Benzo[a]pyrene hapten, artificial antigen and detection kit for benzo[a]pyrene

Abstract

The present invention discloses a benzo[a]pyrene hapten, an artificial antigen and a detection kit for benzo[a]pyrene. The artificial antigen shown by the structural formula as formula (III) is used as the coating antigen, and the artificial antigen shown by the structural formula as formula (IV) is used as the artificial coating antigen. The antibody has good sensitivity to benzo[a]pyrene, with a half-inhibition concentration of 4.07 ng / mL and a minimum detection limit of 0.38 ng / mL. The cross-reaction rates with common polycyclic aromatic hydrocarbon compounds are all lower than 5%, indicating that the antibody has good specificity for benzo[a]pyrene and can effectively exclude the interference of other drugs, providing a core reagent for establishing an immunoassay method for benzo[a]pyrene. The purpose of rapidly and accurately detecting benzo[a]pyrene in related samples is achieved by using the benzo[a]pyrene hapten, artificial antigen and antibody of the present invention.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and specifically, to a benzopyrene hapten, an artificial antigen, and a detection kit for benzopyrene. Background Art

[0002] Benzopyrene is a common polycyclic aromatic hydrocarbon compound with teratogenic, carcinogenic, and mutagenic properties. It is widely distributed in nature and mainly comes from two aspects: one is the waste gas generated by the incomplete combustion of organic compounds, such as coal, petroleum, natural gas, etc.; the other is generated during the high-temperature smoking and baking of food, especially after charring occurs, and its content will reach 10-20 times that of normal food.

[0003] The presence of benzopyrene poses a great threat to human health. It is one of the most toxic strong carcinogens among polycyclic aromatic hydrocarbon compounds and was first noticed in 1775 when there was a high incidence of scrotal cancer in the group of chimney sweeps in London. Benzopyrene can enter the human body through environmental pollution and cause serious harm. Benzopyrene in the waste gas generated by the incomplete combustion of organic compounds will adsorb on atmospheric particles and is easily adhered to the human skin or inhaled by the human body, leading to cancer; it will also pollute water sources and soil and enter foods such as grains, vegetables, fruits, and aquatic products on which humans rely for survival, threatening human health. When benzopyrene is absorbed into the intestine, it will spread to all parts of the body through blood circulation, affecting the human endocrine system and causing lesions and canceration in the human body. Some researchers administered drugs to animals by gavage, intravenous injection, inhalation, etc., and found that benzopyrene can cause various cancers such as gastric cancer, digestive tract cancer, lung cancer, and bladder cancer. Benzopyrene also has the characteristics of long-term and hidden nature. When the human body comes into contact with a low dose of benzopyrene, the body will not immediately produce adverse reactions but will continuously accumulate in the human body, and then cause canceration in the human body. Some experts have shown through animal experiments that it can also affect the next generation through the placenta by the mother, causing fetal malformation and death, and also causing a decline in the immune system function of the offspring, seriously threatening the survival and reproduction of humans. As one of the most representative carcinogens among polycyclic aromatic hydrocarbon compounds, many countries and organizations have strict requirements for its content in food. According to GB 2762-2017 "National Food Safety Standard Limits of Contaminants in Foods", the maximum residue limit of benzopyrene in grains, meats, aquatic animals and their products is 5 μg / kg, and the maximum residue limit in oils and fats and their products is 10 μg / kg. Research shows that the teratogenic, carcinogenic, and mutagenic properties of benzopyrene pose a visible serious threat to the survival and reproduction of humans. Therefore, it is particularly important to develop more advanced, effective, and sensitive benzopyrene detection technologies.

[0004] At present, there are few detection methods for benzo[a]pyrene. Traditional detection methods include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS / MS), etc. Although instrument detection technology has high precision and good repeatability, the required instruments are expensive, sample pretreatment is complex, and the detection time is long, which has limitations in on-site detection. Enzyme-linked immunosorbent assay (ELISA) technology utilizes the specific binding ability of antigen and antibody, and has the advantages of high sensitivity, rapidity, high throughput, etc., and is widely used in the detection of benzo[a]pyrene residues.

[0005] Matschulat et al. (Matschulat, D, Deng, A, Niessner, R, et al. Development of a Highly Sensitive Monoclonal Antibody Based ELISA for Detection of Benzo[a]pyrene in Potable water. Analyst, 2005, 130(7):1078 - 1086.) prepared a monoclonal antibody against benzo[a]pyrene, and based on this monoclonal antibody, the content of benzo[a]pyrene in water was detected by enzyme-linked immunosorbent assay. Its IC50 was 24 ng / mL. The prepared monoclonal antibody had the disadvantage of poor specificity, with a cross-reaction rate of more than 20% with other polycyclic aromatic hydrocarbon compounds. Shao Huifeng et al. prepared a monoclonal antibody against benzo[a]pyrene, and the detection limit of the ELISA method was 3.3 ng / mL, (Shao Huifeng. Synthesis of Benzo[a]pyrene Artificial Antigen and Establishment of Its ELISA Method [D]. Nanchang: Jiangxi Normal University, 2014.) with a cross-reaction rate of 67% with indeno(1,2,3-cd)pyrene and more than 15% with other polycyclic aromatic hydrocarbon compounds. Pschenitza et al. (Pschenitza, M., Hackenberg, R., Niessner, R., et al. Analysis of Benzo[a]pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISPE) Coupled with Enzyme-Linked Immunosorbent Assay (ELISA). Sensors, 2014, 14(6):9720 - 9737) used solid phase extraction combined with enzyme-linked immunosorbent assay to detect the content of benzo[a]pyrene in edible oil, and the detection limit was 0.63 μg / g.

[0006] At present, there are few reports on the preparation of monoclonal antibodies against benzo[a]pyrene, and the sensitivity and specificity are poor, and the drug cross-reactivity rate exceeds 20%. Therefore, it is necessary to prepare a specific monoclonal antibody against benzo[a]pyrene and develop a sensitive, rapid and simple detection method for benzo[a]pyrene. SUMMARY OF THE INVENTION

[0007] The purpose of the present invention is to overcome the above deficiencies of the prior art and provide a benzo[a]pyrene hapten, an artificial antigen and a detection kit for benzo[a]pyrene.

[0008] The first object of the present invention is to provide a benzo[a]pyrene hapten.

[0009] The second object of the present invention is to provide another benzo[a]pyrene hapten.

[0010] The third object of the present invention is to provide a benzo[a]pyrene hapten.

[0011] The fourth object of the present invention is to provide another benzo[a]pyrene hapten.

[0012] The fifth object of the present invention is to provide the application of the benzo[a]pyrene hapten in the preparation of a benzo[a]pyrene artificial antigen.

[0013] The sixth object of the present invention is to provide a composition for detecting benzo[a]pyrene.

[0014] The seventh object of the present invention is to provide the application of the benzo[a]pyrene hapten, the benzo[a]pyrene artificial antigen or the composition in the preparation of a benzo[a]pyrene detection kit.

[0015] The eighth object of the present invention is to provide a detection kit for benzo[a]pyrene.

[0016] The ninth object of the present invention is to provide a detection method for benzo[a]pyrene for non-disease diagnosis purposes.

[0017] In order to achieve the above objects, the present invention is realized by the following solutions:

[0018] A benzo[a]pyrene hapten (benzo[a]pyrene hapten 1), the structural formula of which is shown in formula (I),

[0019]

[0020] The preparation method of the compound with the structural formula shown in (I) includes the following steps:

[0021] S1. 2-Bromo-4-methoxy-1,3-dimethylbenzene and potassium permanganate react fully in a mixed solution of methanol and water, then remove the insoluble substances, adjust the pH to 4, and then extract with ethyl acetate, retain the organic phase, and purify to obtain intermediate 1;

[0022] S2. The anhydrous methanol solution of Intermediate 1 reacts fully with concentrated sulfuric acid, and then is extracted with ethyl acetate. The organic phase is retained, washed with water, then washed with saturated sodium bicarbonate solution, and the organic phase is retained and purified to obtain Intermediate 2;

[0023] S3. Intermediate 2 is mixed with lithium aluminum hydride under the condition of -78 °C in anhydrous tetrahydrofuran, quenched after fully reacting, extracted with ethyl acetate, the organic phase is retained, the organic phase is washed with water, and then washed with saturated sodium bicarbonate solution, and the organic phase is retained and purified to obtain Intermediate 3;

[0024] S4. The dichloromethane solution of Intermediate 3 and sodium acetate reacts fully with Dess-Martin oxidant, and then is extracted with dichloromethane. The organic phase is retained and purified to obtain Intermediate 4;

[0025] S5. The methanol solution of Intermediate 4 and 2-naphthaleneboronic acid reacts fully with sodium bicarbonate. After removing the solvent, it is extracted with dichloromethane, the organic phase is retained and purified; the product is heated and dissolved with a mixed solution of ethyl acetate - dichloromethane, stirred to evaporate the solvent until the solute precipitates, and then recrystallized with petroleum ether to obtain Intermediate 5;

[0026] S6. The tetrahydrofuran solution of (methoxymethyl)triphenylphosphonium chloride reacts fully with n-butyllithium, and then reacts fully with Intermediate 5. After removing the solvent, it is extracted with dichloromethane, the organic phase is retained and purified to obtain Intermediate 6;

[0027] S7. The dichloromethane solution of Intermediate 6 reacts fully with methanesulfonic acid, then the pH is adjusted to neutral, extracted with dichloromethane, the organic phase is retained and purified to obtain Intermediate 7;

[0028] S8. The glacial acetic acid solution of Intermediate 7 reacts fully with hydroiodic acid. After cooling, the solid-liquid separation is carried out to retain the solid, dissolved with ethyl acetate and then extracted with water, washed with saturated sodium chloride, the organic phase is retained and purified to obtain Intermediate 8;

[0029] S9. Intermediate 8, methyl 4-bromobutyrate, anhydrous potassium carbonate and N,N-dimethylformamide react fully, and then are extracted with dichloromethane. The organic phase is retained and purified to obtain Intermediate 9;

[0030] S10. The methanol solution of Intermediate 9 reacts fully with sodium hydroxide, the pH is adjusted to 6, the solid-liquid separation is carried out to retain the solid, and after purification, it is obtained.

[0031] Preferably, the preparation method of the compound shown in the structural formula (I) comprises the following steps:

[0032] S1. 2-Bromo-4-methoxy-1,3-dimethylbenzene and potassium permanganate are dissolved in a round-bottom flask with a mixed solvent of methanol and water, and reacted fully at 70 °C. After the reaction is completed, the insoluble matter is filtered off, and the pH of the filtrate is adjusted to 4 with hydrochloric acid. After extraction with ethyl acetate, the organic phases are combined, dried to remove the solvent, and then purified by silica gel column chromatography and freeze-dried to obtain the white solid powder intermediate 1;

[0033] S2. An anhydrous methanol solution of intermediate 1 is mixed with concentrated sulfuric acid and stirred fully at 80 °C. After the reaction is completed, water is added and extracted with ethyl acetate. The organic phase is washed with water and then washed with saturated sodium bicarbonate solution. After the organic phases are combined, they are dried and dehydrated with anhydrous sodium sulfate, and the solvent is evaporated to dryness to obtain the colorless oily intermediate 2;

[0034] S3. The temperature of the tetrahydrofuran solution of intermediate 2 is lowered to -78 °C, and then mixed with lithium aluminum hydride. After full reaction, ice water is added dropwise to quench the reaction. After extraction with ethyl acetate, the organic phase is washed with water and then washed with saturated sodium bicarbonate solution. After the organic phases are combined, they are dried and dehydrated with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the colorless oily intermediate 3;

[0035] S4. A dichloromethane solution of intermediate 3 and sodium acetate is mixed with Dess-Martin periodinane. After full reaction, water is added and extracted with dichloromethane. After the organic phases are combined, filtered, dried, and purified by silica gel column chromatography to obtain the white solid intermediate 4;

[0036] S5. A methanol solution of intermediate 4 and 2-naphthylboronic acid is fully mixed with a sodium bicarbonate solution and reacted fully at 80 °C. After the solvent is removed by rotary evaporation, water is added and extracted with dichloromethane; after the organic phases are combined, they are dried and dehydrated with anhydrous sodium sulfate and then purified by silica gel column chromatography. The product is dissolved in a mixed solution of ethyl acetate-dichloromethane and heated to 80 °C, stirred to evaporate the solvent until the solute precipitates, and then recrystallized with petroleum ether to obtain the white powder fixed intermediate 5;

[0037] S6. (Methoxymethyl)triphenylphosphonium chloride is dried and placed in a dry three-necked flask. After sealing, the air is replaced with nitrogen. After cooling to room temperature, it is dissolved in tetrahydrofuran. The temperature is lowered to -78 °C and n-butyllithium is added dropwise. After full mixing, a tetrahydrofuran solution of intermediate 5 is dropped into the system, fully mixed, and heated to react fully. After the reaction is completed, the solvent is removed by rotary evaporation, water and dichloromethane are added for extraction. After the organic phases are combined, they are dried and dehydrated with anhydrous sodium sulfate and then evaporated to dryness, and purified by silica gel column chromatography to obtain the colorless oily intermediate 6;

[0038] S7. A dichloromethane solution of intermediate 6 is added dropwise with methanesulfonic acid and reacted fully. After the reaction is completed, water and a dilute sodium bicarbonate solution are added to neutralize the acidity. After extraction with dichloromethane, the organic phases are combined, dried and dehydrated with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the pale yellow solid intermediate 7;

[0039] The solution of intermediate 7 in glacial acetic acid is mixed with hydroiodic acid, and the reaction is carried out at 80 °C until completion. After the reaction is completed, it is cooled, and the system is mixed with water to precipitate a yellow solid. The solid-liquid separation is carried out. The solid is washed with water, dissolved in ethyl acetate, extracted with water, washed with saturated brine, and the organic phase is dried over anhydrous sodium sulfate and dehydrated, and then concentrated by rotary evaporation to obtain a pale yellow solid intermediate 8;

[0040] Intermediate 8, methyl 4-bromobutyrate, anhydrous potassium carbonate and N,N-dimethylformamide are mixed, and the reaction is carried out at 80 °C until completion. After the reaction is completed, water is added, and it is extracted with dichloromethane. The combined organic phases are dried over anhydrous sodium sulfate and dehydrated, and then the solvent is concentrated by rotary evaporation, and purified by silica gel column chromatography to obtain a brown oily substance intermediate 9;

[0041] The methanol solution of intermediate 9 is mixed with an aqueous sodium hydroxide solution, and the reaction is carried out at 80 °C until completion. After the reaction is completed, the pH of the system is adjusted to 6, the aqueous phase is discarded, the solid is washed with dichloromethane, and then purified by silica gel column chromatography to obtain a brown solid, thus obtaining the product.

[0042] The present invention also claims to protect a benzo[a]pyrene artificial antigen (benzo[a]pyrene artificial antigen 1), which is a benzo[a]pyrene hapten (benzo[a]pyrene hapten 1) of the formula (I) coupled with a carrier protein, and its structural formula is as shown in formula (III).

[0043]

[0044] Preferably, the carrier protein is one or more of lactoferrin (LF), bovine serum albumin (BSA), ovalbumin (OVA), or keyhole limpet hemocyanin (KLH).

[0045] The preparation method of the benzo[a]pyrene artificial antigen (benzo[a]pyrene artificial antigen 1) includes the following steps:

[0046] The carboxyl group of the benzo[a]pyrene hapten of the formula (I) is activated to obtain an activated solution of the benzo[a]pyrene hapten. The activated solution of the benzo[a]pyrene hapten reacts fully with the BB buffer solution of the carrier protein, and after purification, the benzo[a]pyrene artificial antigen of the formula (III) is obtained.

[0047] Preferably, the preparation method of the benzo[a]pyrene artificial antigen (benzo[a]pyrene artificial antigen 1) includes the following steps:

[0048] Dissolve the carrier protein in BB buffer solution (boric acid buffer solution) to obtain a carrier protein solution; dissolve the benzo[a]pyrene hapten shown in the structural formula (I) in DMF (N,N-dimethylformamide), add EDC (1-ethyl-3-(3-dimethylaminopropyl)) and NHS (N-hydroxysuccinimide), and react fully in the dark to obtain an activation solution of the benzo[a]pyrene hapten shown in the structural formula (I); slowly drop the activation solution of the benzo[a]pyrene hapten shown in the structural formula (I) into the solution dissolved with the carrier protein, stir evenly, and react fully in the dark for the coupling reaction; dialyze the coupling reaction product to obtain the benzo[a]pyrene artificial antigen shown in the structural formula (III).

[0049] Preferably, the molar ratio of the benzo[a]pyrene hapten shown in the structural formula (I) to the carrier protein is 1:125.

[0050] A benzo[a]pyrene hapten (benzo[a]pyrene hapten 2), the structural formula of which is shown in formula (II),

[0051]

[0052] Its preparation method includes the following steps:

[0053] S1. The ethylene glycol dimethyl ether solution of 2-bromopropiophenone reacts fully with tetrakis(triphenylphosphine)palladium under nitrogen protection, then is mixed with 5-hydroxy-2-naphthaleneboronic acid and ethanol, reacts fully, then is mixed with sodium carbonate, heated under reflux, cooled to remove the solid, the liquid phase is washed with ethyl acetate, the solvent is removed, and the residue is dissolved in ethyl acetate, washed with water, the organic phase is dried and dehydrated with anhydrous sodium sulfate, and then the organic solvent is removed and purified to obtain intermediate 1;

[0054] S2. Under nitrogen protection, the dichloromethane solution of intermediate 1 is mixed with the dichloromethane solution of titanium tetrachloride, after reacting fully, the reaction mixture is quenched and extracted with ethyl acetate, the combined organic phases are washed with water, the organic phases are dried and dehydrated with anhydrous sodium sulfate, and then the organic solvent is removed and purified to obtain intermediate 2;

[0055] S3. Intermediate 2, methyl 4-bromobutyrate, anhydrous potassium carbonate and N,N-dimethylformamide are mixed, react fully, extracted with dichloromethane, the combined organic phases are dried and dehydrated with anhydrous sodium sulfate, and then the organic solvent is removed and purified to obtain the white intermediate 3;

[0056] S4. The methanol solution of intermediate 3 reacts fully with sodium hydroxide, then the pH of the system is adjusted to 6, the solid-liquid separation is carried out to discard the aqueous phase, the solid is washed with dichloromethane, and purified to obtain the brown solid product.

[0057] Preferably, the preparation method of the compound shown in formula (II) includes the following steps:

[0058] S1. A solution of 2-bromopropiophenone in ethylene glycol dimethyl ether is mixed with tetrakis(triphenylphosphine)palladium under nitrogen protection. The resulting solution is thoroughly mixed, then mixed with 5-hydroxy-2-naphthaleneboronic acid and ethanol. After thorough mixing, it is mixed with a sodium carbonate solution, and the mixture is heated to 90 °C and refluxed thoroughly. After the reaction is completed, the solution is cooled, the solid is separated by filtration and the liquid phase is washed with ethyl acetate. The solvent is removed, and the residue is dissolved in ethyl acetate, washed with water, and dried and dehydrated with anhydrous sodium sulfate. Then, the solvent is removed by evaporation under reduced pressure, and the residue is purified by silica gel column chromatography to obtain the white intermediate 1;

[0059] S2. Under nitrogen, the dichloromethane solution of intermediate 1 is cooled to -78 °C, and then a dichloromethane solution of titanium tetrachloride is added dropwise. After the addition is completed, the temperature is raised for sufficient reaction. After the reaction is completed, it is quenched with ice water. The mixture is extracted with ethyl acetate, the organic phases are combined, washed with water, and dried and dehydrated with anhydrous sodium sulfate. After the solvent is evaporated to dryness, it is purified by silica gel column chromatography to obtain intermediate 2;

[0060] S3. Intermediate 2, methyl 4-bromobutyrate, anhydrous potassium carbonate, and N,N-dimethylformamide are mixed and reacted at 80 °C sufficiently. After the reaction is completed, water is added, and it is extracted with dichloromethane. The organic phases are combined, dried and dehydrated with anhydrous sodium sulfate, and then the solvent is evaporated to dryness. After purification by silica gel column chromatography, the brown oily intermediate 3 is obtained;

[0061] S4. The methanol solution of intermediate 3 is mixed with an aqueous sodium hydroxide solution and reacted at 80 °C sufficiently. After the reaction is completed, the pH of the system is adjusted to 6, and the aqueous phase is discarded by centrifugation. The solid is washed with dichloromethane, and then purified by silica gel column chromatography to obtain a brown solid.

[0062] The present invention also claims to protect a benzo[a]pyrene artificial antigen (benzo[a]pyrene artificial antigen 2), which is a benzo[a]pyrene hapten conjugated to a carrier protein as shown in the structural formula of formula (II), and its structural formula is as shown in formula (IV).

[0063]

[0064] The preparation method of the benzo[a]pyrene artificial antigen as shown in the structural formula of formula (IV) includes the following steps:

[0065] The carboxyl group of the benzo[a]pyrene hapten as shown in the structural formula of formula (II) is activated to obtain an activated solution of the benzo[a]pyrene hapten. The activated solution of the benzo[a]pyrene hapten reacts sufficiently with the BB buffer solution of the carrier protein, and after purification, the benzo[a]pyrene artificial antigen as shown in the structural formula of formula (IV) is obtained.

[0066] Preferably, the method for preparing the benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV) includes the following steps: dissolving the carrier protein in BB buffer solution (carbonate buffer solution) to obtain a carrier protein solution; dissolving the benzo[a]pyrene hapten with the structural formula shown in formula (II) in DMF, adding EDC and NHS, and reacting fully in the dark to obtain an activation solution of the benzo[a]pyrene hapten with the structural formula shown in formula (II); slowly dropping the activation solution of the benzo[a]pyrene hapten with the structural formula shown in formula (II) into the carrier protein solution, stirring evenly, and reacting fully in the dark for coupling reaction; dialyzing the coupling reaction product to obtain the benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV).

[0067] Preferably, the molar ratio of the hapten to the carrier protein is 1:125.

[0068] The application of one or more of the above-mentioned benzo[a]pyrene haptens in the preparation of benzo[a]pyrene artificial antigens also belongs to the protection scope of the present invention.

[0069] The present invention also claims to protect a composition for detecting benzo[a]pyrene, which contains the benzo[a]pyrene artificial antigen with the structural formula shown in formula (III) and the benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV). The benzo[a]pyrene artificial antigen with the structural formula shown in formula (III) is used as an immunogen, and the benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV) is used as a coating antigen.

[0070] Preferably, the carrier protein of the benzo[a]pyrene artificial antigen with the structural formula shown in formula (III) is lactoferrin as an immunogen, and the carrier protein of the benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV) is bovine serum albumin as a coating antigen.

[0071] That is, the structural formula of the immunogen is shown in formula (III-1),

[0072]

[0073] The structural formula of the coating antigen is shown in formula (IV-1),

[0074]

[0075] The application of the above-mentioned benzo[a]pyrene hapten, the above-mentioned benzo[a]pyrene artificial antigen, or the above-mentioned composition in the preparation of a benzo[a]pyrene detection kit also belongs to the protection scope of the present invention.

[0076] The present invention also claims to protect a benzo[a]pyrene detection kit containing the above-mentioned composition.

[0077] Preferably, it further contains one or more of an enzyme conjugate, a substrate chromogenic solution, a termination solution, and / or a washing solution.

[0078] As a specific embodiment, the detection kit contains the following components:

[0079] (1) An enzyme-labeled plate coated with a coating antigen (a benzo[a]pyrene artificial antigen with a structural formula as shown in formula (IV-1));

[0080] (2) A benzo[a]pyrene standard solution

[0081] (3) A benzo[a]pyrene monoclonal antibody prepared using a benzo[a]pyrene artificial antigen with a structural formula as shown in (III-1);

[0082] (4) An enzyme conjugate: a goat anti-mouse secondary antibody labeled with horseradish peroxidase;

[0083] (5) A substrate chromogenic solution: composed of solution A and solution B, where solution A is urea peroxide and solution B is tetramethylbenzidine;

[0084] (6) The termination solution is 10% (v / v) H2SO4;

[0085] (7) The washing solution has a pH of 7.4 and contains 0.5% - 1.0% (w / v) Tween-20, 0.01‰ - 0.03‰ sodium azide preservative, and 0.1 - 0.3 mol / L phosphate buffer, where the percentages are weight / volume percentages.

[0086] Its usage method is the same as the detection method of benzo[a]pyrene for non-disease diagnosis purposes described below.

[0087] The present invention also claims to protect a detection method of benzo[a]pyrene for non-disease diagnosis purposes, using the said composition.

[0088] Preferably, for the benzo[a]pyrene artificial antigen with a structural formula as shown in formula (IV), the benzo[a]pyrene artificial antigen with bovine serum albumin as the carrier protein is used as the coating antigen, and the benzo[a]pyrene artificial antigen with a structural formula as shown in formula (III) and lactoferrin as the carrier protein is used as the immunogen to immunize animals, and the prepared antibody is used as the detection antibody for detection.

[0089] Specifically, the structural formula of the immunogen is as shown in formula (III-1),

[0090]

[0091] The structural formula of the coating antigen is as shown in formula (IV-1),

[0092]

[0093] Preferably, the coating concentration is 1 μg / mL, and the dilution factor of the benzo[a]pyrene antibody is 8000-fold.

[0094] The immunoassay methods include, but are not limited to, one or more of enzyme immunoassay, immunochromatography, immunosensing, immunogold colloidal, etc.

[0095] As a specific embodiment, the detection method is based on the indirect competitive ELISA method, and the specific detection method is as follows:

[0096] (1) The benzo[a]pyrene artificial antigen with the structural formula shown in formula (IV-1) is used as the coating antigen, diluted to 1 μg / mL with carbonate buffer (CB, 0.1 M pH = 9.8), and coated on a 96-well enzyme-linked immunosorbent assay (ELISA) plate. 100 μL is added to each well and incubated overnight (12 h) at 37 °C;

[0097]

[0098] (2) Discard the coating solution, wash twice with the washing solution (pH = 7.4, containing 0.6% Tween-20, 0.02% sodium azide preservative, 0.2 mol / L phosphate buffer, and the percentage is weight / volume percentage), and pat dry;

[0099] (3) Add 120 μL of the blocking solution (i.e., 5% skim milk powder by mass ratio) to each well and block at 37 °C for 3 h;

[0100] (4) Discard the blocking solution, pat the plate, take it out after drying at 37 °C for 30 min, and store it in a self-sealing bag for later use;

[0101] (5) Dilute the benzo[a]pyrene monoclonal antibody prepared from the benzo[a]pyrene artificial antigen with the structural formula shown in (III-1) 1:8000 times with phosphate buffer (PBS, 0.01 M, pH = 7.4), and dilute the benzo[a]pyrene drug to be detected 2-fold serially to 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL, 3.91 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.12 ng / mL, 0.06 ng / mL;

[0102] (6) Add 50 μL of the benzo[a]pyrene drug dilution to be detected (in triplicate) to each row, and then add 50 μL / well of the benzo[a]pyrene monoclonal antibody prepared from the benzo[a]pyrene artificial antigen with the structural formula shown in (III-1), incubate at 37 °C for 40 min, wash five times with the washing solution, and pat dry;

[0103] (7) Add the goat anti-mouse secondary antibody-HRP diluted 5000 times with phosphate buffered saline with Tween (PBST, 0.01 M), 100 μL / well, incubate at 37 °C for 30 min, wash five times with the washing solution, and pat dry;

[0104] (8) Add 100 μL of chromogenic solution to each well and incubate at 37 °C for 10 min for color development.

[0105] (9) Add 50 μL of 10% H2SO4 solution to terminate the reaction and read the OD value at 450 nm.

[0106] (10) Operate according to the above steps (1) to (9), replace the diluent of the benzo[a]pyrene to be measured in step (6) with the diluent of the sample to be measured after extraction, and combine with the standard curve drawn to determine the actual content of benzo[a]pyrene drug in the unknown sample.

[0107] Compared with the prior art, the present invention has the following beneficial effects:

[0108] The present invention prepares a benzo[a]pyrene hapten with the structural formula shown in formula (I), and couples it with a carrier protein with the structural formula shown in formula (III) to obtain an artificial antigen; prepares a benzo[a]pyrene hapten with the structural formula shown in formula (II), and couples it with a carrier protein with the structural formula shown in formula (IV) to obtain an artificial antigen, and further prepares a specific antibody for detecting benzo[a]pyrene. Using the artificial antigen with the structural formula shown in formula (III) as the coating antigen and the artificial antigen with the structural formula shown in formula (IV) as the artificial coating antigen, the antibody has good sensitivity to benzo[a]pyrene, with a half-inhibitory concentration of 4.07 ng / mL and a minimum detection limit of 0.38 ng / mL. The cross-reaction rate with common polycyclic aromatic hydrocarbon compounds is less than 5%, indicating that the antibody has good specificity for benzo[a]pyrene, can effectively exclude the interference of other drugs, and provides a core reagent for establishing an immunoassay method for benzo[a]pyrene. Using the benzo[a]pyrene hapten, artificial antigen, and antibody of the present invention, the purpose of quickly and accurately detecting benzo[a]pyrene in related samples is achieved. Description of the Drawings

[0109] Figure 1 It is a synthetic route diagram of benzo[a]pyrene hapten 1 in Example 1.

[0110] Figure 2 It is a mass spectrometry identification diagram of benzo[a]pyrene hapten 1 in Example 1.

[0111] Figure 3 It is an ultraviolet scanning diagram of benzo[a]pyrene hapten 1, benzo[a]pyrene artificial antigen 1 (benzo[a]pyrene hapten 1-LF), and LF in Example 2.

[0112] Figure 4 It is an ultraviolet scanning diagram of benzo[a]pyrene hapten 1, benzo[a]pyrene artificial antigen 2 (benzo[a]pyrene hapten 1-BSA), and BSA in Example 3.

[0113] Figure 5 It is a synthetic route diagram of benzo[a]pyrene hapten 2 in Example 4.

[0114] Figure 6 Mass spectrometry identification chart of benzo[a]pyrene hapten 2 in Example 4.

[0115] Figure 7 UV scanning charts of benzo[a]pyrene hapten 2, benzo[a]pyrene artificial antigen 3 (benzo[a]pyrene hapten 2 - BSA), and BSA in Example 5.

[0116] Figure 8 Inhibitory curve of the antibody prepared with benzo[a]pyrene artificial antigen 1 (benzo[a]pyrene hapten 1 - LF) as the immunogen against benzo[a]pyrene in Example 8. Detailed implementation manners

[0117] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0118] Example 1 Synthesis and identification of benzo[a]pyrene hapten 1

[0119] I. Experimental method

[0120] 1. Synthesis of benzo[a]pyrene hapten 1 (see the synthesis route diagram in Figure 1 )

[0121] Weigh 2 - bromo - 4 - methoxy - 1,3 - dimethylbenzene (40 g, 0.186 mol) and potassium permanganate (73.5 g, 0.465 mol) and dissolve them in a 1 L round - bottom flask with a mixed solvent of 100 mL of methanol and 100 mL of water. React at 70 °C overnight. After the reaction is completed, filter to remove the insoluble substances and adjust the pH of the filtrate to 4 with 6 mol / L hydrochloric acid. Extract with ethyl acetate, combine the organic phases, dry to remove the solvent, and then purify by silica gel column chromatography. Lyophilize to obtain the white solid powder intermediate 1 (2 - bromo - 4 - methoxyisophthalic acid).

[0122]

[0123] Take intermediate 1 (25.2 g, 91.62 mmol) and dissolve it in 100 mL of anhydrous methanol. Then add 10 mL of concentrated sulfuric acid and stir and react at 80 °C overnight. After the reaction is completed, add water, extract with ethyl acetate, wash the organic phase 3 times with water, then wash with saturated sodium bicarbonate solution, retain the organic phase, dry and dehydrate with anhydrous sodium sulfate, and then spin - dry to obtain the colorless oily substance intermediate 2 (dimethyl 2 - bromo - 4 - methoxyisophthalate).

[0124]

[0125] Intermediate 2 (24.25 g, 80 mmol) was dissolved in 200 mL of tetrahydrofuran and cooled to -78 °C. Then, 1 mol / L lithium aluminum hydride was slowly added. After addition, the temperature was raised to room temperature and the reaction was carried out for 1 h. After the reaction was completed, ice water was added dropwise to quench the reaction. The mixture was extracted with ethyl acetate. The organic phase was washed with water three times and then washed with saturated sodium bicarbonate solution. The organic phase was retained, dried over anhydrous sodium sulfate to remove water, and then purified by silica gel column chromatography to obtain colorless oily Intermediate 3 (2-bromo-4-methoxy-1,3-phenylenedimethanol).

[0126]

[0127] Intermediate 3 (6.4 g, 25.90 mmol) and sodium acetate (9.560 g, 116.555 mmol) were dissolved in 200 mL of dichloromethane. Then, Dess-Martin periodinane (32.957 g, 77.703 mmol) was added, and the mixture was stirred at room temperature for 1 h. After the reaction was completed, water was added and the mixture was extracted with dichloromethane three times. The organic phases were combined, filtered, dried over anhydrous sodium sulfate to remove water, and then purified by silica gel column chromatography to obtain white solid Intermediate 4 (2-bromo-4-methoxyisophthalaldehyde).

[0128]

[0129] Intermediate 4 (6.015 g, 24.750 mmol) and 2-naphthaleneboronic acid (4.257 g, 24.750 mmol) were dissolved in 50 mL of methanol. While stirring, 1 mol / L sodium bicarbonate solution was added to the system in its entirety, and the reaction was carried out at 80 °C for 2.5 h. After the solvent was removed by rotary evaporation, an appropriate amount of water was added, and the mixture was extracted with dichloromethane three times. The organic phases were combined, dried over anhydrous sodium sulfate to remove water, and then purified by silica gel column chromatography. The product was dissolved in a 1:1 (v / v) mixed solution of ethyl acetate and dichloromethane and heated to 80 °C. The solvent was evaporated with stirring until the solute precipitated, and then petroleum ether was added for recrystallization to obtain white powder Intermediate 5 (4-methoxy-2-(naphthalen-2-yl)isophthalaldehyde).

[0130]

[0131] Weigh (methoxymethyl)triphenylphosphonium chloride (15.302 g, 44.640 mmol). After drying, pour it into a dry three-necked flask, quickly seal it, and displace the air with nitrogen. After cooling to room temperature, dissolve it in 50 mL of tetrahydrofuran. Cool down to -78 °C and dropwise add 2.5 mol / L n-butyllithium (17.9 mL, 44.640 mmol). After stirring for 15 min, weigh Intermediate 5 (4.32 g, 14.880 mmol), dissolve it in 50 mL of tetrahydrofuran, and then drop it into the system. Continue to stir for 15 min, then take out the three-necked flask and react at room temperature for 1 h. After the reaction is completed, rotary evaporate to remove the solvent, add water and dichloromethane for extraction three times. Combine the organic phases, dry and dehydrate with anhydrous sodium sulfate, then rotary evaporate to dryness, and purify by silica gel column chromatography to obtain a colorless oily substance Intermediate 6 (2-(3-methoxy-2-(E)-2-methoxyvinyl)-6-(Z)-2-methoxyvinyl)phenylnaphthalene).

[0132]

[0133] Dissolve Intermediate 6 (656 mg, 1.893 mmol) in 5 mL of dichloromethane, dropwise add 78 μL of methanesulfonic acid, and stir at room temperature for 1 h. After the reaction is completed, add water and a dilute solution of sodium bicarbonate to neutralize the acidity, extract with dichloromethane three times, and combine the organic phases. Dry and dehydrate with anhydrous sodium sulfate, then rotary evaporate to dryness, and purify by silica gel column chromatography to obtain a light yellow solid Intermediate 7 (1-methoxybenzo[PQR]tetraphenylether).

[0134]

[0135] Dissolve Intermediate 7 (375.9 mg, 1.331 mmol) in 20 mL of glacial acetic acid, then add 20 mL of 55% hydroiodic acid (mass fraction), and react at 80 °C for 3 h. After the reaction is completed, cool to room temperature, pour the system into water to precipitate a yellow solid, filter it through four layers of filter paper. Wash the filter cake with water, dissolve it in ethyl acetate, extract with water, and then wash it once with saturated brine. Dry and dehydrate the organic phase with anhydrous sodium sulfate, then rotary evaporate to dryness to obtain a light yellow solid Intermediate 8 (benzo[PQR]tetraphen-1-ol).

[0136]

[0137] Take Intermediate 8 (367.7 mg, 1.370 mmol), methyl 4-bromobutyrate (496 mg, 2.741 mmol), and anhydrous potassium carbonate (753 mg, 4.111 mmol), dissolve them together in 20 mL of DMF, and react at 80 °C overnight. After the reaction is completed, add an appropriate amount of water, extract with dichloromethane three times, combine the organic phases, dry and dehydrate with anhydrous sodium sulfate, then rotary evaporate the organic phase to dryness, and purify by silica gel column chromatography to obtain a brown oily substance Intermediate 9 (methyl 4-(benzo[PQR]tetraphen-1-yloxy)butyrate).

[0138]

[0139] Take the intermediate 9 (505 mg, 1.370 mmol), use 10 mL of methanol as the solvent, then add 5 mL of 10% sodium hydroxide (mass fraction) aqueous solution, and react at 80 °C for 2 h. After the reaction is completed, adjust the pH of the system to 6, centrifuge and discard the aqueous phase, wash the precipitated solid with dichloromethane, and then purify it by silica gel column chromatography to obtain a brown solid (4-(benzo[PQR]tetraphenyl-1-yloxy)butyric acid). The specific method of silica gel column chromatography purification is as follows: dissolve the solid with a small amount of solvent methanol, add silica gel powder and mix well, dry the solvent by rotary evaporation, and then load the silica gel powder for purification by silica gel column chromatography.

[0140]

[0141] 2. Identification of Benzo[a]pyrene Hapten 1

[0142] Perform mass spectrometry analysis and 1H NMR analysis on benzo[a]pyrene hapten 1 to determine its molecular weight and structural characteristics.

[0143] II. Experimental Results

[0144] 1H NMR results of benzo[a]pyrene hapten 1: 1H NMR (600 MHz, DMSO) δ 12.20 (s, 1H), 8.60 (s, 1H), 8.42 (d, J = 9.1 Hz, 1H), 8.36 - 7.97 (m, 4H), 7.78 (d, J = 2.5 Hz, 2H), 7.52 (dd, J = 9.2, 2.5 Hz, 3H), 4.27 (t, J = 6.5 Hz, 2H), 2.39 (s, 2H), 2.23 - 1.94 (m, 2H).

[0145] The ESI-MS identification results of benzo[a]pyrene hapten 1 are as Figure 2 shown. The MS of this benzo[a]pyrene hapten 1: C 24 H 18 O3: 354.41, ESI-[M+H]+: 355.1.

[0146] The structural formula of benzo[a]pyrene hapten 1 is shown in formula (I):

[0147]

[0148] Example 2 Synthesis and Identification of Benzo[a]pyrene Artificial Antigen 1

[0149] I. Experimental Method

[0150] 1. Synthesis of Benzo[a]pyrene Artificial Antigen 1

[0151] Using the benzo[a]pyrene hapten 1 prepared in Example 1, the benzo[a]pyrene artificial antigen 1 was prepared by coupling lactoferrin (LF) through the active ester method. The specific method is as follows:

[0152] Take 14.2 mg of the benzo[a]pyrene hapten 1 prepared in Example 1 and dissolve it in 0.5 mL of DMF solution. Stir and add 12 mg of EDC and 10 mg of NHS, and stir and react overnight at room temperature in the dark to obtain the activated solution of benzo[a]pyrene hapten 1. Take 16 mg of LF and dissolve it in 1.6 mL of BB buffer solution with pH = 9.0, then stir and add 150 μL of the activated solution of benzo[a]pyrene hapten 1. After stirring evenly, couple at room temperature in the dark for 4 h to obtain a coupling mixture. The coupling mixture was dialyzed with PBS buffer solution at 4 °C for 3 days, and the dialysis solution was changed 2 times a day to obtain the benzo[a]pyrene artificial antigen 1 (benzo[a]pyrene hapten 1-LF). The benzo[a]pyrene artificial antigen 1 was aliquoted at a concentration of 1 mg / mL and stored frozen at -20 °C in the refrigerator.

[0153] 2. Identification of benzo[a]pyrene artificial antigen 1

[0154] Perform ultraviolet scanning measurements (190 - 400 nm) on the carrier protein lactoferrin (LF), benzo[a]pyrene hapten 1, and benzo[a]pyrene artificial antigen 1.

[0155] II. Experimental results

[0156] The measurement results are as Figure 3 shown. It can be seen from Figure 3 that the ultraviolet characteristic absorption peaks of the complete antigen have different degrees of shift relative to the benzo[a]pyrene hapten and the carrier protein (LF), and it is found that the benzo[a]pyrene artificial antigen 1 simultaneously has the characteristic absorption peaks of the benzo[a]pyrene hapten and LF, indicating that the benzo[a]pyrene hapten 1 is successfully coupled with LF, and the benzo[a]pyrene artificial antigen 1 is successfully prepared. Its structural formula is as formula (III-1).

[0157]

[0158] Example 3 Synthesis and identification of benzo[a]pyrene artificial antigen 2

[0159] I. Experimental method

[0160] 1. Synthesis of benzo[a]pyrene artificial antigen 2

[0161] Using the benzo[a]pyrene hapten 1 prepared in Example 1, the benzo[a]pyrene artificial antigen 2 was prepared by coupling bovine serum albumin (BSA) through the active ester method. The specific method is as follows:

[0162] Take 14.2 mg of the benzo[a]pyrene hapten 1 prepared in Example 1 and dissolve it in 0.5 mL of DMF solution. Stir and add 12 mg of EDC and 10 mg of NHS, and stir and react overnight in the dark at room temperature to obtain the activated solution of benzo[a]pyrene hapten 1. Take 24 mg of BSA and dissolve it in 2.4 mL of BB buffer solution with pH = 9.0. Then stir and add 225 μL of the activated solution of benzo[a]pyrene hapten 1. After stirring evenly, couple at room temperature in the dark for 4 h to obtain a coupling mixture. Dialyze the coupling mixture with PBS buffer solution at 4°C for 3 days, changing the dialysis solution twice a day, to obtain the artificial benzo[a]pyrene antigen 2 (benzo[a]pyrene hapten 1-BSA). The artificial benzo[a]pyrene antigen 2 is aliquoted at a concentration of 1 mg / mL and stored frozen at -20°C in a refrigerator.

[0163] 2. Identification of the artificial benzo[a]pyrene antigen 2

[0164] Perform ultraviolet scanning measurements (190 - 400 nm) on the carrier protein bovine serum albumin (BSA), the benzo[a]pyrene hapten 1, and the artificial benzo[a]pyrene antigen 2.

[0165] II. Experimental Results

[0166] The measurement results are as Figure 4 shown. It can be seen from Figure 4 that the ultraviolet characteristic absorption peaks of the complete antigen have different degrees of shift relative to the benzo[a]pyrene hapten and the carrier protein (BSA). Moreover, it is found that the artificial benzo[a]pyrene antigen 2 simultaneously has the characteristic absorption peaks of the benzo[a]pyrene hapten and BSA, indicating that the benzo[a]pyrene hapten 1 is successfully coupled with BSA, and the artificial benzo[a]pyrene antigen 2 is successfully prepared. Its structural formula is as shown in the above formula (III-2).

[0167]

[0168] Synthesis and Identification of Benzo[a]Pyrene Hapten 2 in Example 4

[0169] I. Experimental Method

[0170] 1. Synthesis of Benzo[a]Pyrene Hapten 2 (See the synthesis route diagram in Figure 5 )

[0171] Weigh 2-bromopropiophenone (6.07 g, 28.488 mmol) and dissolve it in 80 ml of ethylene glycol dimethyl ether solution. Under nitrogen protection, add tetrakis(triphenylphosphine)palladium (330 mg, 0.286 mmol). Stir the resulting solution at room temperature for 20 min, then add 5-hydroxy-2-naphthaleneboronic acid (6.430 g, 34.205 mmol) and 100 mL of ethanol. After stirring at room temperature for 20 min, add sodium carbonate solution (2 mol / L, 28.5 mL). Heat the mixture under reflux at 90 °C for 4 h. After the reaction is completed, cool the solution to room temperature to produce a precipitate. Filter out the precipitate and wash the liquid phase with ethyl acetate. Evaporate the filtrate to dryness, dissolve the residue in ethyl acetate, wash with water, dry over anhydrous sodium sulfate to dehydrate, and then evaporate the solvent under reduced pressure. Purify the residue by silica gel column chromatography to obtain the white intermediate 1 (1-(2-(5-hydroxynaphthalen-2-yl)phenyl)-2-propanone).

[0172]

[0173] Under nitrogen, add an 85 mL dichloromethane solution of intermediate 1 (2.760 g, 9.988 mmol) to a dry flask, cool the temperature to -78 °C, and then dropwise add a dichloromethane solution of titanium tetrachloride (1 mol / L, 10 mL). After the addition is completed, raise the temperature to room temperature and react for 3 h. After the reaction is completed, quench with ice water. Extract the mixture with ethyl acetate, combine the organic phases, wash with water, dry over anhydrous sodium sulfate to dehydrate, rotary evaporate the solvent, and then purify by silica gel column chromatography to obtain intermediate 2 (5-methyldibenzosuberan-1-ol).

[0174]

[0175] Take intermediate 2 (938 mg, 3.635 mmol), methyl 4-bromobutyrate (1.316 g, 7.270 mmol), and anhydrous potassium carbonate (1.997 g, 10.905 mmol), and dissolve them together in 10 mL of N,N-dimethylformamide. React at 80 °C overnight. After the reaction is completed, add an appropriate amount of water, extract 3 times with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate to dehydrate, rotary evaporate the solvent, and then purify by silica gel column chromatography to obtain the brown oily intermediate 3 (methyl 4-((5-methyldibenzosuber-1-yl)oxy)butyrate).

[0176]

[0177] Take the intermediate 3 (1.008 g, 2.814 mmol), use 10 mL of methanol as the solvent, then add 5 mL of 10% aqueous sodium hydroxide solution, and react at 80 °C for 2 h. After the reaction is completed, adjust the pH of the system to 6, centrifuge, discard the aqueous phase, wash the solid with dichloromethane, and purify it by silica gel column chromatography to obtain a brown solid (4-((5-methylchrysen-1-yl)oxy)butyric acid), thus obtained.

[0178]

[0179] 2. Identification of Benzo[a]pyrene Hapten 2

[0180] Perform mass spectrometry and 1H NMR analysis on benzo[a]pyrene hapten 2 to determine its molecular weight and structural characteristics.

[0181] II. Experimental Results

[0182] 1H NMR results of benzo[a]pyrene hapten 2: 1H NMR (600 MHz, DMSO) δ 12.19 (s, 1H), 9.05 - 8.86 (m, 2H), 8.18 - 7.83 (m, 1H), 7.48 (s, 6H), 7.49 - 7.20 (m, 1H), 4.23 (dd, J = 38.4, 32.1 Hz, 2H), 2.86 (d, J = 13.4 Hz, 3H), 2.42 (d, J = 43.9 Hz, 2H), 2.10 - 1.98 (m, 2H).

[0183] ESI-MS identification results of benzo[a]pyrene hapten 2: As Figure 6 shown, the MS of this benzo[a]pyrene hapten 2: C 23 H 20 O3: 344.41, ESI-[M + H] + :: 345.1.

[0184] The structural formula of benzo[a]pyrene hapten 2 is shown in Formula (II):

[0185]

[0186] Example 5 Synthesis and Identification of Benzo[a]pyrene Artificial Antigen 3

[0187] I. Experimental Method

[0188] Using the benzo[a]pyrene hapten 2 prepared in Example 4, couple bovine serum albumin (BSA) by the active ester method to prepare benzo[a]pyrene artificial antigen 3, and the method is as follows:

[0189] Take 14.2 mg of the benzo[a]pyrene hapten 2 prepared in Example 4 and dissolve it in 0.5 mL of DMF solution. Stir and add 12 mg of EDC and 10 mg of NHS, and stir the reaction overnight in the dark at room temperature to obtain the activated solution of benzo[a]pyrene hapten 2. Take 24 mg of BSA and dissolve it in 2.4 mL of BB buffer solution with pH = 9.0. Then, slowly add the activated solution of benzo[a]pyrene hapten 2 dropwise while stirring. After stirring evenly, couple overnight in the dark at room temperature to obtain a coupling mixture. Dialyze the coupling mixture with PBS buffer solution at 4°C for 3 days, changing the dialysis solution 2 times a day, to obtain the artificial antigen 3 of benzo[a]pyrene (benzo[a]pyrene hapten 2 - BSA). The artificial antigen 3 of benzo[a]pyrene is aliquoted at a concentration of 1 mg / mL and stored frozen at -20°C in a refrigerator.

[0190] Perform ultraviolet absorption peak scanning measurements (190 - 400 nm) on the carrier protein bovine serum albumin (BSA), benzo[a]pyrene hapten 2, and the artificial antigen 3 of benzo[a]pyrene.

[0191] II. Experimental Results

[0192] The measurement results are as Figure 7 shown. The ultraviolet characteristic absorption peaks of the complete antigen 3 of benzo[a]pyrene have different degrees of shift relative to those of benzo[a]pyrene hapten 2 and the carrier protein BSA. Moreover, it is found that the artificial antigen 3 of benzo[a]pyrene simultaneously has the characteristic absorption peaks of benzo[a]pyrene hapten 2 and BSA, indicating that benzo[a]pyrene hapten 2 and BSA are successfully coupled, and the artificial antigen 3 of benzo[a]pyrene is successfully prepared. Its structural formula is as shown in Formula (IV - 1).

[0193]

[0194] Example 6 Preparation and Identification of Antibodies

[0195] I. Experimental Method

[0196] 1. Immunization of mice: Take 250 μL of the artificial antigen 1 of benzo[a]pyrene (benzo[a]pyrene hapten 1 - LF) prepared in Example 2 and diluted to 1 mg / mL, and emulsify it evenly with an equal amount of immunoadjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations), and immunize the animals.

[0197] Bal b / c mice at 6 - 7 weeks old are immunized by various injection methods including subcutaneous injection in the back, subcutaneous injection at various sites, intraperitoneal injection, and foot injection. The immunization dose is 100 μL per mouse. The second immunization is carried out 2 weeks later, and subsequent booster immunizations are carried out every 2 weeks. One week after the third booster immunization, blood is taken from the tail of the mice, and the serum titer is measured by indirect competitive ELISA. When the titer no longer rises, 100 μL of the artificial antigen 1 of benzo[a]pyrene with a concentration of 1 mg / mL is injected intraperitoneally for boost immunization.

[0198] 2. Cell fusion: Three days after immunization by shock, PEG (polyethylene glycol) was used for cell fusion. The specific steps are as follows:

[0199] a. Collect mouse spleen cells: The mouse was sacrificed by cervical dislocation and immediately immersed in 75% alcohol for disinfection. The spleen of the mouse was aseptically taken out and placed in a 200-mesh cell sieve. The rubber head of a sterile syringe was used to gently grind it, and the spleen cells were washed with a basal medium to obtain a spleen cell suspension. The suspension was collected and centrifuged (1000 rpm, 7 min). The spleen cells were washed three times with the basal medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and reserved for use;

[0200] b. Collect SP2 / 0 cells: Seven to ten days before fusion, the SP2 / 0 myeloma cells were cultured in a complete medium in a 5% CO2 incubator. Before fusion, it was required that the number of SP2 / 0 tumor cells reached 1 - 4×10 7 , ensuring that the SP2 / 0 tumor cells were in the logarithmic growth phase before fusion. At the time of fusion, the myeloma cells were collected, suspended in the basal medium, and cell counting was performed;

[0201] c. Mix the two kinds of cells at a ratio of spleen cells:SP2 / 0 = 5:1, centrifuge, discard the supernatant, and obtain the mixed cells deposited at the bottom of the centrifuge tube;

[0202] d. Fusion: In the first minute, 1 mL of PEG was slowly added dropwise to the cells at the bottom of the centrifuge tube; in the second minute, the centrifuge tube was shaken evenly; in the third minute, 1 mL of preheated basal medium was added dropwise; in the fourth minute, 3 mL of preheated basal medium was added dropwise; in the fifth minute, 8 mL of preheated basal medium was added dropwise; in the sixth minute, 8 mL of preheated basal medium was added dropwise; centrifuge (1000 rpm, 7 min), discard the supernatant, resuspend in the HAT screening culture medium, and add it to a 96-well cell plate at 200 μL / well, and culture it in a 37°C, 5% CO2 incubator.

[0203] 3. Cell screening and cell line establishment: On the 5th day after cell fusion, the fusion cells were semi-replaced with HT medium, and on the 8th day, a full replacement was performed. On the 10th day, the cell supernatant was taken and screened by ic-ELISA, and the inhibitory effect of the titer on positive cells was measured. The cell wells with good inhibition on the benzo[a]pyrene standard were selected, and subcloning was performed by the limiting dilution method, and detection was performed in the same way. Repeat 4 - 5 times to obtain cell line E6.

[0204] 4. Preparation and identification of monoclonal antibodies: Several Bal b / c mice over 10 weeks old were taken, and 500 μL of liquid paraffin was intraperitoneally injected into each mouse; seven days later, about 1×10 6Hybridoma cells. After 7 days, when the abdomen of the mouse is swollen, collect the ascites. Purify the collected ascites by column chromatography. Load 1 mL of protein G packing material into the column, add 50 mL of ascites diluted with PBS buffer, load the flow-through liquid onto the column 7 - 8 times repeatedly, then elute with glycine, and immediately adjust the eluate to neutral with Tris-HCL. Dialyze to remove salts, and finally obtain the purified monoclonal antibody E6. After aliquoting, store it at -20 °C.

[0205] 5. Use indirect competitive ELISA to determine the titer and inhibition rate of the antibody. The specific method is as follows:

[0206] (1) Coating: Dilute the coating antigen to 1 μg / mL with the coating buffer, add 100 μL per well, and coat overnight in a 37 °C water bath;

[0207] (2) Blocking: Discard the coating buffer, wash the plate 2 times with the washing buffer, pat dry on the absorbent paper, add 120 μL of blocking solution per well, incubate for blocking in a 37 °C water bath for 3 h, drain, and dry in an inverted position in a 37 °C oven for 1 h;

[0208] (3) Adding antibody and drug: Dilute the monoclonal antibody E6 by 1K (1000), 2K, 4K, 8K, 16K, 32K, 64K and other multiples with PBS, and dilute the benzo[a]pyrene standard product to 10 ng / mL with PBS for standby.

[0209] Titer column: First add 50 μL of PBS buffer to each well, then add the serially diluted monoclonal antibody E6 to each well at 50 μL per well in sequence, and add PBS to the last well as a blank control;

[0210] Inhibition column: First add 50 μL of the drug diluted with PBS buffer to each well, then add the serially diluted monoclonal antibody E6 to each well at 50 μL per well in sequence, and add PBS to the last well as a blank control;

[0211] Incubate at 37 °C for 40 min, and wash the plate 5 times with the washing buffer;

[0212] (4) Adding secondary antibody: Add goat anti-mouse secondary antibody diluted 5000 times with PBST buffer (100 μL per well), incubate at 37 °C for 30 min, and wash the plate 5 times with the washing buffer;

[0213] (5) Color development: Mix equal volumes of TMB substrate buffer A and B to obtain the substrate solution, add the substrate solution (100 μL per well), and incubate at 37 °C for 10 min;

[0214] (6) Termination: Add the termination solution of 10% H2SO4 (50 μL per well) to the enzyme-linked immunosorbent assay plate to terminate the reaction;

[0215] (7) Reading: Read the absorbance (OD) using an ELISA reader at a wavelength of 450 nm. The antibody dilution factor with an absorbance value in the range of 1.0 to 1.5 is selected as the antibody titer, and the drug recognition performance of the antibody is obtained from its inhibition rate. The inhibition rate is calculated as shown in Formula 1.

[0216]

[0217] 2. Experimental Results

[0218] The antibody titers and results are shown in Table 1.

[0219] Table 1 Antiserum titers and inhibition rates of different coating combinations

[0220]

[0221] As shown in Table 1, homologous coating showed a higher titer of 16K, but the corresponding inhibition rate was low, only 41.21%. After using heterologous coating, the antibody titer was 8K, and the inhibition rate was significantly increased to 73.42%. Therefore, the sensitivity of the detection method can be improved by using heterologous coating. At the same time, the defect of low antibody titer can also be solved by further concentrating the antibody or increasing the coating concentration.

[0222] Example 7 Effect of coating concentration and antibody dilution multiple on IC 50 Impact

[0223] 1. Experimental Methods

[0224] In the immunoassay for detecting benzopyrene, the checkerboard detection method was used to detect the effects of coating concentration and benzopyrene monoclonal antibody concentration on benzopyrene detection.

[0225] The artificial antigen 3 (hapten 2-BSA) prepared in Example 5 was used as the coating source, and the benzopyrene antibody (monoclonal antibody E6) prepared in Example 6 was used as the antibody.

[0226] The coating source was coated on a 96-well ELISA plate at a concentration of 2, 1, 0.5, and 0.25 μg / mL, respectively; the antibody was diluted 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, and the benzopyrene standard was prepared into a solution with a concentration of 10 ng / mL; 50 μL of the serially diluted benzopyrene antibody (monoclonal antibody E6 prepared in Example 6) and 50 μL of 10 ng / mL benzopyrene standard were added to a 96-well ELISA plate, and an indirect competitive ELISA was performed according to the method of Example 5, and the antibody concentration and coating concentration combination with an absorbance value of 1.0 to 1.5 were read by an ELISA reader.

[0227] 2. Experimental Results

[0228] Using the drug concentration as the abscissa and B / B0 as the ordinate, a standard curve was plotted using the normalization method. The half-maximal inhibitory concentration IC50 was calculated based on the standard curve, and the maximum absorbance Amax was selected. The best working concentration was determined by choosing the lowest IC50 value and the highest Amax / IC50 value. The optimal coating concentration of this antibody (monoclonal antibody E6) was 1 μg / mL, and the antibody dilution factor was 8K.

[0229] Example 8 A method for detecting benzo[a]pyrene

[0230] An indirect competitive ELISA method for detecting benzo[a]pyrene, comprising the following steps:

[0231] (1) Using the benzo[a]pyrene artificial antigen 3 (benzo[a]pyrene hapten 2-BSA, whose structural formula is as shown in formula (IV-1)) prepared in Example 5 as the coating antigen, it was diluted to 1 μg / mL with carbonate buffer (CB, 0.1 M, pH = 9.8), and a 96-well enzyme-linked immunosorbent assay (ELISA) plate was coated, with 100 μL added to each well, and incubated overnight (12 h) at 37°C;

[0232]

[0233] (2) Discard the coating solution, wash twice with a washing solution (pH value of 7.4, containing 0.6% Tween-20, 0.02% sodium azide preservative, 0.2 mol / L phosphate buffer, the percentages are weight / volume percentages), and pat dry;

[0234] (3) Add 120 μL of blocking solution (i.e., 5% skim milk powder by mass ratio) to each well, and block at 37°C for 3 h;

[0235] (4) Discard the blocking solution, tap the plate, take it out after drying at 37°C for 30 min, and store it in a self-sealing bag for later use;

[0236] (5) Dilute the monoclonal antibody prepared in Example 6 1:8000-fold with phosphate buffer (PBS, 0.01 M, pH = 7.4), and serially dilute the benzo[a]pyrene drug to be detected 2-fold to 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL, 3.91 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.12 ng / mL, 0.06 ng / mL;

[0237] (6) Add 50 μL of the benzo[a]pyrene drug dilution to be detected (in triplicate) to each row, then add 50 μL / well of the monoclonal antibody dilution prepared in Example 6, incubate at 37°C for 40 min, wash five times with the washing solution, and pat dry;

[0238] (7) Add the enzyme conjugate (goat anti-mouse secondary antibody labeled with horseradish peroxidase) diluted 5000-fold with phosphate-buffered saline with Tween (PBST, 0.01 M), 100 μL per well, incubate at 37 °C for 30 min, wash five times with the washing solution, and pat dry;

[0239] (8) Add the chromogenic solution with a volume ratio of solution A to solution B of 1:1, 100 μL per well, incubate at 37 °C for color development for 10 min;

[0240] (9) Add 50 μL of the termination solution (10% H2SO4 solution) to terminate the reaction, and read the OD value at 450 nm.

[0241] (10) Operate according to the above steps (1) to (9), replace the diluted benzo[a]pyrene solution to be measured in step (6) with the diluted sample solution to be measured after extraction, and combine with the standard curve drawn to determine the actual content of benzo[a]pyrene drug in the unknown sample.

[0242] II. Experimental Results

[0243] The indirect competitive ELISA standard curve of the antibody for detecting benzo[a]pyrene drug is as Figure 8 shown. It can be seen from Figure 8 that the half-inhibition concentration (IC 50 ) of the antibody for detecting benzo[a]pyrene drug is 4.07 ng / mL, the quantitative detection range is 0.91 - 18.22 ng / mL, and the lowest detection limit (LOD) is 0.38 ng / mL; it shows that the antibody for detecting benzo[a]pyrene prepared by the present invention has high sensitivity and can meet the detection requirements.

[0244] Example 9 A kit for detecting benzo[a]pyrene

[0245] I. Composition

[0246] The kit for detecting benzo[a]pyrene contains the following parts:

[0247] (1) Preparation of the enzyme-labeled plate coated with the coating antigen: Use the benzo[a]pyrene artificial antigen 3 prepared in Example 5 (benzo[a]pyrene hapten 2-BSA, whose structural formula is as shown in Formula (IV-1)) as the coating antigen. Dilute the coating antigen to 1 μg / mL with carbonate buffer (CB, 0.1 M, pH = 9.8), add 100 μL to each well, incubate overnight at 37 °C in the dark, pour out the liquid in the wells, wash 2 times with the washing solution, 30 s each time, pat dry, then add 120 μL of the blocking solution to each well, incubate at 37 °C in the dark for 2 h, pour out the liquid in the wells and pat dry, and store in a vacuum-sealed manner with an aluminum film after drying; among them, the coating buffer is 0.05 mol / L carbonate buffer with a pH value of 9.6, and the blocking solution has a pH value of 7.1 - 7.5 and contains 1% - 3% casein by mass ratio and 0.1 - 0.3 mol / L phosphate buffer;

[0248] (2) Benzo[a]pyrene standard solution: 15 concentration gradients, which are 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL, 3.91 ng / mL, 1.95 ng / mL, 0.98 ng / mL, 0.49 ng / mL, 0.24 ng / mL, 0.12 ng / mL, 0.06 ng / mL respectively;

[0249] (3) Benzo[a]pyrene monoclonal antibody prepared in Example 6 (monoclonal antibody E6);

[0250] (4) Enzyme conjugate: Horseradish peroxidase-labeled goat anti-mouse secondary antibody;

[0251] (5) Substrate chromogenic solution: Composed of solution A and solution B in a volume ratio of 1:1. Solution A is urea peroxide, and solution B is tetramethylbenzidine;

[0252] (6) The termination solution is 10% (v / v) H2SO4;

[0253] (7) The washing solution has a pH value of 7.4 and contains 0.6% (w / v) Tween - 20, 0.02% (w / v) sodium azide preservative, and 0.2 mol / L phosphate buffer, and the percentages are weight - volume percentages.

[0254] II. Usage method

[0255] Same as Example 8.

[0256] Sensitivity of the detection of benzo[a]pyrene and other common polycyclic aromatic hydrocarbon compounds in Example 10

[0257] I. Experimental method

[0258] Using the kit of Example 9, 50 μL of benzopyrene standards of a series of concentrations and 50 μL of benzopyrene antibody prepared in Example 6 diluted 8K were added to a 96-well ELISA plate, and the absorbance (OD) was measured by an ELISA analyzer after an indirect competitive ELISA reaction.

[0259] 2. Experimental Results

[0260] With B / B0 as the ordinate and the corresponding standard concentration as the abscissa, the standard curve was drawn using the normalization method. The half-maximal inhibitory concentration was the IC 50 , with IC 10 The detection limit is IC 20 ~IC 80 The standard curve of ELISA was established with benzopyrene as the standard. The results are shown in Figure 8 , the relevant standard curve parameters are shown in Table 2.

[0261] Table 2 Detection parameters of benzopyrene by benzopyrene antibody

[0262]

[0263] Combination Figure 8 As shown in Table 2, the standard curve established with benzopyrene as the standard substance has a typical S-shaped curve, indicating that the detection sensitivity using the antibody of the present invention is good.

[0264] Example 11 Specificity of detection of benzopyrene and other common polycyclic aromatic hydrocarbons

[0265] 1. Experimental Methods

[0266] The kit of Example 9 was used to detect benzopyrene and other common polycyclic aromatic hydrocarbon drugs.

[0267] 2. Experimental Results

[0268] The results are shown in Table 3. The calculation method of the cross-reaction rate is shown in Formula 2.

[0269]

[0270] Table 3:

[0271]

[0272]

[0273] As shown in Table 3, the cross-reactivity rate of the antibody to benzopyrene is 100%, and the IC 50 The value is 4.07 ng / mL, and the cross-reaction rate to other polycyclic aromatic hydrocarbons is less than 5%, indicating that the antibody of the present application can specifically recognize benzopyrene and further detect the content of benzopyrene.

[0274] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description and ideas. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A benzo[a]pyrene hapten, characterized in that, Its structural formula is shown in formula (I).

2. A benzo[a]pyrene artificial antigen, characterized in that, It is the benzo[a]pyrene hapten-conjugated carrier protein described in claim 1, and its structural formula is shown in formula (III).

3. A benzo[a]pyrene hapten, characterized in that, Its structural formula is shown in formula (II). 。 4. A benzo[a]pyrene artificial antigen, characterized in that, It is the benzo[a]pyrene hapten-conjugated carrier protein described in claim 3, and its structural formula is shown in formula (IV).

5. Use of the benzo[a]pyrene hapten described in claim 1 and / or claim 3 in the preparation of a benzo[a]pyrene artificial antigen.

6. A composition for detecting benzo[a]pyrene, characterized in that, Containing the benzo[a]pyrene artificial antigen described in claims 2 and 4, the benzo[a]pyrene artificial antigen described in claim 2 as an immunogen, and the benzo[a]pyrene artificial antigen described in claim 4 as a coating antigen.

7. The composition according to claim 6, wherein The carrier protein of the benzo[a]pyrene artificial antigen described in claim 2 is lactoferrin as an immunogen, and the carrier protein of the benzo[a]pyrene artificial antigen described in claim 4 is bovine serum albumin as a coating antigen.

8. Use of the benzo[a]pyrene hapten described in claim 1 and / or claim 3, the benzo[a]pyrene artificial antigen described in claim 2 and / or claim 3, and / or the composition described in claim 6 in the preparation of a benzo[a]pyrene detection kit.

9. A detection kit for benzopyrene, characterized in that, Containing the composition described in claim 6.

10. A method for detecting benzo[a]pyrene for non-diagnostic purposes, characterized in that, Using the composition described in claim 6.

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