Polycyclic aromatic hydrocarbon antigens and uses thereof

Abstract

The application discloses a polycyclic aromatic hydrocarbon antigen and application thereof, and belongs to the field of immunology. The polycyclic aromatic hydrocarbon antigen is a BKF antigen, wherein the BKF antigen comprises a BKF-BSA coating antigen and a BKF-KLH immunogen; and the BKF hapten is a coupling arm and an active-COC2H4COOH group introduced on a benzene ring of the BKF hapten. The application guarantees that a BKF characteristic structure is recognized by an immunocompetent cell, facilitates the establishment of a specific BKF antibody, is used for rapid screening and detection of the BKF, provides a basis for the application of the BKF commercialized rapid detection antibody, and has a high application prospect.

Description

Technical Field

[0001] This invention relates to a polycyclic aromatic hydrocarbon antigen and its application, specifically to benzo(k)fluoranthene antigen and its preparation method and application, belonging to the field of immunoassay technology. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants that are receiving increasing attention in various fields of environmental and human health. These are aromatic compounds with two or more fused benzene rings and are a class of persistent pollutants widely present in the environment, detected in animal and plant tissues, sediments, soil, air, surface water, drinking water, industrial wastewater, river water, well water, and groundwater. Among them, 4-6 ring PAHs, especially asymmetric PAHs such as fluoranthene and benzo[a]pyrene, are generally carcinogenic. PAHs are not direct carcinogens, but they can be converted into carcinogens in the body through enzymatic action. These carcinogens can then bind to DNA or RNA, causing irreparable damage and leading to cancer. Benzo[k]fluoranthene (BKF), as an important and highly toxic PAH compound, has received increasing attention in recent years and was included in the "List of Toxic and Hazardous Water Pollutants (Second Batch)" in June 2025. Considering the potential harm of BKF to human health and ecosystems, establishing a rapid detection method for BKF is imperative. However, there is currently a lack of commercially available antibodies and rapid detection methods for BKF. Therefore, it is crucial to research a highly sensitive and specific rapid immunological detection method for BKF. Summary of the Invention

[0003] At least to address one of the problems existing in the prior art, the present invention provides a polycyclic aromatic hydrocarbon antigen and its application. By introducing a coupling arm and an active -COC2H4COOH group onto the benzene ring of BKF, and then coupling it with a carrier protein, the characteristic structure of BKF is ensured to be recognized by immune active cells, which facilitates the establishment of specific BKF antibodies for rapid screening and detection of BKF. This provides a foundation for the application of commercial rapid detection antibodies for BKF and has high application prospects.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The present invention also provides a polycyclic aromatic hydrocarbon antigen, namely BKF antigen, wherein the BKF antigen comprises BKF-BSA coating antigen and BKF-KLH immunogen; The BKF hapten is formed by introducing a coupling arm and an active -COC2H4COOH group onto the benzene ring of BKF.

[0005] Preferably, the BKF-BSA coating agent is obtained by conjugating BKF hapten and carrier protein BSA using EDC and Sulfo-NHS conjugates.

[0006] Preferably, the BKF-KLH immunogen is obtained by conjugating the BKF hapten with the carrier protein KLH via EDC and Sulfo-NHS.

[0007] Preferably, the BKF hapten is a BKF benzene ring with an active -COC2H4COOH group replacing the H bond, and its structural formula is shown in formula (1): Equation (1): .

[0008] Preferably, the preparation method of the BKF hapten is as follows: BKF and anhydrous aluminum trichloride are added sequentially to anhydrous dichloromethane under a nitrogen atmosphere and stirred until completely dissolved. Succinic anhydride is added dropwise, and the mixture is stirred after the addition is complete. After the reaction is complete, the reaction solution is placed in crushed ice and stirred continuously until a solid is produced. Hydrochloric acid is added to adjust the pH to 4, and the mixture is heated to 20°C and stirred thoroughly until a light yellow solid is produced. The crude BKF hapten is obtained by filtration and purified to obtain purified BKF hapten.

[0009] Preferably, the molar ratio of BKF to succinic anhydride is 1:1 to 1.5; the molar ratio of BKF to anhydrous aluminum trichloride is 1:2 to 3.

[0010] Preferably, the crude BKF hapten is purified by column chromatography. The column chromatography uses gradient elution with petroleum ether:ethyl acetate = 1:1 (v / v) as the initial mobile phase. Every 3 to 5 column volumes, 10% ethyl acetate is added, and the ratios are adjusted sequentially to petroleum ether:ethyl acetate = 2:3 (v / v), 3:7 (v / v), 1:4 (v / v), and 1:9 (v / v) until pure ethyl acetate is obtained.

[0011] The BKF-KLH immunogen prepared in this invention is used to prepare BKF monoclonal antibodies.

[0012] Preferably, the method for preparing the BKF monoclonal antibody involves first immunizing BALB / c female mice with BKF-KLH immunogen and collecting spleen cells from the immunized BALB / c female mice with high titers; then fusing the spleen cells from the immunized BALB / c female mice with expanded cultured myeloma SP2 / 0 cells, culturing and screening hybridoma cell lines capable of secreting BKF monoclonal antibodies; then injecting the hybridoma cell lines capable of secreting BKF monoclonal antibodies into the peritoneal cavity of BALB / c female mice that have been pretreated with Freund's incomplete adjuvant, collecting the ascites fluid from the mice, and purifying it to obtain the BKF monoclonal antibody.

[0013] Preferably, the immunization is performed via intraperitoneal injection, combined with multiple rounds of immunization including an initial immunization with Freund's complete adjuvant, subsequent immunizations with Freund's incomplete adjuvant, and a shock immunization.

[0014] Preferably, the purification method uses an affinity column packed with Protein G gel and elution with elution buffer; the elution buffer is a 0.1M glycine solution.

[0015] Preferably, the BKF monoclonal antibody is of type IgG1.

[0016] The beneficial effects of this invention are as follows: 1. This invention uses -COC2H4COOH to replace the H bond on the benzene ring of BKF to prepare BKF hapten. While retaining the characteristic structure of BKF, it successfully introduces an active carboxyl coupling arm. This design not only ensures the specificity of the immune response, but also avoids the "carrier effect" caused by excessively long or complex connecting arms, reducing the production of non-specific antibodies and laying a reliable foundation for the subsequent preparation of antigens.

[0017] 2. This invention employs an active ester method to conjugate BKF hapten with KLH and bovine serum albumin (BSA), respectively. The conjugation products exhibit good stability, yielding BKF-KLH immunogen and BKF-BSA coating antigen. Due to the large molecular weight and strong immunogenicity of KLH, it can effectively activate the mouse immune system. The BKF-KLH immunogen can induce the production of high-titer antibodies. The BSA coating antigen is suitable as a BKF-BSA coating antigen for establishing an ELISA detection system.

[0018] 3. The BKF monoclonal antibody prepared by the BKF-KLH immunogen in this invention is of type IgG1, which has the characteristics of high affinity, easy purification and strong stability, and is suitable for the establishment of immunoassay methods.

[0019] 4. Through a series of experimental procedures, including hapten design, artificial antigen synthesis, animal immunization, cell fusion, and antibody purification and identification, a highly sensitive and specific BKF monoclonal antibody was successfully prepared, providing core biomaterials for the subsequent establishment of a rapid immunoassay method for BKF. Attached Figure Description

[0020] Figure 1 This is the NMR image of the BKF hapten in Example 1 of the present invention; Figure 2 This is a full-wavelength ultraviolet scan of Embodiment 2 of the present invention; Figure 3 This is a full-wavelength ultraviolet scan of Embodiment 3 of the present invention; Figure 4 The graphs show the mouse serum titer and inhibition rate determination in Example 4 of the present invention, where (a) is the mouse serum titer determination graph and (b) is the mouse inhibition rate determination graph; Figure 5The inhibition curve and standard curve of the antibody against benzo(k)fluoranthene standard in Example 4 of the present invention are shown, where (a) is the inhibition curve and (b) is the standard curve. Figure 6 The electrophoresis diagram for identifying the purification effect of ascites fluid in Example 6 of the present invention is shown, where lane M is a protein marker; lanes 1 and 2 are purified BKF monoclonal antibodies. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents, instruments, or components are not specified, they are all conventional products that can be purchased commercially.

[0022] Example 1 The preparation method of a polycyclic aromatic hydrocarbon hapten, namely BKF hapten, is as follows: under a nitrogen atmosphere and stirring at 0℃, 30 ml of anhydrous dichloromethane, 0.378 g (1.5 mmol) of benzo(k)fluoranthene and 0.44 g (3.3 mmol) of anhydrous aluminum trichloride are added sequentially to a 100 ml three-necked round-bottom flask. After the aluminum trichloride is completely dissolved, a solution of dichloromethane (15 ml) containing 0.15 g of succinic anhydride (1.5 mmol) is slowly added dropwise over 20 min. The reaction is then stirred for about 24 h (TLC monitoring). Pour the reaction solution into 100 ml of crushed ice and stir continuously with a glass rod until no obvious solid is produced. Add 1 mol / L glacial hydrochloric acid to adjust the pH to 4 to terminate the reaction. Heat to 20°C and stir to ensure complete reaction. A light yellow solid will form. Filter to obtain the crude product, which will be purified by column chromatography: gradient elution is used, with petroleum ether:ethyl acetate = 1:1 (v / v) as the initial mobile phase. Increase the ethyl acetate concentration by 10% every 3-5 column volumes, successively adjusting to petroleum ether:ethyl acetate = 2:3 (v / v), 3:7 (v / v), 1:4 (v / v), and 1:9 (v / v) until... The mixture was transitioned to pure ethyl acetate (gradient elution, ethyl acetate: petroleum ether = 1:1 → ethyl acetate; using petroleum ether: ethyl acetate = 1:1 (v / v) as the initial mobile phase, ethyl acetate was added by 10% every 3-5 column volumes, successively adjusting to petroleum ether: ethyl acetate = 2:3 (v / v), petroleum ether: ethyl acetate = 3:7 (v / v), petroleum ether: ethyl acetate = 1:4 (v / v), petroleum ether: ethyl acetate = 1:9 (v / v), until pure ethyl acetate was obtained), yielding purified BKF hapten in 35% yield; this BKF hapten is formed by the active -COC2H4COOH group replacing the H bond on the BKF benzene ring, and the reaction equation is as follows: .

[0023] through 1 Characterization and verification by HNMR and ESI-MS, NMR identification as follows: Figure 1 As shown. 1HNMR (DMSO-d6, 400MHz): δ12.22 (s,1H,OH), 8.64 (t,J=8.0Hz,2H,Ar-H), 8.56 (d,J=8.8Hz,1H,Ar-H), 8.49 (d,J=7.4Hz,1H,Ar-H), 8.30-8.20 (m,2 H, Ar-H), 8.04 (q, J = 3.2Hz, 2H, Ar-H), 7.84-7.79 (m, 1H, Ar-H), 7.58 (t, J = 3.8Hz, 2H, Ar-H), 3.45 (t, J = 6.1Hz, 2H, CH2), 2.70 (t, J = 6.2Hz, 2H, CH2). ESI-MS: m / zcalcdforC 24 H 16 O3[MH]-: 351.10; found: 351.2. This indicates it is a BKF hapten.

[0024] Example 2 The preparation method of a polycyclic aromatic hydrocarbon antigen, namely BKF antigen, is as follows: (1) Equilibrate the carboxyl activator EDC to room temperature; (2) Add 2 mg of lyophilized KLH (keyhole hemocyanin) to 200 μl of coupling buffer to make a carrier protein KLH solution with a final concentration of 10 mg / ml; (3) Dissolve 2 mg of the BKF hapten prepared in Example 1 above in 50 μl of pure DMF, then add coupling buffer to make up to 500 μl to form a BKF solution with a final concentration of 4 mg / ml, and then add the above-mentioned carrier protein KLH solution to form a carrier protein KLH-small molecule solution. (4) Dissolve 10 mg EDC in 1 ml of ultrapure water, immediately add 100 μl of solution to the carrier protein KLH-small molecule solution, add the prepared coupling agent sμlfo-NHS to make the final concentration of sμlfo-NHS 5 mM, shake well at room temperature for 2 h to obtain BKF antigen, i.e. BKF-KLH immunogen; (5) The BKF antigen was dialyzed three times with different solutions to remove impurities and small molecules. The solution was changed every 3-4 hours. The BKF antigen concentration was diluted to 1 mg / ml with PBS buffer at pH 7.4 and then aliquoted and stored at -20℃.

[0025] In this embodiment, the keyhole hemocyanin (KLH) is manufactured by Beijing Solarbio Technology Co., Ltd.

[0026] Coupling buffer, i.e. carbonate buffer (CBS) pH=9.6: 2.93g NaHCO3, 1.59g Na2CO3, dissolved in 1L deionized water, shaken to mix well, and sealed for later use.

[0027] Phosphate-buffered saline (PBS) pH=7.4: Weigh 80g NaCl, 2g KCl, 29g Na2HPO4·12H2O, and 2g KH2PO4, dissolve them in 1L of deionized water, and sonicate to mix well before use.

[0028] For conjugate identification, 200 ppm BKF hapten (denoted as BKF hapten) was prepared with methanol, 200 ppm KLH (denoted as KLH) was prepared with PBS buffer, and 100 ppm BKF antigen (denoted as BKF-KLH) was prepared with PBS buffer. Ultraviolet spectrophotometry was used, and the corresponding blank was subtracted, with a full-wavelength scan of 200–400 nm. Results are as follows: Figure 2 As shown, the overlay analysis revealed that the characteristic absorption peak position of the BKF-KLH immunogen shifted relative to the characteristic absorption peaks of the BKF hapten and the carrier protein KLH, indicating that the BKF hapten had successfully coupled with the carrier protein KLH, thus demonstrating the successful coupling of the BKF hapten with the carrier protein KLH.

[0029] Example 3 The preparation method of a polycyclic aromatic hydrocarbon antigen, namely BKF antigen, is as follows: (1) Equilibrate the carboxyl activator EDC to room temperature; (2) Add 2 mg of lyophilized BSA (fetal bovine serum albumin) to 200 μl of coupling buffer to make a carrier protein BSA solution with a final concentration of 10 mg / ml; (3) Dissolve 2 mg of BKF prepared in Example 1 above in 50 μl of pure DMF, then add coupling buffer to make up to 500 μl to form a BKF solution with a final concentration of 4 mg / ml, and then add the above-mentioned carrier protein BSA solution to form a carrier protein BSA-small molecule solution. (4) Dissolve 10 mg EDC in 1 ml of ultrapure water, immediately add 100 μl of solution to the carrier protein KLH-small molecule solution, add the prepared coupling agent sμlfo-NHS to make the final concentration of sμlfo-NHS 5 mM, shake well at room temperature for 2 h to obtain BKF antigen, i.e. BKF-BSA. (5) The BKF antigen was dialyzed three times with different solutions to remove impurities and small molecules. The solution was changed every 3-4 hours. The BKF antigen concentration was diluted to 1 mg / ml with PBS buffer at pH 7.4 and then aliquoted and stored at -20℃.

[0030] In this embodiment, the bovine serum albumin (BSA) is manufactured by Sigma-Aldrich, Inc.

[0031] Coupling buffer, i.e. carbonate buffer (CBS) pH=9.6: 2.93g NaHCO3, 1.59g Na2CO3, dissolved in 1L deionized water, shaken to mix well, and sealed for later use.

[0032] PBS buffer pH=7.4: Weigh 80g NaCl, 2g KCl, 29g Na2HPO4·12H2O, and 2g KH2PO4, dissolve them in 1L of deionized water, and sonicate to mix well before use.

[0033] For conjugate identification, 200 ppm BKF hapten (denoted as BKF hapten) was prepared with methanol, 200 ppm BSA (denoted as BSA) was prepared with PBS buffer, and 100 ppm BKF antigen (denoted as BKF-BSA) was prepared with PBS buffer. Ultraviolet spectrophotometry was used, and the corresponding blank was subtracted, followed by a full-wavelength ultraviolet scan from 200 to 400 nm. Results are as follows: Figure 3 As shown, the overlay analysis revealed that the characteristic absorption peak position of the BKF-BSA coating antigen shifted relative to the characteristic absorption peaks of the BKF hapten and the carrier protein BSA, indicating that the BKF hapten had successfully coupled with the carrier protein BSA, thus demonstrating the successful coupling of the BKF hapten with the carrier protein BSA.

[0034] Example 4: Mouse Immunization and Serum Titer Detection Mouse immunization: Female Blab / c mice (from the Comparative Medicine Center of Yangzhou University) aged approximately 6-8 weeks were selected and immunized with the BKF-KLH immunogen prepared in Example 2. The specific procedure was as follows: The BKF-KLH immunogen was diluted with PBS buffer to a concentration of 1 mg / ml, and then initially mixed with Freund's complete adjuvant in an equal proportion. Subsequent immunizations used Freund's incomplete adjuvant. The emulsifier was repeatedly pumped using a 2.5 ml syringe for 40 minutes to ensure complete emulsification. Complete emulsification was indicated when the water-in-oil emulsion, when dropped into water, remained intact and floated on the surface as a droplet. Five mice were immunized. The immunization and blood collection protocols are shown in Table 1.

[0035] Table 1. Mouse immunization and blood collection protocols

[0036] Freund's complete adjuvant and Freund's incomplete adjuvant are manufactured by Sigma-Aldrich, Inc., USA.

[0037] PBS buffer pH=7.4: Weigh 80g NaCl, 2g KCl, 29g Na2HPO4·12H2O, and 2g KH2PO4, dissolve them in 1L of deionized water, and sonicate to mix well before use.

[0038] Serum titer assay: Serum from immunized mice was collected and diluted twofold starting from 1000-fold. The titer of each tail serum sample was determined by an indirect non-competitive ELISA method. The serum dilution factor (i.e., titer) with an absorbance (OD450 value) of around 1.0 at 450 nm and the original concentration of BKF-BAS coating were selected as the optimal working parameters.

[0039] The serum titer was detected by indirect non-competitive ELISA. The specific operation is as follows: (1) Coating: Dilute BKF-BAS coating agent to 2 μg / ml with CBS buffer, 100 μl / well, coat ELISA plate, and incubate overnight at 4℃. (2) Blocking: Wash the plate 3 times with PBST, pat dry, block the microplate with 3% M skim milk powder blocking solution, 220 μl / well, and incubate at 37℃ for 1 h; (3) Add primary antibody: Wash the plate 3 times with PBST, pat dry, and then add the antiserum to the plate at an initial dilution of 1000 times, with 50 μl / well of serum diluted 2 times. Then add 50 μl of PBS and incubate at 37°C for 1 h. (4) Add secondary antibody: Wash the plate 3 times with PBST, pat dry, add 5000 times diluted HRP-goat anti-mouse secondary antibody, 100 μl / well, and incubate at 37℃ for 1 h; (5) Color development: Wash the plate 3 times with PBST, pat dry, add substrate color development solution, 100 μl / well, and incubate at 37℃ for 10 min; (6) Stop reading: Add 50 μl of stop solution per well; read the OD450 value of the microplate reader.

[0040] The following solutions were prepared: CBS buffer (pH=9.6): 2.93g NaHCO3, 1.59g Na2CO3 dissolved in 1L deionized water, vortexed and mixed thoroughly, then sealed for later use; PBS buffer (pH=7.4): 80g NaCl, 2g KCl, 29g Na2HPO4·12H2O, 2g KH2PO4 dissolved in 1L deionized water, sonicated and mixed thoroughly for later use; PBST washing buffer: 100ml 0.1M PBS buffer mixed with 900ml double-distilled water, then 500μl Tween-20 added, mixed thoroughly for later use; 3% M skim milk powder blocking solution: 0.3g skim milk powder dissolved in 10ml PBS buffer, vortexed and mixed thoroughly for later use (prepared in the same proportion as needed for the experiment); 5% DMF-PBS buffer: taking 10ml 5% DMF-PBS buffer as an example, 500μl DMF added to 9.5ml PBS, mixed thoroughly for later use.

[0041] A checkerboard titration method was established using indirect non-competitive ELISA to determine the optimal working concentrations of BKF-BSA and BKF monoclonal antibodies, as shown in Table 2.

[0042] Table 2. Identification of the optimal working concentrations of BKF-BSA coating antigen and BKF antibody.

[0043] Table 2 shows that the optimal working concentrations are the original BKF-BSA coating concentration and the BKF antibody dilution factor corresponding to an OD450 value of approximately 1.0. The original BKF-BSA coating concentration for antibody a is 0.25 μg / ml, and the antibody dilution factor is 8000. Under these conditions, the OD450 value is 0.943, which is closest to 1.0. This can reduce the error of subsequent ELISA experiments and ensure the accuracy of the experiment.

[0044] Under the conditions of serum dilution of 8000 times and original BKF-BAS coating concentration of 0.25 μg / ml, the specific binding of BKF monoclonal antibody produced in serum to BKF standard was determined, and the mouse with the highest titer and the best specific binding effect to BKF was selected for cell fusion experiment.

[0045] The steps for indirect competitive ELISA are basically the same as those for the indirect competitive non-ELISA described above, except for the primary antibody loading step: the antiserum is directly diluted 8000 times.

[0046] For the indirect competitive ELISA method, each well requires 50 μl of BKF standard and 50 μl of diluted mouse serum. The mouse serum is diluted with PBS buffer, and the BKF is diluted with a 5% DMF-PBS mixture. Simultaneously, 50 μl of mouse serum and 50 μl of 5% DMF-PBS mixture serve as positive controls, with the positive OD450 value recorded as B. 50 μl of 5% DMF-PBS mixture and 50 μl of PBS solution serve as negative controls, with the negative OD450 value recorded as B0. The inhibition rate H is calculated.

[0047] Inhibition rate H = (B - B0) / B0 × 100%. Mice that were effective in the blank control and had the highest inhibition rate and titer were selected as cell fusion experimental mice and subjected to shock immunization according to the immunization protocol.

[0048] Starting with the third immunization, blood was collected from the tail of the mice every week to measure serum titer and the inhibition rate against BKF. The results are as follows: Figure 4 The results show the determination of mouse serum titer and inhibition rate, where (a) is the determination of mouse serum titer and (b) is the determination of mouse inhibition rate.

[0049] from Figure 4The study found that the serum titer and inhibition rate of immunized mouse 1 reached the highest level after six immunizations. After six immunizations, the highest titer of the mouse was 48,000, and the highest inhibition rate against BKF was 71.2%. Considering all factors, the immunization effect was higher than that of other immunized mice. Therefore, immunization was immediately stopped and mouse 1 was selected as the subject of cell fusion.

[0050] In addition, the concentration (C) of the small molecule substance BKF standard is set. BKF logC BKF The x-axis represents the horizontal axis, and the y-axis represents the corresponding OD450 binding ratio (B / B0). ELISA standard curves of the antibody against different concentrations of BKF were plotted accordingly. The inhibition curve relationship of the BKF monoclonal antibody against BKF is shown below: IC50 is 0.9742 ng / mL, and the linear range is 0.48–1.88 ng / mL. Figure 5 As shown, (a) is the inhibition curve and (b) is the standard curve.

[0051] from Figure 5 It can be seen that the BKF monoclonal antibody has good efficacy and sensitivity, indicating that the BKF monoclonal antibody has good sensitivity and specificity, and can realize the quantitative detection of trace amounts of BKF.

[0052] Example 5 Cell Fusion 1. Culture of myeloma SP2 / 0 cells: A crucial prerequisite for cell fusion is myeloma cells in good condition. First, the cells are revived. The procedure is as follows: (1) Take out the SP 2 / 0 myeloma cells (from Wuhan Boster Biological Co., Ltd.) frozen in liquid nitrogen and place them in a 37 ℃ water bath to thaw. Transfer them to a 15 ml cell-specific centrifuge tube and immediately centrifuge at 1000 g for 7 min. (2) After centrifugation, discard the supernatant and resuspend the cells in complete DMEM high-glucose liquid culture medium (from Wuhan Boster Biological Co., Ltd.). Transfer the medium to a cell culture flask and control the solution volume to about 5 ml. Incubate at 37 ℃ in a 5% CO2 incubator. After 24 h, observe the cell condition (cells that are uniform in size, round and translucent are ready for cell fusion). (3) Passage: When the cells have grown to 2 / 3 of the bottom area of ​​the culture flask, they can be passaged. First, discard the liquid in the flask, and wash the cells attached to the bottom of the cell flask with 2 ml of 37 ℃ DMEM high glucose liquid culture medium. Transfer 1 ml to a new culture flask and culture. Repeat the above operation when the cells have grown to the top. Expand the culture to 7-8 flasks for cell fusion and keep 1-2 flasks for cryopreservation.

[0053] 2. Collection of spleen cells: Blood was collected from the orbital vein of the immunized mice in Example 4. The mice were euthanized by cervical spine debridement and sterilized in 75% ethanol for 5 min. Mice with higher serum titers were transferred to a clean bench, and the spleens were removed and placed in a sieve. The spleens were ground in the sieve, and the sieve was washed with 15 ml of DMEM high-glucose liquid culture medium to collect spleen cells. The cells were centrifuged at 1200 r / min for 5 min. The supernatant was discarded, and the cells were resuspended in 10 ml of DMEM high-glucose liquid culture medium.

[0054] 3. Cell fusion: 1~2×0 7 SP2 / 0 cells / ml and 10⁸ cells / ml immune spleen cells were mixed in a 50 ml centrifuge tube and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and the centrifuge tube was shaken to loosen the cell pellet. The centrifuge tube containing the cell mixture was placed in a 37 ℃ water bath, and 0.8 ml of 50% PEG-2000 at 37 ℃ was slowly added dropwise over 1 min. The mixture was stirred for another 1 min, and then 10 ml of DMEM culture medium at 37 ℃ was slowly added. The specific timing of the addition was as follows: 1 ml was added gradually over the first min, another 1 ml over the second min, another 3 ml over the third and fourth mins, and the remaining 5 ml of culture medium was added over the fifth min. Each time the culture medium was added, it was added slowly and gently. Finally, 30 ml of DMEM culture medium was slowly added, and the mixture was centrifuged at 800 rpm for 5 min. The supernatant was discarded and the mixture was placed at 37 ℃ for 5-8 min.

[0055] 4. Culture of fused cells and screening of hybridoma cells 24 h after fusion, observe the fused cells under a microscope for contamination, cell growth and culture medium color; one week later, replace with HAT medium (50×) (Sigma, USA), wait for the fused cells to adapt to growth, and change the culture medium every three days; when the bottom of the 96-well plate is filled to 1 / 3, count and record the cell growth of each well, and screen for positive cell lines.

[0056] Following the ELISA procedure, the cells were coated with HAT-selected supernatant for ELISA detection. Positive clones were picked and expanded to 24 wells, and cultured statically at 37 ℃ and 5% CO2 for 1-2 days. The supernatant was then used for ELISA detection.

[0057] After cell fusion, positive wells were screened and their inhibitory effect on BKF was further measured. Subcloning was performed using the limiting dilution method, and finally a hybridoma cell line numbered 12-7-11 that could stably secrete BKF monoclonal antibody was screened out. The hybridoma cells were stored in liquid nitrogen for later use. The stored hybridoma cells were expanded and cultured to prepare ascites fluid.

[0058] Example 6: Preparation, purification, and titer determination of BKF monoclonal antibody Six 6-week-old female Balb / c mice were selected and sensitized by intraperitoneal injection of Freund's incomplete adjuvant at a dose of 250 μL per mouse; 5 days later, each mouse was injected intraperitoneally with 1×10 6 ~2×10 6 Hybridoma cells that stably secrete BKF monoclonal antibodies were preserved in Example 5. The production of ascites in mice was observed daily. Ascites was collected when the abdomen of the immunized mice was significantly distended and there was obvious accumulation of ascites. Multiple collections could be made. Usually, 5-10 ml of ascites could be collected from each mouse. The ascites was released by inserting a 10 ml syringe needle into the peritoneal cavity of the mouse. The ascites was centrifuged at 3000 rpm for 10 min at 4°C to remove the surface fat layer and collect the supernatant. The supernatant was stored at -20°C.

[0059] The purification steps for BKF monoclonal antibody in ascites fluid are as follows: (1) Centrifuge the ascites at 4℃ and 10000 rpm for 10 min, discard the precipitate and take the supernatant; remove the lipids from the upper layer of the ascites sample; (2) Filter the supernatant into centrifuge tubes using a 0.22 μm filter membrane; (3) Add 1 / 10 of the supernatant volume of 0.01 M PBS buffer to the filtered supernatant to adjust the pH and ionic strength of the supernatant and prepare for sample loading; (4) Load Protein G gel into the affinity column tube, place it vertically on the support, remove the plugs at the top and bottom, and let the liquid inside flow out by gravity. (5) Use a syringe to draw 10 column volumes of 0.01 M PBS buffer to wash the affinity column, allowing the binding buffer to flow out by gravity; (6) Take the sample to be purified and add it to the affinity column, so that the sample flows out slowly by gravity.

[0060] (7) After all the samples have entered the affinity column, use a syringe to draw 10 column volumes of 0.01M PBS buffer and wash the affinity column; (8) Take a 10 ml centrifuge tube and add 0.5 ml of neutralization buffer to the centrifuge tube; (9) Draw 10 ml of elution buffer into a syringe and place the 10 ml centrifuge tube containing 0.5 ml of neutralization buffer into the affinity column; (10) Elute the antibody at the lower outlet and collect the elution product using a 1 ml centrifuge tube. Replace the centrifuge tube after collecting 500 μl each time. After all the product has been collected, rinse the column with elution buffer and binding buffer. Finally, wash once with 20% ethanol, freeze dry, and store at 4 °C to obtain purified BKF monoclonal antibody.

[0061] Elution buffer: Dissolve 3.028g of glycine in 200ml of double-distilled water to obtain a 0.2M glycine solution. Adjust the pH to 2.7 with hydrochloric acid and then dilute it by 1 time to obtain the elution buffer.

[0062] Binding buffer: Solution A: Weigh 3.12 g of NaH2PO4·2H2O and add 100 ml of deionized water. Solution B: Weigh 14.34 g of Na2HPO4·12H2O and dissolve in 200 ml; take 78 ml of solution A and 122 ml of solution B and mix thoroughly. Dilute the mixture 10 times with double-distilled water to obtain 20 mM sodium phosphate buffer. Adjust the pH to 7.0, which is the binding buffer.

[0063] Identification of BKF monoclonal antibody purification efficiency: SDS-PAGE electrophoresis was used. (1) Load the 12% protein precast gel into the electrophoresis tank and secure it with wedge-shaped slots; (2) Add electrophoresis buffer, pull up the comb, and adjust the sample well to keep both sides vertical; (3) Sample pretreatment: Mix the sample with 4× protein loading buffer at a ratio of 1:3 and boil in boiling water for 5 minutes; (4) Loading: Load 3 μl of 250 KD protein marker and load 10 μl of sample according to the number; (5) Add electrode buffer to 1 / 2 of the electrophoresis tank, connect it to the electrophoresis apparatus, turn on the power, and electrophore for 55 min at a constant voltage of 140 V. When the protein loading buffer indicator reaches the bottom of the glass plate, turn off the power. (6) Remove the electrophoresis plate, take out the gel, rinse it with water, and stain it with "Instant Blue" staining solution.

[0064] The purified BKF monoclonal antibody from the ascites fluid was identified by SDS-PAGE electrophoresis, and the results are as follows: Figure 6 As shown (lane M: protein marker; lanes 1 and 2 are purified BKF monoclonal antibodies).

[0065] from Figure 6 As can be seen, after high-temperature heating, the BKF monoclonal antibody breaks the disulfide bonds and splits into two chains: a heavy chain (molecular weight approximately 45 kD) and a light chain (molecular weight approximately 22 kD). Therefore, the SDS-PAGE electrophoresis results show two clear bands, indicating that the BKF monoclonal antibody has a good purification effect.

[0066] The concentration of BKF monoclonal antibody in the purified ascites fluid was determined by ultraviolet spectrophotometry to be 0.6 mg / ml, which meets the requirements for subsequent experiments.

[0067] In addition, the purified BKF monoclonal antibody was identified using a commercially available mouse monoclonal antibody subtype identification kit, and the identification results are shown in Table 3.

[0068] Table 3. Identification of BKF monoclonal antibody subtypes

[0069] Table 3 shows that the prepared BKF monoclonal antibody is of the IgG1 type, which has the characteristics of high affinity, easy purification and strong stability, and is suitable for the establishment of immunoassay methods.

[0070] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit and essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0071] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A polycyclic aromatic hydrocarbon antigen, characterized in that, The polycyclic aromatic hydrocarbon antigen is a BKF antigen, which includes BKF-BSA coating antigen and BKF-KLH immunogen; The BKF hapten is formed by introducing a coupling arm and an active -COC2H4COOH group onto the benzene ring of BKF.

2. The polycyclic aromatic hydrocarbon antigen according to claim 1, characterized in that, The BKF hapten is a BKF benzene ring with an active -COC2H4COOH group replacing the H bond, and its structural formula is shown in formula (1): Equation (1): .

3. A polycyclic aromatic hydrocarbon antigen according to claim 1 or 2, characterized in that, The preparation method of the BKF hapten is as follows: BKF and anhydrous aluminum trichloride are added sequentially to anhydrous dichloromethane under a nitrogen atmosphere and stirred at 0°C until completely dissolved. Succinic anhydride is added dropwise, and the reaction is stirred after the addition is complete. After the reaction is complete, the reaction solution is placed in crushed ice and stirred continuously until a solid is produced. Hydrochloric acid is added to adjust the pH to 4, and the mixture is heated to 20°C and stirred thoroughly until a light yellow solid is produced. The crude BKF hapten is obtained by filtration and purified to obtain purified BKF hapten.

4. A polycyclic aromatic hydrocarbon antigen according to claim 3, characterized in that, The molar ratio of BKF to succinic anhydride is 1:1 to 1.5; the molar ratio of BKF to anhydrous aluminum trichloride is 1:2 to 3.

5. A polycyclic aromatic hydrocarbon antigen according to claim 3, characterized in that, The crude BKF hapten was purified by column chromatography. The column chromatography used gradient elution with a petroleum ether:ethyl acetate volume ratio of 1:1 as the initial mobile phase. Every 3-5 column volumes, 10% ethyl acetate was added, and the ratio was adjusted to 2:3, 3:7, 1:4, and 1:9, until pure ethyl acetate was obtained.

6. The polycyclic aromatic hydrocarbon antigen according to claim 1, characterized in that, The BKF-BSA coating agent is obtained by conjugating BKF hapten with carrier protein BSA using EDC and Sulfo-NHS conjugates. The BKF-KLH immunogen is obtained by conjugating the BKF hapten with the carrier protein KLH using EDC and Sulfo-NHS.

7. An application of a polycyclic aromatic hydrocarbon antigen, wherein the BKF-KLH immunogen of claim 1 is used to prepare BKF monoclonal antibodies.

8. The application of a polycyclic aromatic hydrocarbon antigen according to claim 7, characterized in that, The method for preparing the BKF monoclonal antibody involves first immunizing BALB / c female mice with BKF-KLH immunogen and collecting spleen cells from the immunized BALB / c female mice with high titers; then fusing the spleen cells from the immunized BALB / c female mice with expanded cultured myeloma SP2 / 0 cells, culturing and screening hybridoma cell lines capable of secreting BKF monoclonal antibodies; finally, injecting the hybridoma cell lines capable of secreting BKF monoclonal antibodies into the peritoneal cavity of BALB / c female mice that have been pretreated with Freund's incomplete adjuvant, collecting the ascites fluid from the mice, and purifying it to obtain the BKF monoclonal antibody.

9. The application of a polycyclic aromatic hydrocarbon antigen according to claim 8, characterized in that, The immunization was administered via intraperitoneal injection, combined with multiple rounds of immunization including initial immunization with Freund's complete adjuvant, subsequent immunizations with Freund's incomplete adjuvant, and pulse immunization. The purification method uses an affinity column packed with Protein G gel and elution with elution buffer; the elution buffer is a 0.1M glycine solution.

10. The application of a polycyclic aromatic hydrocarbon antigen according to claim 7, characterized in that... The BKF monoclonal antibody is of type IgG1.

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