Hybridoma cell line BBBE1H1, anti-benzo[a]pyrene monoclonal antibody produced thereby, and application thereof

Abstract

The present invention relates to a hybridoma cell line BBBE1H1, an anti-benzo[a]pyrene monoclonal antibody produced thereby, and applications thereof. The hybridoma cell line BBBE1H1 provided by the present invention can be used to prepare a high-titer anti-benzo[a]pyrene monoclonal antibody, the titer of which can reach 1.2×10 5 The anti-benzo[a]pyrene monoclonal antibody provided by the present invention has high sensitivity and good specificity, and its 50% inhibitory concentration IC 50 It is 0.013ng / mL.

Description

Technical Field

[0001] The present invention relates to a hybridoma cell line BBBE1H1 and an anti-benzo[a]pyrene monoclonal antibody produced by the hybridoma cell line. Background Art

[0002] Benzo[a]pyrene (BaP), also known as 3,4-benzopyrene, is a fused-ring aromatic hydrocarbon containing a benzene ring. It is a stable yellow powder at room temperature and is a strong carcinogen, potentially causing lung, respiratory, stomach, digestive tract, bladder, and skin cancers. Benzo[a]pyrene is also teratogenic and mutagenic, and can affect offspring through the mother's placenta, causing embryonic malformations or death and decreased immune function in offspring. Benzo[a]pyrene in food primarily comes from two sources: first, waste gases from incomplete combustion enter vegetables, fruits, grains, and aquatic products through water, the atmosphere, and soil; second, during cooking processes such as smoking, baking, and frying, when scorching occurs, the benzo[a]pyrene content increases 10 to 20 times compared to normal food. Therefore, benzo[a]pyrene contamination poses a significant risk to the quality and safety of agricultural products and the development of the agricultural industry. my country has established limits on benzo[a]pyrene in food.

[0003] Existing benzo[a]pyrene detection methods primarily rely on laboratory confirmatory testing techniques using large-scale instrumentation. Immunological detection methods rely on antigen-antibody specific recognition, converting immune signals into detectable signals through enzyme, fluorescent, and other labeling techniques, thereby qualitatively or quantitatively indicating the target's content. These methods offer advantages such as simple sample preparation, high sensitivity, strong specificity, and short experimental cycles. The sensitivity and specificity of the antibody directly determine the accuracy of the immunoassay. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a hybridoma cell line BBBE1H1, an anti-benzo[a]pyrene (BaP) monoclonal antibody produced by the hybridoma cell line, and applications thereof.

[0005] The present invention provides a variable region sequence of a monoclonal antibody that specifically binds to benzo[a]pyrene, wherein the heavy chain variable region of the monoclonal antibody specifically binding to benzo[a]pyrene is the amino acid sequence shown in SEQ ID NO:3 in the sequence listing, or a conservative mutant thereof obtained by adding, deleting, replacing, or modifying one or more amino acids through conservative mutation; and the light chain variable region is the amino acid sequence shown in SEQ ID NO:4 in the sequence listing, or a conservative mutant thereof obtained by adding, deleting, replacing, or modifying one or more amino acids through conservative mutation. The variable region sequence of the monoclonal antibody provided by the present invention can specifically bind to benzo[a]pyrene.

[0006] The present invention also provides a gene sequence encoding the variable region sequence of the above-mentioned monoclonal antibody specifically binding to benzo[a]pyrene.

[0007] Preferably, as an embodiment of the present invention, the monoclonal antibody that specifically binds to benzo[a]pyrene is an anti-benzo[a]pyrene monoclonal antibody secreted and produced by the hybridoma cell line BBBE1H1.

[0008] A hybridoma cell line BBBE1H1 was provided. The hybridoma cell line was deposited with the China Center for Type Culture Collection (CCTCC) on April 3, 2018, at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: C201882. The hybridoma cell line comprises the gene sequence encoding the heavy chain variable region of an anti-benzo[a]pyrene monoclonal antibody as shown in SEQ ID NO: 1 in the sequence listing, and the gene sequence encoding the light chain variable region of an anti-benzo[a]pyrene monoclonal antibody as shown in SEQ ID NO: 2 in the sequence listing.

[0009] An anti-benzo[a]pyrene monoclonal antibody secreted by the hybridoma cell line BBBE1H1, deposited with CCTCC NO: C201882. The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing; the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.

[0010] The anti-benzo[a]pyrene monoclonal antibody provided by the present invention has high sensitivity and high specificity. Its 50% inhibition concentration for benzo[a]pyrene (BaP), IC 50 The cross-reactivity with benzo[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene and pyrene was less than 15%.

[0011] Application of anti-benzo[a]pyrene monoclonal antibodies in the preparation of immunoassay products for benzo[a]pyrene or benzo[a]pyrene affinity columns.

[0012] According to the above scheme, the immunoassay product for benzo[a]pyrene is a benzo[a]pyrene immunoassay test strip or a benzo[a]pyrene immunoassay ELISA kit.

[0013] Provided are benzo[a]pyrene immunomagnetic beads, including magnetic beads and anti-benzo[a]pyrene monoclonal antibodies coupled with COOH groups on the magnetic beads.

[0014] According to the above scheme, the mass ratio of anti-benzo[a]pyrene monoclonal antibody to magnetic beads in the immunomagnetic beads is 2:1 to 1:5.

[0015] A preparation method for benzo[a]pyrene immunomagnetic beads is provided, which comprises washing the magnetic beads to remove impurities; adding anti-benzo[a]pyrene monoclonal antibodies for coupling, and magnetic separation; adding a blocking solution to the coupled magnetic beads for blocking; after the blocking reaction is completed, magnetic separation is performed, and the supernatant is discarded and stored for future use.

[0016] According to the above scheme,

[0017] Provided is a benzo[a]pyrene immunosorbent, which comprises a solid phase carrier and an anti-benzo[a]pyrene monoclonal antibody coupled to the solid phase carrier.

[0018] An immunoaffinity column loaded with the above-described [a]pyrene immunoadsorbent is provided.

[0019] Provided is a method for preparing a benzo[a]pyrene immunosorbent:

[0020] a) Matrix treatment

[0021] The CNBr-activated agarose gel matrix powder was redissolved and activated;

[0022] b) Ligand coupling

[0023] Dissolve the anti-benzo[a]pyrene monoclonal antibody to be coupled in a coupling buffer to obtain an antibody solution, and quickly transfer the activated agarose gel matrix in step a) into the antibody solution for coupling;

[0024] c) Ligand blocking

[0025] Block all remaining active groups;

[0026] d) removing excess ligands that are not coupled after coupling;

[0027] And further a method for obtaining an immunoaffinity column:

[0028] The benzo[a]pyrene immunoadsorbent is loaded onto a column to obtain a benzo[a]pyrene immunoaffinity column loaded with the benzo[a]pyrene immunoadsorbent.

[0029] The hybridoma cell line BBBE1H1 provided by the present invention is obtained by a two-step screening method, which specifically comprises the following steps: BALB / c mice are immunized 4-6 times with a complete benzo[a]pyrene antigen BaP-BSA, followed by a booster immunization with a complete antigen BaP-BSA dose 1 / 2 times that of the previous immunization, cell fusion is performed 3 days later, and cells are cultured in a HAT semi-solid medium for 1-2 weeks. Single cell colonies are then transferred to a 96-well cell culture plate for culture, and when the cells grow to 1 / 2 of the well, screening is performed using an indirect competitive ELISA method, using a benzo[a]pyrene standard as a competitor, selecting wells with higher absorbance values ​​and inhibition rates, performing subcloning 4-5 times using a limiting dilution method, and finally screening to obtain the hybridoma cell line BBBE1H1.

[0030] The present invention further provides a method for preparing an anti-benzo[a]pyrene monoclonal antibody, comprising the following steps: injecting the hybridoma cell line BBBE1H1 obtained above into the abdomen of a BALB / c mouse that has been pre-treated with Freund's incomplete adjuvant; collecting ascites into a centrifuge tube after the ascites grows; and purifying the ascites using the octanoic acid-ammonium sulfate method to obtain the anti-benzo[a]pyrene monoclonal antibody.

[0031] According to the above scheme, the antibody purification method using octanoic acid-ammonium sulfate is as follows: filter the mouse ascites with double filter paper, centrifuge at 4°C, 12000r / min for more than 15 minutes, aspirate the supernatant, mix the obtained ascites supernatant with 4 volumes 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 30-60 minutes, and let it 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;

[0032] 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.

[0033] Beneficial effects of the present invention:

[0034] (1) The hybridoma cell line BBBE1H1 provided by the present invention can be used to prepare high-titer benzo[a]pyrene monoclonal antibodies. The antibody titer measured by enzyme-linked immunosorbent assay (ELISA) can reach 1.2×10 5 .

[0035] (2) The anti-benzo[a]pyrene monoclonal antibody provided by the present invention has high sensitivity and good specificity. Its 50% inhibitory concentration for benzo[a]pyrene (BaP), IC 50 The cross-reactivity to benzo[a]anthracene, benzo[b]fluoranthene, benzo[e]pyrene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene, fluoranthene, and pyrene was less than 15%.

[0036] (3) The anti-benzo[a]pyrene monoclonal antibody provided by the present invention can be used to determine the content of benzo[a]pyrene. DETAILED DESCRIPTION

[0037] Example 1: Screening of hybridoma cell line BBBE1H1

[0038] 1. Animal immunization

[0039] 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.

[0040] 2. Cell Fusion

[0041] 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.

[0042] Cell line screening and cloning

[0043] 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.

[0044] Example 2: Sequence determination of the variable region of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1.

[0045] (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;

[0046] (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;

[0047] (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).

[0048] 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.

[0049] Example 3: Preparation, purification, subtype and characterization of anti-benzo[a]pyrene monoclonal antibodies

[0050] The anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BBBE1H1 obtained in Example 1 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 antibody was 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 12000 rpm at 4°C for more than 15 minutes, and the supernatant was aspirated. The obtained 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;

[0051] 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.

[0052] 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.

[0053] 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 5The 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:

[0054] CR%=(IC 50 BaP / IC 50 other analogs) × 100.

[0055] 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%.

[0056] Detailed results are shown in Table 1:

[0057] Table 1. Cross-reactivity of BBBE1H1 with other structural analogs

[0058]

[0059]

[0060] Affinity determination of BBBE1H1 using indirect non-competitive ELISA:

[0061] 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.

[0062] Example 4: Preparation of Benzo[a]pyrene Immunomagnetic Beads:

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Example 5: Establishment of the Benzo[a]pyrene ic-ELISA Detection Method

[0068] 1. Sample pretreatment:

[0069] (1) Take a 10 mL plastic centrifuge tube as a reaction vessel, accurately weigh 1 g of vegetable oil sample, add 5 mL of n-hexane solution, and sonicate for 10 min. Then add another 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 the above-mentioned benzo[a]pyrene immunomagnetic beads coupled with benzo[a]pyrene monoclonal antibody to specifically bind to benzo[a]pyrene in the sample, vortex mix for 14 minutes, and discard the supernatant.

[0070] (2) Add 1 mL of methanol solution and vortex mix for 2 minutes to elute the benzo[a]pyrene bound to the magnetic beads. Dilute the solution 5-fold with detection solution (0.5% Tween 20 / PBS, pH 7.4) as the test solution for ELISA detection.

[0071] 2. The specific steps are as follows:

[0072] Coat a 96-well microtiter plate with complete benzo[a]pyrene antigen overnight at 4°C. Wash the plate three times with PBST and block with 3.5%-5% skim milk powder at 37°C for 1 hour. Add 50 μl of benzo[a]pyrene monoclonal antibody at the appropriate working concentration and a serially diluted benzo[a]pyrene standard solution, incubating at 37°C for 1 hour. Wash the plate three times with PBST, add HRP-conjugated secondary antibody, wash the plate three times with PBST, stop color development, and read the OD value on a microtiter plate reader.

[0073] The concentration of benzo[a]pyrene complete antigen was 1 μg / mL; the concentration of benzo[a]pyrene monoclonal antibody was 0.1 μg / mL.

[0074] The antibody diluent has a salt ion concentration of 10 mM, a pH of 7.4, and a Tween 20 concentration of 0.05%. The sensitivity of the anti-benzo[a]pyrene monoclonal antibody of the present invention was detected by IC-ELISA (50% inhibition concentration for benzo[a]pyrene (BaP), IC 50 ) is 0.013ng / mL.

[0075] The content of benzo[a]pyrene in the sample solution to be tested was obtained based on the relationship curve between the obtained OD value and the benzo[a]pyrene concentration:

[0076] A series of benzo[a]pyrene (BaP) standards were spiked into blank edible vegetable oil matrix samples to give final concentrations of 200, 66.6, 22.2, 7.4, 2.4, 0.8, and 0.2 ng / mL. Pretreatment was performed according to step 1 and detection was performed according to step 2. A calibration curve of Bx / B0 versus BaP (log 10) concentration was constructed using Origin 8.6 (Origin Lab Corporation, Northampton, MA, USA) using four-parameter logistic regression. Each data point is the average of three independent measurements.

[0077] 3. Spike recovery experiment

[0078] To evaluate the accuracy of the established benzo[a]pyrene ic-ELISA (indirect competitive ELISA) method, a matrix standard curve was constructed and spike recovery experiments were conducted on blank soybean oil samples. The specific steps were: Benzo[a]pyrene standards were spiked into the samples at concentrations of 5 ng / mL, 50 ng / mL, and 100 ng / mL. Extraction was performed according to the aforementioned sample pretreatment method. A standard curve was constructed using the ic-ELISA method established in step 2 above, and the spiked sample content was determined. The results showed that the spiked sample recoveries for the three concentrations were 104.1%, 89.6%, and 96.8%, respectively.

[0079] 4. Actual sample testing

[0080] The established ic-ELISA method was used to detect 6 soybean oil samples collected and compared with the liquid chromatography method for verification. The test results showed (Table 2) that the test results of ic-ELISA and HPLC were highly correlated, with the correlation curve being y=1.0481x-0.2124, R 2 is 0.9974.

[0081] Table 2. Actual sample testing

[0082]

[0083] Example 6: Preparation of the Benzo[a]pyrene Immunoaffinity Column

[0084] Preparation of Benzo[a]pyrene Immunoaffinity Column

[0085] Matrix preparation

[0086] Weigh out 10 g of CNBr-activated Sepharose lyophilized matrix powder (each gram of lyophilized matrix powder will yield a final volume of 4-5 mL of swollen matrix) and dissolve it in 20% methanol-water. The matrix will swell immediately. Wash the matrix three times in a fritted funnel with 20% methanol-water.

[0087] Antibody conjugation

[0088] Wash the agarose gel 2-3 times with coupling buffer (0.2 mol / L NaHCO3, pH 8.3). Dissolve the anti-benzo[a]pyrene monoclonal antibody to be coupled in the coupling buffer to a concentration of 20 mg / mL. Add the agarose gel and antibody solution to a conical flask and place on a temperature-controlled shaker at 10°C, 250 rpm, and incubate for 20 hours.

[0089] b. Calculation of coupling efficiency: Centrifuge at 2,000 rpm until the agarose gel reaches the bottom of the tube. Transfer the supernatant to a new tube and measure the protein content. The coupling efficiency is calculated to be 98.5%.

[0090] Blocking: Transfer the matrix to 0.1 mol / L Tris-HCl buffer. Place on a temperature-controlled shaker at 10°C, 250 rpm, and incubate for 4 hours to block any remaining active groups.

[0091] Storage: Wash the gel three times with 0.01 mol / L PBS to remove excess uncoupled ligand, using 5-10 times the gel volume of PBS buffer each time. Then, wash once with 3-5 times the gel volume of 0.01% NaN3-PBS and store in 0.01% NaN3-PBS.

[0092] Column Packing: Rinse the column with ultrapure water and then install the lower frit to rinse again. Load each column with 0.5 mL of the immunoaffinity adsorbent prepared in the previous step. After filling the column, install the plug and fill the column completely with 0.01 mol / L PBS. Install the upper frit and gently push it downward, pressing the immunoaffinity adsorbent lightly between the upper and lower frits, but do not compact it. Finally, filter the column with sterile-filtered 0.01% NaN₃-PBS and store in 0.01% NaN₃-PBS. The benzopyrene affinity column is now loaded and equilibrated and ready for use.

Claims

1. Hybridoma cell line BBBE1H1 was deposited with the China Center for Type Culture Collection on April 3, 2018, at Wuhan University, Wuhan, China, with the accession number CCTCC NO: C201882.

2. An anti-benzo[a]pyrene monoclonal antibody, characterized in that: The protein is secreted and produced by the hybridoma cell line BBBE1H1 with a deposit number of CCTCC NO: C201882.

3. Use of the anti-benzo[a]pyrene monoclonal antibody according to claim 2 in the preparation of a benzo[a]pyrene immunoassay test strip, a benzo[a]pyrene immunoassay ELISA kit, or a benzo[a]pyrene affinity column.

4. Benzo[a]pyrene immunomagnetic beads, characterized in that: The invention comprises magnetic beads and the anti-benzo[a]pyrene monoclonal antibody according to claim 2, wherein the COOH group on the magnetic beads is coupled with the amino group of the anti-benzo[a]pyrene monoclonal antibody.

5. The method for preparing benzo[a]pyrene immunomagnetic beads according to claim 4, characterized in that: The magnetic beads are washed and impurities are removed; anti-benzo[a]pyrene monoclonal antibodies are added for coupling and magnetic separation is performed; the coupled magnetic beads are blocked with blocking solution; after the blocking reaction is completed, magnetic separation is performed, the supernatant is discarded and stored for later use.

6. A benzo[a]pyrene immunoadsorbent, characterized in that: The immunosorbent comprises a solid phase carrier and the anti-benzo[a]pyrene monoclonal antibody according to claim 2 coupled to the solid phase carrier.

7. An immunoaffinity column loaded with the poly(a)pyrene immunoadsorbent according to claim 6.

8. The method for preparing the benzo[a]pyrene immunoadsorbent according to claim 6, characterized in that: The preparation steps are: a) Matrix treatment The CNBr-activated agarose gel matrix powder was redissolved and activated; b) Ligand coupling Dissolve the anti-benzo[a]pyrene monoclonal antibody to be coupled in a coupling buffer to obtain an antibody solution, and quickly transfer the activated agarose gel matrix in step a) into the antibody solution for coupling; c) Ligand blocking Block all remaining active groups; d) Remove excess uncoupled ligand after coupling.

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