Preparation method of acenaphthene or fluorene artificial antigen

Abstract

The invention belongs to the technical field of polycyclic aromatic hydrocarbon artificial antigen synthesis, and relates to a novel synthesis method of a complete antigen for detecting polycyclic aromatic hydrocarbon (PAHs)-acenaphthene or fluorene. The hapten is successfully prepared through Friedel-Crafts acylation reaction, and the hapten is esterified and then coupled with carrier protein to prepare the complete antigen. These complete antigens have been identified. The invention provides a novel and efficient immunoassay technique for the detection of acenaphthene or fluorene, and achieves highly specific polyclonal antibody generation by immunoassay methods for optimizing antibody production in diagnostic and therapeutic applications associated with acenaphthene or fluorene exposure.

Description

Technical Field

[0001] The present invention belongs to the field of polycyclic aromatic hydrocarbon artificial antigen synthesis, and specifically relates to a method for preparing an acenaphthene or fluorene artificial antigen. The present invention prepares an immunogen through a specific chemical reaction, aims to improve the sensitivity and accuracy of acenaphthene or fluorene detection, and is suitable for environmental monitoring, public health assessment and related disease diagnosis and treatment. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are organic compounds containing at least two aromatic rings. Due to their carcinogenic potential, even trace levels in the environment can pose a significant risk to human health. PAHs enter the human body mainly through breathing and skin contact. Traditional PAHs detection methods mainly include high-performance liquid chromatography (HPLC) and gas chromatography (GC), which require complex sample pretreatment steps such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and supercritical fluid extraction (SFE). Nevertheless, the application of these methods is limited by their operational complexity and time consumption.

[0003] Immunoassay is an analytical technique that utilizes the specific recognition and binding between antigens and antibodies. It is widely used due to its high sensitivity, simple operation and low cost.

[0004] Acenaphthene and fluorene are both polycyclic aromatic hydrocarbons with small molecular weight and similar structure. They do not have obvious reactivity and immunogenicity, and lack active groups such as amino or carboxyl groups, which limits their direct coupling with carrier proteins and their ability to induce specific antibodies in animals. This makes it challenging to directly use acenaphthene or fluorene for immunoassay.

[0005]

[0006] In order to effectively detect acenaphthene or fluorene, it is necessary to synthesize acenaphthene or fluorene antigens with high titer. Although there are no extensive reports on acenaphthene or fluorene antigens, the design and synthesis of effective acenaphthene or fluorene or fluorene antigens and related antibodies is still a technical difficulty in immunoassay technology. Summary of the invention

[0007] The purpose of the present invention is to synthesize artificial antigens against acenaphthene or fluorene.

[0008] The purpose of the present invention can be achieved by the following measures:

[0009] A method for preparing an artificial antigen of acenaphthene or fluorene, characterized in that it is prepared according to the following steps:

[0010] a. The acenaphthene or fluorene, aluminum chloride and succinic anhydride, glutaric anhydride or adipic anhydride are subjected to Friedel-Crafts acylation to obtain the compound shown in formula I

[0011] Compounds;

[0012] b. The compound shown in formula Ⅰ is added with a coupling agent and a polar solvent to be mixed for activation to obtain an activated hapten solution; wherein the coupling agent

[0013] are dicyclohexylcarbodiimide and N-hydroxysuccinimide;

[0014] c. mixing the activated hapten solution and the carrier protein buffer solution to perform a coupling reaction to obtain the acenaphthene or fluorene artificial antigen;

[0016]

[0017]

[0018] The method for preparing an artificial antigen of acenaphthene or fluorene is characterized in that in step a, the molar ratio of acenaphthene or fluorene, aluminum chloride and succinic anhydride, glutaric anhydride or adipic anhydride is 1:2-3:1-2; the aluminum chloride is anhydrous aluminum chloride; the reaction temperature of the Friedel-Crafts acylation is 0-25°C, and the reaction time is 12-24h. The aluminum chloride is preferably anhydrous aluminum chloride; the temperature of the Friedel-Crafts acylation reaction is preferably 0-25°C, and the reaction time is preferably 12-24h;

[0019] The method for preparing an artificial antigen of acenaphthene or fluorene is characterized in that the polar solvent in step b is dimethyl sulfoxide, the dosage ratio of the acenaphthene or fluorene hapten to the polar solution is 10 mg: 100-300 μL; and the activation temperature is 0-25°C.

[0020] The method for preparing an artificial antigen of acenaphthene or fluorene is characterized in that the carrier protein in step c is at least one of bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.

[0021] The method for preparing an artificial antigen of acenaphthene or fluorene is characterized in that the molar ratio of the carrier protein and the acenaphthene or fluorene hapten in step c is preferably 1:20-50; the buffer solution is PBS buffer solution; the activated hapten solution is added dropwise to the buffer solution of the carrier protein, the temperature of the coupling reaction is 0-27°C; and the time of the coupling reaction is preferably 12-24h.

[0022] Further optimization methods:

[0023] In an ice bath and mechanically stirred, acenaphthene or fluorene, aluminum trichloride, succinic anhydride, glutaric acid liver or adipic anhydride and dichloromethane are mixed for Friedel-Crafts acylation reaction, wherein the molar ratio of acenaphthene or fluorene, aluminum trichloride and succinic anhydride, glutaric acid liver or adipic anhydride is preferably 1:(2-3):(1-2), more preferably; the aluminum trichloride is preferably anhydrous aluminum trichloride; the temperature of the Friedel-Crafts acylation reaction is preferably 0-25°C, and the time is preferably 12-24h; in a specific embodiment of the present invention, the Friedel-Crafts acylation reaction is preferably carried out under N2 protection conditions, and the reaction progress is monitored by TLC. Purification by column chromatography to obtain a pure product. The temperature of the Friedel-Crafts acylation reaction is preferably 0-25°C, and the time is preferably 12-24h.

[0024] The acenaphthene or fluorene hapten, coupling agent and polar solvent described in the above scheme are mixed for activation to obtain an activated hapten solution; the activated hapten solution and the buffer solution of the carrier protein are mixed for coupling reaction to obtain the acenaphthene or fluorene antigen. The present invention mixes the acenaphthene or fluorene hapten, coupling agent and polar solvent described in the above scheme for activation to obtain an activated hapten solution. In the present invention, the polar solvent is preferably dimethyl sulfoxide, and the amount ratio of the acenaphthene or fluorene hapten and the polar solution is preferably 10 mg: 100-300 μL; the coupling agent is preferably dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), the activation temperature is preferably 0-25°C, more preferably 4°C, and the activation time is preferably 12-24h. After the activation is completed, it is preferably centrifuged at 4000-8000r / min for 15-20min, more preferably 15min, 8000r / min to obtain the activated hapten solution. After obtaining the activated hapten solution, the present invention mixes the activated hapten solution with a buffer solution of a carrier protein to perform a coupling reaction to obtain the acenaphthene or fluorene antigen.

[0025] In the present invention, when preparing the artificial antigen, the molar ratio of the carrier protein to the acenaphthene or fluorene hapten is preferably 1:(20-50); the buffer solution of the carrier protein is preferably obtained by dissolving the carrier protein in a PBS buffer solution, and the concentration of the carrier protein PBS buffer is preferably 0.02 mol / L; the present invention preferably adds the activated hapten solution dropwise to the buffer solution of the carrier protein, the temperature of the coupling reaction is preferably 0-27°C, more preferably 4°C, and the time of the coupling reaction is preferably 12-24h.

[0026] After the coupling reaction is completed, the present invention preferably centrifuges the obtained reaction solution, draws the supernatant, and dialyzes the supernatant to obtain the acenaphthene or fluorene antigen. In the present invention, the molecular weight cutoff of the dialysis bag for dialysis is preferably 8000-12000, and the dialysis bag is preferably treated with EDTA solution and washed with distilled water before use; the concentration of the EDTA solution is preferably 0.2 mol / L; when preparing the acenaphthene or fluorene coating source, the dialysis solution is preferably CBS buffer, and the concentration of the PBS buffer is preferably 0.02 mol / L; when preparing the acenaphthene or fluorene immunogen, the dialysis solution is preferably PBS buffer, and the concentration of the PBS buffer is preferably 0.02 mol / L; the dialysis time is preferably 3-5 days, and the dialysis solution is replaced twice a day. After the dialysis is completed, it is preferred to precipitate the solution in the dialysis bag and freeze it at -20°C for standby use.

[0027] Beneficial effects:

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. The present invention obtains acenaphthene or fluorene artificial antigens through a series of multi-step reactions: Friedel-Crafts acylation reaction and activated ester reaction coupled with carrier protein, and improves the coupling efficiency and specificity with carrier protein by introducing active groups, wherein the generated artificial antigens have different carbon positions, different spacer arm lengths, and different carrier proteins, which will affect the coupling efficiency and specificity of the antigen; for this reason, the inventors comprehensively considered the results of DFT and molecular docking, and found that the hapten with a four-carbon link arm length is optimal when the 2nd position of fluorene is substituted in the present invention. And the hapten with a four-carbon link arm length is optimal when the 3rd position of acenaphthene is substituted. Therefore, the substitution position and the length of the substituent in the present invention affect the effect of the antigen, which is superior to other position substituents. ELSIA confirmed that the artificial antigen of acenaphthene or fluorene obtained by the present invention has good antibody titer, so the experimental results are in line with expectations, that is, as an artificial antigen, it can induce specific antibodies in animals. The present invention has a short synthetic route, readily available raw materials, mild conditions, simple operation, and high product purity.

[0030] 2. The present invention effectively simplifies the synthesis process of acenaphthene or fluorene artificial antigens through Friedel-Crafts acylation reaction and activated ester reaction. These reaction conditions are mild and the operation is simple, which greatly reduces the complexity of the synthesis process and improves the convenience of operation and the repeatability of the experiment. DETAILED DESCRIPTION

[0031] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0032] Example 1

[0033]

[0034] Preparation method of compound I: 30 mL of anhydrous dichloromethane, 0.924 g (6 mmol) of acenaphthene and 1.76 g (13.2 mmol) of anhydrous aluminum trichloride were added to a 100 mL three-necked round-bottom flask at 0°C, under N2 protection and mechanical stirring. After the aluminum trichloride was completely dissolved, a dichloromethane solution containing 0.6 g (6 mmol) of succinic anhydride was slowly added dropwise. The reaction solution changed from brown to dark green after about 30 minutes of dripping. The reaction was then stirred for about 24 hours. The pure compound I was obtained by column chromatography separation and purification.

[0035] Preparation method of acenaphthene artificial antigen II: Stir a mixture of 76.8 mg (0.32 mmol) II and 1.5 mL anhydrous DMF at 0 ° C, and then add 1.5 mL anhydrous DMF containing 98.9 mg (0.48 mmol) DCC and 55.2 mg, (0.48 mmol) NHS within 10 minutes. React for 24 hours under magnetic stirring at 4 ° C. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 664.3 mg (0.1 mmol) BSA and dissolve it in 8 mL PBS buffer (pH 7.4), stir and dissolve to prepare the carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution and react at 4 ° C for 12 hours. After centrifugation, take the supernatant, dialyze with PBS at 4 ° C for 3 days, change the dialysate twice a day, and centrifuge to obtain the supernatant after the end to obtain the artificial antigen, which is freeze-dried for use.

[0036] Example 2

[0037]

[0038] Preparation method of compound III: 30 mL of anhydrous dichloromethane, 0.924 g (6 mmol) of acenaphthene and 1.76 g (13.2 mmol) of anhydrous aluminum trichloride were added to a 100 mL three-necked round-bottom flask at 0°C, under N2 protection and mechanical stirring. After the aluminum trichloride was completely dissolved, a dichloromethane solution containing 0.684 g (6 mmol) of glutaric anhydride was slowly added dropwise. The reaction solution changed from brown to dark green after about 30 minutes of dripping. The reaction was then stirred for about 24 hours. The pure compound III was obtained by column chromatography separation and purification.

[0039] Preparation method of acenaphthene artificial antigen IV: Stir a mixture of 85.77 mg (0.32 mmol) IV and 1.5 mL anhydrous DMF at 0 ° C, and then add 1.5 mL anhydrous DMF containing 98.9 mg (0.48 mmol) DCC and 55.2 mg, (0.48 mmol) NHS within 10 minutes. React for 24 hours under magnetic stirring at 4 ° C. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 445 mg (0.1 mmol) OVA and dissolve it in 8 mL CBS buffer (pH 9.6), stir and dissolve to prepare a carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution and react at 4 ° C for 12 hours. After centrifugation, take the supernatant, dialyze with CBS at 4 ° C for 3 days, change the dialysate twice a day, and centrifuge to obtain the artificial antigen after the end, and freeze-dry for use.

[0040] Example 3

[0041]

[0042] Preparation method of compound V: 30 mL of anhydrous dichloromethane, 0.924 g (6 mmol) of acenaphthene and 1.76 g (13.2 mmol) of anhydrous aluminum chloride were added to a 100 mL three-necked round-bottom flask at 0 ° C. Under N2 protection and mechanical stirring, after the aluminum chloride was completely dissolved, a dichloromethane solution containing 0.768 g (6 mmol) of adipic anhydride was slowly added dropwise. The reaction solution changed from brown to dark green after about 30 minutes of dripping. The reaction was stirred for about 24 hours. The pure compound V was obtained by column chromatography separation and purification.

[0043] Preparation method of acenaphthene artificial antigen VI: Stir a mixture of 1.34 mg (5 μmol) VII and 200 μL anhydrous DMF at 0°C, then add 300 μL anhydrous DMF containing 1.54 μL DCC and 0.69 mg (6 μmol) NHS within 10 minutes. React under magnetic stirring at 4°C for 24 hours. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 10 mg KLH and dissolve it in 1 mL PBS buffer (pH 7.4), stir and dissolve to prepare a carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution and react at 4°C for 12 hours. After centrifugation, take the supernatant, dialyze with PBS at 4°C for 3 days, change the dialysate twice a day, centrifuge and take the supernatant to obtain the artificial antigen, and freeze-dry for use.

[0044] Example 4

[0045]

[0046] Preparation method of compound VII: 30 mL of anhydrous dichloromethane, 1.00 g (6 mmol) of fluorene and 1.76 g (13.2 mmol) of anhydrous aluminum trichloride were added to a 100 mL three-necked round-bottom flask at 0 ° C, under N2 protection and mechanical stirring. After the aluminum trichloride was completely dissolved, a dichloromethane solution containing 0.6 g (6 mmol) of succinic anhydride was slowly added dropwise. The reaction solution changed from brown to dark green after about 30 minutes of dripping. The reaction was stirred for about 24 hours. The pure compound VII was obtained by column chromatography separation and purification.

[0047] Preparation method of artificial fluorene antigen VIII: Stir a mixture of 81 mg (0.32 mmol) of II and 1.5 mL of anhydrous DMF at 0°C, then drop 1.5 mL of anhydrous DMF containing 98.9 mg (0.48 mmol) of DCC and 55.2 mg (0.48 mmol) of NHS within 10 minutes. React under magnetic stirring at 4°C for 24 hours. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 664.3 mg (0.1 mmol) of BSA and dissolve it in 8 mL of PBS buffer (pH 7.4), stir and dissolve to prepare a carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution, and react at 4°C for 12 hours. After centrifugation, take the supernatant, dialyze with PBS at 4°C for 3 days, change the dialysate twice a day, and centrifuge to obtain the artificial antigen. The artificial antigen is freeze-dried for use.

[0048] Example 5

[0049]

[0050] Preparation method of compound IX: 30 mL of anhydrous dichloromethane, 1.00 g (6 mmol) of fluorene and 1.76 g (13.2 mmol) of anhydrous aluminum chloride were added to a 100 mL three-necked round-bottom flask at 0°C under N2 protection and mechanical stirring. After the aluminum chloride was completely dissolved, a dichloromethane solution containing 0.684 g (6 mmol) of glutaric anhydride was slowly added dropwise. The reaction solution changed from brown to dark green after about 30 minutes of dripping. The reaction was then stirred for about 24 hours. The pure compound IX was obtained by column chromatography separation and purification.

[0051] Preparation method of artificial fluorene antigen X: Stir a mixture of 90 mg (0.32 mmol) IV and 1.5 mL anhydrous DMF at 0°C, then drop 1.5 mL anhydrous DMF containing 98.9 mg (0.48 mmol) DCC and 55.2 mg (0.48 mmol) NHS within 10 minutes. React under magnetic stirring at 4°C for 24 hours. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 445 mg (0.1 mmol) OVA and dissolve it in 8 mL CBS buffer (pH 9.6), stir and dissolve to prepare a carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution, and react at 4°C for 12 hours. After centrifugation, take the supernatant, dialyze with CBS at 4°C for 3 days, and change the dialysate twice a day. After the end, centrifuge and take the supernatant to obtain the artificial antigen, which is freeze-dried for use.

[0052] Example 6

[0053]

[0054] Preparation method of compound XI: 30 mL of anhydrous dichloromethane, 1.00 g (6 mmol) of fluorene and 1.76 g (13.2 mmol) of anhydrous aluminum chloride were added to a 100 mL three-necked round-bottom flask in sequence under N2 protection and mechanical stirring at 0°C. After the aluminum chloride was completely dissolved, a dichloromethane solution containing 0.768 g (6 mmol) of adipic anhydride was slowly added dropwise. The reaction solution changed from brown to dark green in about 30 minutes. The reaction was stirred for about 24 hours. The pure compound XI was obtained by column chromatography separation and purification.

[0055] Preparation method of artificial fluorene antigen XII: Stir a mixture of 1.47 mg (5 μmol) VII and 200 μL anhydrous DMF at 0°C, then add 300 μL anhydrous DMF containing 1.54 μL DCC and 0.69 mg (6 μmol) NHS dropwise within 10 minutes. React under magnetic stirring at 4°C for 24 hours. Remove the formed precipitate by centrifugation at 8000 r / min for 15 minutes, and take the supernatant as the activated ester solution. Weigh 10 mg KLH and dissolve it in 1 mL PBS buffer (pH 7.4), stir and dissolve to prepare the carrier protein solution. Under magnetic stirring, gradually drop the activated ester solution into the carrier protein solution and react at 4°C for 12 hours. After centrifugation, take the supernatant, dialyze with PBS at 4°C for 3 days, change the dialysate twice a day, centrifuge and take the supernatant to obtain the artificial antigen, and freeze-dry for use.

[0056] Example 7

[0057] Fluorene hapten

[0058] 1. Computer-aided modeling

[0059] a. In the present invention, density functional theory (DFT) is used based on the B3LYP / 6-31G** basis set to design a variety of hapten structures based on fluorene with different substitution sites and different linker arm lengths. The electronic properties, energy levels of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), and the HOMO-LUMO energy gap ΔE of these hapten structures are calculated and analyzed to evaluate their potential for preparing completely artificial antigens after esterification and combination with carrier proteins.

[0060] b. Using Auto Dock Tools (Ver1.5.7), the carrier protein underwent dehydration, desolvation, hydrogenation and charge addition operations. The screened structure was used as the molecular docking simulation verification with the carrier protein after hapten esterification. Its two-dimensional structure was constructed using ChemDraw software. It was pre-optimized by the MM2 module of Chem3D, fully optimized by DFT (Gaussian09, B3LYP / 6-31G** basis set), and hydrogenated. During docking, semi-flexible technology was used to take into account the flexibility of receptor and ligand molecules. The optimal docking configuration determined by the lowest energy is visualized in the three-dimensional molecular docking diagram.

[0061] Based on the results of DFT and molecular docking, the hapten with a four-carbon linker arm length and a 2-position substitution of fluorene in the present invention is optimal. Its structure is as follows

[0062]

[0063] Acenaphthene hapten

[0064] 1. Computer-aided modeling

[0065] a. In the present invention, density functional theory (DFT) was used to design a variety of hapten structures based on acenaphthene with different substitution sites and different linker arm lengths based on acenaphthene based on the B3LYP / 6-31G** basis set. The electronic properties, energy levels of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), and the HOMO-LUMO energy gap ΔE of these hapten structures were calculated and analyzed to evaluate their potential for preparing completely artificial antigens after esterification and combination with carrier proteins.

[0066] b. Using Auto Dock Tools (Ver1.5.7), the carrier protein underwent dehydration, desolvation, hydrogenation and charge addition operations. The screened structure was used as the molecular docking simulation verification with the carrier protein after hapten esterification. Its two-dimensional structure was constructed using ChemDraw software. It was pre-optimized by the MM2 module of Chem3D, fully optimized by DFT (Gaussian09, B3LYP / 6-31G** basis set), and hydrogenated. During docking, semi-flexible technology was used to take into account the flexibility of receptor and ligand molecules. The optimal docking configuration determined by the lowest energy is visualized in the three-dimensional molecular docking diagram.

[0067] Based on the results of DFT and molecular docking, the hapten with acenaphthene substituted at position 3 and a four-carbon linker length is optimal. Its structure is as follows

[0068]

[0069] Example 8 Antigen-antibody titer detection

[0070] Entrust Detai Biotechnology to conduct antigen and antibody titer testing:

[0071] 1. Antigen:

[0072] Immunogen: γ-Carboxyl-2-fluorenebutyric acid-BSA

[0073] Screening agent: γ-Carboxyl-2-fluorenylbutyric acid-OVA

[0074] Immune host:

[0075] New Zealand White Rabbit

[0076] Antibody concentration:

[0077] Anti-y-carbonyl-2-fluorenebutyric acid-A: 0.37 mg / ml;

[0078] Anti-y-carbonyl-2-fluorenebutyric acid-B: 0.65 mg / ml; the antibody concentration was determined by a microspectrophotometer.

[0079] The antibody detection method is the conventional ELSIA method:

[0080] The results are as follows:

[0081] ELISA titer:

[0082] Anti-y-carbonyl-2-fluorenebutyric acid Antibody-A:>1:256,000; S / C.0=4.62 (Standard S / C.0>1)

[0083] Anti-y-carbonyl-2-fluorenebutyric acid Antibody-B:>1:256.000; S / C.0=4.95 (Standard S / C.0>1)

[0084] Quality Assurance

[0085]

[0086] Titer: the maximum dilutiin when Sample OD / NCOD≥2.1;

[0087] NC, Negative Control; S, Sample OD value; CO, Cut-Off OD value = 2.1*NC

[0088] 2. Antigen:

[0089] Immunogen:ε-Carbonyl-2-fluorenylhexanoic acid-BSA

[0090] Screening source: ε-Carbonyl-2-fluorenylhexanoic acid-KIH

[0091] Immune host:

[0092] New Zealand White Rabbit

[0093] Antibody concentration:

[0094] Anti-ε-carbonyl-2-fluorenylhexanoic acid-A: 0.57 mg / ml;

[0095] Anti-ε-carbonyl-2-fluorenehexanoic acid-B: 0.51 mg / ml; Antibody concentration was determined by micro-spectrophotometer; ELISA titer:

[0096] Anti-ε-carbonyl-2-fluorenehexanoic acid Antibody-A:>1:256,000; S / C.0=2.99 (Standard S / C.0>1) Anti-s-carbonyl-2-fluorenehexanoic acid Antibody-B>1:256.000; S / C.0=2.77 (Standard S / C.0>1)

[0097] Quality Assurance

[0098]

[0099] Titer: the maximum dilution when Sample OD / NCOD≥2.1;

[0100] NC, Negative Control; S, Sample OD value; CO, Cut-Off OD value = 2.1*NC

[0101] Although the present invention has been described in detail with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art should be able to make various modifications and changes without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be deemed to be the scope defined by the attached claims.

Claims

1. A method for preparing an artificial antigen of acenaphthene or fluorene, characterized in that: It is prepared according to the following steps: a. The compound Ⅰ shown in formula is prepared by Friedel-Crafts acylation reaction of acenaphthene or fluorene, aluminum chloride and succinic anhydride, glutaric anhydride or adipic anhydride; b. adding a coupling agent and a polar solvent to the compound represented by formula Ⅰ and mixing them for activation to obtain an activated hapten solution; in The coupling agents are dicyclohexylcarbodiimide and N-hydroxysuccinimide; c. The activated hapten solution and the carrier protein buffer solution are mixed to perform a coupling reaction to obtain the acenaphthene or fluorene artificial antigen 2. The method for preparing an artificial antigen of acenaphthene or fluorene according to claim 1, characterized in that: In step a, the molar ratio of acenaphthene or fluorene, aluminum chloride and succinic anhydride, glutaric anhydride or adipic anhydride is 1:2-3:1-2; the aluminum chloride is anhydrous aluminum chloride; the reaction temperature of the Friedel-Crafts acylation is 0-25°C, and the reaction time is 12-24h. The aluminum chloride is preferably anhydrous aluminum chloride; the temperature of the Friedel-Crafts acylation reaction is preferably 0-25°C, and the reaction time is preferably 12-24h.

3. The method for preparing an artificial antigen of acenaphthene or fluorene according to claim 1, characterized in that: In step b, the polar solvent is dimethyl sulfoxide, the usage ratio of the acenaphthene or fluorene hapten to the polar solution is 10 mg: 100-300 μL; and the activation temperature is 0-25°C.

4. The method for preparing an artificial antigen of acenaphthene or fluorene according to claim 1, characterized in that: The carrier protein in step c is at least one of bovine serum albumin, ovalbumin or keyhole limpet hemocyanin.

5. The method for preparing an artificial antigen of acenaphthene or fluorene according to claim 1, characterized in that: In step c, the molar ratio of the carrier protein to the acenaphthene or fluorene hapten is preferably 1:20-50; the buffer solution is a PBS buffer solution; the activated hapten solution is added dropwise to the buffer solution of the carrier protein, the temperature of the coupling reaction is 0-27° C.; the time of the coupling reaction is preferably 12-24 hours.

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