A benzopyrene immunoaffinity column, its preparation method and application

Abstract

The application provides a benzopyrene immunoaffinity column and a preparation method and application thereof, and a filler of the benzopyrene immunoaffinity column comprises a solid carrier coupled with a benzopyrene monoclonal antibody. The benzopyrene monoclonal antibody in the benzopyrene immunoaffinity column is prepared by using a benzopyrene hapten-carrier protein conjugate as an immunogen, so that the antibody can specifically adsorb benzopyrene substances in petroleum chemicals, effectively improves the recovery rate of benzopyrene, reduces the interference of other impurities such as lipids, proteins and pigments in the petroleum chemicals, and improves the accuracy of benzopyrene detection.

Description

Technical Field

[0001] This invention belongs to the field of benzo[a]pyrene detection technology, specifically relating to a benzo[a]pyrene immunoaffinity column, its preparation method, and its application. Background Technology

[0002] Petroleum, as the most important energy resource and industrial raw material, provides the most fundamental guarantee for social development. With the depletion of conventional oil and gas resources and the increased development of tight oil, the number and depth of drilling have grown rapidly, generating massive amounts of drilling waste. Pollutants in drilling waste can harm the environment. Among these pollutants, benzo[a]pyrene has "three-way" effects (carcinogenic, mutagenic, and teratogenic) and is usually a key focus of monitoring as a representative environmental carcinogen. The implemented DB65 / T3997—2017 "Pollution Control Requirements for Comprehensive Utilization of Solid Waste from Oil and Gas Field Drilling" for the first time listed benzo[a]pyrene as a pollution control indicator for drilling waste, with a maximum allowable limit of 0.7 mg / kg.

[0003] CN105548382A discloses a method for detecting benzo[a]pyrene in soil, comprising the following steps: accurately weighing a soil sample into a stoppered conical flask, adding 8-10 times the amount of organic solvent A, stopping and shaking, then ultrasonically extracting for at least 30 minutes, centrifuging and collecting the supernatant, adding another 8-10 times the amount of organic solvent A to the remaining soil sample, ultrasonically extracting for at least 30 minutes, centrifuging, combining the supernatants, adding organic solvent A to a solid-phase extraction column for activation, and after activation, transferring the combined supernatant to the activated solid-phase extraction column and eluting with organic solvent A. This detection method is simple to operate and has straightforward steps, effectively reducing the probability of human error leading to inhalation or skin contact with benzo[a]pyrene, and improving the safety of laboratory personnel.

[0004] CN103472170A discloses a method for detecting benzo[a]pyrene in edible oils, comprising the following steps: weighing the oil sample, adding potassium hydroxide ethanol aqueous solution, shaking until clear, saponifying in a water bath at 50°C for 1 hour, cooling to room temperature, adding water to the sample centrifuge tube, shaking well, adding 15 mL of n-hexane, mixing well, centrifuging, washing twice with 5 mL of ultrapure water, concentrating the extract to dryness in a nitrogen blower, making up to volume with a tetrahydrofuran + acetonitrile = 2 + 8 mixed solution, filtering, and determining the result using high performance liquid chromatography. This analytical method has the advantages of high stability and high recovery rate.

[0005] Due to the carcinogenicity of benzo[a]pyrene, its detection and separation in petrochemical agents are of paramount importance. Although existing technologies have disclosed some methods for detecting benzo[a]pyrene-like substances, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS), benzo[a]pyrene is usually present in trace or even ultra-trace amounts in oilfield chemicals, and the complex matrix of these chemicals contains many interfering substances, making direct measurement difficult. Therefore, instrumental determination requires complex pretreatment. Currently available solid-phase extraction column products suffer from low recovery rates, significant sample matrix interference, and the need for large amounts of organic solvents, greatly reducing the applicability of these methods. Therefore, developing a detection method with high recovery efficiency, good sensitivity, and the ability to effectively reduce the influence of lipids, proteins, pigments, and other components in the petrochemical matrix is ​​a key research focus in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a benzo[a]pyrene immunoaffinity column, its preparation method, and its applications. The benzo[a]pyrene immunoaffinity column provided by the present invention can effectively enrich residual benzo[a]pyrene in petrochemical agents, effectively separate benzo[a]pyrene from interfering components, improve the recovery rate of benzo[a]pyrene, and enhance the accuracy of detection.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a benzo[a]pyrene immunoaffinity column, wherein the filling material of the benzo[a]pyrene immunoaffinity column comprises a solid-phase carrier coupled with a benzo[a]pyrene monoclonal antibody.

[0009] The benzo[a]pyrene immunoaffinity column packing material provided by this invention is a conjugation product of benzo[a]pyrene monoclonal antibody and solid-phase support. This packing material can effectively enrich residual benzo[a]pyrene in petrochemical agents, effectively separate benzo[a]pyrene from interfering components, improve the recovery rate of benzo[a]pyrene, and improve the accuracy of detection.

[0010] Preferably, the benzo[a]pyrene monoclonal antibody is prepared from a benzo[a]pyrene hapten-carrier protein conjugate as an immunogen.

[0011] Preferably, the structure of the benzo[a]pyrene hapten is as follows: The benzo[a]pyrene hapten defined in this invention includes a hydrogenated benzo[a]pyrene-derived structure.

[0012] Preferably, the carrier protein includes any one of keyhole hemocyanin (KLH), chicken ovalbumin (OVA), or bovine serum albumin (BSA).

[0013] Preferably, the solid support comprises any one of agarose gel, polystyrene, and silicone rubber.

[0014] Preferably, the filling material of the benzo[a]pyrene immunoaffinity column further includes a preservation solution.

[0015] Preferably, the preservation solution includes any one of PBS buffer, HEPES buffer, and Tris buffer.

[0016] The benzo[a]pyrene monoclonal antibody in the benzo[a]pyrene immunoaffinity column of this invention is prepared by using a benzo[a]pyrene hapten-carrier protein conjugate as an immunogen. Therefore, this antibody can specifically adsorb benzo[a]pyrene-like substances in petrochemical agents, effectively improving the recovery rate of benzo[a]pyrene. At the same time, it can reduce the interference of other impurities such as lipids, proteins, and pigments in petrochemical agents, thereby improving the accuracy of benzo[a]pyrene detection.

[0017] Preferably, the benzo[a]pyrene hapten is prepared by the following method, the method comprising:

[0018] 7,8,9,10-Tetrahydrobenzo[a]pyrene-7-ol was dissolved in a solvent, succinic anhydride and 4-dimethylaminopyridine (DMAP) were added, and the mixture was heated to reflux until the reaction was complete. The pH was then adjusted to 4 with an acidic pH adjuster to obtain the benzo[a]pyrene hapten. The synthetic route is shown below:

[0019]

[0020] Preferably, the benzo[a]pyrene hapten-carrier protein conjugate is prepared by the following method, the method comprising:

[0021] The benzo[a]pyrene hapten is mixed with an activator to obtain a benzo[a]pyrene hapten activation solution; the benzo[a]pyrene hapten activation solution is reacted with a carrier protein to obtain the benzo[a]pyrene hapten-carrier protein conjugate.

[0022] Preferably, the activator comprises a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0023] Preferably, the carrier protein is dissolved in a buffer solution, and the buffer solution is preferably a carbonate buffer solution.

[0024] Preferably, the benzo[a]pyrene monoclonal antibody is prepared by the following method, the method comprising:

[0025] The benzo[a]pyrene hapten-carrier protein conjugate was injected into an animal. After an immune response was initiated, spleen cells from the animal were fused with myeloma cells. Hybridoma cells that stably secrete monoclonal antibodies were screened from the fused cells. After expansion culture and in vivo culture in animals, the benzo[a]pyrene monoclonal antibody was obtained.

[0026] Preferably, the screening method is the indirect competitive ELISA method.

[0027] Preferably, the myeloma cells are Sp2 / 0 myeloma cells in the logarithmic growth cycle.

[0028] Preferably, the fusion ratio of spleen cells to myeloma cells is (8-12):1, for example, it can be 8:1, 9:1 or 10:1, etc.

[0029] Preferably, the expanded culture involves subcloning the selected hybridoma cells 2-4 times, for example, 2, 3, or 4 times, until the positive rate of the resulting hybridoma cells is 100%.

[0030] Preferably, during the in vivo culture of the animal, 1×10⁻⁶ is injected into the peritoneal cavity of the animal. 6 -2×10 6 The number of hybridoma cells mentioned above can be, for example, 1.2 × 10⁶. 6 1.4×10 6 1.6×10 6 Or 1.8×10 6 wait.

[0031] Preferably, sterilized paraffin is injected into the peritoneum of the animal one week before the in vivo culture operation.

[0032] In a second aspect, the present invention provides a method for preparing a benzo[a]pyrene immunoaffinity column as described in the first aspect, the preparation method comprising the following steps:

[0033] The solid-phase support was activated and then coupled with benzo[a]pyrene monoclonal antibody to obtain a solid-phase support coupled with benzo[a]pyrene monoclonal antibody; the solid-phase support coupled with benzo[a]pyrene monoclonal antibody was washed and mixed with preservation solution, and then loaded into a column tube to obtain the benzo[a]pyrene immunoaffinity column.

[0034] Preferably, the activator for activating the solid support includes cyanogen bromide.

[0035] Preferably, the detergent used for washing includes Tris-HCl buffer and acetate buffer.

[0036] Thirdly, the present invention provides the application of the benzo[a]pyrene immunoaffinity column as described in the first aspect in the detection or separation of benzo[a]pyrene.

[0037] Preferably, the benzo[a]pyrene immunoaffinity column is used for the detection of benzo[a]pyrene in petrochemical agents.

[0038] Preferably, the petrochemical agent includes any one of humic acid, polyanionic cellulose, benzene-based polyether, and modified polyacrylamide.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The benzo[a]pyrene monoclonal antibody in the benzo[a]pyrene immunoaffinity column provided by this invention is prepared by using a benzo[a]pyrene hapten-carrier protein conjugate as an immunogen. Therefore, this antibody can specifically adsorb benzo[a]pyrene, improving the detection sensitivity of the benzo[a]pyrene immunoaffinity column of this invention. At the same time, the effective enrichment of benzo[a]pyrene in petrochemicals can greatly improve the recovery rate of benzo[a]pyrene. The specificity of the benzo[a]pyrene immunoaffinity column can also reduce the interference of other impurities such as lipids, proteins, and pigments in petrochemicals, effectively improving the accuracy of benzo[a]pyrene detection. The preferred benzo[a]pyrene immunoaffinity column provided by this invention achieves a recovery rate of over 85% for benzo[a]pyrene-like substances. Attached Figure Description

[0041] Figure 1 The high-performance liquid chromatogram of the benzo[a]pyrene hapten in Example 1;

[0042] Figure 2 The mass spectrum of the benzo[a]pyrene hapten in Example 1. Detailed Implementation

[0043] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0044] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not limited to those elements and may also include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0045] "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event will occur and the possibility that the event will not occur.

[0046] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0047] The terms "one embodiment," "some embodiments," "exemplary," "specific example," or "some examples," etc., used in this invention refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this document, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example.

[0048] Preparation Example 1

[0049] This preparation example provides a benzo[a]pyrene monoclonal antibody, and the specific steps are as follows:

[0050] (1) Dissolve 0.50 g of 7,8,9,10-tetrahydrobenzo[a]pyrene-7-ol in 2 mL of pyridine. Add 0.55 g of succinic anhydride and 0.022 g of 4-dimethylaminopyridine (DMAP) with stirring. Heat to reflux and stir at 110 °C for 48 h. After the reaction is complete, stop heating and let the reaction solution cool to room temperature. Concentrate the solution. Add 2 mL of pure water and adjust the pH to 4 with 1 mol / L HCl solution. Extract with CH2Cl2, dry with anhydrous Na2SO4, and concentrate. Purify by silica gel column chromatography, concentrate, and dry to obtain benzo[a]pyrene hapten, which is stored at 4 °C for later use. The synthetic route is shown below:

[0051]

[0052] (2) Weigh 4.5 mg of benzo[a]pyrene hapten, 2 mg of EDC, and 1 mg of NHS, add N,N-dimethylformamide (DMF) to dissolve, stir at room temperature, and activate for 4 h to obtain benzo[a]pyrene hapten activation solution; take 5 mg of keyhole hemocyanin (KLH) and dissolve it in 2 mL of carbonate buffer solution with a concentration of 0.05 mol / L and a pH of 9.6. Add the benzo[a]pyrene hapten activation solution dropwise to the keyhole hemocyanin solution, stir at room temperature overnight, and dialyze at 4 °C for three days to obtain benzo[a]pyrene hapten-KLH conjugate, which is the immunogen.

[0053] (3) Female 8-week-old BALB / c mice were selected. 100 μg of immunogen was mixed with an equal amount of Freund's complete adjuvant, emulsified, and then injected subcutaneously and intraperitoneally. Immunization was boosted every 2 weeks. During the booster, 100 μg of immunogen and an equal amount of incomplete adjuvant were injected. Blood was collected on the 7th day after each immunization injection, and the serum antibody titer was determined by the interval ELISA method. 3 days before cell fusion, mice were injected intraperitoneally with 100 μg of immunogen without adjuvant.

[0054] (4) Spleen cells from immunized BALB / c mice were mixed with Sp2 / 0 myeloma cells in the logarithmic growth phase at a ratio of 10:1. The mixture was centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded. The centrifuge tube was inverted, and the culture medium at the opening was aspirated with a pipette. The bottom of the tube was gently tapped to loosen the precipitated cells into a paste. The tube was incubated at 37°C for 2 minutes. Preheated 50% PEG-1000 (at 37°C) was slowly added over 40 seconds. The tube was incubated at 37°C for 1.5 minutes, and 1 mL of RPMI 1640 basal medium was slowly added. After 30 seconds, 3 mL of RPMI 1640 basal medium was added, followed by 16 mL of RPMI 1640 basal medium. The tube was incubated at 37°C for 2 minutes, centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded. RPMI 1640 basal medium was then slowly added. Fill centrifuge tubes with 1640 basal medium, centrifuge at 1000 r / min for 4 min, discard the supernatant, suspend the cells in HAT medium, and seed them into 96-well plates with pre-coated feeder cells at 120 μL / well. Incubate in a 5% CO2 incubator at 37°C.

[0055] (5) After cell fusion, when the cell colonies have grown to about 1 / 3 of the bottom of the well, select wells with higher positive values ​​for screening specific antibodies using an indirect competitive ELISA method. The steps of the indirect competitive ELISA method are as follows: a. Coat the ELISA plate with the prepared benzo[a]pyrene hapten-KLH conjugate, 100 μL / well, incubate at 37°C for 2 h, and wash the plate 3 times with PBS buffer, 2 min each time; b. Add 0.1 mol / L potassium-free PBS buffer, 200 μL / well, incubate at 37°C for 2 h, and wash 3 times with PBS buffer, 2 min each time; c. Add the supernatant of the culture medium to the wells to be tested, incubate at 37°C for 30 min, wash 3 times with PBS buffer, 2 min each time, add horseradish peroxidase (HRP) labeled goat anti-mouse IgG at a labeling ratio of 1:3000, and incubate at 37°C for 30 min; d. Wash 3 times with PBS buffer, 2 min each time, and add substrate for color development for 15 min.

[0056] Cloning was performed using limiting dilution with wells exhibiting good inhibition. After three subcloning cycles, the positive rate reached 100%. Hybridoma cells were expanded and cultured, then cryopreserved in liquid nitrogen. Upon thawing, the cryovials were removed and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture flasks and cultured at 37°C. BALB / c mice were intraperitoneally injected with 1 mL of sterile paraffin, and one week later, 1.5 × 10⁻⁶ g of paraffin was injected intraperitoneally. 6 Seven days after the aseptic collection of ascites from a hybridoma cell, benzo[a]pyrene monoclonal antibody was obtained.

[0057] Preparation Example 2

[0058] This preparation example provides a benzo[a]pyrene monoclonal antibody, and the specific steps are as follows:

[0059] The only difference between this example and Preparation Example 1 is that step (2) is different. In step (2), the keyhole hemocyanin is replaced with an equal mass of bovine serum albumin (BSA). The other raw materials, amounts and preparation methods are the same as in Preparation Example 1.

[0060] Preparation Example 3

[0061] This preparation example provides a benzo[a]pyrene monoclonal antibody, and the specific steps are as follows:

[0062] The only difference between this preparation example and preparation example 1 is that step (2) is different. In step (2), the keyhole hemocyanin is replaced with an equal mass of chicken ovalbumin (OVA). The other raw materials, amounts and preparation methods are the same as in preparation example 1.

[0063] Comparative Preparation Example 1

[0064] This comparative preparation example provides a benzo[a]pyrene monoclonal antibody, and the specific steps are as follows:

[0065] The only difference between this preparation example and preparation example 1 is that it does not include step (1) and replaces the benzo[a]pyrene hapten in step (2) with an equal mass of the following compound (preparation method refers to CN114933544A). The other raw materials, amounts and preparation methods are the same as in preparation example 1.

[0066]

[0067] Comparative Preparation Example 2

[0068] This comparative preparation example provides a benzo[a]pyrene monoclonal antibody, and the specific steps are as follows:

[0069] The only difference between this preparation example and preparation example 1 is that it does not include step (1) and replaces the benzo[a]pyrene hapten in step (2) with an equal mass of the following compound (preparation method refers to CN114933544A). The other raw materials, amounts and preparation methods are the same as in preparation example 1.

[0070]

[0071] Test Example 1

[0072] High performance liquid chromatography

[0073] The benzo[a]pyrene hapten in Preparation Example 1 was detected by high performance liquid chromatography. The chromatographic column was a 4.6 mm × 250 mm ODS-C18 (octadecylsilane-bonded silica gel) column with a packing material of 5.0 μm.

[0074] Column oven temperature: 35℃; injection volume: 10μL; excitation wavelength (λex) / emission wavelength (λem) of fluorescence detector: 305nm / 430nm;

[0075] The gradient elution program is shown in the table below. Mobile phase A: acetonitrile; Mobile phase B: water.

[0076] 0 1.2 65 35 27 1.2 65 35 41 1.2 100 0 45 1.2 65 35

[0077] Test results are as follows Figure 1 As shown, there is an independent chromatographic peak at a retention time of 8.5 min, with a good peak shape and no significant forward or tailing phenomenon. It is determined that the peak time of benzo[a]pyrene hapten is 8.50 min.

[0078] The benzo[a]pyrene hapten in Preparation Example 1 was detected by mass spectrometry.

[0079] Extraction ion map results are as follows Figure 2 As shown, the peak at 371 m / z is where the benzo[a]pyrene hapten is located.

[0080] Test Example 2

[0081] Antibody titers were tested on the benzo[a]pyrene monoclonal antibodies provided in Preparation Examples 1-3 and Comparative Preparation Example 1-2. The results are shown in Table 1.

[0082] Table 1

[0083] Preparation Example 1 2.83 Preparation Example 2 1.93 Preparation Example 3 1.04 Comparative Preparation Example 1 1.27 Comparative Preparation Example 2 0.65

[0084] Based on the data in Table 1, it can be seen from Preparation Example 1 and Comparative Preparation Examples 1-2 that the benzo[a]pyrene monoclonal antibody prepared from the benzo[a]pyrene hapten with the specific structure provided by the present invention can more effectively reduce the influence of impurities in petrochemical agents and effectively improve the titer compared with the prior art. Comparative Preparation Examples 1-3 show that the present invention uses a specific carrier protein, preferably keyhole hemocyanin, which, combined with the benzo[a]pyrene hapten with the specific structure provided by the present invention, can further improve the titer of the benzo[a]pyrene monoclonal antibody, thereby improving the sensitivity of the benzo[a]pyrene monoclonal antibody provided by the present invention.

[0085] Test Example 3

[0086] Cross-reactivity of the benzo[a]pyrene monoclonal antibody provided in Preparation Example 1 was tested. The benzo[a]pyrene antibody obtained in Preparation Example 1 and its corresponding coating were used for plate coating. Benzo[a]pyrene structural analogs, benzo[a]anthracene, benzo[a]fluoranthracene, and benzo[a]perylene, were used as target substances, and the cross-reactivity of the antibody with them was measured. The structures of benzo[a]anthracene, benzo[a]fluoranthracene, and benzo[a]perylene are shown below:

[0087]

[0088] The test results are shown in Table 2:

[0089] Table 2

[0090] Benzo[a]pyrene 100 Benzanthracene ＜10 Benzoanthracene ＜10 Benzo[a]perylene ＜10

[0091] According to the data in Table 2, the benzo[a]pyrene monoclonal antibody provided in Preparation Example 1 has good specificity for benzo[a]pyrene, but low reaction rate for benzo[a]anthracene, benzo[a]fluoranthracene, and benzo[a]perylene compounds. This indicates that the benzo[a]pyrene monoclonal antibody provided by the present invention can effectively enrich benzo[a]pyrene compounds in petrochemical agents with high separation, thereby effectively improving the accuracy of benzo[a]pyrene detection in petrochemical agents.

[0092] Example 1

[0093] This embodiment provides a benzo[a]pyrene immunoaffinity column, which is prepared as follows:

[0094] (1) Weigh 2g of agarose gel 4B dry powder activated by cyanogen bromide and swell it in 25mL of 1mmol / L HCl solution. After 30min, transfer the agarose gel 4B solution into a sintered funnel, add 200mL of 1mmol / L HCl solution to wash it, and then wash it thoroughly with 200mL of 0.1mol / L sodium bicarbonate buffer. After that, filter it to obtain the activated agarose gel carrier.

[0095] (2) The activated agarose gel carrier, the benzo[a]pyrene monoclonal antibody provided in Preparation Example 1, and the buffer solution were stirred at room temperature for 2 hours. The buffer solution was a mixture of 0.1 mol / L sodium bicarbonate solution and 0.5 mol / L sodium chloride solution with a pH of 8.3. After the reaction was completed, the mixture was allowed to stand, and the supernatant was collected to obtain the conjugate. The conjugate ratio of the benzo[a]pyrene monoclonal antibody to the activated agarose gel carrier in the conjugate was determined by the BCA method. The conjugate ratio was calculated as follows: the conjugate ratio of the benzo[a]pyrene monoclonal antibody to the activated agarose gel carrier in the conjugate was 100%.

[0096] (3) The conjugate was washed with 15 mL of Tris-HCl buffer (0.1 mol / L, pH 8.0), and gently stirred at room temperature for 2 h. After filtration, it was washed with 600 mL of 0.1 mol / L acetate buffer (pH 4.5). The Tris-HCl buffer and acetate buffer were washed alternately 5 times.

[0097] (4) After washing, the conjugate is thoroughly mixed with 250 mL of PBS buffer and packed into a column. Take a 3 mL empty column tube and press the bottom sieve plate. Then, pack the conjugate evenly into 10 3 mL column tubes, plug the top and bottom plugs, label them, and store them at 5 °C to obtain benzo[a]pyrene immunoaffinity columns.

[0098] Example 2-3

[0099] This embodiment provides a benzo[a]pyrene immunoaffinity column, and the preparation method is as follows:

[0100] The only difference between this example and Example 1 is that the benzo[a]pyrene monoclonal antibody provided in Preparation Example 1 is replaced with an equal mass of the benzo[a]pyrene monoclonal antibody provided in Preparation Examples 2-3. All other raw materials, amounts, and preparation methods are the same as in Example 1.

[0101] Example 4

[0102] This embodiment provides a benzo[a]pyrene immunoaffinity column, which is prepared as follows:

[0103] The only difference between this example and Example 1 is that the agarose gel 4B in the preparation method is replaced with an equal mass of polystyrene, while the other raw materials, amounts and preparation methods are the same as in Example 1.

[0104] Comparative Examples 1-2

[0105] This comparative example provides a benzo[a]pyrene immunoaffinity column, prepared by the following methods:

[0106] The only difference between this example and Example 1 is that the benzo[a]pyrene monoclonal antibody provided in Preparation Example 1 is replaced with an equal mass of the benzo[a]pyrene monoclonal antibody provided in Comparative Preparation Examples 1-2. All other raw materials, amounts, and preparation methods are the same as in Example 1.

[0107] Application Example 1

[0108] This application example provides a method for detecting benzo[a]pyrene using a benzo[a]pyrene immunoaffinity column, with the specific steps as follows:

[0109] (1) Sample preparation: Accurately weigh 2.0 g of homogenized sample into a 50 mL stoppered test tube, add 6 mL of extraction solvent (i.e., 80% n-hexane aqueous solution), vortex for 1 min, sonicate for 8 min, centrifuge at 6000 r / min for 4 min, and transfer the supernatant to a graduated glass tube; then add 6 mL of 80% n-hexane aqueous solution to repeat the extraction once, combine the two extracts, and make up to 16 mL with 80% n-hexane aqueous solution; transfer 4 mL of the extract and dilute with 20 mL of PBS buffer solution to obtain the sample solution for later use.

[0110] (2) Purification of benzo[a]pyrene immunoaffinity column: Remove the top stopper from the benzo[a]pyrene immunoaffinity column provided in Example 1, install the adapter, and connect and fix the other end of the adapter to the syringe. Connect and fix the column directly to the syringe on the pneumatic control frame, load the sample solution, and after loading, rinse the immunoaffinity column with 6 mL of 20% n-hexane aqueous solution at a flow rate of 2 drops / second. Drain the residual liquid in the column and discard all the above eluent. Finally, elute with elution buffer (3 mL n-hexane) at a flow rate of 2 drops / second, collect all the eluent, filter it through a 0.22 μm organic microporous membrane, and then perform the determination.

[0111] (3) Recovery rate determination: Take different blank petrochemical samples: humic acid, polyanionic cellulose PAC-LV, benzene polyether, modified polyacrylamide and other petrochemical agents (provided by China Petroleum Exploration and Development Research Institute), add benzo[a]pyrene to each sample to a concentration of 10 μg / kg, prepare the samples according to the above method and purify them by immunoaffinity column, and then detect them according to the method of "Determination of Polycyclic Aromatic Hydrocarbons in Soil and Sediments by High Performance Liquid Chromatography" (HJ784-2016) to obtain the recovery rate of benzo[a]pyrene.

[0112] Application Example 2-4

[0113] This application example provides a method for detecting benzo[a]pyrene using a benzo[a]pyrene immunoaffinity column, with the specific steps as follows:

[0114] The only difference between this example and Application Example 1 is that the benzo[a]pyrene immunoaffinity column provided in Example 1 in specific step (2) is replaced with the benzo[a]pyrene immunoaffinity column provided in Examples 2-4. The other raw materials, dosages and preparation methods are the same as in Application Example 1.

[0115] Comparative Application Examples 1-2

[0116] This comparative application example provides a method for detecting benzo[a]pyrene using a benzo[a]pyrene immunoaffinity column, with the specific steps as follows:

[0117] The only difference between this example and Application Example 1 is that the benzo[a]pyrene immunoaffinity column provided in Example 1 in specific step (2) is replaced with the benzo[a]pyrene immunoaffinity column provided in Comparative Examples 1-2. The other raw materials, dosages and preparation methods are the same as in Application Example 1.

[0118] The detection results of Application Example 1-4 and Comparison Example 1-2 are shown in Table 3:

[0119] Table 3

[0120]

[0121]

[0122] According to the experimental data in Table 3, the sample recovery rate in the application examples of this invention is better than that in the comparative application examples, indicating that the benzo[a]pyrene hapten prepared and used in this invention has a better adsorption effect on benzo[a]pyrene-like substances in petrochemical agents such as humic acid, polyanionic cellulose, benzene polyether, and modified polyacrylamide. The sample recovery rate of application example 1 is between 85.2% and 93.7%, which is higher than that of application examples 2, 3, and 4. This indicates that the adsorption effect of the immunoaffinity column prepared using keyhole hemocyanin in the preparation of the immunogen in this invention is better than that using bovine serum albumin and chicken ovalbumin, and the effect of using agarose gel 4B as the solid phase carrier is better than that of using polystyrene. Therefore, the benzo[a]pyrene immunoaffinity column provided by this invention can effectively separate and recover benzo[a]pyrene-like substances in petrochemical agents such as humic acid, polyanionic cellulose, benzene polyether, and modified polyacrylamide. Moreover, the recovery rate of benzo[a]pyrene in application example 1 for the four petrochemical agents all reached more than 85%.

[0123] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A benzo[a]pyrene immunoaffinity column, characterized in that, The packing material of the benzo[a]pyrene immunoaffinity column includes a solid-phase carrier coupled with a benzo[a]pyrene monoclonal antibody. The benzo[a]pyrene monoclonal antibody was prepared by using a benzo[a]pyrene hapten-carrier protein conjugate as an immunogen. The structure of the benzo[a]pyrene hapten is The benzo[a]pyrene hapten was prepared by the following method: 7,8,9,10-tetrahydrobenzo[a]pyrene-7-ol was dissolved in a solvent, succinic anhydride and 4-dimethylaminopyridine were added, the mixture was heated to reflux, and after the reaction was complete, an acidic pH adjuster was added to adjust the pH to 4 to obtain the benzo[a]pyrene hapten. The carrier protein includes any one of keyhole hemocyanin, chicken ovalbumin, or bovine serum albumin.

2. The benzo[a]pyrene immunoaffinity column according to claim 1, characterized in that, The solid support includes any one of agarose gel, polystyrene, or silicone rubber.

3. The benzo[a]pyrene immunoaffinity column according to claim 1, characterized in that, The filling material of the benzo[a]pyrene immunoaffinity column also includes a preservation solution.

4. The benzo[a]pyrene immunoaffinity column according to claim 3, characterized in that, The preservation solution includes any one of PBS buffer, HEPES buffer, and Tris buffer.

5. The benzo[a]pyrene immunoaffinity column according to claim 1, characterized in that, The benzo[a]pyrene hapten-carrier protein conjugate is prepared by the following method, the method comprising: The benzo[a]pyrene hapten is mixed with an activator to obtain a benzo[a]pyrene hapten activation solution; the benzo[a]pyrene hapten activation solution is reacted with a carrier protein to obtain the benzo[a]pyrene hapten-carrier protein conjugate.

6. The benzo[a]pyrene immunoaffinity column according to claim 5, characterized in that, The activator includes a combination of EDC and NHS.

7. The benzo[a]pyrene immunoaffinity column according to claim 1, characterized in that, The benzo[a]pyrene monoclonal antibody is prepared by the following method, the method comprising: The benzo[a]pyrene hapten-carrier protein conjugate was injected into an animal. After an immune response was initiated, spleen cells from the animal were fused with myeloma cells. Hybridoma cells that stably secrete monoclonal antibodies were screened from the fused cells. After expansion culture and in vivo culture in animals, the benzo[a]pyrene monoclonal antibody was obtained.

8. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, The screening method is the indirect competitive ELISA method.

9. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, The myeloma cells are Sp2 / 0 myeloma cells in the logarithmic growth cycle.

10. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, The fusion ratio of spleen cells to myeloma cells was (8-12):

1.

11. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, The expanded culture involves performing 2-4 subclonings on the selected hybridoma cells until the positive rate of the resulting hybridoma cells is 100%.

12. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, During in vivo culture of the animal, 1×10⁻⁶ mmol / L was injected intraperitoneally into the animal. 6 -2×10 6 One hybridoma cell.

13. The benzo[a]pyrene immunoaffinity column according to claim 7, characterized in that, One week before the in vivo culture procedure, sterile paraffin is injected into the animal's abdominal cavity.

14. A method for preparing a benzo[a]pyrene immunoaffinity column as described in any one of claims 1-13, characterized in that, The preparation method includes the following steps: The solid-phase support was activated and then coupled with benzo[a]pyrene monoclonal antibody to obtain a solid-phase support coupled with benzo[a]pyrene monoclonal antibody. The solid-phase support coupled with benzo[a]pyrene monoclonal antibody was washed and mixed with preservation solution, and then loaded into a column tube to obtain the benzo[a]pyrene immunoaffinity column.

15. The preparation method according to claim 14, characterized in that, The activator for activating the solid support includes cyanide bromide.

16. The preparation method according to claim 14, characterized in that, The washing process uses Tris-HCl buffer and acetate buffer as detergents.

17. The use of a benzo[a]pyrene immunoaffinity column as described in any one of claims 1-13 in the detection or separation of benzo[a]pyrene.

18. The application according to claim 17, characterized in that, Application of the benzo[a]pyrene immunoaffinity column in the detection of benzo[a]pyrene in petrochemical agents.

19. The application according to claim 18, characterized in that, The petrochemical agent includes any one of humic acid, polyanionic cellulose, benzene-based polyether, or modified polyacrylamide.

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