Benzophenanthrene polycyclic aromatic hydrocarbon hapten, monoclonal antibody, hybridoma cell strain and application

Abstract

The invention provides a benzophenanthrene polycyclic aromatic hydrocarbon hapten, a monoclonal antibody, a hybridoma cell strain and application, and relates to the technical field of immunodetection. According to the invention, triphenylene hapten is synthesized, triphenylene complete antigen is prepared, cell fusion and indirect competitive enzyme-linked immunosorbent assay screening are carried out, a hybridoma cell strain is obtained, the hybridoma cell strain is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.46732, and the hybridoma cell strain can be used for preparing a triphenylene monoclonal antibody. The monoclonal antibody secreted by the hybridoma cell strain has good detection sensitivity to benzo [a] pyrene, benzo [a] anthracene, benzo [b] anthracene, dibenzo [a, h] anthracene and benzo [k] anthracene, the IC50 values are 6.3 ng / mL, 19.4 ng / mL, 6.8 ng / mL, 55.5 ng / mL and 21.7 ng / mL respectively, and the monoclonal antibody can be used for establishing an immunological detection method for benzophenanthrene polycyclic aromatic hydrocarbons.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to benzo[a]phenanthrene polycyclic aromatic hydrocarbon haptens, monoclonal antibodies, hybridoma cell lines and their applications. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants widely present in food processing and environmental pollution, primarily produced by the incomplete combustion of carbon-containing materials. Among them, benzo[a]pyrene, benzo[a]anthracene, benzo[b]anthracene, dibenzo[a,h]anthracene, and benzo[k]anthracene, among others, are benzo[a]phenanthrene PAHs. Due to their typical "bay" structure, they can form highly reactive electrophilic epoxides after metabolic activation in vivo. These epoxides can covalently bind to DNA to form adducts, thus exhibiting strong carcinogenic, teratogenic, and mutagenic effects. The International Agency for Research on Cancer (IARC) has classified benzo[a]pyrene and benzo[a]anthracene as Group 2A, possibly carcinogenic to humans, while benzo[b]anthracene, dibenzo[a,h]anthracene, and benzo[k]anthracene are classified as Group 2B, possibly carcinogenic to humans. These contaminants primarily originate from high-temperature processing methods in food, such as grilling, smoking, and frying, and tend to accumulate in oils, the surface of grilled meat, and contaminated aquatic products and crops, posing a serious threat to human health. Therefore, establishing rapid, sensitive, and high-throughput detection methods is crucial for food safety control and occupational exposure risk assessment.

[0003] Currently, the detection of benzo[a]phenanthrene polycyclic aromatic hydrocarbons (PAHs) mainly relies on instrumental analytical methods, such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). While these methods offer advantages in terms of high precision and accuracy, they also have significant limitations: complex sample pretreatment, time-consuming procedures, expensive equipment, and the need for specialized technical personnel. Furthermore, they struggle to meet the rapid screening needs of market regulators and food companies for large batches of samples. Therefore, establishing a rapid and convenient detection method for benzo[a]phenanthrene PAHs is of great importance. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method suitable for rapid on-site detection of large numbers of samples, providing a new detection pathway for benzo[a]phenanthrene PAHs. Crucially, since benzo[a]phenanthrene PAHs often coexist in complex mixtures in real samples, detection methods that develop specific antibodies against single compounds have limitations in practical applications. Therefore, developing a broad-spectrum antibody capable of simultaneously recognizing multiple structural analogs is essential for the practical application of ELISA technology. These broad-spectrum antibodies can simultaneously screen for multiple benzo[a]phenanthrene polycyclic aromatic hydrocarbons, which not only greatly improves detection efficiency and reduces detection costs, but also more accurately reflects the actual situation of complex contamination, providing strong technical support for the comprehensive and rapid assessment of the overall toxicity equivalent of such contaminants in food samples. Summary of the Invention

[0004] To solve the above-mentioned technical problems, this invention synthesizes benzo[a]phenanthrene hapten, prepares benzo[a]phenanthrene complete antigen, and obtains hybridoma cell lines through cell fusion and screening by indirect competitive enzyme-linked immunosorbent assay (ELISA). These hybridoma cell lines are then deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46732.

[0005] The first objective of this invention is to provide a hybridoma cell line that secretes monoclonal antibodies against benzo[a]phenanthrene polycyclic aromatic hydrocarbons, the hybridoma cell line having the accession number CGMCC NO.46732.

[0006] Furthermore, the benzo[a]phenanthrene polycyclic aromatic hydrocarbons include benzo[a]pyrene, benzo[a]anthracene, benzo[b]anthracene, dibenzo[a,h]anthracene, and benzo[k]anthracene.

[0007] Furthermore, the method for preparing the hybridoma cell line includes the following steps:

[0008] S1. Use complete antigens to immunize animals;

[0009] S2. Blood samples are collected from immunized animals to screen for serum immunogenicity and immunosuppressive capacity.

[0010] S3. The spleen cells and myeloma cells of the selected immunized animals were fused and cultured to obtain a hybridoma cell line that secretes benzo[a]phenanthrene-type polycyclic aromatic hydrocarbon monoclonal antibodies.

[0011] Further, in step S1, the animal immunization process includes primary immunization, booster immunization and sprint immunization. Primary immunization uses complete antigen and complete Freund's adjuvant, booster immunization uses complete antigen and incomplete Freund's adjuvant, and sprint immunization uses complete antigen.

[0012] Furthermore, the animal in question is a mouse.

[0013] The second objective of this invention is to provide a benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten, the structural formula of which is shown below:

[0014] .

[0015] Furthermore, the preparation method of the benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten includes the following steps:

[0016] In a mixed solvent of dichloromethane and N-methylpyrrolidone, benzo[k]anthracene was reacted with N-bromosuccinimide at 0°C for 6 h to give 3-bromobenzo[k]fluoranthracene; 3-bromobenzo[k]fluoranthracene and methyl 4-pentenoate were reacted at 80°C for 12 h under palladium acetate catalysis to generate (E)-5-(benzo[k]fluoranthracene-3-yl)pent-4-enoate methyl ester; the olefin bond was saturated by palladium carbon-catalyzed hydrogenation at room temperature for 4 h to give methyl 5-(benzo[k]fluoranthracene-3-yl)pentanoate; finally, the ester group was hydrolyzed with lithium hydroxide in 1,4-dioxane for 5 h, and acidified to obtain the final product 5-(benzo[k]fluoranthracene-3-yl)pentanoic acid, i.e., benzophenanthrene polycyclic aromatic hydrocarbon hapten.

[0017] Furthermore, the volume ratio of dichloromethane to N-methylpyrrolidone is 10:1.

[0018] Furthermore, the molar ratio of benzo[k]anthracene to N-bromosuccinimide, 3-bromobenzo[k]fluoranthracene to methyl 4-pentenoate is 1:1-1.5 (preferably 1.2).

[0019] Preferably, the molar ratio of benzo[k]anthracene to N-bromosuccinimide, 3-bromobenzo[k]fluoranthracene to methyl 4-pentenoate is 1:1.2.

[0020] A third objective of this invention is to provide a complete antigen of benzo[a]phenanthrene polycyclic aromatic hydrocarbons, which is obtained by conjugating the above-mentioned benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten with a carrier protein.

[0021] Furthermore, it includes the following steps:

[0022] (1) The above-mentioned benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten was activated to obtain an activated solution;

[0023] (2) Add the activation solution obtained in step (1) above into the carrier protein solution and react to obtain the complete antigen of benzo[a]phenanthrene polycyclic aromatic hydrocarbons.

[0024] Further, in step (1), the activation is to dissolve the benzophenanthrene hapten and add 1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to react.

[0025] Furthermore, benzophenanthrene haptens were dissolved using N,N-dimethylformamide.

[0026] Furthermore, in step (2), the solution after the reaction is dialyzed and separated to obtain benzo[a]phenanthrene complete antigen.

[0027] Further, in step (2), the carrier protein solution is obtained by dissolving the carrier protein in a carbonate buffer solution.

[0028] Furthermore, the carbonate buffer solution has a concentration of 0.01-0.5 mol / L and a pH of 8.0-10.0.

[0029] Preferably, the carbonate buffer solution is 0.05 mol / L (preferably 0.05 mol / L) and has a pH of 9.6.

[0030] A fourth objective of this invention is to provide the use of the above-mentioned hybridoma cell line, the above-mentioned benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten, or the above-mentioned benzo[a]phenanthrene polycyclic aromatic hydrocarbon complete antigen in the preparation of benzo[a]phenanthrene polycyclic aromatic hydrocarbon monoclonal antibodies.

[0031] A fifth objective of this invention is to provide a monoclonal antibody against benzo[a]phenanthrene polycyclic aromatic hydrocarbons, said monoclonal antibody being secreted by the aforementioned hybridoma cell line.

[0032] The sixth objective of this invention is to provide the application of the described hybridoma cell line or the above-mentioned monoclonal antibody in the preparation of benzo[a]phenanthrene polycyclic aromatic hydrocarbon detection products.

[0033] A seventh objective of this invention is to provide a detection product for benzo[a]phenanthrene polycyclic aromatic hydrocarbons, wherein the detection product contains the aforementioned monoclonal antibody.

[0034] Furthermore, the detection product is a reagent kit, test strip, or biochip.

[0035] The eighth object of the present invention is to provide the application of the above-mentioned monoclonal antibody or the above-mentioned detection product in the detection of benzo[a]phenanthrene polycyclic aromatic hydrocarbons.

[0036] The beneficial effects of this invention are:

[0037] The benzo[a]phenanthrene hapten synthesis procedure provided by this invention is simple and efficient, and can be fully utilized in immunoassays, providing a convenient approach for future research. The monoclonal antibodies secreted by the hybridoma cell lines screened by this invention exhibit good detection sensitivity against benzo[a]pyrene, benzo[a]anthracene, benzo[b]anthracene, dibenzo[a,h]anthracene, and benzo[k]anthracene, with an IC50 of [missing value]. 50 The values ​​were 6.3 ng / mL, 19.4 ng / mL, 6.8 ng / mL, 55.5 ng / mL, and 21.7 ng / mL, respectively. These values ​​can be used to establish an immunological detection method for benzo[a]phenanthrene polycyclic aromatic hydrocarbons (PAHs), providing a basic tool for the immunological detection of PAHs. This method can be applied to the detection of PAH residues in food, which is of great significance in the field of food safety and has practical application value.

[0038] Preservation of biological materials

[0039] Hybridoma cell lines secreting monoclonal antibodies against benzo[a]phenanthrene polycyclic aromatic hydrocarbons are deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The cell line is classified as a monoclonal cell line, deposited on November 12, 2025, with accession number CGMCCNO.46732. Attached Figure Description

[0040] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0041] Figure 1 The synthetic route of the benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten in the examples is shown.

[0042] Figure 2 The synthetic route for the benzo[a]phenanthrene polycyclic aromatic hydrocarbon complete antigen in the examples is shown below;

[0043] Figure 3 This is a standard inhibition curve of the benzo[a]phenanthrene polycyclic aromatic hydrocarbon monoclonal antibody in the examples. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0045] The solutions involved in the following examples are prepared as follows:

[0046] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0047] Phosphate-buffered saline (PBS): Dissolve 8.0 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, and 2.9 g Na2HPO4·12H2O in 800 mL of pure water. Adjust the pH to 7.2-7.4 with NaOH or HCl and bring the volume to 1000 mL.

[0048] PBST: PBS containing 0.05% Tween 20.

[0049] TMB colorimetric solution: Solution A: Na2HPO4 . 12H₂O 18.43 g, citric acid 9.33 g, diluted to 1000 mL with pure water; Solution B: 60 mg TMB dissolved in 100 mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution, and mix fresh before use.

[0050] Example 1: Synthesis of benzo[a]phenanthrene polycyclic aromatic hydrocarbon haptens

[0051] Starting with benzo[k]anthracene, 5-(benzo[k]fluoranthracene-3-yl)valerate, a benzo[k]phenanthrene hapten, is finally obtained through four steps: bromination, Heck reaction, hydrogenation, and hydrolysis. The synthetic route is as follows: Figure 1 As shown.

[0052] The specific steps are as follows:

[0053] Benzo[K]anthracene (1.00 g, 4.28 mmol) was dissolved in a mixed solvent of 40 mL anhydrous dichloromethane and 4 mL N-methylpyrrolidone. Under nitrogen protection and an ice bath at 0°C, N-bromosuccinimide (0.84 g, 4.71 mmol) was added in portions and reacted for 6 hours to give 3-bromobenzo[K]fluoranthracene. 3-bromobenzo[K]fluoranthracene (1.00 g, 3.18 mmol) was reacted with methyl 4-pentenoate (0.55 g, 4.77 mmol), palladium acetate (0.036 g, 0.16 mmol), and triethylamine (1.32 mL, 9.54 mmol) in 50 mL degassed acetonitrile and reacted at 80°C for 12 hours to give methyl (E)-5-(benzo[K]fluoranthracene-3-yl)pent-4-enoate. Methyl (E)-5-(benzo[k]fluoranthen-3-yl)pent-4-enoate (1.00 g, 2.54 mmol) was dissolved in 30 mL of ethyl acetate, and 50 mg of 10% palladium on carbon was added. The mixture was hydrogenated at room temperature for 4 hours under a hydrogen atmosphere to obtain methyl 5-(benzo[k]fluoranthen-3-yl)pentanoate. Finally, methyl 5-(benzo[k]fluoranthen-3-yl)pentanoate (0.90 g, 2.27 mmol) was placed in a mixed solvent of 20 mL of 1,4-dioxane and 10 mL of water, and lithium hydroxide (0.29 g, 6.81 mmol) was added. The mixture was heated to 85°C and refluxed for 5 hours. After acidification, 0.82 g of the final product, 5-(benzo[k]fluoranthen-3-yl)pentanoic acid, was obtained, which is the benzo[k]phenanthrene hapten. The structural formula is shown below.

[0054] .

[0055] Example 2: Preparation of complete antigen

[0056] The benzo[a]phenanthrene hapten is coupled to a carrier protein using the carbodiimide method to obtain the benzo[a]phenanthrene artificial antigen. Specifically, the benzo[a]phenanthrene hapten prepared in Example 1 is coupled with bovine serum albumin (BSA) to obtain the complete antigen benzo[a]phenanthrene-EDC-BSA; or coupled with chicken oocyte albumin (OVA) to obtain the complete antigen benzo[a]phenanthrene-EDC-OVA. The synthetic route is as follows: Figure 2 As shown.

[0057] The method for preparing the complete antigen benzo[a]phenanthrene-EDC-BSA is as follows:

[0058] (1) Weigh 0.631 mg of benzo[a]phenanthrene hapten, 0.618 mg of N-hydroxysuccinimide, and 1.0 mg of 1-ethylcarbodiimide hydrochloride prepared in Example 1, and dissolve them in 300 μL of N,N-dimethylformamide (referred to as solution A). Stir the mixture at room temperature for 4-6 h. Weigh 6.0 mg of BSA (the molar ratio of benzo[a]phenanthrene to BSA is 20:1), add 2 mL of carbonate buffer solution (the solution after BSA protein dissolution is referred to as solution B), and add solution A dropwise to solution B at room temperature. Adjust the pH of the mixture to 8-9 with 1 M NaOH solution and react at room temperature overnight to obtain the conjugate benzo[a]phenanthrene-EDC-BSA. The method for conjugating benzo[a]phenanthrene-EDC-OVA is similar to the above method.

[0059] (2) Cut an 8 cm section of the dialysis bag, boil it in boiling water for 3 min and cool it, then store it in deionized water at 4℃ for later use; place the conjugate benzo[a]phenanthrene-EDC-BSA / OVA conjugate solution into the dialysis bag and dialyze it in 0.01 mol / L PBS, changing the solution every 8 h for 3 days to obtain the complete antigen benzo[a]phenanthrene-EDC-BSA / OVA, remove it and store it at -20℃. The structural formula of the obtained complete antigen is shown below, where Protein is BSA or OVA:

[0060] .

[0061] Example 3: Immunization in mice

[0062] For the initial immunization, BALB / c mice were immunized with a mixture of the complete benzo[a]phenanthrene antigen benzo[a]phenanthrene-EDC-BSA and an equal volume of complete Freund's adjuvant at a dose of 100 μg / mouse. The mixture was emulsified and administered via multiple subcutaneous injections into the neck and back. Four weeks later, a booster immunization was performed with half the dose of the complete antigen (50 μg / mouse) emulsified with incomplete Freund's adjuvant. Subsequent booster immunizations were administered at 3-week intervals. For the final sprint immunization, the dose was again halved (25 μg / mouse), and the complete antigen was diluted with physiological saline and administered via intraperitoneal injection. After the third immunization, tail-disconnected blood samples were collected for testing. Serum titers and IC50 values ​​were determined using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). 50 Choose high-performance ICs 50 Low-grade mice were fused.

[0063] Example 4: Cell Fusion and Screening

[0064] (1) Three days after the sprint immunization, cell fusion was performed according to the conventional PEG 1500 (polyethylene glycol) method. The specific steps are as follows:

[0065] a. Collection of SP2 / 0 tumor cells: 7-10 days before fusion, culture SP2 / 0 tumor cells in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. The required number of SP2 / 0 tumor cells before fusion should reach 1-4 × 10⁻⁴ cells / year. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion. During fusion, tumor cells are collected, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0066] b. After euthanizing mice by cervical dislocation, immediately sterilize them in 75% alcohol for about 5 minutes. Aseptically remove the spleen, gently grind it with a syringe tip, and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect 50 mL of the suspension in a sterile centrifuge tube, centrifuge at 1200 r / min for 8 minutes, wash the spleen cells with RPMI-1640 medium, remove any large tissue impurities, and repeat the process three times. After the final centrifugation, dilute the spleen cells to a specific volume, count them, and set aside for later use.

[0067] c. Fusion process (7 min): At min 1, add 1 mL of PEG 1500 dropwise to the cells, gradually increasing the speed. At min 2, allow the centrifuge tube to stand and hold it firmly with both hands. At min 3 and min 4, add 1 mL of RPMI-1640 medium dropwise every 1 min. At min 5 and min 6, add 1 mL of RPMI-1640 medium dropwise every 30 s. At min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s. Then incubate at 37°C for 5 min. Centrifuge at 800 r / min for 10 min, discard the supernatant, gently break up the cells in the centrifuge tube, and add RPMI-1640 selective medium (HAT medium) containing 20% ​​fetal bovine serum and 2% 50×HAT to the medium. Add 200 μL / well to a 96-well cell plate and incubate at 37°C in a 5% CO2 incubator.

[0068] (2) Cell screening and cell line establishment: On day 3 after cell fusion, the fused cells were partially replaced with HAT medium; on day 5, the medium was completely replaced with RPMI-1640 transition medium (HT medium) containing 20% ​​fetal bovine serum and 1% 100×HT; on day 7, the cell supernatant was collected for screening. The screening was carried out in two steps: first, positive cell wells were screened by ic-ELISA; second, the inhibitory effect of benzo[a]phenanthrene standard on positive cells was determined by ic-ELISA. Cell wells that showed good inhibition of benzo[a]phenanthrene standard were selected and subcloned using the limiting dilution method. The same method was used for detection seven days later. Subcloning was performed four times according to the above method to finally obtain the benzo[a]phenanthrene polycyclic aromatic hydrocarbon monoclonal antibody cell line LQN.

[0069] Example 5: Preparation and Identification of Monoclonal Antibodies

[0070] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 2 × 10⁻⁶ g of paraffin oil. 6 Ascites fluid was collected from benzo[a]phenanthrene-based polycyclic aromatic hydrocarbon hybridoma cells starting on day 7. The ascites fluid was then purified using the caprylic acid-saturated ammonium sulfate method. Under slightly acidic conditions, caprylic acid precipitates other proteins in the ascites fluid besides IgG immunoglobulins. The precipitate was then discarded after centrifugation. Next, an equal volume of saturated ammonium sulfate solution was used to precipitate IgG-type monoclonal antibodies. After centrifugation and discarding the supernatant, the antibodies were dissolved in 0.01 M PBS solution (pH 7.4), dialyzed to desalt, and finally the purified monoclonal antibodies were stored at -20°C.

[0071] (1) Coating: The complete antigen benzophenanthrene-EDC-OVA was used as the coating agent and diluted 3 times from 1 µg / mL with 0.05M (pH 9.6) carbonate buffer. The solution was 100 μL / well and reacted at 37℃ for 2 h.

[0072] (2) Washing: Pour off the solution in the plate and wash with washing solution 3 times, 3 minutes each time.

[0073] (3) Sealing: After patting dry, add 200 μL / well sealing solution and react at 37℃ for 2 h. Wash and dry for later use.

[0074] (4) Sample addition: The antiserum (antiserum obtained by diluting the blood from the tail of mice with antibody diluent) was serially diluted from 1:1000 and added to each well of the coating at 100 μL / well. The reaction was carried out at 37℃ for 30 min. After thorough washing, HRP-goat anti-mouse IgG diluted at 1:3000 was added at 100 μL / well. The reaction was carried out at 37℃ for 30 min.

[0075] (5) Color development: Take out the microplate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37°C in the dark for 15 min.

[0076] (6) Termination and measurement: Add 50 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0077] The IC50 of monoclonal antibody against benzo[a]pyrene was determined by ic-ELISA. 50 The concentration was 6.3 ng / mL, indicating good sensitivity to benzo[a]phenanthrene polycyclic aromatic hydrocarbons (PAHs), making it suitable for immunoassay detection of PAHs.

[0078] Example 6: Application of Monoclonal Antibodies

[0079] The IC50 of the monoclonal antibody against benzo[a]pyrene was determined using an indirect competitive ELISA method. 50 The IC50 of benzo[a]anthracene was 6.3 ng / mL. 50 The IC50 of benzo[b]anthracene was 19.4 ng / mL. 50 The IC50 of dibenzo[a,h]anthracene was 6.8 ng / mL. 50 The IC50 values ​​were 55.5 ng / mL and benzo[k]anthracene. 50 The concentration was 21.7 ng / mL, and its IC50 for compounds such as phenanthrene, anthracene, and pyrene was verified. 50 The cross-reactivity rate and cross-reactivity value are calculated as follows:

[0080] (IC of benzo[a]pyrene) 50 IC of other compounds 50 () × 100%, as shown in Table 1.

[0081] Table 1 IC50 of monoclonal antibodies against structural analogues 50 and cross-reactivity

[0082] Compound Name <![CDATA[IC 50 (ng / mL)]]> Cross-reactivity Benzo[a]pyrene 6.3 100% Benzo[a]anthracene 19.4 32.5% Benz[b]anthracene 6.8 92.6% Dibenzo[a,h]anthracene 55.5 29.0% Benz[k]anthracene 21.7 ＜1% Philippines ＞500 ＜1% Anthracene ＞500 ＜1% pyrene ＞500 ＜1%

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line that secretes monoclonal antibodies against benzo[a]phenanthrene polycyclic aromatic hydrocarbons, characterized in that: The hybridoma cell line has the accession number CGMCC NO.46732.

2. The hybridoma cell line according to claim 1, characterized in that: The benzo[a]phenanthrene polycyclic aromatic hydrocarbons include benzo[a]pyrene, benzo[a]anthracene, benzo[b]anthracene, dibenzo[a,h]anthracene and benzo[k]anthracene.

3. A benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten, characterized in that, The structural formula is as follows: 。 4. A complete antigen of benzo[a]phenanthrene polycyclic aromatic hydrocarbons, characterized in that: The complete antigen is obtained by conjugating the benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten of claim 3 with a carrier protein.

5. The use of the hybridoma cell line of claim 1 or 2, the benzo[a]phenanthrene polycyclic aromatic hydrocarbon hapten of claim 3, or the benzo[a]phenanthrene polycyclic aromatic hydrocarbon complete antigen of claim 4 in the preparation of benzo[a]phenanthrene polycyclic aromatic hydrocarbon monoclonal antibodies.

6. A monoclonal antibody against benzo[a]phenanthrene polycyclic aromatic hydrocarbons, characterized in that: The monoclonal antibody is obtained from the hybridoma cell line described in claim 1 or 2.

7. The use of the hybridoma cell line of claim 1 or 2 or the monoclonal antibody of claim 6 in the preparation of benzo[a]phenanthrene polycyclic aromatic hydrocarbon detection products.

8. A detection product for benzo[a]phenanthrene-based polycyclic aromatic hydrocarbons, characterized in that: The testing product contains the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that: The detection products are reagent kits, test strips, or biochips.

10. The application of the monoclonal antibody of claim 6 or the detection product of claim 8 in the detection of benzo[a]phenanthrene polycyclic aromatic hydrocarbons.

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