Immunochromatographic test strip for rapid detection of benzo[a]pyrene contamination, preparation method and application thereof

Abstract

The present invention discloses an immunochromatographic test strip for rapid detection of benzo[a]pyrene, and its preparation method and application. This test strip includes a water-absorbing pad, a detection pad, a gold-labeled pad, and a sample pad. Adjacent pads are overlapped and connected at the joint and are sequentially affixed to a base plate from top to bottom. The detection pad is based on a nitrocellulose membrane. A quality control line and a detection line are horizontally arranged on the nitrocellulose membrane. The quality control line is coated with a rabbit anti-mouse polyclonal antibody. The detection line is located below the quality control line. The detection line is coated with a benzo[a]pyrene-ovalbumin conjugate; the gold-labeled pad is horizontally sprayed with a nanogold-labeled anti-benzo[a]pyrene monoclonal antibody; the anti-benzo[a]pyrene monoclonal antibody is secreted by BBBE1H1, which has a deposit number of CCTCC NO: C201882. It can achieve rapid and highly sensitive detection of benzo[a]pyrene.

Description

Technical Field

[0001] The present invention relates to a target immunochromatographic test strip, and in particular to an immunochromatographic test strip for rapidly detecting benzo[a]pyrene contamination, a preparation method and application thereof. Background Art

[0002] Benzo[a]pyrene, also known as benzo[a]pyrene, is a polycyclic aromatic hydrocarbon compound composed of a benzene ring and a pyrene molecule. Its molecular formula is C 20 H 12 Benzo[a]pyrene has a relative molecular mass of 252.32 and exists in a crystalline state at room temperature. It is insoluble in water, slightly soluble in ethanol and methanol, and soluble in organic solvents such as benzene, toluene, xylene, chloroform, ether, and acetone. It is stable in alkaline environments but unstable in acidic environments. Benzo[a]pyrene enters the human body primarily through food or drinking water, accumulating in mammary glands and adipose tissue. It is a strong irritant to the skin and eyes and is a mutagen and carcinogen that causes human diseases. Animal studies have shown that polycyclic aromatic hydrocarbons, especially 3,4-benzo[a]pyrene, are associated with lung cancer in animals and humans and are a cause of lung cancer. Benzo[a]pyrene is excreted in feces after passing through detoxification organs such as the liver and kidneys. Feces are then applied to the land as fertilizer, contaminating crops and ultimately affecting human health. Benzo[a]pyrene (B[a]pyrene) is more toxic than aflatoxin and is not only the most toxic of the PAHs but also one of the most abundant, accounting for approximately one-fifth of all carcinogenic PAHs in the environment. With rising living standards, people are increasingly demanding food quality and safety. To improve food quality and safety in my country and better meet people's demand for safe consumption, accurate and efficient detection technologies for B[a]pyrene in food are urgently needed. Currently, the main methods for detecting B[a]pyrene are instrumental analysis and immunoassays. Instrumental analysis methods, such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), offer high sensitivity and accuracy, but they are expensive, require complex and time-consuming sample pretreatment, and require a high laboratory environment, making rapid detection difficult. Immunoassays, based on the specific recognition and reversible binding reaction between antigens and antibodies, offer high selectivity and sensitivity. Compared to instrumental analysis, they significantly simplify and shorten sample processing time, saving testing costs. Immunochromatographic test strips, based on the specific binding reaction of colloidal gold-labeled antibodies with antigens, offer visible results, low cost, and short analysis times. In recent years, they have been widely used for the qualitative and rapid detection of trace compounds such as target substances. However, colloidal gold immunochromatographic test strips for benzo[a]pyrene (B[a]pyrene) are currently unavailable on the market. Given the increasing trend of B[a]pyrene contamination reported in numerous publications in recent years, there is an urgent need for rapid on-site detection technologies for B[a]pyrene contamination, providing technical support for rapid screening of B[a]pyrene in food. Summary of the Invention

[0003] The present invention aims to provide an immunochromatographic test strip for rapid detection of benzo[a]pyrene, a preparation method and its application. The test strip is simple to operate and can accurately and rapidly determine the benzo[a]pyrene content in a sample.

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

[0005] An immunochromatographic test strip for rapid benzo[a]pyrene detection comprises a water-absorbing pad, a detection pad, a gold-labeled pad, and a sample pad. Adjacent pads are overlapped and connected at a connection point and sequentially adhered to a base plate from top to bottom. The detection pad uses a nitrocellulose membrane as a base pad, a quality control line and a detection line are transversely arranged on the nitrocellulose membrane, the quality control line is coated with a rabbit anti-mouse polyclonal antibody, and the detection line is located below the quality control line and is coated with a benzo[a]pyrene-ovalbumin conjugate. The gold-labeled pad is transversely sprayed with an anti-benzo[a]pyrene monoclonal antibody labeled with nanogold. The anti-benzo[a]pyrene monoclonal antibody is secreted and produced by the BBBE1H1 hybridoma cell line with a deposit number of CCTCC NO: C201882.

[0006] According to the above scheme, the coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter on the test line is 100-300 ng; the coating amount of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 50-200 ng.

[0007] According to the above scheme, the amount of nano-gold labeled anti-benzo[a]pyrene monoclonal antibody required for each centimeter of spraying length on the gold label pad is 100-200 ng.

[0008] According to the above solution, the distance between the detection line and the upper edge of the detection pad is 15 to 20 mm, and the distance between the detection line and the quality control line is 5 to 7 mm.

[0009] The method for preparing the immunochromatographic test strip for rapid detection of benzo[a]pyrene as described above comprises the following steps:

[0010] (1) Preparation of absorbent pad

[0011] Cut the absorbent paper to make an absorbent pad;

[0012] (2) Preparation of detection pad

[0013] Test line coating:

[0014] Benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) is prepared into 0.25-0.5 mg / mL coating solutions using coating buffer, and the solutions are coated on nitrocellulose membranes to obtain test lines, followed by drying at 37-40° C. for 30-60 minutes; the coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter of the test line is 100-300 ng; and the distance between the test line and the upper edge of the nitrocellulose membrane is 15-20 mm;

[0015] Coating of quality control line:

[0016] Prepare a 0.2-0.4 mg / mL coating solution of rabbit anti-mouse polyclonal antibody using coating buffer, coat it horizontally on a nitrocellulose membrane to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per centimeter of the quality control line is 50-200 ng, and then dry it at 37-40°C for 1-2 hours.

[0017] (3) Preparation of sample pad

[0018] 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;

[0019] (4) Preparation of gold label pad

[0020] The glass fiber membrane is soaked in a blocking solution, taken out, and dried at 37-40° C. for 6-10 hours. A solution of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold is sprayed horizontally onto the dried glass fiber membrane by a dot spraying method, wherein the amount of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold is 100-200 ng per centimeter of spraying length. The membrane is then freeze-dried in a vacuum for 2-4 hours and stored in a desiccator at room temperature.

[0021] (5) Assembly of test strips

[0022] The absorbent pad, the detection pad, the gold label pad, and the sample pad are pasted on the bottom plate from top to bottom, overlapping each other by 1 to 2 mm, thereby obtaining an immunochromatographic test strip for detecting benzo[a]pyrene.

[0023] According to the above scheme, the blocking solution used contains 2-5 g of sucrose, 0.8 g of sodium chloride, 0.02 g of potassium chloride, 0.02 g of sodium azide, 0.02 g of potassium dihydrogen phosphate, 1-2 g of bovine serum albumin, and 0.29 g of disodium hydrogen phosphate dodecahydrate per 100 mL.

[0024] According to the above scheme, each 10 mL of the coating buffer contains: 0.08 g of sodium chloride, 0.002 g of potassium chloride, 0.002 g of potassium dihydrogen phosphate, 0.1-0.2 g of ovalbumin, and 0.029 g of disodium hydrogen phosphate dodecahydrate.

[0025] According to the above scheme, the nanogold-labeled anti-benzo[a]pyrene monoclonal antibody was prepared by an unsaturated labeling method, which is as follows: 50.0 mL of a commercially available nanogold solution with a mass concentration of 0.01% was taken, the pH value was adjusted with 0.6 mL of a 0.1 mol / L potassium carbonate aqueous solution, and 2.0 mL of A 0.1 mg / mL aqueous solution of anti-benzo[a]pyrene monoclonal antibody was stirred for 30 minutes; a 10% aqueous solution of bovine serum albumin was added to a final concentration of 1% bovine serum albumin, 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;

[0026] The 0.1 mol / L potassium carbonate aqueous solution is prepared by dissolving 13.8 g of 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 0.2 g of sodium azide, 0.1235 g of boric acid, and 2.0 g of polyethylene glycol-20000 in pure water to a volume of 1000 mL and filtering through a 0.22 μm filter membrane.

[0027] The application method of the benzo[a]pyrene immunochromatographic test strip in the detection of benzo[a]pyrene content is as follows: after the sample extract is diluted with the sample sustained-release solution, it is added dropwise to the sample pad of the immunochromatographic test strip for rapid detection of benzo[a]pyrene for detection, which serves as the detection test strip; another equal volume of the sample sustained-release solution is taken as the negative control solution, which is added dropwise to the sample pad of another immunochromatographic test strip for rapid detection of benzo[a]pyrene, which serves as the control test strip; after 15-20 minutes, the detection strip and The control test strip is used for color development control: when the color of the test line 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 benzo[a]pyrene in the test sample solution is less than 5 ng / mL; when the color is lighter than the color of the corresponding test line on the control test strip, it indicates that the content of benzo[a]pyrene 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 content of benzo[a]pyrene in the test sample solution is equal to or higher than 50 ng / mL;

[0028] 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;

[0029] Finally, the content of benzo[a]pyrene in the sample to be tested is obtained by conversion.

[0030] 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 coupled with anti-benzo[a]pyrene monoclonal antibodies, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with sample release solution to obtain the supernatant, which is the test solution.

[0031] According to the above scheme, the sample sustained-release solution is PBS containing 0.5% Tween 20, pH 7.4.

[0032] According to the above scheme, the immunomagnetic beads coupled with benzo[a]pyrene monoclonal antibodies include magnetic beads and anti-benzo[a]pyrene monoclonal antibodies coupled with COOH groups on the magnetic beads.

[0033] According to the above scheme, the mass ratio of anti-benzo[a]pyrene monoclonal antibody to magnetic beads is 2:1 to 1:5.

[0034] The preparation method of immunomagnetic beads coupled with benzo[a]pyrene monoclonal antibodies comprises the following steps: washing and removing impurities from the magnetic beads; adding anti-benzo[a]pyrene monoclonal antibodies for coupling and magnetic separation; blocking the coupled magnetic beads with a blocking solution; and after the blocking reaction is completed, performing magnetic separation, discarding the supernatant, and storing for later use.

[0035] The working principle of the immunochromatographic test strip provided by the present invention in the application of detecting benzo[a]pyrene contamination is as follows: 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 label pad, the nanogold-labeled anti-benzo[a]pyrene monoclonal antibody is dissolved. When the sample contains benzo[a]pyrene, benzo[a]pyrene will bind to the anti-benzo[a]pyrene monoclonal antibody labeled with nanogold on the gold pad and swim upward together. When it reaches the detection line where the benzo[a]pyrene-ovalbumin conjugate antigen is fixed, the antigen coated on the detection line will compete with benzo[a]pyrene for binding to the limited antigen binding sites on the anti-benzo[a]pyrene monoclonal antibody labeled with nanogold. The higher the benzo[a]pyrene content in the sample, the fewer nanogold-labeled anti-benzo[a]pyrene monoclonal antibodies that the antigen on the detection line can bind to, and the lighter the color of the color band formed on the detection line. When the number of nanogold-labeled corresponding antibodies bound to the antigen on the detection line is less than a certain number, no red line will appear at the detection line. Regardless of whether the sample contains the target, the gold nanoparticle-labeled anti-target antibody or the conjugate of the gold nanoparticle-labeled anti-target antibody and the target that is not captured by the antigen on the test line 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. Based on this, by comparing the color of the test line coated with the benzo[a]pyrene-ovalbumin conjugate on the test strip with the corresponding test line on the control strip, the contamination status of the target benzo[a]pyrene in the sample can be determined.

[0036] Beneficial effects of the present invention:

[0037] (1) One-step detection of benzo[a]pyrene contamination. The immunochromatographic test strip for detecting benzo[a]pyrene contamination provided by the present invention can achieve rapid detection of benzo[a]pyrene.

[0038] (2) High sensitivity. The 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A front view of an immunochromatographic test strip for detecting benzo[a]pyrene contamination according to the present invention;

[0040] Figure 2 This is a result judgment chart for detecting benzo[a]pyrene contamination in immunochromatographic test strips;

[0041] In the figure: 1 cardboard; 2 absorbent pad; 3 test pad; 4 quality control line; 5 test line; 6 gold label pad; 7 sample pad; DETAILED DESCRIPTION

[0042] Example 1: Preparation of anti-benzo[a]pyrene monoclonal antibodies

[0043] 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:

[0044] 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 / LPBS 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;

[0045] 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.

[0046] 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.

[0047] 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:

[0048] CR%=(IC 50 BaP / IC 50 other analogs) × 100.

[0049] 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%.

[0050] Detailed results are shown in Table 1:

[0051] Table 1. Cross-reactivity of BBBE1H1 with other structural analogs

[0052]

[0053]

[0054] Affinity determination of BBBE1H1 using indirect non-competitive ELISA:

[0055] 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. 450The 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.

[0056] Screening of hybridoma cell line BBBE1H1

[0057] 1. Animal immunization

[0058] 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.

[0059] 2. Cell Fusion

[0060] 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.

[0061] Cell line screening and cloning

[0062] 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.

[0063] Sequence determination of the variable region of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1.

[0064] (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;

[0065] (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;

[0066] (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).

[0067] 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.

[0068] Example 2: Preparation of Benzo[a]pyrene Immunomagnetic Beads:

[0069] a. Washing: Weigh the carboxyl-modified magnetic bead powder 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.

[0070] b Coupling: Add 4 mL of coupling buffer to the washed magnetic beads, add 2.5 mg of anti-benzo[a]pyrene 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.

[0071] 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.

[0072] 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.

[0073] Example 3

[0074] The preparation method of the immunochromatographic test strip for detecting benzo[a]pyrene contamination comprises the following steps:

[0075] (1) Preparation of absorbent pad

[0076] Cut the absorbent paper into 16mm long and 4mm wide specifications to make the absorbent pad;

[0077] (2) Preparation of detection pad

[0078] Benzo[a]pyrene-ovalbumin conjugate was prepared into a 0.4 mg / mL coating solution using coating buffer. The solution was applied to the nitrocellulose membrane by dot spraying at a distance of 15 mm from the top edge of the membrane to form a test line. The coating amount of benzo[a]pyrene-ovalbumin conjugate required per cm of the test line was 150 ng. The membrane was then dried at 37°C for 30 minutes.

[0079] The nitrocellulose membrane is 22 mm long and 4 mm wide;

[0080] Coating of quality control line:

[0081] Prepare a 0.25 mg / mL coating solution of rabbit anti-mouse polyclonal antibody using coating buffer and apply it horizontally to a nitrocellulose membrane at a distance of 6 mm from the test line 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.

[0082] The coating buffer is: 0.1 g ovalbumin, 0.08 g sodium chloride, 0.002 g potassium chloride, 0.002 g potassium dihydrogen phosphate, 0.029 g disodium hydrogen phosphate dodecahydrate, and water is added to make up to 10 mL;

[0083] (3) Preparation of sample pad

[0084] Cut the glass fiber membrane into 12 mm long and 4 mm wide specifications, 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;

[0085] The blocking solution is prepared by adding water to 100 mL of 1 g bovine serum albumin, 2 g sucrose, 0.8 g sodium chloride, 0.02 g potassium chloride, 0.02 g sodium azide, 0.02 g potassium dihydrogen phosphate, and 0.29 g disodium hydrogen phosphate dodecahydrate;

[0086] (4) Preparation of gold label pad

[0087] 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, and dry it at 37°C for 8 hours. Spray a solution of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold onto the dried glass fiber membrane in a dot-spraying manner, with the amount of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold being 125 ng per centimeter of spraying length. Then, freeze-dry it in a vacuum for 2 hours and store it in a desiccator at room temperature.

[0088] The nano-gold labeled anti-benzo[a]pyrene monoclonal antibody solution is prepared by an unsaturated labeling method, wherein 50.0 mL of a commercially available nano-gold solution with a mass concentration of 0.01% is taken, 0.4 mL of a 0.1 mol / L potassium carbonate aqueous solution is added to adjust the pH value, and 2 mL of the solution is slowly added under stirring. 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 solution was added; the solution was centrifuged again at 12000 rpm for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in labeled washing solution to obtain 5.0 mL of concentrate, which was placed in a 4°C refrigerator for use. The mass concentration of the gold nanoparticle-labeled anti-benzo[a]pyrene monoclonal antibody solution was 0.06 mg / mL;

[0089] The particle size of the nano-gold in the nano-gold solution is 15 nm;

[0090] The 0.1 mol / L potassium carbonate aqueous solution is: 13.8 g potassium carbonate is dissolved in pure water to a volume of 1000 mL, 0.22 μm

[0091] The labeled washing and preservation solution is: 0.1235g boric acid, 0.2g sodium azide, 2.0g polyethylene glycol-20000, pure water is diluted to 1000mL, and filtered through a 0.22μm filter membrane;

[0092] (5) Assembly of test strips

[0093] The absorbent pad, test pad, gold label pad and sample pad overlap each other by 1-3mm and are pasted on one side of the cardboard in sequence. Figure 1 .

[0094] Example 4

[0095] Application of the above-mentioned immunochromatographic test strip for detecting benzo[a]pyrene contamination in soybean oil sample testing:

[0096] Six randomly selected soybean oil samples were tested. The specific steps are as follows:

[0097] 1. Sample Pretreatment: Use a 10mL plastic centrifuge tube as a reaction vessel, accurately weigh 1g of soybean oil, 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 benzo[a]pyrene monoclonal antibody, vortex for 14 minutes, and discard the supernatant. Add 1mL of methanol, vortex for 2 minutes, and dilute 5-fold with sample release solution. The supernatant is used as the test solution.

[0098] 2. Sample detection steps: Take 100 μL of the sample solution to be tested as the sample release solution and add it dropwise to the sample pad of the benzo[a]pyrene-contaminated immunochromatographic test strip, which serves as the test test strip. At the same time, take an equal volume of the sample release solution as the negative control solution and add it dropwise to the sample pad of another immunochromatographic test strip, which serves as the control test strip. Read the results after 15 minutes.

[0099] Test result description:

[0100] The quality control line of the test strip shows a red strip, and the color of the test line is lighter than that of the control test strip. Figure 2-1 , judgment: the content of benzo[a]pyrene is equal to or higher than 5ng / mL and lower than 50ng / mL.

[0101] The quality control line of the test strip shows a red strip, and the color of the test line is close to the color of the test line in the control test strip. Figure 2-2 , judgment: the content of benzo[a]pyrene is less than 5ng / mL.

[0102] The control line of the test strip shows a red strip, while the test line does not show any color. Figure 2 -3, determine that the content of benzo[a]pyrene is equal to or higher than 50 ng / mL.

[0103] Test results and comparison with HPLC results:

[0104] The test results of the immunoassay strips were compared with those of the high performance liquid chromatography method. The results showed that the two methods had a good correlation, indicating that the benzo[a]pyrene immunoassay strips can be used to detect the benzo[a]pyrene content in actual soybean oil samples.

[0105] Table 1. Actual sample testing

[0106]

Claims

1. An immunochromatographic test strip for rapid detection of benzo[a]pyrene, characterized by: The invention comprises a water-absorbing pad, a detection pad, a gold-labeled pad and a sample pad. Adjacent pads are overlapped and connected at the connection points and are sequentially adhered to a bottom plate from top to bottom. The detection pad uses a nitrocellulose membrane as a base pad. A quality control line and a detection line are horizontally arranged on the nitrocellulose membrane. The quality control line is coated with a rabbit anti-mouse polyclonal antibody. The detection line is located below the quality control line and is coated with a benzo[a]pyrene-ovalbumin conjugate. The gold-labeled pad is horizontally sprayed with an anti-benzo[a]pyrene monoclonal antibody labeled with nanogold. The anti-benzo[a]pyrene monoclonal antibody is secreted and produced by the BBBE1H1 hybridoma cell line with a deposit number of CCTCC NO: C201882.

2. The immunochromatographic test strip for rapid detection of benzo[a]pyrene according to claim 1, characterized in that: The coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter on the test line is 100-300 ng; the coating amount of rabbit anti-mouse polyclonal antibody required per centimeter on the quality control line is 50-200 ng.

3. The immunochromatographic test strip for rapid detection of benzo[a]pyrene according to claim 1, characterized in that: The amount of the nano-gold labeled anti-benzo[a]pyrene monoclonal antibody required for each centimeter of spraying length on the gold label pad is 100-200 ng.

4. The immunochromatographic test strip for rapid detection of benzo[a]pyrene according to claim 1, characterized in that: The distance between the detection line and the upper edge of the detection pad is 15 to 20 mm, and the distance between the detection line and the quality control line is 5 to 7 mm.

5. The method for preparing the immunochromatographic test strip for rapid detection of benzo[a]pyrene 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: Benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) is prepared into 0.25-0.5 mg / mL coating solutions using coating buffer, and the solutions are coated on nitrocellulose membranes to obtain test lines, followed by drying at 37-40° C. for 30-60 minutes; the coating amount of benzo[a]pyrene-ovalbumin conjugate (BAP-OVA) required per centimeter of the test line is 100-300 ng; and the distance between the test line and the upper edge of the nitrocellulose membrane is 15-20 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, coat it horizontally on a nitrocellulose membrane to obtain a quality control line. The coating amount of rabbit anti-mouse polyclonal antibody required per centimeter of the quality control line is 50-200 ng, and then dry it 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 solution of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold is sprayed horizontally onto the dried glass fiber membrane by a dot spraying method, wherein the amount of anti-benzo[a]pyrene monoclonal antibody labeled with nanogold is 100-200 ng per centimeter of spraying length. The membrane is then freeze-dried in a vacuum for 2-4 hours and stored in a desiccator at room temperature. (5) Assembly of test strips The absorbent pad, the detection pad, the gold label pad and the sample pad are pasted on the bottom plate from top to bottom, overlapping each other by 1 to 2 mm, thereby obtaining an immunochromatographic test strip for detecting benzo[a]pyrene.

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

7. The method for using the benzo[a]pyrene immunochromatographic test strip according to claim 1 in the detection of benzo[a]pyrene content is as follows: after the sample extract is diluted with the sample sustained-release solution, it is added dropwise to the sample pad of the immunochromatographic test strip for rapid detection of benzo[a]pyrene for detection, which serves as the detection test strip; another equal volume of the sample sustained-release solution is taken as a negative control solution, which is added dropwise to the sample pad of another immunochromatographic test strip for rapid detection of benzo[a]pyrene, which serves as the control test strip; after 15-20 minutes, the detection strip is The test strip and the control strip are subjected to color contrast: when the color of the test line on the test strip is close to the color of the corresponding test line on the control strip, it indicates that the content of benzo[a]pyrene in the test sample solution is less than 5 ng / mL; when the color of the test line is lighter than the color of the corresponding test line on the control strip, it indicates that the content of benzo[a]pyrene 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 content of benzo[a]pyrene in the test sample solution is equal to or higher than 50 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 in the sample to be tested is obtained by conversion.

8. The use according to claim 7, characterized in that The sample is edible vegetable oil, and the pretreatment steps are: ultrasonic extraction of 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, vortex mixing, discarding the supernatant, adding methanol and vortex mixing, and diluting it with a sample sustained-release solution to obtain the supernatant, which is the test solution.

9. The use according to claim 8, characterized in that The mass ratio of anti-benzo[a]pyrene monoclonal antibody to immunomagnetic beads is 1:1 to 1:5.

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