Rapid detection kit for evaluating the safety of edible vegetable oils, preparation method and application thereof

Abstract

The present invention relates to a time-resolved fluorescence kit for evaluating the safety of edible vegetable oils, a preparation method, and its application. The kit comprises a fluorescent test strip and a sample reaction bottle containing a europium-labeled anti-benzo[a]pyrene monoclonal antibody, a europium-labeled anti-aflatoxin B1 monoclonal antibody, and a europium-labeled anti-capsaicinoid universal monoclonal antibody. One side of the fluorescent test strip comprises, from top to bottom, an absorbent pad, a test pad, and a sample pad, with adjacent pads overlapping and connecting at the junction. The test pad is based on a nitrocellulose membrane, which is provided with a transverse quality control line and a test line. The quality control line is coated with a rabbit anti-mouse polyclonal antibody. The test lines are located below the quality control line and are spaced apart. There are two or three test lines, each coated with a test substance-protein conjugate. The kit can achieve simultaneous and rapid detection of multiple targets on a single test strip. The kit is used for evaluating the quality and safety of edible vegetable oils, identifying waste cooking oils, and discovering strong carcinogens in edible vegetable oils.

Description

Technical Field

[0001] The present invention relates to the field of edible vegetable oil safety, and in particular to a rapid detection kit for detecting and evaluating the safety of edible vegetable oil, a preparation method and an application thereof. Background Art

[0002] The quality and safety issues of edible oil caused by the return of waste cooking oil to the dining table, and the pollution problems caused by strong carcinogens such as benzo[a]pyrene and aflatoxin in the oil raw materials and processing processes, have become hot and difficult research topics in the field of global food safety.

[0003] Benzo[a]pyrene can be produced during oil processing and is teratogenic, carcinogenic, and mutagenic to humans. Aflatoxin B1 is the most toxic mycotoxin and a Class I carcinogen. It can occur in all stages of the oilseed production process, including the planting, harvesting, storage, transportation, and processing of raw materials. Capsaicinoids are the primary chemical compounds that give chili peppers their spicy flavor. Their main components include capsaicin, dihydrocapsaicin, homodihydrocapsaicin, nordihydrocapsaicin, and homocapsaicinoids. 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. Summary of the Invention

[0004] The present invention relates to a time-resolved fluorescence kit for evaluating the safety of edible vegetable oils and its preparation method. The kit features simple operation, rapid detection, and high sensitivity. It can simultaneously detect the levels of benzo[a]pyrene, aflatoxin B1, and capsaicinoids in samples and is used for quality and safety assessment of edible vegetable oils.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A time-resolved fluorescence kit for evaluating the safety of edible vegetable oils includes a fluorescent test strip and a sample reaction bottle containing a europium-labeled anti-benzo[a]pyrene monoclonal antibody, a europium-labeled anti-aflatoxin B1 monoclonal antibody, and a europium-labeled anti-capsaicinoid universal monoclonal antibody. One side of the fluorescent test strip is composed of an absorbent pad, a detection pad, and a sample pad from top to bottom, with adjacent pads overlapping and connected at the connection point. The detection pad is based on a nitrocellulose membrane, and the nitrocellulose membrane is provided with a horizontal quality control line and a detection line. The quality control line is coated with a rabbit anti-mouse polyclonal antibody. The detection line is located below the quality control line and there are two detection lines. The test line comprises one or three test lines arranged at intervals, each test line being coated with a test substance-protein conjugate, the test substances being benzo[a]pyrene, aflatoxin B1, and capsaicinoids, the test substance-protein conjugates being benzo[a]pyrene-ovalbumin conjugate (BAP-OVA), aflatoxin B1-ovalbumin conjugate (AFB1-OVA), and capsaicinoids-ovalbumin conjugate (CAP-OVA), and the quality control line being coated with a rabbit anti-mouse polyclonal antibody; the anti-benzo[a]pyrene monoclonal antibody is secreted by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882; the hybridoma cell line BBBE1H1 was deposited in the China Center for Type Culture Collection (CCTCC) on April 3, 2018, and the deposit address is Wuhan University, Wuhan, China.

[0007] According to the above scheme, the preparation method of the europium-labeled anti-benzo[a]pyrene monoclonal antibody is as follows: the europium labeling reagent is added to the boric acid buffer and sonicated, and then the EDC solution is added, vortexed and mixed, centrifuged, the supernatant is removed, the boric acid buffer is added for re-dissolution, vortexed and mixed, the anti-benzo[a]pyrene monoclonal antibody is added, the reaction is shaken, the supernatant is removed by centrifugation, the solution is blocked, and the solution is packaged and lyophilized.

[0008] According to the above scheme, the preparation method of the europium-labeled anti-aflatoxin B1 monoclonal antibody is as follows: the europium labeling reagent is added to the boric acid buffer and sonicated, then the EDC solution is added, the mixture is shaken and mixed, centrifuged, the supernatant is removed, the boric acid buffer is added for re-dissolution, the mixture is shaken and mixed, the anti-benzo[a]pyrene monoclonal antibody is added, the reaction is shaken, the supernatant is removed by centrifugation, the mixture is blocked, and the mixture is packaged and freeze-dried.

[0009] According to the above scheme, the preparation method of the europium-labeled universal monoclonal antibody against capsaicinoids is as follows: the europium labeling reagent is added to the boric acid buffer and sonicated, and then the EDC solution is added, shaken and mixed, centrifuged, the supernatant is removed, the boric acid buffer is added for re-dissolution, shaken and mixed, the anti-benzo[a]pyrene monoclonal antibody is added, the reaction is shaken, the supernatant is removed by centrifugation, the solution is blocked, and the solution is packaged and lyophilized.

[0010] According to the above scheme, the europium labeling reagent is europium oxide latex fluorescent microspheres.

[0011] According to the above scheme, the EDC solution is activated by oscillation for 15 to 30 minutes, the centrifugal speed is 10,000 to 15,000 rpm, and the reaction is carried out on a shaker for 2 to 4 hours; the blocking is to add borate buffer containing 0.5 to 1% BSA to the precipitate after centrifugation to re-dissolve it and block the excess binding sites on the surface of the europium labeling reagent.

[0012] 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.

[0013] According to the above scheme, the IC50 of the universal monoclonal antibody against capsaicinoids 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.

[0014] According to the above scheme, the distance between the detection line close to the quality control line on the detection pad and the upper edge of the nitrocellulose membrane is 8-20 mm, the spacing between each two adjacent detection lines is 1.0-4.5 mm, the spacing between the detection line close to the quality control line and the quality control line is 3-10 mm, and the sample reaction bottle is a 1-5 mL snap-top bottle.

[0015] According to the above scheme, the coating amount of benzo[a]pyrene-ovalbumin conjugate on the detection line of the time-resolved fluorescent immunochromatographic test strip is 0.4-0.8 μg / cm, the coating amount of aflatoxin B1-ovalbumin conjugate is 0.4-0.8 μg / cm, and the coating amount of capsaicinoid-ovalbumin conjugate is 0.8-1.0 μg / cm; the content of the europium-labeled anti-benzo[a]pyrene monoclonal antibody freeze-dried product in the sample reaction bottle is 0.1-0.3 μg, the content of the anti-aflatoxin B1 monoclonal antibody freeze-dried product is 0.15-0.4 μg, and the content of the anti-capsaicin monoclonal antibody freeze-dried product is 0.2-0.4 μg.

[0016] According to the above scheme, the time-resolved fluorescence immunochromatographic test strip for simultaneous detection of mixed contamination of benzo[a]pyrene, aflatoxin B1, and capsaicin further comprises a sample release solution and a sample release solution pipette, wherein the sample release solution is a PBS solution containing 0.01% to 0.30% Tween-20, 0.5 to 1.5% sucrose, and 0.1 to 1% bovine serum albumin;

[0017] According to the above scheme, the preparation method of the time-resolved fluorescent immunochromatographic test strip is as follows:

[0018] (1) Cut absorbent paper into absorbent pads;

[0019] (2) Preparation of test pad:

[0020] Benzo[a]pyrene-ovalbumin conjugate, aflatoxin B1-ovalbumin conjugate, and capsaicinoid-ovalbumin conjugate are prepared into coating solutions with a concentration of 0.25-2 mg / mL. Depending on the type and quantity of the test substance to be tested, the coating solutions are streaked onto a nitrocellulose membrane to obtain two or three test lines, and then dried at 37-40°C for 30-60 minutes. The coating amount for the benzo[a]pyrene-ovalbumin conjugate is 0.4-0.8 μg, the coating amount of the aflatoxin B1-ovalbumin conjugate required per centimeter of the test line is 0.4-0.8 μg, and the coating amount required for the capsaicin-ovalbumin conjugate is 0.8-1.0 μg.

[0021] Prepare a coating solution of rabbit anti-mouse polyclonal antibody at a concentration of 0.1-0.45 mg / mL and coat it horizontally on a nitrocellulose membrane at a position 5-10 mm away from the test line by streaking to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the quality control line is 0.4-0.8 μg, and then dry at 37-40°C for 30-60 minutes.

[0022] (3) Preparation of sample pad:

[0023] Soak the glass fiber membrane in the blocking solution, take it out, dry it at 37-40℃ for 4-10 hours to obtain the sample pad, and then store it in a desiccator at room temperature;

[0024] (4) Assembly of immunochromatographic time-resolved fluorescence test strips:

[0025] On one side of the cardboard, a water-absorbing pad, a detection pad, and a sample pad are sequentially pasted from top to bottom. Adjacent pads are overlapped at the joints with an overlap length of 1-3 mm to obtain an immunochromatographic time-resolved fluorescence test strip.

[0026] According to the above scheme, the distance between the detection line close to the quality control line on the detection pad in the immunochromatographic time-resolved fluorescence test strip and the upper edge of the nitrocellulose membrane is 8-20 mm, the distance between each two adjacent detection lines is 1.0-4.5 mm, and the distance between the detection line close to the quality control line and the quality control line is 3-10 mm.

[0027] According to the above scheme, the coating buffer used in the preparation of the aflatoxin B1-ovalbumin conjugate coating solution, the benzo[a]pyrene-ovalbumin conjugate coating solution, and the capsaicinoid-ovalbumin conjugate coating solution in the preparation of the immunochromatographic time-resolved fluorescence test strip is as follows: per 10 mL, it contains 0.1 g of ovalbumin, 0.002 g of sodium azide, 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] The coating buffer used in the preparation of rabbit anti-mouse polyclonal antibody coating solution is as follows: per 10 mL, it contains 0.002 g of sodium azide, 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;

[0029] The blocking solution used in the preparation of the immunochromatographic time-resolved fluorescence test strip is as follows: per 100 mL, it contains 0.5-2 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;

[0030] The above-mentioned immunochromatographic time-resolved fluorescence rapid test kit is used in the detection of benzo[a]pyrene, aflatoxin B1, and capsaicin content: after the sample to be tested is pre-treated to obtain a sample solution to be tested, the sample is added to a sample reaction bottle, mixed, and a time-resolved fluorescence test strip is inserted. After reacting at 37°C for 6 minutes, the sample is tested using a time-resolved fluorescence tester to obtain the ratio of the time-resolved fluorescence intensity of the test line (T) on the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the control line (C); based on the relationship curve of the pre-obtained ratio of the time-resolved fluorescence intensity to the time-resolved fluorescence intensity of the control line (T / C) and the concentration of benzo[a]pyrene, aflatoxin B1, and capsaicin, the contents of benzo[a]pyrene, aflatoxin, and capsaicin in the sample solution to be tested are obtained, and finally, the contents of benzo[a]pyrene, aflatoxin B1, and capsaicin in the sample to be tested are obtained by conversion.

[0031] According to the above scheme, the relationship curves of the ratio of the time-resolved fluorescence intensity of the detection line to the time-resolved fluorescence intensity of the quality control line (T / C) of the immunochromatographic time-resolved fluorescence test strip and the concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicin are obtained by the following method:

[0032] (1) preparing a series of benzo[a]pyrene standard solutions; preparing a series of aflatoxin B1 standard solutions; and preparing a series of capsaicin standard solutions;

[0033] (2) Add appropriate amounts of the above-mentioned benzo[a]pyrene, aflatoxin B1, and capsaicin standard solutions of each concentration to the sample reaction bottle, mix well, insert the immunochromatographic time-resolved fluorescence test strip, react at 37°C for 10 minutes, and use a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values ​​of the test line (T) and the control line (C) on each immunochromatographic time-resolved fluorescence test strip, thereby obtaining the ratio of the time-resolved fluorescence intensity of the test line of each immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the control line (T / C);

[0034] (3) The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line (T / C) and the concentration of aflatoxin B1 was obtained by fitting; the relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line (T / C) and the concentration of benzo[a]pyrene was obtained by fitting.

[0035] Beneficial effects of the present invention:

[0036] (1) Rapid and simultaneous detection of benzo[a]pyrene, aflatoxin B1, and capsaicinoids. The immunochromatographic time-resolved fluorescence kit provided by the present invention can achieve simultaneous and rapid detection of multiple targets, including benzo[a]pyrene, aflatoxin B1, and capsaicinoids, on a single test strip. The antibodies used are all monoclonal antibodies, offering excellent specificity and high sensitivity. There is no interference between the three, making the assay simple and rapid. It is used for the quality and safety evaluation of edible vegetable oils, identification of waste cooking oils, and discovery of strong carcinogens in edible vegetable oils.

[0037] (2) High sensitivity. The immunochromatographic time-resolved fluorescence kit provided by the present invention has a minimum detection limit of 0.5 ng / mL for benzo[a]pyrene, 0.01 ng / mL for aflatoxin B1, and 1.91 ng / mL for capsaicinoids in the test solution. These detection limits meet the EU's food limit requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the structure of the time-resolved fluorescence immunochromatographic test strip for benzo[a]pyrene, aflatoxin B1, and capsaicinoids provided by the present invention. In the figure: 1: absorbent pad, 2: test pad, 3: sample pad, 4: quality control line, 5: capsaicinoid test line, 6: aflatoxin B1 test line, 7: benzo[a]pyrene test line.

[0039] Figure 2This is a schematic diagram of the structure of the time-resolved fluorescence immunochromatographic test strip for benzo[a]pyrene and capsaicinoids provided by the present invention. In the figure: 1 absorbent pad, 2 detection pad, 3 sample pad, 4 quality control line, 5 benzo[a]pyrene detection line, 6 capsaicinoid detection line. DETAILED DESCRIPTION

[0040] Example 1: Preparation of anti-benzo[a]pyrene monoclonal antibodies

[0041] 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:

[0042] The anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1 was injected into BALB / c mice that had been pre-treated with Freund's incomplete adjuvant. The ascites of the mice were collected, and the antibodies were purified using the octanoic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered through double filter paper, centrifuged at 12,000 rpm at 4°C for more than 15 minutes, and the supernatant was aspirated. The resulting ascites supernatant was mixed with 4 volumes of acetate buffer, and octanoic acid was slowly added with stirring. The volume of octanoic acid required per milliliter of ascites was 30-35 μL. The mixture was mixed at room temperature for 30-60 minutes and allowed to stand at 4°C for more than 2 hours. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, add 1 / 10 volume of filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of ammonium sulfate of 0.277g / mL, let it stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, and resuspend the obtained precipitate with 1 / 10 volume of original ascites water with a molar concentration of 0.01mol / L and pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days. Remove the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then freeze-dry it in a freeze dryer. Collecting the lyophilized powder is the purified anti-benzo[a]pyrene monoclonal antibody;

[0043] 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.

[0044] 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.

[0045] The antibody titer 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 test 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 competition 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:

[0046] CR%=(IC 50 BaP / IC 50 other analogs) × 100.

[0047] The cross-reactivity of the BBBE1H1 monoclonal antibody provided by the present invention with other structural analogs, 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%.

[0048] Detailed results are shown in Table 1:

[0049] Table 1. Cross-reactivity of BBBE1H1 with other structural analogs

[0050]

[0051]

[0052] Affinity determination of BBBE1H1 using indirect non-competitive ELISA:

[0053] The ELISA plate was coated with BaP-OVA 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 with 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. Draw 4 S-shaped curves with 4 concentrations. Find the maximum OD value at the top of each S curve, i.e. ODmax, and find the antibody concentration corresponding to 50% ODmax value of each curve. Pair any two of the 4 concentrations together and calculate the affinity constant of the antibody 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). 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.

[0054] Example 2 Screening of hybridoma cell line BBBE1H1

[0055] 1. Animal immunization

[0056] 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 mouse's 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 immunization method. The fourth immunization was performed three weeks after the third, also using the same intraperitoneal injection. The same dose of 100 μg was administered per mouse for all four immunizations. 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.

[0057] 2. Cell Fusion

[0058] Three days after the booster immunization, cell fusion was performed using 50% polyethylene glycol (PEG) (molecular weight 1450) as a fusion agent according to conventional methods. The following steps were performed: mice were sacrificed by cervical dislocation under sterile conditions, the spleens were removed and crushed with a homogenizer, and splenocytes were separated using a filter. The cells were mixed with murine myeloma SP2 / 0 cells at a cell population ratio of 5:1-10:1, centrifuged at 1000 rpm for 5 minutes, and resuspended in RPMI-1640 basal medium. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 1 mL of 50% PEG was added, and the mixture was incubated for 1 minute. Once adhered, 20 mL of RPMI-1640 basal medium was added to resuspend the cells. The cells were centrifuged and the supernatant was discarded. The fused cells at the bottom of the tube were resuspended in 20 mL of complete cell culture medium containing 1% HAT. The suspended cells were added to 80 mL of semi-solid culture medium, mixed thoroughly, and plated into 6-well cell culture plates at a rate of 1-2 mL / well. The plates were then incubated 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, i.e., HAT, and methylcellulose, which were purchased from Sigma-Aldrich.

[0059] Cell line screening and cloning

[0060] After 1-2 weeks of cell fusion, when cell colonies grow to be visible to the naked eye, clones are picked out from the culture medium with a micropipette and transferred to a 96-well cell culture plate using HAT liquid for 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 uses the indirect ELISA method to screen positive wells that are resistant to benzo[a]pyrene but not to the carrier protein BSA. The second step uses 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 refers to wells with zero competitor, i.e., positive control wells, with higher final measured values; higher sensitivity refers to the competitor concentration at which the inhibition rate is 50%, also known as IC). 50 The 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.

[0061] Example 3 Determination of the variable region sequence of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1.

[0062] (1) Extraction of total RNA: Total RNA from hybridoma cell line BBBE1H1 was extracted using the total RNA extraction kit from Tiangen Company according to the instructions;

[0063] (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;

[0064] (3) PCR cloning of variable region genes: Primers were designed based on the conserved sites of mouse antibody gene sequences 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 seconds, 58°C for 45 seconds, and 72°C for 1 minute, with 30 cycles of amplification and a final extension at 72°C for 10 minutes. 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 sequences of the primers are: heavy chain variable region primers are 5'-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCAGTG GAT AGA CAG ATG GGG G-3' (28mer), where S, M, R and W are merged bases, M = A / C, R = A / G, S = G / C, W = A / T; light chain variable region primers are 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24mer).

[0065] 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.

[0066] Example 4 Obtaining anti-aflatoxin B1 monoclonal antibodies

[0067] The anti-aflatoxin monoclonal antibody was prepared from the hybridoma cell line 3G1 with a deposit number of CCTCC NO. C201014, specifically according to the method reported in the patent disclosed in 201210117614.9. The preparation method is as follows: the obtained hybridoma cell line 1C11 was injected into BALB / c mice that had been pre-treated with Freund's incomplete adjuvant, the ascites of the mice was collected, and the anti-aflatoxin monoclonal antibody was obtained after purification. Among them, the purification method is the octanoic acid-ammonium sulfate method, and the specific operation is as follows: filter the mouse ascites with double filter paper, centrifuge the filtered ascites at 4°C and 12000r / min for more than 15 minutes, 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, let it stand at 4°C for more than 2h, then centrifuge at 4°C and 12000r / min for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, and add 1 / 10 volume of filtrate with a molar concentration of 0.1mol / L and a pH value of 7.4. The mixture was diluted with 1% phosphate buffer, and the pH value of the mixture was adjusted to 7.4 with 2 mol / L sodium hydroxide solution. 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 hours, and then centrifuged at 4°C and 12000 r / min for more than 30 minutes. The supernatant was discarded, and the resulting precipitate was resuspended with 0.01 mol / L phosphate buffer with a volume of 1 / 10 of the original ascites water, 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, and the antibody was placed in a -20°C refrigerator for use;

[0068] 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.

[0069] Example 5 Obtaining a universal monoclonal antibody against capsaicinoids

[0070] The universal monoclonal antibody against capsaicinoids is a monoclonal antibody secreted by the hybridoma cell line YQQD8 with a deposit number of CCTCC NO. C201534, and is prepared in advance according to the method reported in the patent disclosed in 201610079095.X. The specific preparation method is as follows:

[0071] The hybridoma cell line YQQD8 was injected into the abdomen of BALB / c mice pre-treated with incomplete Freund's adjuvant. The ascites was collected and purified to obtain a universal monoclonal antibody against capsaicinoids. The purification method employed the octanoic acid-ammonium sulfate method, which involved filtering the mouse ascites through double-layer filter paper, centrifuging at 12,000 rpm for at least 15 minutes at 4°C, aspirating the supernatant, and mixing it with four volumes of acetate buffer. With stirring, octanoic acid (30-35 μL per mL of ascites) was slowly added. The mixture was mixed at room temperature for 30-60 minutes and allowed to stand at 4°C for at least 2 hours. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double filter paper, add 1 / 10 volume of filtrate with a molar concentration of 0.1mol / L and a pH of 7.4 phosphate buffer, adjust the pH of the mixture to 7.4 with 2mol / L sodium hydroxide solution, slowly add ammonium sulfate in an ice bath to a final concentration of ammonium sulfate of 0.277g / mL, let it stand at 4℃ for more than 2h, then centrifuge at 12000r / min, 4℃ for more than 30min, discard the supernatant, and resuspend the obtained precipitate with 1 / 10 volume of original ascites water with a molar concentration of 0.01mol / L and pH of 7.4 phosphate buffer, put it into a dialysis bag, dialyze it with 0.01mol / L PBS for two days, and then dialyze it with PB for two days. Remove the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then freeze-dry it in a freeze dryer. Collecting the lyophilized powder is the purified universal monoclonal antibody against capsaicinoids;

[0072] 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.

[0073] 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 6High 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%.

[0074] Example 6: Preparation of Benzo[a]pyrene, Capsaicinoids, and Aflatoxin B1 Immunomagnetic Beads:

[0075] 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.

[0076] 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, place on a shaker at 8°C, 200 rpm overnight for coupling. After the reaction is complete, place on a magnetic stand for magnetic separation.

[0077] 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.

[0078] 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.

[0079] Example 7 Synchronous Detection of Benzo[a]pyrene, Aflatoxin B1, and Benzo[a]pyrene Time-Resolved Fluorescence Kit and Its Application

[0080] A time-resolved fluorescent immunochromatographic test strip for quantitatively detecting benzo[a]pyrene, aflatoxin B1, and capsaicin comprises a fluorescent test strip, a reaction bottle containing europium-labeled anti-benzo[a]pyrene monoclonal antibody, europium-labeled aflatoxin B1 monoclonal antibody, europium-labeled capsaicin monoclonal antibody, and a sample diluent. The fluorescent test strip comprises a cardboard, one side of which is sequentially adhered with an absorbent pad, a detection pad, and a sample pad from top to bottom. Adjacent pads are overlapped at the joints, with an overlap length of 1 mm. The absorbent pad in the immunochromatographic time-resolved fluorescent test strip is 18 mm long and 4 mm wide; the detection pad is 25 mm long and 4 mm wide; the sample pad is 15 mm long and 4 mm wide, and the overlap length of adjacent pads is 1 mm. The detection pad is made of nitrocellulose. The cellulose membrane is used as the base pad, and a horizontal quality control line is set from top to bottom on the nitrocellulose membrane. The quality control line is coated with rabbit anti-mouse polyclonal antibody, and its coating amount is 0.25μg / cm3 quality control line, as well as the first test line, the second test line and the third test line. The coating amount of benzo[a]pyrene-ovalbumin conjugate on the test line is 0.4μg / cm3, the coating amount of aflatoxin B1-ovalbumin conjugate is 0.4μg / cm3; the coating amount of capsaicinoid-ovalbumin conjugate is 1.0μg / cm3 test line. The distance between the first test line and the upper edge of the nitrocellulose membrane is 10mm, the distance between the quality control line and the first test line is 4mm, the distance between the quality control line and the second test line is 8mm, and the distance between the quality control line and the third test line is 12mm.

[0081] Obtaining the fluorescent test strips:

[0082] (1) Preparation of absorbent pad

[0083] Cut the absorbent paper into 18mm long and 4mm wide specifications to make the absorbent pad;

[0084] (2) Preparation of detection pad

[0085] Test line coating:

[0086] Benzo[a]pyrene-coated antigen was prepared with coating buffer to a concentration of 1.25 mg / mL; the coating solution was horizontally coated on a nitrocellulose membrane by streaking to form a test line. The coating amount required for each centimeter of the test line was 0.4 μg, and then dried at 37°C for 60 minutes.

[0087] Aflatoxin coating antigen was prepared with coating buffer to a concentration of 0.25 mg / mL; the coating solution was horizontally coated on a nitrocellulose membrane by streaking to obtain a test line. The coating amount of the coating antigen required per centimeter of the test line was 0.4 μg, and then dried at 37°C for 60 minutes.

[0088] Prepare capsaicin-coated antigen with coating buffer to a concentration of 1 mg / mL; coat the solution horizontally on a nitrocellulose membrane by streaking to form a test line. The coating amount required for each centimeter of the test line is 1.0 μg of coating antigen, and then dry at 37°C for 60 minutes.

[0089] The coating buffer is as follows: each 10 mL contains 0.1 g of ovalbumin BSA, 0.002 g of sodium azide, 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;

[0090] Coating of quality control line:

[0091] Prepare a 0.25 mg / mL coating solution of rabbit anti-mouse polyclonal antibody in coating buffer; coat the solution horizontally on a nitrocellulose membrane by streaking to obtain a control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the control line is 0.4 μg, and then dry at 37°C for 2 hours.

[0092] The coating buffer is:

[0093] Each 10 mL contains 0.1 g of ovalbumin, 0.002 g of sodium azide, 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;

[0094] The nitrocellulose membrane is 25 mm long and 4 mm wide.

[0095] (3) Preparation of sample pad:

[0096] Cut the glass fiber membrane into 15 mm long and 4 mm wide specifications, 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;

[0097] The blocking solution is prepared by adding water to 100 mL of 2.9 g disodium hydrogen phosphate dodecahydrate, 0.3 g sodium dihydrogen phosphate dihydrate, 1.0 g Tween-20, 1.0 g polyvinylpyrrolidone (PVPK-30), 0.25 g EDTA, 0.5 g ovalbumin BSA, and 0.02 g sodium azide.

[0098] (4) Assembly of fluorescent test strips:

[0099] On one side of the cardboard, a water-absorbing pad, a detection pad and a sample pad are pasted in sequence from top to bottom. Adjacent pads are overlapped at the joints with an overlapping length of 1 mm to obtain a fluorescent test strip.

[0100] Obtaining the europium-labeled anti-benzo[a]pyrene monoclonal antibody probe:

[0101] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium labeling reagent (100 nm particle size, 1% solids content) and vortex to mix. Sonicate for 3 seconds. Add 40 μL of 15 mg / mL EDC solution and vortex to mix for 15 minutes. Centrifuge at 13,000 rpm at 10°C for 10 minutes, remove the supernatant, reconstitute in 1 mL of borate buffer, vortex to mix, and sonicate for 3 seconds. Add 30 μg of benzo[a]pyrene monoclonal antibody, mix, and shake at 250 rpm at 25°C overnight. Centrifuge again, remove the supernatant, and reconstitute in 1 mL of borate buffer containing 0.5% BSA. Vortex to mix, sonicate for 10 minutes, and shake at 25°C for 2 hours to obtain the target europium-labeled anti-benzo[a]pyrene monoclonal antibody.

[0102] Obtaining the europium-labeled anti-aflatoxin B1 cloned antibody probe:

[0103] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium labeling reagent (particle size 100 nm, solids content 1%) and vortex to mix. Ultrasonicate for 3 seconds. Add 40 μL of 15 mg / mL EDC solution and vortex to mix for 15 minutes. Centrifuge at 13,000 rpm at 10°C for 10 minutes, remove the supernatant, add 1 mL of borate buffer to reconstitute, vortex to mix, and ultrasonicate for 3 seconds. Add 20 μg of anti-aflatoxin B1 monoclonal antibody, mix, and shake at 250 rpm and 25°C overnight. Centrifuge again, remove the supernatant, add 1 mL of borate buffer containing 0.5% BSA to reconstitute, vortex to mix, ultrasonicate for 10 minutes, and shake at 25°C for 2 hours to obtain the target product, europium-labeled anti-aflatoxin B1 monoclonal antibody.

[0104] Obtaining the europium-labeled anti-capsaicin monoclonal antibody probe:

[0105] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium labeling reagent (100 nm particle size, 1% solids content), and vortex to mix. Ultrasonicate for 3 seconds. Add 40 μL of 15 mg / mL EDC solution, and vortex to mix for 15 minutes.

[0106] Centrifuge at 13,000 rpm at 10°C for 10 min, remove the supernatant, add 1 mL of borate buffer to reconstitute, vortex to mix, and sonicate for 3 seconds. Add 20 μg of capsaicin monoclonal antibody, mix, and shake at 250 rpm at 25°C overnight. Centrifuge again, remove the supernatant, add 1 mL of borate buffer containing 0.5% BSA to reconstitute, vortex to mix, sonicate for 10 minutes, and shake at 25°C for 2 hours to obtain the target product, europium-labeled anti-capsaicin monoclonal antibody.

[0107] Application of the above-mentioned time-resolved fluorescence immunochromatographic kit in the quantitative detection of benzo[a]pyrene, aflatoxin B1, and capsaicinoids:

[0108] 1. Establishment of relationship curve between the ratio of time-resolved fluorescence intensity of the test line of the fluorescent test strip to the time-resolved fluorescence intensity of the control line (T / C) and the concentrations of aflatoxin B1, capsaicinoids, and benzo[a]pyrene:

[0109] (1) Soybean oil that was negative for benzo[a]pyrene, aflatoxin B1, and capsaicinoids by high performance liquid chromatography (HPLC) was added with a benzo[a]pyrene / aflatoxin B1 / capsaicinoid standard (1:1:1), where the capsaicinoid standard was a 1:1:1 mixture of natural capsaicin, dihydrocapsaicin, and synthetic capsaicinoids.

[0110] Sample Pretreatment: Accurately weigh 1g of soybean oil in a 10mL plastic centrifuge tube as a reaction vessel. Add 200ng of the standard stock solution and vortex mix for 1 minute. Add 5mL of n-hexane solution and sonicate for 10 minutes. Then add another 5mL of n-hexane solution and sonicate for 10 minutes. Take 1mL of the extract and dilute to 10mL with 9mL of ddH2O. Vortex for 2 minutes. Add immunomagnetic beads conjugated with monoclonal antibodies to benzo[a]pyrene, aflatoxin B1, and capsaicin. Vortex mix for 10 minutes and discard the supernatant. Add 1mL of methanol solution and vortex mix for 2 minutes. Dilute 5-fold with the detection solution and use the supernatant as the test solution.

[0111] (2) Take 300 μL of a mixed standard solution of benzo[a]pyrene, aflatoxin B1, and capsaicin at different concentrations and add it to the sample bottle. Mix well. Insert one end of the fluorescent test strip sample pad into the reaction bottle. Incubate at 37°C for 10 minutes and then test on the instrument. The detection instrument is a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. The ratio of the time-resolved fluorescence intensity of the test line of each fluorescent test strip to the time-resolved fluorescence intensity of the quality control line (T / C) is obtained.

[0112] (3) The concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicinoids were plotted as the horizontal axis, and the time-resolved fluorescence intensity ratios of the test and control lines (T / C values) of the standard solutions at each concentration were plotted as the vertical axis. The effective detection ranges of this method were 1.9–62.5 ng / mL for capsaicinoids, 0.01–5 ng / mL for aflatoxin B1, and 0.5–28.7 ng / mL for benzo[a]pyrene.

[0113] 2. Recovery experiment of benzo[a]pyrene, aflatoxin B1 and capsaicin in samples:

[0114] Soybean oil negative for benzo[a]pyrene, capsaicinoids, and aflatoxin B1 by high-performance liquid chromatography (HPLC) was supplemented with 10 ng / mL, 20 ng / mL, and 40 ng / mL of benzo[a]pyrene / aflatoxin B1 / capsaicinoid standards (1:1:1). Mix thoroughly before sample pretreatment and extraction. Sample pretreatment steps were as follows: Accurately weigh 1 g of soybean oil in a 10 mL plastic centrifuge tube as a reaction vessel, add 200 ng of the standard stock solution, and vortex mix for 1 minute. Add 5 mL of n-hexane solution, sonicate for 10 minutes, then add another 5 mL of n-hexane solution, and sonicate for 10 minutes. Take 1 mL of the extract, dilute to 10 mL with 9 mL of ddH2O, vortex for 2 minutes, and add immunomagnetic beads conjugated with monoclonal antibodies to benzo[a]pyrene, aflatoxin B1, and capsaicin. Vortex mix for 10 minutes, and discard the supernatant. Add 1 mL of methanol solution, vortex mix for 2 minutes, dilute 5-fold with the test solution, and use the supernatant as the test solution. Add 300 μL of the test solution to a sample reaction vial containing europium-labeled monoclonal antibodies, mix thoroughly, insert one end of a fluorescent test strip sample pad into the reaction vial, incubate at 37°C for 10 minutes, and analyze on a time-resolved fluorescence immunoassay instrument with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. Measure the ratio (T / C) of the time-resolved fluorescence intensity of each fluorescent test strip's test line to the time-resolved fluorescence intensity of the control line. Substitute this value into the curve of the relationship between the time-resolved fluorescence intensity of the fluorescent test strip's test line and the time-resolved fluorescence intensity of the control line (T / C) and the concentration of the standard substance to determine the concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicinoids in the sample solution. The concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicinoids in the sample can then be calculated based on the dilution factor. The recoveries of benzo[a]pyrene in soybean oil samples were 92.5%, 93.2%, and 103.5% respectively; the recoveries of capsaicin were 91.2%, 89.6%, and 110.2% respectively; and the recoveries of aflatoxin B1 were 95.2%, 93.1%, and 108.2% respectively.

[0115] Example 8: Preparation of Benzo[a]pyrene and Capsaicinoid Immunomagnetic Beads

[0116] 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.

[0117] 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 rpm overnight for coupling. After the reaction is complete, place on a magnetic stand for magnetic separation.

[0118] 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.

[0119] 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.

[0120] Example 9: Time-resolved fluorescence kit for simultaneous detection of benzo[a]pyrene and capsaicinoids and its application

[0121] The invention relates to a time-resolved fluorescent immunochromatographic test strip for quantitatively detecting benzo[a]pyrene and capsaicinoids, comprising a fluorescent test strip, an anti-benzo[a]pyrene monoclonal antibody containing europium labeling, a universal anti-capsaicinoid monoclonal antibody, a reaction bottle, and a sample diluent. The fluorescent test strip comprises a cardboard, one side of which is sequentially pasted with an absorbent pad, a detection pad, and a sample pad from top to bottom. Adjacent pads are overlapped at the joints, and the overlap length is 1 mm. The absorbent pad in the time-resolved fluorescent immunochromatographic test strip is 18 mm long and 4 mm wide; the detection pad is 25 mm long and 4 mm wide; and the sample pad is 15 mm long. The test pad is a nitrocellulose membrane with a thickness of 4 mm and a width of 4 mm. The overlapping length of adjacent pads is 1 mm. The test pad is based on a nitrocellulose membrane. A horizontal quality control line, a first test line, and a second test line are arranged from top to bottom on the nitrocellulose membrane. The quality control line is coated with a rabbit anti-mouse polyclonal antibody with a coating amount of 0.25 μg / cm3. The test line is coated with a capsaicin-coated antigen with a coating amount of 0.4 μg / cm3. The spacing between the first test line and the upper edge of the nitrocellulose membrane is 9 mm. The spacing between the quality control line and the first test line is 4 mm, and the spacing between the quality control line and the second test line is 8 mm.

[0122] Obtaining the fluorescent test strips:

[0123] (1) Preparation of absorbent pad

[0124] Cut the absorbent paper into 18mm long and 4mm wide specifications to make the absorbent pad;

[0125] (2) Preparation of detection pad

[0126] Test line coating:

[0127] Prepare capsaicin-coated antigen with coating buffer to a concentration of 1 mg / mL; coat the solution horizontally on a nitrocellulose membrane by streaking to form a test line. The coating amount required for each centimeter of the test line is 1.0 μg of coating antigen, and then dry at 37°C for 60 minutes.

[0128] Benzo[a]pyrene-coated antigen was prepared with coating buffer to a concentration of 1.25 mg / mL; the coating solution was horizontally coated on a nitrocellulose membrane by streaking to form a test line. The coating amount required for each centimeter of the test line was 0.4 μg, and then dried at 37°C for 60 minutes.

[0129] The coating buffer is as follows: each 10 mL contains 0.1 g of ovalbumin BSA, 0.002 g of sodium azide, 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;

[0130] Coating of quality control line:

[0131] Prepare a 0.25 mg / mL coating solution of rabbit anti-mouse polyclonal antibody in coating buffer; coat the solution horizontally on a nitrocellulose membrane by streaking to obtain a control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the control line is 0.4 μg, and then dry at 37°C for 2 hours.

[0132] The nitrocellulose membrane is 25 mm long and 4 mm wide.

[0133] (3) Preparation of sample pad:

[0134] Cut the glass fiber membrane into 15 mm long and 4 mm wide specifications, 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 100 mL of 2.9 g disodium hydrogen phosphate dodecahydrate, 0.3 g sodium dihydrogen phosphate dihydrate, 1.0 g Tween-20, 1.0 g polyvinylpyrrolidone (PVPK-30), 0.25 g EDTA, 0.5 g ovalbumin BSA, and 0.02 g sodium azide.

[0136] (4) Assembly of fluorescent test strips:

[0137] On one side of the cardboard, a water-absorbing pad, a detection pad and a sample pad are pasted in sequence from top to bottom. Adjacent pads are overlapped at the joints with an overlapping length of 1 mm to obtain a fluorescent test strip.

[0138] Obtaining the europium-labeled anti-benzo[a]pyrene monoclonal antibody:

[0139] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium labeling reagent (100 nm particle size, 1% solids content) and vortex to mix. Sonicate for 3 seconds. Add 40 μL of 15 mg / mL EDC solution and vortex to mix for 15 minutes. Centrifuge at 13,000 rpm at 10°C for 10 minutes, remove the supernatant, reconstitute in 1 mL of borate buffer, vortex to mix, and sonicate for 3 seconds. Add 20 μg of benzo[a]pyrene monoclonal antibody, mix, and shake at 250 rpm at 25°C overnight. Centrifuge again, remove the supernatant, and reconstitute in 1 mL of borate buffer containing 0.5% BSA. Vortex to mix, sonicate for 10 minutes, and shake at 25°C for 2 hours to obtain the target europium-labeled anti-benzo[a]pyrene monoclonal antibody.

[0140] Obtaining the europium-labeled anti-capsaicinoid monoclonal antibody:

[0141] To 800 μL of 0.2 mol / L borate buffer (pH 8.18), add 200 μL of europium labeling reagent (100 nm particle size, 1% solids content) and vortex to mix. Sonicate for 3 seconds. Add 40 μL of 15 mg / mL EDC solution and vortex to mix for 15 minutes. Centrifuge at 13,000 rpm at 10°C for 10 minutes, remove the supernatant, reconstitute with 1 mL of borate buffer, vortex to mix, and sonicate for 3 seconds. Add 20 μg of benzo[a]pyrene monoclonal antibody, mix, and shake at 250 rpm at 25°C overnight. Centrifuge again, remove the supernatant, and reconstitute with 1 mL of borate buffer containing 0.5% BSA. Vortex to mix, sonicate for 10 minutes, and shake at 25°C for 2 hours to obtain the target europium-labeled anti-capsaicinoid monoclonal antibody.

[0142] Application of the above-mentioned time-resolved fluorescence immunochromatographic test strips in the quantitative detection of benzo[a]pyrene and capsaicinoids:

[0143] 1. Establishment of the relationship curve between the ratio of the time-resolved fluorescence intensity of the test line of the fluorescent test strip to the time-resolved fluorescence intensity of the quality control line (T / C) and the concentration of benzo[a]pyrene and capsaicinoids:

[0144] (1) Add benzo[a]pyrene / capsaicinoid standard (1:1) to soybean oil that is negative for benzo[a]pyrene and capsaicinoids by high performance liquid chromatography (HPLC). The capsaicinoid standard is a 1:1:1 mixture of natural capsaicinoid, dihydrocapsaicinoid, and synthetic capsaicinoid. The sample pretreatment steps are as follows: Take a 10 mL plastic centrifuge tube as a reaction container, accurately weigh 1 g of soybean oil, add 200 ng of the standard stock solution, and vortex mix for 1 min. Add 5 mL of n-hexane solution, sonicate for 10 min, then add 5 mL of n-hexane solution, and sonicate for 10 min. Take 1 mL of the extract, dilute to 10 mL with 9 mL of ddH2O, vortex for 2 min, add immunomagnetic beads coupled with monoclonal antibodies to benzo[a]pyrene and capsaicinoids, vortex mix for 10 minutes, and discard the supernatant. 1 mL of methanol solution was added, vortexed for 2 minutes, and diluted 5 times with sample release solution (PBS solution with a volume fraction of 0.20% Tween-20, 0.5% sucrose and 1% bovine serum albumin), and the supernatant was used as the test solution.

[0145] (2) Take 300 μL of benzo[a]pyrene and capsaicin extract and add it to a sample vial. Then add it to the sample reaction vial containing europium-labeled monoclonal antibody, mix well, insert one end of the fluorescent test strip sample pad into the reaction vial, react at 37°C for 10 minutes, and then test on the instrument. The detection instrument is a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. The detection obtains the ratio of the time-resolved fluorescence intensity of each fluorescent test strip test line to the time-resolved fluorescence intensity of the quality control line (T / C).

[0146] (3) The concentrations of benzo[a]pyrene and capsaicinoid standards were plotted as the horizontal axis, and the time-resolved fluorescence intensity ratios of the test and control lines (T / C values) for each standard solution were plotted as the vertical axis. The effective detection range of this method was 1.9–62.5 ng / mL for capsaicinoids and 0.5–28.7 ng / mL for benzo[a]pyrene.

[0147] 2. Recovery experiment of benzo[a]pyrene and capsaicinoids in soybean oil samples:

[0148] Soybean oil negative for benzo[a]pyrene and capsaicinoids by high-performance liquid chromatography (HPLC) was spiked with 10 ng / mL, 20 ng / mL, and 40 ng / mL of benzo[a]pyrene / capsaicinoid standards (1:1). Mix thoroughly before sample pretreatment and extraction. Accurately weigh 1 g of peanut oil in a 10 mL plastic centrifuge tube as a reaction vessel. Add 200 ng of the standard stock solution and vortex mix for 1 minute. Add 5 mL of n-hexane solution to the spiked vegetable oil and sonicate for 10 minutes. Then, add another 5 mL of n-hexane solution and sonicate for 10 minutes. Take 1 mL of the extract, dilute to 10 mL with 9 mL of ddH2O, vortex for 2 minutes, and add immunomagnetic beads conjugated to monoclonal antibodies against benzo[a]pyrene and capsaicinoids. Vortex mix for 14 minutes, and discard the supernatant. Add 1 mL of methanol solution, vortex mix for 2 minutes, and dilute 5-fold with sample release solution (0.20% Tween-20, 0.5% sucrose, and 1% bovine serum albumin in PBS). The supernatant is used as the test solution. Add 300 μL of the test solution to a sample reaction vial containing europium-labeled monoclonal antibodies and mix thoroughly. Insert the sample pad of a fluorescent test strip into the reaction vial. Incubate at 37°C for 10 minutes before testing on a time-resolved fluorescence immunoassay analyzer with an excitation wavelength of 365 nm and an emission wavelength of 615 nm. The time-resolved fluorescence intensity ratio (T / C) of the test line of each fluorescent test strip was measured to determine the time-resolved fluorescence intensity of the control line. This ratio was then substituted into the curve of the relationship between the time-resolved fluorescence intensity ratio (T / C) of the test line of the fluorescent test strip and the time-resolved fluorescence intensity of the control line and the concentration of the standard substance to determine the concentration of benzo[a]pyrene and capsaicinoids in the sample solution. The benzo[a]pyrene and capsaicinoid content in the sample was then calculated based on the dilution factor. The spiked recoveries of benzo[a]pyrene in the soybean oil sample were 92.5%, 93.2%, and 103.5%, respectively; and the spiked recoveries of capsaicinoids were 91.2%, 89.6%, and 110.2%, respectively.

Claims

1. A time-resolved fluorescence kit for evaluating the safety of edible vegetable oils, characterized by: The invention comprises a fluorescent test strip and a sample reaction bottle containing europium-labeled anti-benzo[a]pyrene monoclonal antibody, europium-labeled anti-aflatoxin B1 monoclonal antibody, and europium-labeled anti-capsaicinoids universal monoclonal antibody. One side of the fluorescent test strip is provided with a water-absorbing pad, a detection pad, and a sample pad from top to bottom. Adjacent pads overlap and connect at the connection. The detection pad is based on a nitrocellulose membrane. The nitrocellulose membrane is provided with a horizontal quality control line and a detection line. The quality control line is coated with a rabbit anti-mouse polyclonal antibody. The detection line is located below the quality control line and there are two of them. The test line comprises one or three test lines arranged at intervals, each test line being coated with a test substance-protein conjugate, the test substances being benzo[a]pyrene and aflatoxin B1 and / or capsaicinoids, the test substance-protein conjugates being benzo[a]pyrene-ovalbumin conjugate and aflatoxin B1-ovalbumin conjugate and / or capsaicinoids-ovalbumin conjugate, the quality control line being coated with a rabbit anti-mouse polyclonal antibody; the anti-benzo[a]pyrene monoclonal antibody is secreted by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882.

2. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, characterized in that: The europium-labeled anti-benzo[a]pyrene monoclonal antibody is formed by coupling the surface-modified carboxyl group with the amino terminus of the anti-benzo[a]pyrene monoclonal antibody. The preparation method comprises the following steps: adding a europium labeling reagent to a boric acid buffer solution and sonicating the solution, then adding an EDC solution, oscillating and mixing the solution, centrifuging, removing the supernatant, re-dissolving the solution in a boric acid buffer solution, oscillating and mixing the solution, adding the anti-benzo[a]pyrene monoclonal antibody, reacting the solution on a shaking table, centrifuging and removing the supernatant, blocking the solution, and aliquoting and lyophilizing the solution. The preparation method of the europium-labeled anti-aflatoxin B1 monoclonal antibody is as follows: europium labeling reagent is added to boric acid buffer and sonicated, then EDC solution is added, vortexed and mixed, centrifuged, supernatant removed, boric acid buffer is added for re-dissolution, vortexed and mixed, anti-benzo[a]pyrene monoclonal antibody is added, shaken for reaction, centrifuged and supernatant removed, blocked, and aliquoted and lyophilized; The preparation method of the europium-labeled universal monoclonal antibody against capsaicinoids is as follows: europium labeling reagent is added to boric acid buffer and sonicated, then EDC solution is added, oscillated and mixed, centrifuged, supernatant removed, boric acid buffer is added for re-dissolution, oscillated and mixed, anti-benzo[a]pyrene monoclonal antibody is added, shaken for reaction, centrifuged and supernatant removed, blocked, and aliquoted and lyophilized.

3. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, wherein: The coating amount of benzo[a]pyrene-ovalbumin conjugate on the detection line of the time-resolved fluorescent immunochromatographic test strip is 0.4-0.8 μg / cm, the coating amount of aflatoxin B1-ovalbumin conjugate is 0.4-0.8 μg / cm, and the coating amount of capsaicinoid-ovalbumin conjugate is 0.8-1.0 μg / cm; the content of the europium-labeled anti-benzo[a]pyrene monoclonal antibody freeze-dried product in the sample reaction bottle is 0.1-0.3 μg, the content of the anti-aflatoxin B1 monoclonal antibody freeze-dried product is 0.15-0.4 μg, and the content of the anti-capsaicin monoclonal antibody freeze-dried product is 0.2-0.4 μg.

4. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, wherein: The IC50 of the anti-aflatoxin B1 monoclonal antibody is less than or equal to 1.6 ppb; the IC50 of the anti-capsaicinoid universal monoclonal antibody is less than or equal to 8.5 ppb.

5. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, wherein: The device also includes a sample release solution and a sample release solution pipette. The sample release solution is a PBS solution containing 0.01% to 0.30% Tween-20 (v / v), 0.5 to 1.5% sucrose (w / w) and 0.1 to 1% bovine serum albumin (w / w).

6. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, characterized in that: The preparation method of the time-resolved fluorescent immunochromatographic test strip is as follows: (1) Cut the absorbent paper into absorbent pads; (2) Preparation of test pad: Benzo[a]pyrene-ovalbumin conjugate, aflatoxin B1-ovalbumin conjugate, and capsaicinoid-ovalbumin conjugate were prepared into coating solutions with a concentration of 0.25-2 mg / mL. Depending on the type and quantity of the test substance, the coating was performed on the nitrocellulose membrane by streaking to obtain two or three test lines. The membrane was then incubated at 37-40 o Dry under C conditions for 30-60 minutes; the coating amount for coating benzo[a]pyrene-ovalbumin conjugate is 0.4-0.8 μg, and the coating amount of aflatoxin B1-ovalbumin conjugate required per centimeter of the detection line for aflatoxin B1-ovalbumin conjugate is 0.4-0.8 μg; the coating amount required for coating capsaicin-ovalbumin conjugate is 0.8-1.0 μg; Prepare a coating solution of rabbit anti-mouse polyclonal antibody at a concentration of 0.1-0.45 mg / mL and coat it horizontally on a nitrocellulose membrane at a position 5-10 mm away from the test line by streaking to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per cm of the quality control line is 0.4-0.8 μg, and then dry at 37-40°C for 30-60 minutes. (3) Preparation of sample pad: Soak the glass fiber membrane in the blocking solution, take it out, and incubate at 37-40 o Dry under C conditions for 4-10 hours to obtain the sample pad, and then store it in a desiccator at room temperature; (4) Assembly of immunochromatographic time-resolved fluorescence test strips: On one side of the cardboard, a water-absorbing pad, a detection pad, and a sample pad are sequentially pasted from top to bottom. Adjacent pads are overlapped at the joints with an overlap length of 1-3 mm to obtain an immunochromatographic time-resolved fluorescence test strip.

7. The time-resolved fluorescence kit for evaluating the safety of edible vegetable oils according to claim 1, wherein: In the time-resolved fluorescent immunochromatographic test strip, the distance between the detection line close to the quality control line on the detection pad and the upper edge of the nitrocellulose membrane is 8-20 mm, the spacing between each two adjacent detection lines is 1.0-4.5 mm, and the spacing between the detection line close to the quality control line and the quality control line is 3-10 mm. The sample reaction bottle is a 1-5 mL snap-top bottle.

8. Use of the time-resolved fluorescence test kit for evaluating the safety of edible vegetable oils according to claim 1 in the detection of benzo[a]pyrene, aflatoxin B1, and capsaicinoids: pre-treating the sample to be tested to obtain a sample solution, adding the solution to the sample reaction bottle, mixing, inserting a time-resolved fluorescence test strip, 37 o After 6 minutes of C reaction, the test is performed using a time-resolved fluorescence tester to obtain the ratio of the time-resolved fluorescence intensity of the test line to the time-resolved fluorescence intensity of the quality control line on the immunochromatographic time-resolved fluorescence test strip; based on the relationship curves of the ratio of the time-resolved fluorescence intensity to the time-resolved fluorescence intensity of the quality control line and the concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicinoids obtained in advance, the contents of benzo[a]pyrene and aflatoxin B1 and / or capsaicinoids in the sample solution to be tested are obtained, and finally, the contents of benzo[a]pyrene and aflatoxin B1 and / or capsaicinoids in the sample to be tested are obtained through conversion.

9. The use according to claim 8, characterized in that The relationship curves of the ratio of the time-resolved fluorescence intensity of the detection line of the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line and the concentrations of benzo[a]pyrene, aflatoxin B1, and capsaicinoids are obtained by the following method: (1) Prepare a series of benzo[a]pyrene standard solutions at different concentrations; prepare a series of aflatoxin B1 standard solutions at different concentrations; prepare a series of capsaicinoid standard solutions at different concentrations; (2) Add appropriate amounts of the above-mentioned standard solutions of benzo[a]pyrene, aflatoxin B1, and capsaicinoids to the sample reaction bottle, mix well, insert the immunochromatographic time-resolved fluorescence test strip, react at 37°C for 10 minutes, and use a time-resolved fluorescence immunoassay to obtain the time-resolved fluorescence intensity values ​​of the test line and the quality control line on each immunochromatographic time-resolved fluorescence test strip, thereby obtaining the ratio of the time-resolved fluorescence intensity of the test line of each immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line; (3) The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line and the aflatoxin B1 concentration was obtained by fitting; The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line and the time-resolved fluorescence intensity of the quality control line of the immunochromatographic time-resolved fluorescence test strip and the capsaicin concentration was obtained by fitting. The relationship curve between the ratio of the time-resolved fluorescence intensity of the detection line of the immunochromatographic time-resolved fluorescence test strip to the time-resolved fluorescence intensity of the quality control line and the benzo[a]pyrene concentration was obtained by fitting.

Images