Hybridoma cell strain BAP1C12, anti-benzo [a] pyrene monoclonal antibody generated by hybridoma cell strain BAP1C12 and application
By developing the BAP1C12 of hybrid tumor cells and the anti -benzene and [A] single cloning antibody it produced, it solves the problem of high -throughput of food safety on -site high -throughput. Detection effect of specific and high sensitivity.
Patent Information
- Application Number
- CN202411379852.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art is difficult to meet the demand for high-throughput rapid detection of benzo[a]pyrene in food safety site, and there are challenges in the creation of antibodies in immunoassay methods, which affects the sensitivity and accuracy of the detection.
The BAP1C12 of hybrid tumor cells was developed and the anti-benzene was produced by it. And [a] 芘 test.
A high -specific and high -sensitivity anti -benzene is provided [A] 芘 monoclonal antibody. The IC50 is 0.45ng/ml. It can quickly and accurately detect benzene and [A] 芘 to meet the high -throughput detection needs of food safety.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and specifically relates to a hybridoma cell line BAP1C12, an anti-benzo[a]pyrene monoclonal antibody produced by the hybridoma cell line, and applications thereof. Background Art
[0002] Benzo(a)pyrene (BaP), also known as 3,4-benzopyrene, has a molecular structure composed of benzene rings and pyrene molecules fused together, and is a polycyclic aromatic hydrocarbon compound. The pollution of benzo(a)pyrene comes from a wide range of sources, mainly from two aspects. First, the waste gas produced by the incomplete combustion of industrial fuels such as coal, oil, and natural gas is discharged into water sources, air, and soil, and enters the food chain through the migration, transformation, metabolism, and absorption of animals and plants, contaminating foods such as grains, oilseeds, vegetables, fruits, and livestock and poultry products; second, during the baking, frying, and smoking of primary edible agricultural products, their main components such as proteins, sugars, oils, and organic acids are prone to chemical reactions such as oxidation-reduction, cracking, and fusion under high temperature and oxygen-free conditions, generating polycyclic aromatic hydrocarbon compounds such as benzo(a)pyrene. Benzo[a]pyrene is a strong carcinogen, more toxic than aflatoxin. Long-term contact or accidental ingestion of contaminated food can cause strong irritation to the eyes and skin. It enters the human body through digestion and absorption, accumulates in adipose tissue and breast, and causes skin cancer, digestive tract cancer, stomach cancer, and lung cancer. Benzo[a]pyrene is also teratogenic, mutagenic, and reproductively toxic. It can affect offspring through the mother's placenta, causing embryonic malformation, death, and fetal immunodeficiency. Countries and organizations around the world have established strict limit requirements for polycyclic aromatic hydrocarbons and benzo[a]pyrene. my country promulgated and implemented the "GB 2762-2022 National Food Safety Standard Contaminants in Food" on June 30, 2023, stipulating that the limit value of benzo[a]pyrene in oils and fats and products is 10μg kg -1 .
[0003] At present, the determination of benzo[a]pyrene mainly adopts precision instrument methods such as high-performance liquid chromatography, liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry, but the steps are cumbersome, the instruments and equipment are expensive, and the process is time-consuming, which makes it difficult to meet the regulatory needs of high-throughput rapid detection of food safety on-site. The immunoassay method is a rapid detection technology based on the specific recognition of antigens and antibodies, which uses specific antibodies as recognition elements. It has the characteristics of simple operation, accurate results and low cost. The core of the immunoassay method is to create antibodies. High-quality antibodies can specifically identify small molecule pollutants such as benzo[a]pyrene, significantly improve the sensitivity and accuracy of immunoassay technology, and provide key technical support for the accurate and rapid quantification of benzo[a]pyrene in plants.
[0004] The purpose of the present invention is to provide an anti-benzo[a]pyrene monoclonal antibody and a hybridoma cell line thereof with high specificity and detection sensitivity for benzo[a]pyrene, so as to provide a key core reagent for establishing a rapid immune detection technology for benzo[a]pyrene in vegetable oils. Summary of the invention
[0005] The object of the present invention is to provide a highly specific and sensitive anti-benzo[a]pyrene monoclonal antibody and a hybridoma cell line thereof. Specifically, the present invention provides a hybridoma cell line BAP1C12, an anti-benzo[a]pyrene monoclonal antibody produced thereby and applications thereof.
[0006] The first aspect of the present invention provides a hybridoma cell line BAP1C12, which was deposited in the China Center for Type Culture Collection (CCTCC) on October 18, 2023, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2023315. It has an anti-benzo[a]pyrene monoclonal antibody heavy chain variable region encoding gene sequence shown in SEQ ID NO: 1 in the sequence list and an anti-benzo[a]pyrene monoclonal antibody light chain variable region encoding gene sequence shown in SEQ ID NO: 2 in the sequence list.
[0007] The second aspect of the present invention provides a monoclonal antibody that specifically binds to benzo[a]pyrene, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:3 in the sequence list or a conservative mutant obtained by adding, deleting, replacing or modifying one or more amino acids through conservative mutation; the light chain variable region is the amino acid sequence shown in SEQ ID NO:4 in the sequence list or a conservative mutant obtained by adding, deleting, replacing or modifying one or more amino acids through conservative mutation. The variable region of the monoclonal antibody provided by the present invention can specifically bind to benzo[a]pyrene.
[0008] Furthermore, the anti-benzo[a]pyrene monoclonal antibody provided by the present invention has a heavy chain variable region having an amino acid sequence shown in SEQ ID NO:3 in the sequence listing; and a light chain variable region having an amino acid sequence shown in SEQ ID NO:4 in the sequence listing.
[0009] Furthermore, the anti-benzo[a]pyrene monoclonal antibody of the present invention is secreted and produced by the hybridoma cell line BAP1C12 with a deposit number of CCTCC NO: C2023315.
[0010] The third aspect of the present invention also provides a method for preparing an anti-benzo[a]pyrene monoclonal antibody, wherein the hybridoma cell line BAP1C12 is injected into the abdomen of a BALB / c mouse that has been treated with Freund's incomplete adjuvant in advance, the ascites of the mouse is collected, and the anti-benzo[a]pyrene monoclonal antibody is obtained by purification.
[0011] According to the above scheme, the purification method is the caprylic acid-ammonium sulfate method.
[0012] The fourth aspect of the present invention provides the use of the above-mentioned anti-benzo[a]pyrene monoclonal antibody in the determination of benzo[a]pyrene content.
[0013] The fifth aspect of the present invention provides the use of the above-mentioned anti-benzo[a]pyrene monoclonal antibody in the preparation of a product for detecting benzo[a]pyrene content.
[0014] A sixth aspect of the present invention provides a product for detecting benzo[a]pyrene comprising the above-mentioned anti-benzo[a]pyrene monoclonal antibody.
[0015] According to the above scheme, the products are benzo[a]pyrene immunoassay ELISA kit, colloidal gold test strip kit, immunoaffinity column and other products.
[0016] The anti-benzo[a]pyrene monoclonal antibody provided by the present invention has high sensitivity and strong specificity. Its 50% inhibition concentration for benzo[a]pyrene, IC 50 It is 0.45ng / mL; the cross-reactions with its structural analogues fluoranthene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]anthracene, benzo[k]fluoranthene, anthracene, phenanthrene, acenaphthene, fluorene and naphthalene are all less than 3.5%. The cross-reaction rates to fluoranthene are 3.5%, benzo[b]fluoranthene is 2.7%, indeno[1,2,3-cd]pyrene is 1.8%, benzo[a]anthracene is 1.3%, benzo[k]fluoranthene is 0.3%, anthracene is 0.2%, and the cross-reaction rates to phenanthrene, acenaphthene, fluorene and naphthalene are all less than 0.01%.
[0017] The hybridoma cell line BAP1C12 provided by the present invention is obtained by a two-step screening method, and the specific steps are: synthesizing the complete antigen BBP1-BSA by esterification reaction of the hapten BBP1 and bovine serum albumin (BSA). After immunizing female BALB / c mice 4-5 times after emulsification, the immunization is strengthened with the same immunization dose of non-emulsified BBP1-BSA, and cell fusion is performed 3 days later. After culturing with HAT semi-solid culture medium for 1-2 weeks, a single cell colony is transferred to a 96-well cell culture plate for culturing. When the cells grow to 1 / 2 of the well, an indirect competitive enzyme-linked immunosorbent assay (icELISA) method is used for screening, using a benzo[a]pyrene standard as a competitor, selecting wells with higher absorbance values and inhibition rates, and performing subcloning 3-4 times by a limiting dilution method, and finally screening to obtain the hybridoma cell line BAP1C12.
[0018] Beneficial effects of the present invention:
[0019] (1) The hybridoma cell line BAP1C12 provided by the present invention can be used to prepare high-titer anti-benzo[a]pyrene monoclonal antibodies, and the antibody titer measured by enzyme-linked immunosorbent assay (ELISA) can reach 2×10 5 .
[0020] (2) The anti-benzo[a]pyrene monoclonal antibody provided by the present invention has high sensitivity and good specificity, and the 50% inhibitory concentration (IC 50 ) was 0.45ng / mL, and the cross-reaction with its structural analogs fluoranthene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]anthracene, benzo[k]fluoranthene, anthracene, phenanthrene, acenaphthene, fluorene, and naphthalene was low and specific. The cross-reaction rate to fluoranthene was 3.5%, the cross-reaction rate to benzo[b]fluoranthene was 2.7%, the cross-reaction rate to indeno[1,2,3-cd]pyrene was 1.8%, the cross-reaction rate to benzo[a]anthracene was 1.3%, the cross-reaction rate to benzo[k]fluoranthene was 0.3%, the cross-reaction rate to anthracene was 0.2%, and the cross-reaction rates to phenanthrene, acenaphthene, fluorene, and naphthalene were all <0.01%.
[0021] (3) The anti-benzo[a]pyrene monoclonal antibody provided by the present invention can be used to determine the content of benzo[a]pyrene. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the affinity identification curve of the anti-benzo[a]pyrene 1C12 antibody.
[0023] Figure 2 This is the sensitivity standard curve of the anti-benzo[a]pyrene 1C12 antibody ELISA method. DETAILED DESCRIPTION
[0024] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.
[0025] Example 1: Screening of hybridoma cell line BAP1C12
[0026] 1. Preparation of Haptens
[0027] Dissolve 5.0 mg of 7,8,9,10-tetrahydrobenzo[a]pyrene-7-ol and 3.67 mg of succinic anhydride in 5 mL of pyridine, heat to 80°C for 4 h, blow nitrogen to dryness, dissolve the reaction product in chloroform, wash the organic phase four times with 20 mL of ultrapure water, add anhydrous sodium sulfate, let stand overnight, filter, and vacuum dry. The purified product is the hapten BBP1.
[0028] 2. Animal Immunization
[0029] The hapten BBP1 bovine serum protein (BSA) was synthesized into the complete antigen BBP1-BSA through esterification reaction. For the first immunization, it was emulsified with Freund's complete adjuvant in a ratio of 1:1, and 6-week-old female BALB / c mice were selected for subcutaneous multiple injections in the back of the neck. Three weeks after the second immunization, Freund's incomplete adjuvant was emulsified with the complete antigen in a ratio of 1:1 and injected subcutaneously in the back of the neck of the mice at multiple points. The 3rd and 4th immunization methods were the same as the previous ones, and the immunizations were performed two weeks after the previous immunizations. The four immunizations were the same in dosage, all 100μg / mouse. On the 7th day after each of the first three immunizations, blood was collected from the tail vein of the mice, serum was separated, and the mouse serum titer was monitored by conventional non-competitive enzyme-linked immunosorbent assay, and the mouse serum sensitivity was determined by icELISA. Mice corresponding to serum with relatively high titer and sensitivity were selected for booster immunization, and the immunization dose was twice the previous amount.
[0030] 3. Cell Fusion
[0031] Three days after the last booster immunization, 50% (wt%) polyethylene glycol, PEG (molecular weight 1450) was used as a fusion agent, and cell fusion was performed according to conventional methods. The specific steps were aseptically dislocated the cervical septum to be fused, and spleen cells were separated and mixed with mouse myeloma cells SP2 / 0 at a ratio of 5:1. The mixed cells were washed with DMEM basal culture medium, and centrifuged at 1200rpm for 5min. The supernatant was discarded, the cells were drained, 1mL PEG was added, the cells were fused for 1 minute, DMEM basal culture medium was slowly added, the cells were centrifuged, the supernatant was discarded, and the precipitate was the fused cells. The cells were resuspended with 20mL complete culture medium, and the suspended cells were added to 80mL semi-solid culture medium. After mixing, the cells were added to a 6-well cell culture plate, 2mL / well, and placed in a 37°C carbon dioxide incubator for culture.
[0032] The complete cell culture medium containing 1% HAT contains 20% (V / V) fetal bovine serum, 75% (V / V) DMEM basal culture fluid, 1% (wt%) L-glutamine, 1% (V / V) HEPES, 1% (V / V) double antibody (10,000 units per milliliter of penicillin and 10,000 micrograms per milliliter of streptomycin), 2% (V / V) growth factor (HFCS) and 1% (wt%) hypoxanthine-aminopterin-thymidine and methylcellulose.
[0033] 4. Screening and cloning of cell lines
[0034] 2-3 weeks after cell fusion, when the cell colonies grow to be visible to the naked eye, use a micropipette to pick out the clones from the culture medium and transfer them to a 96-well cell culture plate for HAT liquid culture. When the cells grow to 2 / 3 of the bottom of the well, aspirate the culture supernatant for detection. A two-step screening method is used. In the first step, conventional non-competitive enzyme-linked immunosorbent assay is used to screen out positive wells that are resistant to benzo[a]pyrene but not to the carrier protein BSA; in the second step, the icELISA method is used to detect the positive wells screened in the first step, using benzo[a]pyrene as the competitor, and selecting wells with higher absorbance and sensitivity (higher absorbance means that the final measured value of the well with a competitor of 0, i.e., the positive control well, is higher, and higher sensitivity means that the competitor concentration when the inhibition rate is 50% is also IC 50 The value was small), and subcloning was performed by limiting dilution method. After subcloning, the same two-step method was used for detection. After repeating subcloning 3-4 times, the hybridoma cell line BAP1C12 was obtained.
[0035] Example 2: Determination of the sequence of the variable region of the antibody of the anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BAP1C12.
[0036] (1) Extraction of total RNA: A total RNA extraction kit was used to extract total RNA from the hybridoma cell line BAP1C12;
[0037] (2) cDNA synthesis: Using the total RNA obtained in step 1 as a template and oligo(dT)15 as a primer, the cDNA was synthesized according to SuperScript TM -2II reverse transcriptase instructions for reverse transcription to synthesize the first chain of cDNA;
[0038] (3) PCR method for cloning 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 antibody heavy chain and light chain variable region genes. The PCR program was: 94°C for 30 seconds, 58°C for 45 seconds, 72°C for 1 minute, 30 cycles of amplification, and finally 72°C for 10 minutes of extension. After the PCR products were separated by 1% (weight percent) agarose gel electrophoresis, the DNA fragments were purified and recovered using a kit, connected to the vector pMD18-T, transformed into E. coli DH5α competent cells, and the positive clones were picked for sequencing. The sequences of the primers are: the heavy chain variable region primers are 5'-CAG GTS MARCTG 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; the 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 GGTCCC-3' (24mer).
[0039] The gene sequence results obtained are as follows: the heavy chain variable region encoding gene sequence is 348 bp long, and the sequence is shown in SEQ ID NO: 1. According to the obtained gene sequence, it is deduced that the heavy chain variable region encoded by the gene sequence consists of 116 amino acids, and the sequence is shown in SEQ ID NO: 3. The light chain variable region encoding gene sequence is 321 bp long, and the sequence is shown in SEQ ID NO: 2. According to the obtained gene sequence, it is deduced that the light chain variable region encoded by the gene sequence consists of 107 amino acids, and the sequence is shown in SEQ ID NO: 4.
[0040] SEQ ID NO:1
[0041]
[0042] SEQ ID NO:2
[0043]
[0044]
[0045] SEQ ID NO:3
[0046] EVQLQESGAE LVKPGASVKI SCKSTGNTFS FYWIEWIKQR PGHGLEWIGE ILPGSDSTHY
[0047] NEKFRDKATFTTETSSNTAY MQLSGLTSED SAVYYCARKS FDTSYWGQGT TLTVSS
[0048] SEQ ID NO:4
[0049] DIVITQSPSS MYASLGERVT ITCKASQDIN NCLIWFQQKP GKSPKTLIYR ANRFVDGVPS
[0050] RFSGSGFGQD YSLTISSLEF EDMGIYYCLQ YDDFPYTFGG GTKLEIK
[0051] Example 3: Preparation, purification, subtype and characterization of anti-benzo[a]pyrene monoclonal antibodies
[0052] The anti-benzo[a]pyrene monoclonal antibody hybridoma cell line BAP1C12 obtained in Example 1 was injected into BALB / c mice that had been treated with Freund's incomplete adjuvant in advance, and the ascites of the mice was collected. The antibodies were purified by the octanoic acid-ammonium sulfate method. The specific operation was as follows: the mouse ascites was filtered through a double-layer filter paper, centrifuged at 4°C and 12000r / min for more than 15min, the supernatant was aspirated, and the obtained ascites supernatant was mixed with 4 times the volume of acetate buffer, and octanoic acid was slowly added under stirring, the volume of octanoic acid required per milliliter of ascites was 30-35μL, mixed at room temperature for 30-60min, and allowed to stand at 4°C for more than 2h. 12000r / min, centrifuge at 4℃ for more than 30min, discard the precipitate, filter the obtained supernatant with double-layer filter paper, add 1 / 10 volume of filtrate with phosphate buffer having a molar concentration of 0.1mol / L and a pH of 7.4, 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 0.277g / mL of ammonium sulfate, let stand at 4℃ for 2h, then centrifuge at 12000r / min, 4℃ for 30min, discard the supernatant, resuspend the obtained precipitate with 1 / 10 volume of the original ascites water with a molar concentration of 0.01mol / L and a pH of 7.4, put it into a dialysis bag, dialyze it with 0.01mol / L, pH 7.4 phosphate buffer for two days, take out the protein solution in the dialysis bag, centrifuge, collect the supernatant, discard the precipitate, pre-freeze it at -70℃, and then put it into a freeze dryer for freeze drying. Collect the lyophilized powder, which is the purified anti-benzo[a]pyrene monoclonal antibody.
[0053] The acetate buffer is obtained by adding water to 0.29g sodium acetate and 0.141mL acetic acid to make the volume to 100mL; the 0.01mol / L phosphate buffer is obtained by adding water to 0.8g sodium chloride, 0.29g disodium hydrogen phosphate dodecahydrate, 0.02g potassium chloride, and 0.02g potassium dihydrogen phosphate to make the volume to 100mL; the 0.1mol / L phosphate buffer is obtained by adding water to 8g sodium chloride, 2.9g disodium hydrogen phosphate dodecahydrate, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, and pure water to make the volume to 100mL.
[0054] The subtype of the anti-benzo[a]pyrene monoclonal antibody secreted by the hybridoma cell line BAP1C12 was identified as IgG1 using a commercially available subtype identification kit.
[0055] The antibody titer purified from mouse ascites was measured by conventional non-competitive ELISA and reached 2×10 5 , i.e. antibody dilution 2×10 5 The sensitivity of icELISA to benzo[a]pyrene was 0.45 ng / mL.
[0056] The specificity of monoclonal antibodies can be evaluated by the cross-reaction rate. The icELISA method was used to determine the BAP1C12 monoclonal antibody. A series of standard solutions of concentrations of benzo[a]pyrene, fluoranthene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]anthracene, benzo[k]fluoranthene, anthracene, phenanthrene, acenaphthene, fluorene, and naphthalene were prepared and added to the ELISA plate together with an equal volume of antibody. The solution was incubated at 37°C for 45 minutes. The other steps were the same as the icELISA method. The competitive inhibition curve was drawn with the concentration of the above-mentioned standard as the horizontal axis and the OD value B / B0 at 450nm measured by the microplate reader as the vertical axis. The IC 50 The cross-reaction rate is determined by the ratio of . The calculation formula is as follows:
[0057] Cross-reaction rate % = (IC 50 Benzo[a]pyrene / IC 50 other analogs) × 100%.
[0058] The cross-reactivity of the BAP1C12 monoclonal antibody provided by the present invention to its structural analogs fluoranthene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]anthracene, benzo[k]fluoranthene, anthracene, phenanthrene, acenaphthene, fluorene and naphthalene is less than 3.5%.
[0059] The detailed results are shown in Table 1:
[0060] Table 1 Cross-reaction rate of BAP1C12 monoclonal antibody and polycyclic aromatic hydrocarbons
[0061]
[0062] The affinity of BAP1C12 was determined by conventional non-competitive ELISA:
[0063] The ELISA plate was coated with BBP1-OVA at 2.0, 1.0, 0.5, and 0.25 μg / mL concentrations, 100 μL / well, 4°C, overnight; after blocking with blocking solution for 1 hour, the antibody diluted with phosphate buffer (dilution factor 1:0000) was added to the ELISA plate, and the remaining steps were the same as the conventional non-competitive ELISA. With the measured OD450 value as the ordinate and the logarithm of the antibody concentration (mol / L) as the abscissa, 4 S-shaped curves of 4 concentrations were made. The maximum OD value at the top of each S curve, i.e., ODmax, was found, and the antibody concentration corresponding to 50% ODmax value of each curve was found. The affinity constant of the antibody was calculated by grouping any two of the four concentrations according to the formula Ka = (n-1) / 2(n[Ab']t-[Ab]t), where [Ab']t and [Ab]t are the fusion nanoantibody concentrations corresponding to the two 50% maximum OD values in each group, and n is the multiple of the coating antigen concentration in each group (including three ratios of 1:2, 1:4, and 1:8), and a total of 6 Ka values were obtained.
[0064] like Figure 1 As shown in Figure 2, the six Ka values were averaged to obtain the affinity of BAP1C12 monoclonal antibody enzyme-linked immunosorbent assay (ELISA) of 1.49×10 9 L / mol.
[0065] Example 4: Establishment of benzo[a]pyrene icELISA detection method
[0066] The anti-benzo[a]pyrene monoclonal antibody secreted by the hybridoma cell line BAP1C12 was used in the benzo[a]pyrene addition recovery test, which specifically includes the following steps:
[0067] (1) Coat the ELISA plate with 2 μg / mL BBP1-OVA antigen, 100 μL per well, incubate at 4°C overnight, wash with 0.01 mol / L pH 7.4 0.5% Tween 20 in 0.01 mol / L pH 7.4 phosphate buffer solution and dry;
[0068] (2) Block with 4% skim milk powder, 200 μL per well, incubate at 37°C for 1 h, wash and dry;
[0069] (3) Prepare 0, 0.013, 0.04, 0.11, 0.34, 1.03, 3.09, 9.26, 27.78, 83.34, and 250 ng / mL benzo[a]pyrene standard solutions (phosphate buffer containing 70% methanol), add 50 μL of the benzo[a]pyrene standard solution to be tested to each well in the first row, and use the other wells as detection wells. Each sample is repeated three times, and each well is added with 50 μL of anti-benzo[a]pyrene monoclonal antibody (diluted 8000 times), incubate at 37°C for 1 h, and wash and dry;
[0070] (4) Add 100 μL of 1:5000 goat anti-mouse HRP enzyme-labeled secondary antibody to each well, incubate at 37°C for 1 h, wash and dry;
[0071] (5) Substrate color development reaction: add 100 μL of substrate color development solution to each well (the preparation of the substrate color development solution: 0.5 mL of 1 mg / mL tetramethylbenzidine, 9.5 mL of substrate buffer, 37 μL of 1% H2O2, prepared and used immediately), react at 37°C in the dark for 15 min, terminate the reaction with 50 μL of 2 mol / L H2SO4 solution to each well, and measure the absorbance at 450 nm;
[0072] (6) Draw a standard curve and calculate the concentration of benzo[a]pyrene that inhibits the antigen-antibody binding reaction by 50% (IC 50 ). The result is Figure 2 As shown, it shows that the sample has a small matrix effect on ELISA, making the matrix curve close to that in the absence of matrix.
[0073] Example 5: Analysis of the content of benzo[a]pyrene in actual samples using the icELISA method
[0074] (1) Addition recovery test:
[0075] Weigh 1g of sample, add benzo[a]pyrene standard at three concentrations of 1ng / mL, 5ng / mL, and 20ng / mL to the sample, add 5mL of n-hexane, vortex mix, ultrasonically extract for 150min, centrifuge at 4500r / min for 5min, and take the supernatant. Use benzo[a]pyrene molecular imprinting column, add 5mL of dichloromethane and 5mL of n-hexane to activate the small column. Transfer the sample to be purified into the column, elute with 6mL of n-hexane, and discard the effluent. Add 6mL of dichloromethane to elute, and collect the purified solution into a test tube. Blow dry with nitrogen at 40℃, re-dissolve with 700μL of methanol, dilute with 300μL of 0.01mol / L, pH 7.4 phosphate buffer, and use it as the test solution for ELISA method loading test. The icELISA method was used for the addition recovery test, and the addition recovery rate of the vegetable oil sample was determined to be in the range of 92.7% to 103.9%, with good repeatability and accuracy.
[0076] (2) Actual sample testing
[0077] The icELISA method was used to analyze the benzopyrene content of 12 soybean oil samples, and the results were compared and verified with the liquid chromatography method. The results (Table 2) showed that the ELISA and HPLC analysis methods had a good correlation, and the ELISA method could be applied to the detection of benzopyrene content in vegetable oil samples.
[0078] Preparation of solution required for icELISA:
[0079] Phosphate buffer (pH 7.4): Na2HPO4·12H2O 8.7 g, NaCl 24 g, KCl 0.6 g, KH2PO4 0.6 g, dilute to 3000 mL with ultrapure water.
[0080] Coating buffer (pH 9.6 carbonate buffer) for coating: 1.59 g Na2CO3, 2.93 g NaHCO3, dilute to 1000 mL with ultrapure water.
[0081] 0.5% Tween 20 phosphate buffer solution: Na2HPO4.12H2O 8.7g, NaCl 24g, KCl 0.6g, KH2PO4 0.6g, Tween-20 1.5mL, make up to 3000mL with ultrapure water.
[0082] Blocking solution: 4 g skim milk powder dissolved in 100 mL 0.5% Tween 20 phosphate buffer.
[0083] Substrate buffer: Na2HPO4·12H2O 1.843 g, citric acid 0.933 g, dilute to 100 mL with ultrapure water.
[0084] Table 2. Detection of benzo[a]pyrene content in soybean oil by icELISA and HPLC
[0085]
[0086] Note: Each result is the average of three replicates.
Claims
1. A hybridoma cell line BAP1C12, characterized in that: The deposit number is CCTCC NO:C2023315.
2. A monoclonal antibody that specifically binds to benzo[a]pyrene, characterized in that: The heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing or a conservative mutant thereof obtained by one or more amino acid additions, deletions, substitutions or modified conservative mutations; 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 one or more amino acid additions, deletions, substitutions or modified conservative mutations.
3. The monoclonal antibody against pyrene secreted and produced by the hybridoma cell line BAP1C12 according to claim 1.
4. The method for preparing an anti-benzo[a]pyrene monoclonal antibody according to claim 3, characterized in that: The hybridoma cell line BAP1C12 described in claim 1 is injected into the abdomen of BALB / c mice treated with Freund's incomplete adjuvant in advance, and the ascites of the mice is collected and purified to obtain anti-benzo[a]pyrene monoclonal antibodies.
5. Use of the anti-benzo[a]pyrene monoclonal antibody according to claim 2 or 3 in the determination of benzo[a]pyrene content.
6. Use of the anti-benzo[a]pyrene monoclonal antibody according to claim 2 or 4 in the preparation of a product for detecting benzo[a]pyrene content.
7. A product for detecting benzo[a]pyrene comprising the anti-benzo[a]pyrene monoclonal antibody according to claim 2 or 3.
8. The product according to claim 7, characterized in that: The product is a benzo[a]pyrene immunoassay ELISA kit, a colloidal gold test strip kit or an immunoaffinity column.