dry it. Collect the freeze-dried powder, which is the purified anti-benzo[a]pyrene monoclonal antibody;

[0058] The acetate buffer is prepared by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol / L phosphate buffer is prepared by adding water to 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to 100 mL; the 0.1 mol / L phosphate buffer is prepared by adding water to 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.2 g of potassium dihydrogen phosphate to 100 mL.

[0059] The subtype of the anti-benzo[a]pyrene monoclonal antibody secreted by the hybridoma cell line BBBE1H1 was identified as IgG1 using a commercially available subtype identification kit.

[0060] The titer of the antibody purified from mouse ascites was measured by conventional non-competitive enzyme-linked immunosorbent assay (ELISA) to be 1.2×10 5 , that is, the antibody dilution is 1.2×10 5 The result of the solution assay was positive when the concentration of the solution was doubled. The IC50 of its sensitivity to benzo[a]pyrene was determined by conventional indirect competitive ELISA to be 0.013 ng / mL. The specificity of the antibody can be evaluated by the cross-reaction rate. The indirect competitive ELISA method was used to determine the BBBE1H1 monoclonal antibody. A series of standard solutions of BaP, benzo[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene were prepared and added to the enzyme-labeled plate together with an equal volume of antibody. The solution was incubated at 37°C for 1 hour. The other steps were the same as the indirect competitive ELISA method. The competitive inhibition curve was drawn with the concentration of the above-mentioned standard as the horizontal axis and the OD value B / B0 at 450 nm measured by the enzyme reader as the vertical axis. The IC 50 The cross-reaction rate is determined by the ratio of the values. The calculation formula is as follows:

[0061] CR%=(IC 50 BaP / IC 50 other analogs) × 100.

[0062] The cross-reactivity of the BBBE1H1 monoclonal antibody provided by the present invention with other structural analogs, including benz[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene, is less than 15%, and some are as low as less than 1%.

[0063] Detailed results are shown in Table 1:

[0064] Table 1. Cross-reactivity of BBBE1H1 with other structural analogs

[0065]

[0066] Affinity determination of BBBE1H1 using indirect non-competitive ELISA:

[0067] BaP-OVA was coated on the ELISA plate at concentrations of 2.0, 1.0, 0.5, and 0.25 μg / mL, 100 μL / well, at 37°C for 2 h. After blocking with blocking solution for 1 h, the antibody diluted in PBS (dilution factor 1:2) was added to the ELISA plate. The remaining steps were the same as those of the indirect non-competitive ELISA method. 450 The value is the vertical axis, and the logarithm of the antibody concentration (mol / L) is the horizontal axis, and 4 S-shaped curves of 4 concentrations are made. Find the maximum OD value at the top of each S curve, that is, ODmax, and find the antibody concentration corresponding to the 50% ODmax value of each curve. The 4 concentrations are randomly grouped in pairs, and the affinity constant of the antibody is calculated according to the formula Ka=(n-1) / 2(n[Ab']t-[Ab]t), where [Ab']t and [Ab]t are the antibody concentrations corresponding to the two 50% maximum OD values in each group, and n is the multiple of the coating antigen concentration in each group (including three ratios of 1:2, 1:4, and 1:8), and a total of 6 Ka values are obtained. The average of the six Ka values obtained shows that the affinity of the anti-benzo[a]pyrene mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method can reach 1.6×10 9 L / moL.

[0068] Screening of hybridoma cell line BBBE1H1

[0069] 1. Animal immunization

[0070] Six-week-old female BALB / c mice were immunized with laboratory-prepared complete benzo[a]pyrene antigen, BaP-BSA. For the first immunization, the complete benzo[a]pyrene antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at five points on the back of the neck. The second immunization was performed 21 days later with an equal volume of the complete benzo[a]pyrene antigen emulsified with Freund's incomplete adjuvant and injected intraperitoneally. The third immunization was performed two weeks after the second, using the same protocol. The fourth immunization was performed three weeks after the third, also using the same protocol, intraperitoneally. The dose for all four immunizations was the same: 100 μg per mouse. Eight to ten days after each of the first three immunizations, blood was collected by tail-clip, and serum was isolated and assayed for serum titer using an indirect ELISA. Eight days after the third immunization, blood was collected by tail-clip, and mice with sera exhibiting relatively high titers and sensitivity were selected for a final booster immunization, using half the dose of the previous immunization.

[0071] Cell fusion booster immunization Three days after the immunization, 50% polyethylene glycol (PEG) (molecular weight 1450) was used as a fusion agent, and cell fusion was performed according to conventional methods. The specific steps were aseptically killed by cervical dislocation, the spleen was removed, and the spleen was crushed with a homogenizer. The spleen cells were separated using a filter and mixed with murine myeloma SP2 / 0 cells at a cell ratio of 5:1-10:1. The mixture was centrifuged at 1000 rpm for 5 min, and the mixed cells were resuspended in RPMI-1640 basal culture medium and centrifuged at 1000 rpm for 5 min. The supernatant was discarded. Add 1 mL of 50% PEG and incubate for 1 minute. Once adhered, add 20 mL of RPMI-1640 basal culture medium to resuspend the cells. Centrifuge and discard the supernatant. Resuspend the fused cells at the bottom of the tube with 20 mL of complete cell culture medium containing 1% HAT. Add the suspended cells to 80 mL of semi-solid culture medium, mix well, and add to a 6-well cell culture plate at 1-2 mL / well. Place the plate in a 37°C CO2 incubator for static culture. The complete cell culture medium containing 1% HAT contains 20% (volume percentage) fetal bovine serum, 75% (volume percentage) RPMI-1640 basal culture medium, 1% (weight percentage) L-glutamine, 1% (volume percentage) HEPES, 1% (volume percentage) double antibody (10,000 units per milliliter penicillin and 10,000 micrograms per milliliter streptomycin), 1% (volume percentage) growth factor (clone easy), and 1% (weight percentage) hypoxanthine-aminopterin-thymidine (HAT) and methylcellulose purchased from Sigma-Aldrich.

[0072] Cell line screening and cloning

[0073] After 1-2 weeks of cell fusion, when the cell colonies grow to be visible to the naked eye, the clones are picked out from the culture medium with a micropipette and transferred to a 96-well cell culture plate using HAT liquid culture. When the cells grow to 2 / 3 of the bottom of the well, the culture supernatant is aspirated for detection. A two-step screening method is used. The first step is to use the indirect ELISA method to screen out positive wells that are resistant to benzo[a]pyrene but not to the carrier protein BSA; the second step is to use the indirect competitive ELISA method to detect the positive wells screened in the first step, using benzo[a]pyrene as the competitor, and selecting wells with higher absorbance and sensitivity (higher absorbance means that the final measured value of the well with zero competitor, i.e., the positive control well, is higher; higher sensitivity means that the competitor concentration at which the inhibition rate is 50%, also known as IC 50The hybridoma cell line BBBE1H1 was obtained by subcloning using the limiting dilution method and testing using the same two-step method after subcloning 4-5 times. This hybridoma cell line was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on April 3, 2018, under the CCTCC accession number C201882.

[0074] Sequence determination of the variable region of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1.

[0075] (1) Extraction of total RNA: Use the total RNA extraction kit of Tiangen Company and follow the instructions to extract the total RNA of hybridoma cell line BBBE1H1;

[0076] (2) Synthesize cDNA: Use the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer. TM Reverse transcription was performed using the instructions of the 2II reverse transcriptase to synthesize the first-strand cDNA; the primer oligo(dT)15 was purchased from Invitrogen;

[0077] (3) PCR cloning of variable region genes: Primers were designed based on the conserved sites of the mouse antibody gene sequence in GENBANK, and cDNA was used as a template to amplify the variable region genes of the antibody heavy and light chains. The PCR program was as follows: 94°C for 30 s, 58°C for 45 s, and 72°C for 1 min, with 30 cycles of amplification and a final extension at 72°C for 10 min. The PCR products were separated by 1% (weight percent) agarose gel electrophoresis, and the DNA fragments were purified and recovered using a kit. The DNA fragments were ligated into the vector pMD18-T and transformed into Escherichia coli DH5α competent cells. Positive clones were picked and sent to Suzhou Hongxun Biotechnology Co., Ltd. for sequencing. The primer sequences were: heavy chain variable region primers 5'- CAG GTS MAR CTG MAG GAG TCW G-3' (22 mer) and 5'- CAGGGG CCA GTG GAT AGA CAG ATG GGG G -3' (28 mer), where S, M, R and W are degenerate bases, M=A / C, R=A / G, S=G / C, W=A / T; light chain variable region primers 5'- GAC ATC AAG ATG ACC CAG TCT CCA-3' (24 mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24 mer).

[0078] The resulting gene sequence results showed that the heavy chain variable region encoding gene sequence was 360 bp long, as shown in SEQ ID NO: 1. Based on the obtained gene sequence, it was deduced that the heavy chain variable region encoded by this gene sequence consists of 120 amino acids, as shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence was 321 bp long, as shown in SEQ ID NO: 2. Based on the obtained gene sequence, it was deduced that the light chain variable region encoded by this gene sequence consists of 107 amino acids, as shown in SEQ ID NO: 4.

[0079] Example 2 Obtaining anti-aflatoxin B1 monoclonal antibodies

[0080] The anti-aflatoxin monoclonal antibody was prepared from the hybridoma cell line 3G1, deposited with CCTCC No. C201014, according to the method reported in Patent Publication No. 201210117614.9. The preparation method comprises injecting the obtained hybridoma cell line 1C11 into BALB / c mice pre-treated with Freund's incomplete adjuvant, collecting the ascites from the mice, and purifying the anti-aflatoxin monoclonal antibody. The purification method is the octanoic acid-ammonium sulfate method, which specifically involves filtering the mouse ascites through double-layer filter paper, and then purifying the filtered ascites at 4°C. o C, centrifuge at 12000r / min for more than 15min, aspirate the supernatant, mix the supernatant with 4 times the volume of acetate buffer, slowly add octanoic acid while stirring, the volume of octanoic acid required for each milliliter of ascites is 30-35μL, mix at room temperature for 30-60min, 4 o C for more than 2 hours, then 4 o C, centrifuge at 12000r / min for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / 10 volume of filtrate with a molar concentration of 0.1mol / L and a pH value of 7.4 phosphate buffer, and adjust the pH value of the mixture to 7.4 with 2mol / L sodium hydroxide solution. o The mixture was pre-cooled at 4°C, and ammonium sulfate was slowly added to a final concentration of 0.277 g / mL. The mixture was allowed to stand at 4°C for more than 2 h, and then centrifuged at 12,000 rpm for more than 30 min at 4°C. The supernatant was discarded, and the resulting precipitate was resuspended in 0.01 mol / L phosphate buffer at a volume of 1 / 10 of the original ascites fluid, placed in a dialysis bag, and dialyzed with pure water. The fully dialyzed protein solution was frozen in a -70°C refrigerator, and then lyophilized in a freeze vacuum dryer. The lyophilized powder was collected to obtain the purified anti-aflatoxin monoclonal antibody, which was stored in a -20°C refrigerator for later use.

[0081] The acetate buffer solution is prepared by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to make the volume to 100 mL; the 0.01 mol / L phosphate buffer solution is prepared by adding water to 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to make the volume to 100 mL; the 0.1 mol / L phosphate buffer solution is prepared by adding water to 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.2 g of potassium dihydrogen phosphate to make the volume to 100 mL.

[0082] Example 3 Obtaining monoclonal antibodies against capsaicinoids

[0083] The anti-capsaicinoid monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell line YQQD8 with a deposit number of CCTCC NO.C201534, and is specifically prepared in advance according to the method reported in the patent disclosed in 201610079095.X. The specific preparation method is as follows:

[0084] The hybridoma cell line YQQD8 was injected into the abdomen of BALB / c mice that had been treated with Freund's incomplete adjuvant. The ascites of the mice were collected and purified to obtain monoclonal antibodies against capsaicinoids. The purification method was the octanoic acid-ammonium sulfate method. The specific steps were as follows: the mouse ascites was filtered through double-layer filter paper, 4 o C, centrifuge at 12000 r / min for more than 15 min, aspirate the supernatant, mix the obtained ascites supernatant with 4 times the volume of acetate buffer, slowly add octanoic acid under stirring, the volume of octanoic acid required for each milliliter of ascites is 30-35 μL, mix at room temperature for 30-60 min, 4 o C for more than 2 h. 12000 r / min, 4 o The mixture was centrifuged at 4°C for more than 30 min, and the precipitate was discarded. The supernatant was filtered with double-layer filter paper, and 1 / 10 of the filtrate volume was added with a phosphate buffer with a molar concentration of 0.1 mol / L and a pH of 7.4. The pH of the mixture was adjusted to 7.4 with a 2 mol / L sodium hydroxide solution. Ammonium sulfate was slowly added in an ice bath to a final concentration of 0.277 g / mL. o C for more than 2 h, then 12000 r / min, 4 o C centrifugation for more than 30 min, discard the supernatant, resuspend the precipitate in phosphate buffer with a molar concentration of 0.01 mol / L and a pH of 7.4, which is 1 / 10 of the original ascites volume, put it into a dialysis bag, dialyze it with 0.01 mol / L PBS for two days, and then dialyze it with PB for two days. Take out the protein solution in the dialysis bag, centrifuge it, collect the supernatant, discard the precipitate, and put it into -70 oAfter pre-freezing C, place it in a freeze dryer and freeze-dry it. Collect the freeze-dried powder, which is the purified anti-capsaicinoid monoclonal antibody;

[0085] The acetate buffer is prepared by adding water to 0.29 g of sodium acetate and 0.141 mL of acetic acid to 100 mL; the 0.01 mol / L phosphate buffer is prepared by adding water to 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to 100 mL; the 0.1 mol / L phosphate buffer is prepared by adding water to 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 0.2 g of potassium dihydrogen phosphate to 100 mL.

[0086] The hybridoma cell line YQQD8 provided by the present invention can be used to prepare high-titer capsaicin monoclonal antibodies. The titer of the antibody measured by enzyme-linked immunosorbent assay (ELISA) can reach 2.56×10 6 High sensitivity and good specificity, 50% inhibition concentration IC of capsaicin 50 The 50% inhibitory concentration IC for capsaicin, dihydrocapsaicin, and synthetic capsaicin is 5.8 ng / mL. 50 They were 8.5 ng / mL, 5.0 ng / mL, and 13.5 ng / mL, respectively. The cross-reaction rates to capsaicin, dihydrocapsaicin, and synthetic capsaicin ranged from 62.9% to 170%.

[0087] Example 4

[0088] A method for preparing an immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids comprises the following steps:

[0089] (1) Preparation of absorbent pad

[0090] Cut the absorbent paper into 16mm long and 4mm wide specifications to make the absorbent pad;

[0091] (2) Preparation of test pad

[0092] Test line coating:

[0093] Capsaicinoid-ovalbumin conjugate (CAP-OVA) was prepared into a 0.4 mg / mL coating solution with coating buffer, and coated on the nitrocellulose membrane by spot spraying to obtain detection line III. The coating amount of capsaicinoid-ovalbumin conjugate (CAP-OVA) required for each centimeter of detection line III was 160 ng; aflatoxin B1-ovalbumin conjugate (AFB1-OVA) was prepared into a 0.4 mg / mL coating solution with coating buffer, and coated on the nitrocellulose membrane by spot spraying at a position 2 mm away from the detection line III to obtain To test line II, the coating amount of aflatoxin B1-ovalbumin conjugate (AFB1-OVA) required per centimeter of test line II is 200 ng; Benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) is prepared into a 0.5 mg / mL solution with coating buffer, and coated on a nitrocellulose membrane by dot spraying at a position 2 mm away from test line II to obtain test line I, the coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter of test line I is 200 ng, and then dried at 37°C for 30 minutes;

[0094] Coating of quality control line:

[0095] Prepare a 0.25 mg / mL coating solution of rabbit anti-mouse polyclonal antibody with coating buffer and apply it horizontally to a nitrocellulose membrane at a position 3 mm away from the test line III by dot spraying to obtain a quality control line. The required coating amount of rabbit anti-mouse polyclonal antibody per cm of the quality control line is 80 ng, and then dry at 37°C for 1 hour.

[0096] The coating buffer is prepared by adding water to 10 mL of 0.1 g bovine serum albumin, 0.08 g sodium chloride, 0.029 g disodium hydrogen phosphate dodecahydrate, 0.002 g potassium chloride, and 0.002 g potassium dihydrogen phosphate.

[0097] The nitrocellulose membrane is 24 mm long and 4 mm wide.

[0098] (3) Preparation of sample pad

[0099] Cut the glass fiber membrane into a size of 12 mm in length and 4 mm in width, soak it in the blocking solution, take it out, dry it at 37°C for 8 hours to obtain the sample pad, and then store it in a desiccator at room temperature.

[0100] The blocking solution is prepared by adding water to 100 mL of 1 g of ovalbumin, 2 g of sucrose, 0.02 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate.

[0101] (4) Preparation of gold label pad

[0102] Cut the glass fiber membrane into a size of 10 mm in length and 4 mm in width, soak it in the blocking solution described in step (3), take it out, dry it at 37°C for 8 hours, and spray a mixed solution of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution, colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody solution and colloidal gold-labeled anti-capsaicinoid monoclonal antibody solution on the dried glass fiber membrane in a dot spraying manner, wherein the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length is 150 ng, the amount of colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody required is 120 ng, and the amount of anti-capsaicinoid monoclonal antibody required is 150 ng, and the vacuum freeze-drying is carried out for 2 hours, and the membrane is stored in a desiccator at room temperature.

[0103] The colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution is prepared by an unsaturated labeling method, wherein 50.0 mL of a commercially available colloidal gold solution with a mass concentration of 0.01% is taken, the pH value is adjusted with 0.4 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2 mL of A 0.1 mg / mL aqueous solution of anti-benzo[a]pyrene monoclonal antibody was stirred for 30 minutes; a 10% bovine serum albumin aqueous solution was added to a final bovine serum albumin concentration of 1%, and stirring was continued for 30 minutes; after standing at 4°C for 2 hours, the solution was centrifuged at 1500 rpm for 15 minutes, the supernatant was collected, and the precipitate was discarded; the supernatant was centrifuged at 12000 rpm for 30 minutes, the supernatant was discarded, and 40.0 mL of labeled washing and preservation solution was added; the solution was centrifuged at 12000 rpm for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in labeled washing and preservation solution to obtain 5.0 mL of concentrate, which was placed in a 4°C refrigerator for use. The mass concentration of the colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution was 0.04 mg / mL;

[0104] The colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody is prepared by an unsaturated labeling method, which is as follows: 50.0 mL of a commercially available colloidal gold solution with a mass concentration of 0.01% is taken, the pH value is adjusted with 0.4 mL of a 0.1 mol / L potassium carbonate aqueous solution, 1.5 mL of a 0.1 mg / mL anti-aflatoxin B1 monoclonal antibody aqueous solution is slowly added under stirring, and stirring is continued for 30 minutes; a 10% bovine serum albumin aqueous solution is added to a final bovine serum albumin mass concentration of 1%, and stirring is continued. Stir for 30 minutes; after placing at 4°C for 2 hours, centrifuge at 1500 rpm for 15 minutes, take the supernatant, and discard the precipitate; centrifuge the supernatant at 12000 rpm for 30 minutes, discard the supernatant, and add 40.0 mL of labeled washing and preservation solution; centrifuge again at 12000 rpm for 30 minutes, discard the supernatant, and resuspend the precipitate in labeled washing and preservation solution to obtain 5.0 mL of concentrate, which is placed in a 4°C refrigerator for use. The mass concentration of the colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody solution is 0.03 mg / mL;

[0105] The colloidal gold labeled anti-capsaicinoid monoclonal antibody solution is prepared by an unsaturated labeling method, wherein 50.0 mL of a commercially available colloidal gold solution with a mass concentration of 0.01% is taken, the pH value is adjusted with 0.425 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2.5 Add 0.1 mg / mL of an aqueous solution of anti-capsaicinoid monoclonal antibody and continue stirring for 30 minutes; add a 10% bovine serum albumin aqueous solution to a final bovine serum albumin concentration of 1% and continue stirring for 30 minutes; after standing at 4°C for 2 hours, centrifuge at 1500 rpm for 15 minutes, collect the supernatant, and discard the precipitate; centrifuge the supernatant at 12000 rpm for 30 minutes, discard the supernatant, add 40.0 mL of labeled washing storage solution; centrifuge again at 12000 rpm for 30 minutes, discard the supernatant, and resuspend the precipitate in labeled washing storage solution to obtain 5.0 mL of concentrate, which is refrigerated at 4°C until use. The mass concentration of the colloidal gold-labeled anti-capsaicinoid monoclonal antibody solution is 0.05 mg / mL;

[0106] The particle size of the colloidal gold in the colloidal gold solution is 15 nm;

[0107] The 0.1 mol / L potassium carbonate aqueous solution is prepared by dissolving 13.8 g potassium carbonate in pure water to a volume of 1000 mL and filtering through a 0.22 μm filter membrane. The labeled washing and preservation solution is prepared by dissolving 2.0 g polyethylene glycol-20000, 0.2 g sodium azide, and 0.1235 g boric acid in pure water to a volume of 1000 mL and filtering through a 0.22 μm filter membrane.

[0108] (5) Assembly of test strips

[0109] On one side of the cardboard, stick the absorbent pad, detection pad, gold label pad and sample pad in order from top to bottom. The adjacent pads are overlapped at the joints with an overlap length of 1 to 3 mm to obtain an immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1 and capsaicinoids. Figure 1 and Figure 2 .

[0110] Application of the above-mentioned immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicinoids in the detection of corn samples:

[0111] Preparation of immunomagnetic beads for monoclonal antibodies against benzo[a]pyrene, capsaicinoids, and aflatoxin:

[0112] The steps for coupling magnetic beads to antibodies are as follows:

[0113] a. Washing: Weigh 2.5 mg of carboxyl-modified magnetic beads into a 15 mL centrifuge tube, add 2 mL of washing solution and wash twice, place on a magnetic stand for magnetic separation, discard the supernatant, then wash twice with coupling buffer, magnetically separate, and discard the supernatant.

[0114] b Coupling: Add 4 mL of coupling buffer to the washed magnetic beads, add 2.5 mg of anti-benzo[a]pyrene monoclonal antibody, 4 mg of anti-capsaicinoid monoclonal antibody, and 1 mg of anti-aflatoxin monoclonal antibody, and place on a shaker at 8°C, 200 r / min overnight for coupling. After the reaction is complete, place on a magnetic rack for magnetic separation.

[0115] c Blocking: Add Tris-HCl buffer to the coupled magnetic beads and wash twice, then add 4 mL of Tris-HCl blocking solution and react in a shaker at 8°C and 200 rpm for 2 h.

[0116] d. Storage: After the blocking reaction is complete, perform magnetic separation, discard the supernatant, dilute to 5 mL with 0.01% NaN3 in PBS, and store in a refrigerator at 4°C until use. This will be used for subsequent sample pretreatment.

[0117] Use a 10 mL plastic centrifuge tube as a reaction vessel. Accurately weigh 1 g of soybean oil and add 5 mL of n-hexane solution. Ultrasonicate for 10 minutes, then add another 5 mL of n-hexane solution and ultrasonicate for another 10 minutes. Take 1 mL of the extract and dilute to 10 mL with 9 mL of ddH2O. Vortex for 2 minutes. Add immunomagnetic beads conjugated with anti-benzo[a]pyrene monoclonal antibodies, anti-capsaicinoid monoclonal antibodies, and anti-aflatoxin B1 monoclonal antibodies. Vortex for 10 minutes and discard the supernatant. Add 1 mL of methanol and vortex for 2 minutes. Dilute 5-fold with sample release buffer. The supernatant is used as the test solution. Then take 100 μL of the diluted sample solution as the sample sustained-release liquid and add it dropwise to the sample pad of the immunochromatographic test strip, which serves as the detection test strip. At the same time, take the sample sustained-release liquid as the negative control liquid and add it dropwise to the sample pad of another immunochromatographic test strip used for simultaneous detection of mixed contamination of benzo[a]pyrene and capsaicinoids, which serves as the control test strip. Read the results after 15 minutes.

[0118] Test results: The quality control line of the 001 test strip showed a red strip, the colors of the test lines I and II were close to those of the test lines I and III in the control test strip, and the test line II did not show any color. Figure 2-1 , thus determining: the content of benzo[a]pyrene in the test sample solution 001 is less than 5 ng / mL; the content of capsaicinoids is less than 5 ng / mL; the content of aflatoxin B1 is equal to or greater than 20 ng / mL;

[0119] The quality control line of the 002 test strip shows a red strip. The color of the test line Ⅰ is close to the color of the test line Ⅰ in the control test strip. The colors of the test lines Ⅱ and Ⅲ are lighter than those of the control test strip. Figure 2-2 , thus determining that: the content of benzo[a]pyrene in the test sample solution 002 is lower than 5 ng / mL, the content of aflatoxin B1 is higher than 1 ng / mL and lower than 10 ng / mL, and the content of capsaicinoids is higher than 5 ng / mL and lower than 80 ng / mL;

[0120] The quality control line of the 003 test strip shows a red strip, and the colors of the test lines I, II and III are close to the colors of the test lines in the control test strip. Figure 2 -3, indicating that: the content of benzo[a]pyrene in the test sample solution is less than 5 ng / mL; the content of aflatoxin B1 is less than 1 ng / mL; and the content of capsaicinoids is less than 5 ng / mL.

[0121] The above samples 001, 002 and 003 were tested by high performance liquid chromatography, and the test results showed that the contents of capsaicinoids, benzo[a]pyrene and aflatoxin B1 in the test solution of sample 001 were 2.8 ng / mL, 1.9 ng / mL and 25 ng / mL, respectively; the contents of capsaicinoids, benzo[a]pyrene and aflatoxin B1 in the test solution of sample 002 were 15.2 ng / mL, 4.1 ng / mL and 8.6 ng / mL, respectively; the contents of capsaicinoids, benzo[a]pyrene and aflatoxin B1 in the test solution of sample 003 were 4.6 ng / mL, 0.61 ng / mL and 3.2 ng / mL, respectively. The results showed that the test strip method had a good correlation with the liquid chromatography method, had good accuracy, and could meet the qualitative detection requirements of benzo[a]pyrene, capsaicinoids, and aflatoxins.

[0122] Example 5

[0123] A method for preparing an immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene and capsaicinoids comprises the following steps:

[0124] (1) Preparation of absorbent pad

[0125] Cut the absorbent paper into 18mm long and 3mm wide specifications to make the absorbent pad;

[0126] (2) Preparation of test pad

[0127] Test line coating:

[0128] Benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) was prepared into a 0.5 mg / mL coating solution with coating buffer and coated on the nitrocellulose membrane by spot spraying at a position 18 mm from the top edge of the nitrocellulose membrane to form test line I. The coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per cm of test line I was 200 ng. Capsaicinoid-ovalbumin conjugate (CAP-OVA) was prepared into a 0.25 mg / mL solution with coating buffer and coated on the nitrocellulose membrane by spot spraying at a position 3 mm from the test line I to form test line II. The coating amount of capsaicinoid-ovalbumin conjugate (CAP-OVA) required per cm of test line II was 300 ng. The membrane was then dried at 40°C for 30 minutes.

[0129] Coating of quality control line:

[0130] Prepare a 0.25 mg / mL coating solution of rabbit anti-mouse polyclonal antibody with coating buffer and apply it horizontally to a nitrocellulose membrane at a position 6 mm away from the test line I by dot spraying to obtain a quality control line. The required coating amount of rabbit anti-mouse polyclonal antibody per cm quality control line is 80 ng, and then dry at 37°C for 1 hour.

[0131] The coating buffer is prepared by adding water to 10 mL of 0.2 g bovine serum albumin, 0.08 g sodium chloride, 0.029 g disodium hydrogen phosphate dodecahydrate, 0.002 g potassium chloride, and 0.002 g potassium dihydrogen phosphate.

[0132] The nitrocellulose membrane is 30 mm long and 3 mm wide.

[0133] (3) Preparation of sample pad

[0134] Cut the glass fiber membrane into a size of 15 mm in length and 3 mm in width, soak it in the blocking solution, take it out, dry it at 37°C for 6 hours to obtain the sample pad, and then store it in a desiccator at room temperature.

[0135] The blocking solution is prepared by adding water to 2 g of ovalbumin, 4 g of sucrose, 0.05 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate to make the volume to 100 mL.

[0136] (4) Preparation of gold label pad

[0137] Cut the glass fiber membrane into a size of 12 mm in length and 3 mm in width, soak it in the blocking solution described in step (3), take it out, dry it at 40°C for 6 hours, and spray a mixed solution of a colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution and a colloidal gold-labeled anti-capsaicinoid monoclonal antibody solution on the dried glass fiber membrane in a dot spraying manner, wherein the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length is 200 ng, and the amount of colloidal gold-labeled anti-capsaicinoid monoclonal antibody required is 200 ng. Then, freeze-dry it in a vacuum for 2 hours and store it in a desiccator at room temperature.

[0138] The preparation method of the colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution and the colloidal gold-labeled anti-capsaicinoid monoclonal antibody solution is the same as that in Example 4, except that the colloidal gold particle size of the colloidal gold solution used is 20 nm;

[0139] (5) Assembly of test strips

[0140] On one side of the cardboard, a water-absorbing pad, a detection pad, a gold-labeled pad, and a sample pad are sequentially pasted from top to bottom, and adjacent pads are overlapped at the joints with an overlap length of 1 mm, thereby obtaining an immunochromatographic test strip for simultaneously detecting mixed contamination of benzo[a]pyrene and capsaicinoids.

[0141] Preparation of benzo[a]pyrene and capsaicinoids immunomagnetic beads:

[0142] a. Washing: Weigh 2.5 mg of carboxyl-modified magnetic beads into a 15 mL centrifuge tube, add 2 mL of washing solution and wash twice, place on a magnetic stand for magnetic separation, discard the supernatant, then wash twice with coupling buffer, magnetically separate, and discard the supernatant.

[0143] b Coupling: Add 4 mL of coupling buffer to the washed magnetic beads, add 2.5 mg of anti-benzo[a]pyrene monoclonal antibody and 4 mg of anti-capsaicinoid monoclonal antibody, place on a shaker at 8°C, 200 r / min overnight for coupling. After the reaction is complete, place on a magnetic stand for magnetic separation.

[0144] c Blocking: Add Tris-HCl buffer to the coupled magnetic beads and wash twice, then add 4 mL of Tris-HCl blocking solution and react in a shaker at 8°C and 200 rpm for 2 h.

[0145] d. Storage: After the blocking reaction is complete, perform magnetic separation, discard the supernatant, dilute to 5 mL with 0.01% NaN3 in PBS, and store in a refrigerator at 4°C until use. This will be used for subsequent sample pretreatment.

[0146] Use a 10 mL plastic centrifuge tube as a reaction vessel. Accurately weigh 1 g of soybean oil and add 5 mL of n-hexane solution. Ultrasonicate for 10 minutes. Then add another 5 mL of n-hexane solution and ultrasonicate for 10 minutes. Take 1 mL of the extract and dilute to 10 mL with 9 mL of ddH2O. Vortex for 2 minutes. Add immunomagnetic beads conjugated to monoclonal antibodies against benzo[a]pyrene and capsaicinoids. Vortex for 10 minutes and discard the supernatant. Add 1 mL of methanol solution, vortex mix for 2 minutes, dilute 5 times with sample release solution, use the supernatant as the test solution, take 100 μL of the diluted sample solution as the sample release solution and add it dropwise to the sample pad of an immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene and capsaicinoids, which is used as the test test strip. At the same time, take 100 μL of the sample release solution as the negative control solution and add it dropwise to the sample pad of another immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene and capsaicinoids, which is used as the control test strip. Read the result after 15 minutes.

[0147] Test results: The quality control line of the test strip shows a red band, and the colors of test line I and test line II are close to the colors of the test lines in the control test strip. Therefore, it can be determined that the content of capsaicinoids in the test sample solution is less than 10 ng / mL; the content of benzo[a]pyrene is less than 5 ng / mL.

[0148] High-performance liquid chromatography (HPLC) was used to detect the above samples and found that the contents of capsaicinoids and benzo[a]pyrene were 8.5 ng / mL and 3.2 ng / mL, respectively. This shows that the test strip method has a good correlation with liquid chromatography and has good accuracy, which can meet the qualitative detection needs of benzo[a]pyrene and capsaicinoids.

Claims

1. A rapid test strip for the main harmful factors in edible vegetable oils, characterized in that: The invention comprises a bottom plate, on one side of which a water-absorbing pad, a detection pad, a gold-labeled pad and a sample pad are sequentially attached from top to bottom, and adjacent pads are overlapped and connected at the connection point, the detection pad is based on a nitrocellulose membrane, and a quality control line and a detection line are horizontally arranged on the nitrocellulose membrane, the detection line is located below the quality control line, and there are two or three detection lines, which are distributed at intervals, and each detection line is coated with a test substance-protein conjugate, the test substance is benzo[a]pyrene and aflatoxin B1 and / or capsaicinoids, and the test substance-protein conjugate is coated on the test substance-protein conjugate. The conjugates are benzo[a]pyrene-ovalbumin conjugate, aflatoxin B1-ovalbumin conjugate, and capsaicinoid-ovalbumin conjugate; the quality control line is coated with rabbit anti-mouse polyclonal antibodies; the gold label pad is sprayed laterally with gold-labeled monoclonal antibodies corresponding to the analyte; the monoclonal colloids of the analyte are anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin B1 monoclonal antibodies, and anti-capsaicinoid monoclonal antibodies; the anti-benzo[a]pyrene monoclonal antibodies are secreted by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882.

2. The rapid test strip for detecting major hazard factors in edible vegetable oil according to claim 1, characterized in that: The absorbent pad is 16-18 mm long and 3-4 mm wide; the detection pad is 18-30 mm long and 3-4 mm wide; the gold mark pad is 10-12 mm long and 3-4 mm wide; the sample pad is 12-15 mm long and 3-4 mm wide, and the overlapping length of adjacent pads is 1-3 mm.

3. The rapid test strip for detecting major hazard factors in edible vegetable oil according to claim 1, characterized in that: The distance between each two adjacent detection lines on the detection pad is 2-3 mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7 mm.

4. The rapid test strip for detecting major hazard factors in edible vegetable oils according to claim 1, characterized in that: The coating amount of benzo[a]pyrene-ovalbumin conjugate required per centimeter on the detection line coated with benzo[a]pyrene-ovalbumin conjugate is 100-300 ng; the coating amount of aflatoxin B1-ovalbumin conjugate required per centimeter on the detection line coated with aflatoxin B1-ovalbumin conjugate is 100-300 ng; the coating amount of capsaicinoid-ovalbumin conjugate required per centimeter on the detection line coated with capsaicinoid-ovalbumin conjugate is 100-300 ng; and the coating amount of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 50-200 ng.

5. The rapid test strip for detecting major hazard factors in edible vegetable oil according to claim 1, characterized in that: The particle size of the colloidal gold used in the gold label pad is 15 to 20 nm; the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length on the gold label pad is 100 to 200 ng, the amount of colloidal gold-labeled anti-aflatoxin B1 cloned antibody required is 100 to 200 ng, and the amount of colloidal gold-labeled anti-capsaicinoid monoclonal antibody required is 100 to 600 ng.

6. The rapid test strip for detecting major hazard factors in edible vegetable oils according to claim 1, characterized in that: The IC50 value of the anti-aflatoxin B1 monoclonal antibody is less than or equal to 1.6 ppb; the IC50 value of the anti-capsaicinoid monoclonal antibody is less than or equal to 8.5 ppb.

7. The method for preparing a rapid test strip for detecting major hazard factors in edible vegetable oils according to claim 1, characterized in that: The following steps are involved: (1) Preparation of absorbent pad Cut the absorbent paper to make an absorbent pad; (2) Preparation of detection pad Test line coating: The benzo[a]pyrene-ovalbumin conjugate, the aflatoxin B1-ovalbumin conjugate and the capsaicinoid-ovalbumin conjugate were prepared into 0.25-0.5 mg / mL coating solutions with coating buffer, and coated on nitrocellulose membranes by spraying. Two or three test lines were obtained according to the test object, and then dried at 37-40°C for 30-60 minutes. The test line coated with the benzo[a]pyrene-ovalbumin conjugate had a concentration of 0.25-0.5 mg / mL. The required coating amount of benzo[a]pyrene-ovalbumin conjugate is 100-300 ng; the required coating amount of aflatoxin B1-ovalbumin conjugate per centimeter on the test line coated with aflatoxin B1-ovalbumin conjugate is 100-300 ng; the required coating amount of capsaicinoids-ovalbumin conjugate on the test line coated with capsaicinoids-ovalbumin conjugate is 100-300 ng, and the distance between each two adjacent test lines is 2-3 mm; Coating of quality control line: Prepare a 0.2-0.4 mg / mL coating solution of rabbit anti-mouse polyclonal antibody using coating buffer, and coat it horizontally on a nitrocellulose membrane by dot spraying at a position 5-7 mm away from the test line close to the quality control line to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the quality control line is 50-200 ng, and then dry at 37-40°C for 1-2 hours. (3) Preparation of sample pad Soak the glass fiber membrane in the blocking solution, take it out, dry it at 37-40℃ for 6-10 hours to obtain the sample pad, and then store it in a desiccator at room temperature; (4) Preparation of gold label pad The glass fiber membrane is soaked in a blocking solution, taken out, and dried at 37-40° C. for 6-10 hours. A mixed solution of a monoclonal antibody to be detected labeled with colloidal gold is sprayed horizontally on the dried glass fiber membrane in a dot spraying manner according to the object to be detected. The monoclonal antibody to be detected labeled with colloidal gold is a colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody solution, a colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody solution, or a colloidal gold-labeled anti-capsaicinoid substance. A monoclonal antibody solution is prepared, wherein the amount of colloidal gold-labeled anti-benzo[a]pyrene monoclonal antibody required per centimeter of spraying length is 100-200 ng, the amount of colloidal gold-labeled anti-aflatoxin B1 monoclonal antibody required is 100-200 ng, and the amount of anti-capsaicinoid monoclonal antibody required is 100-600 ng, followed by vacuum freeze-drying for 2-4 hours and storage in a desiccator at room temperature; the anti-benzo[a]pyrene monoclonal antibody is secreted and produced by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882; (5) Assembly of test strips On one side of the cardboard, a water-absorbing pad, a detection pad, a gold label pad and a sample pad are pasted in sequence from top to bottom, and adjacent pads are overlapped at the connection points with an overlapping length of 1 to 3 mm, thereby obtaining a colloidal gold immunochromatographic test strip for simultaneously detecting mixed contamination of benzo[a]pyrene, aflatoxin B1 and capsaicinoids.

8. The preparation method according to claim 7, characterized in that: The coating buffer contains the following in every 10 mL: 0.1-0.2 g of bovine serum albumin, 0.08 g of sodium chloride, 0.029 g of disodium hydrogen phosphate dodecahydrate, 0.002 g of potassium chloride, and 0.002 g of potassium dihydrogen phosphate; and the blocking solution in step (3) and step (4) contains in every 100 mL: 1-2 g of ovalbumin, 2-5 g of sucrose, 0.02-0.05 g of sodium azide, 0.8 g of sodium chloride, 0.29 g of disodium hydrogen phosphate dodecahydrate, 0.02 g of potassium chloride, and 0.02 g of potassium dihydrogen phosphate.

9. Use of the test strip for rapid detection of major hazard factors in edible vegetable oils according to claim 1, characterized in that: The sample extract was diluted with the sample sustained-release solution and added to the sample pad of one of the rapid detection strips for major hazard factors in edible vegetable oils for detection, which served as the detection strip. Another equal volume of the sample sustained-release solution was taken as a negative control solution and added dropwise to the sample pad of another rapid detection strip for major hazard factors in edible vegetable oils, which served as a control strip. After 15-20 minutes, the detection strip and the control strip were subjected to color contrast: When the color of the test line coated with the benzo[a]pyrene-ovalbumin conjugate on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the benzo[a]pyrene content in the test sample solution is less than 5 ng / mL; when the color is lighter than the color of the corresponding test line, it indicates that the benzo[a]pyrene content in the test sample solution is equal to or higher than 5 ng / mL and lower than 50 ng / mL; when no color is developed, it indicates that the benzo[a]pyrene content in the test sample solution is equal to or higher than 50 ng / mL; When the color of the test line coated with the aflatoxin B1-ovalbumin conjugate on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the aflatoxin B1 content in the test sample solution is less than 1 ng / mL; when the color is lighter than the corresponding test line, it indicates that the aflatoxin B1 content in the test sample solution is equal to or higher than 1 ng / mL and lower than 20 ng / mL; when no color is developed, it indicates that the aflatoxin B1 content in the test sample solution is equal to or higher than 20 ng / mL; When the color of the test line coated with the capsaicinoid-ovalbumin conjugate on the test strip is close to the color of the corresponding test line on the control test strip, it indicates that the content of capsaicinoids in the test sample solution is less than 5 ng / mL; when the color is lighter than the color of the corresponding test line, it indicates that the content of capsaicinoids in the test sample solution is equal to or higher than 5 ng / mL and lower than 80 ng / mL; when no color is developed, it indicates that the content of capsaicinoids in the test sample solution is equal to or higher than 80 ng / mL; When the quality control line does not show color, the test strip is considered invalid regardless of whether the test line of the test strip shows color; Finally, the content of benzo[a]pyrene, aflatoxin B1 and / or capsaicinoids in the sample to be tested is obtained through conversion.

10. The use according to claim 9, wherein the sample is edible vegetable oil, and the pretreatment steps are: ultrasonically extracting the edible vegetable oil with n-hexane, taking the supernatant, diluting it 8-12 times with water, then adding immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin monoclonal antibodies, and / or anti-capsaicinoid monoclonal antibodies, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with a sample release solution to obtain a supernatant, i.e., a test solution; When detecting benzo[a]pyrene and aflatoxin B1, the immunomagnetic beads used are those coupled with anti-benzo[a]pyrene monoclonal antibodies and anti-aflatoxin monoclonal antibodies; When detecting benzo[a]pyrene and capsaicinoids, the immunomagnetic beads used are those coupled with anti-benzo[a]pyrene monoclonal antibodies and anti-capsaicinoid monoclonal antibodies; When detecting benzo[a]pyrene, aflatoxin B1 and capsaicinoids, magnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin monoclonal antibodies and anti-capsaicinoid monoclonal antibodies are used. The immunomagnetic beads coupled with anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin monoclonal antibodies and / or anti-capsaicinoid monoclonal antibodies include magnetic beads and anti-benzo[a]pyrene monoclonal antibodies, anti-aflatoxin B1 monoclonal antibodies and / or anti-capsaicinoid monoclonal antibodies coupled based on the COOH groups on the magnetic beads and the amino groups of the antibodies.

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