An arecoline artificial hapten, an artificial antigen, and a preparation method and application thereof

Abstract

The application discloses a kind of areca alkaloid artificial hapten, artificial antigen and its preparation method and application, with norarecoline hydrochloride as the starting material for synthesizing artificial hapten.This areca alkaloid artificial hapten retains the characteristic structure of areca alkaloid to the greatest extent, and has active groups that can couple with carrier proteins, which can be used as antigenic determinants;Further, the areca alkaloid artificial antigen obtained can be used to immunize to obtain an anti-areca alkaloid artificial antibody with high affinity, high sensitivity and strong specificity.The titer of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:70000, and can be used for rapid and accurate immunodetection and immunoassay of areca alkaloid.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to an arecoline artificial hapten, an artificial antigen, its preparation method, and its application. Background Technology

[0002] Arecoline is an amine alkaloid extracted from the seeds of the areca palm (Areca catechu), a plant in the palm family. It is the main component of areca nut and has a certain degree of toxicity. Arecoline's chemical name is "N-methyl-1,2,5,6-tetrahydronicotinic acid methyl ester". In medicine, arecoline is used to treat glaucoma and can paralyze tapeworms, so it is also used as an anti-tapeworm agent. Its effect is enhanced when used with pumpkin seeds.

[0003] Arecoline is clinically used to treat postpartum uterine bleeding, poor uterine involution, and menorrhagia, and it also has a certain stimulating effect. However, it has carcinogenic and cancer-promoting properties. As more and more people enjoy eating areca nuts in recent years, more and more people are dying from cancer.

[0004] However, studies have shown that arecoline not only has carcinogenic and cancer-promoting properties, but also has a certain degree of addictiveness. Furthermore, different concentrations of arecoline can lead to delayed embryonic development and have mutagenic effects on mammals.

[0005] Currently, most domestic methods for analysis and determination include high performance liquid chromatography, gas chromatography, mass spectrometry, proton nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy, and infrared spectroscopy. However, due to the high cost of use and maintenance, these methods are not suitable for large-scale sampling and analysis. On the other hand, using artificially synthesized antigens for immunoassay has the advantages of convenient operation, high specificity, and fast measurement speed. Therefore, it is necessary to synthesize an artificial hapten or artificial antigen of arecoline to meet the needs of immunoassay.

[0006] Therefore, it is necessary to develop an economical, simple, and rapid detection method to detect arecoline levels in the human body, and the preparation of arecoline artificial antigen is the foundation for the realization of this method. Summary of the Invention

[0007] The first objective of this invention is to address the shortcomings of the prior art by providing an artificial hapten of arecoline.

[0008] An artificial hapten of arecoline, the molecular structure of which is shown in formula (Ⅰ):

[0009]

[0010] The second objective of this invention is to provide a method for preparing the aforementioned arecoline artificial hapten. This invention uses demethylarecoline hydrochloride as the starting material for synthesizing the artificial hapten, which not only retains the characteristic structure of arecoline to the greatest extent but also possesses active groups that can couple with carrier proteins, thus serving as antigenic determinants.

[0011] A method for preparing an artificial arecoline hapten includes the following steps:

[0012] Step (1): Dilute concentrated ammonia to obtain DAS solution;

[0013] Step (2): Dissolve demethylarecoline hydrochloride in deionized water, add the DAS solution obtained in step (1) to adjust the pH to alkaline, extract with dichloromethane multiple times, collect the dichloromethane phase, dry, filter, and dry to obtain colorless oily substance A.

[0014] Preferably, in step (2), DAS solution is added to adjust the pH to 9.

[0015] As a preferred option, the dichloromethane extraction in step (2) is performed three times. Under these reaction conditions, the yield of the colorless oily product A is relatively high, the post-processing procedure is relatively simple, and it is easier to purify.

[0016] Step (3): The colorless oily substance A obtained in step (2) is mixed with succinic anhydride in a molar ratio of 1:(1.5-2.5) in a solvent. 4-Dimethylaminopyridine is added as a catalyst, and the mixture is stirred and refluxed at 100-110℃ for 20-21 hours. After the reaction is complete, the arecoline artificial hapten is obtained by thin-layer chromatography.

[0017] Preferably, the solvent in step (3) is either pyridine or dichloromethane.

[0018] Preferably, in step (3), the molar ratio of colorless oily substance A to succinic anhydride is 1:2.

[0019] Preferably, the thin-layer chromatography in step (3) uses a developing solvent with a volume ratio of dichloromethane, 95% ethanol, 1,4-dioxane, and concentrated ammonia of 8:10:1:1. Under these reaction conditions, the yield of arecoline artificial hapten is high, the subsequent processing procedure is relatively simple, and it is easier to purify.

[0020] By using the above method, a linker arm is introduced at the N-position of arecoline. Introducing a linker arm at this modification site can preserve the arecoline-specific structure to the greatest extent.

[0021] Compared with using a circular linker arm, the present invention uses a straight chain as the linker arm, which can reduce non-specific binding during immunoassay and reduce the immune response against the linker arm during immunization, thereby increasing the probability of generating specific antibodies.

[0022] The third objective of this invention is to provide an artificial arecoline antigen, the molecular structure of which is shown in formula (II):

[0023]

[0024] BSA stands for bovine serum albumin.

[0025] A fourth objective of this invention is to provide a method for preparing the above-mentioned arecoline artificial antigen, comprising: binding the arecoline artificial hapten to bovine serum albumin via an active ester method to obtain the arecoline artificial antigen.

[0026] Specifically, the preparation of arecoline artificial antigen using the active ester method includes the following steps:

[0027] Step (1): Mix arecoline artificial hapten, N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in a molar ratio of 1:(1.35~1.5):(1.35~1.5) in N,N-dimethylformamide, stir and react at 20~30℃ for 18~20 hours, centrifuge after the reaction is completed, and take the supernatant.

[0028] Step (2): Add the supernatant to the bovine serum albumin solution, place the resulting mixture at 3-5°C and let it stand overnight. After dialysis and centrifugation, take the supernatant to obtain the arecoline artificial antigen.

[0029] Unless otherwise specified, the bovine serum albumin solution described in this invention is prepared by dissolving bovine serum albumin in 0.01M PBS buffer (pH 7.2-7.4).

[0030] Preferably, in step (2), the concentration of the bovine serum albumin solution is 5 mg / mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5.

[0031] This invention uses bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared to bovine gamma globulin (BGG): ① BSA can bind arecoline artificial hapten better and more extensively, thus preparing artificial antigens with different coupling ratios; ② Experiments show that after BSA binds to arecoline artificial hapten, the arecoline artificial hapten is not easy to preserve for a long time, is greatly affected by temperature, and is easily detached during subsequent processing, exhibiting poor stability. In contrast, the binding of BSA to arecoline artificial hapten can be preserved for a long time under different pH and temperature conditions and is less prone to detachment, demonstrating greater stability; ③ BSA is cheaper and more readily available than BGG, which can reduce costs to some extent.

[0032] A fifth object of the present invention is to provide the use of the arecoline artificial antigen in the preparation of anti-arecoline antibodies.

[0033] The sixth objective of this invention is to provide an anti-arecoline artificial antibody, obtained by animal immunization with the arecoline artificial antigen, which can produce a specific immune response to arecoline.

[0034] Experiments showed that immunizing New Zealand white rabbits with the arecoline artificial antigen yielded an immune serum titer of 1:70000. This indicates that the arecoline artificial antigen of the present invention can produce anti-arecoline antibodies with high affinity, high sensitivity, and strong specificity, which can be used for the immunodetection and analysis of arecoline.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The arecoline artificial hapten of the present invention retains the characteristic structure of arecoline to the greatest extent and has an active group that can couple with carrier proteins, and can serve as an antigenic determinant. The arecoline artificial antigen obtained by further preparation can be used to immunize and obtain anti-arecoline antibodies with high titer, high sensitivity and strong specificity. The polyclonal antibody obtained by immunizing New Zealand white rabbits can be used for rapid and accurate immunodetection and analysis of arecoline. Attached Figure Description

[0037] Figure 1 This is a liquid chromatogram of arecoline artificial hapten I of the present invention; where mV represents signal intensity and min represents minutes;

[0038] Figure 2 This is the mass spectrum of the arecoline artificial hapten I of the present invention; where Intens represents intensity and m / z represents mass-to-charge ratio.

[0039] Figure 3 The images show the ultraviolet scans of bovine serum albumin, arecoline artificial hapten I, and arecoline artificial antigen II; where Abs represents the ultraviolet-visible absorption spectrum and WL(nm) represents the wavelength (nm). Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0041] In this embodiment of the invention, the formulation of the MOP is as follows:

[0042] The mixture consisted of dichloromethane, 95% ethanol, 1,4-dioxane, and concentrated ammonia in a volume ratio of 8:10:1:1.

[0043] Example 1

[0044] This embodiment provides a method for preparing arecoline artificial antigen II, including the following steps:

[0045] (1) Preparation of arecoline artificial hapten I:

[0046] ① Dilute 5ml of concentrated ammonia solution with deionized water to 13ml to obtain DAS solution.

[0047] ② Weigh 150 mg (0.845 mmol) of demethylarecoline hydrochloride, dissolve it in 10 ml of deionized water, and adjust the pH to 9 with DAS solution. No significant change was observed. The aqueous phase was extracted with 15 ml of dichloromethane three times. The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 98 mg (0.695 mmol) of colorless, transparent oily substance A. TLC analysis was performed on this colorless oily substance A. The chromatographic solvent was MOP, and the product was R. f =0.8~0.9;

[0048] ③ The colorless oily substance A was dissolved in 4 ml of pyridine, and 10 mg (0.082 mmol) of 4-dimethylaminopyridine and 139 mg (1.39 mmol) of succinic anhydride were added. The mixture was reacted at 100 °C for 20 h. TLC detection showed that the reaction was basically complete, and the product R was obtained. f =0.2~0.3, and after separation by TLC (developing solvent is MOP, solvent and eluent are anhydrous ethanol), 150 mg (0.622 mmol) of arecoline artificial hapten I (as shown in Formula I) was obtained.

[0049] The liquid chromatogram of arecoline artificial hapten I is shown below. Figure 1 The mass spectrum of arecoline artificial hapten I is shown below. Figure 2 .

[0050] from Figure 1 It can be seen that the purity of the arecoline artificial hapten I obtained after purification reaches over 99%. Figure 2 It can be seen that the mass-to-charge ratio (m / z) of the M+H ion peak of the arecoline artificial hapten I obtained in this embodiment is 242.10, which is consistent with its theoretical relative molecular mass of 241. Therefore, it can be determined that the final compound obtained in step ② is the arecoline artificial hapten designed in this invention.

[0051] (2) Preparation of arecoline artificial antigen II:

[0052] ④ In a 50ml single-necked round-bottom flask, dissolve 150mg (0.622mmol) arecoline artificial hapten I in 7.5ml DMF, add 107mg (0.933mmol) NHS and 192mg (0.933mmol) DCC, stir and react at 20-25℃ for 18h, then stop the reaction. Centrifuge the reaction product and take the supernatant for later use.

[0053] ⑤ Weigh 14.5g (40.503mol) disodium hydrogen phosphate dodecahydrate, 43.875g (750mol) sodium chloride, and 1.495g (9.583mol) sodium dihydrogen phosphate dihydrate, dissolve them in deionized water, and bring the volume to 5.0L to obtain 0.01M PBS buffer with pH 7.4.

[0054] ⑥ Weigh 187.5 mg of bovine serum albumin and dissolve it in 37.5 ml of PBS buffer from step ④ to obtain a bovine serum albumin solution with a concentration of 5 mg / ml.

[0055] ⑦ Under rapid stirring, the supernatant from step ④ is slowly added dropwise to the bovine serum albumin solution from step ⑥. The volume ratio of the supernatant to the bovine serum albumin solution is 1:5. The resulting mixture is stored at 4°C overnight to obtain the artificial antigen mixture.

[0056] ⑧ Transfer the artificial antigen mixture into a dialysis bag and dialyze seven times with the PBS buffer from step ⑤. After dialysis, centrifuge and collect the supernatant to obtain the artificial antigen: arecoline-bovine serum albumin conjugate (as shown in Formula II). Its synthetic route is as follows:

[0057]

[0058] Wherein, DAS represents prepared ammonia solution, Pyridine represents pyridine, NHS represents N-hydroxysuccinimide, DCC represents cyclohexylcarbodiimide, DMF represents N,N-dimethylformamide, DMAP represents 4-dimethylaminopyridine, BSA represents bovine serum albumin, and the same applies below.

[0059] UV scan images of arecoline artificial antigen II before and after preparation are shown below. Figure 3 .

[0060] Figure 3 In the diagram, curve a represents the UV scan of arecoline artificial hapten I, curve b represents the UV scan of arecoline artificial antigen II, and curve c represents the UV scan of bovine serum albumin. The maximum absorption wavelength of arecoline artificial hapten I is 388 nm, while that of arecoline artificial antigen II is 368 nm. Compared with arecoline artificial hapten I and bovine serum albumin, the maximum absorption wavelength of arecoline artificial antigen II shows a significant change, indicating that arecoline artificial hapten I was successfully conjugated to bovine serum albumin.

[0061] Comparative Example 1

[0062] This embodiment provides a method for preparing arecoline artificial antigen IV, including the following steps:

[0063] (1) Preparation of arecoline artificial hapten III:

[0064] ① Dilute 5ml of concentrated ammonia solution with deionized water to 13ml to obtain DAS solution.

[0065] ② Weigh 150 mg (0.845 mmol) of demethylarecoline hydrochloride, dissolve it in 10 ml of deionized water, and adjust the pH to 9 with DAS solution. No significant change was observed. The aqueous phase was extracted with 15 ml of dichloromethane three times. The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 96 mg (0.680 mmol) of colorless, transparent oily substance A. TLC analysis was performed on this colorless oily substance A. The chromatographic solvent was MOP, and the product was R. f =0.8~0.9.

[0066] ③ Dissolve the colorless oily substance A in 3 ml of acetonitrile, add 286 μL (1.962 mmol) of tert-butyl bromoacetate and 256 μL (1.472 mmol) of N,N-diisopropylethylamine, and react at room temperature for more than 16 h. TLC (developing solvent: ethyl acetate, product R) f =0.8~0.9) The test showed that the reaction was basically complete. The reaction was stopped, and the product was dried to obtain a yellow impure oily product B, which was then directly introduced into the next step of the reaction.

[0067] ④ Dissolve the yellow oily substance B in 1.27 ml of dichloromethane, add 1.27 ml of trifluoroacetic acid, stir and react overnight at room temperature, and perform TLC (developing solvent: methanol, product spot R). f =0.3) The test showed that the reaction was basically complete. The reaction was stopped, and the trifluoroacetic acid was removed by azeotropic extraction three times with 10 ml × 3 toluene. 20 ml of deionized water was added and then extracted with 20 ml × 3 dichloromethane. The organic phase was collected, dried with anhydrous magnesium sulfate, filtered, and dried. The product was separated by TLC (the developing solvent was anhydrous methanol, and the solvent and eluent were anhydrous ethanol). Product R f =0.3) yielded 102 mg (0.513 mmol) of arecoline artificial hapten III (as shown in formula III).

[0068] (2) Preparation of arecoline artificial antigen:

[0069] ⑤ In a 50ml round-bottom flask, dissolve 102mg (0.513mmol) arecoline artificial hapten in 5.1ml DMF, add 89mg (0.770mmol) NHS and 159mg (0.770mmol) DCC, stir and react at 20-25℃ for 18h, stop the reaction, centrifuge, and take the supernatant for later use.

[0070] ⑥ Weigh 14.5g (40.503mol) disodium hydrogen phosphate dodecahydrate, 43.875g (750mol) sodium chloride, and 1.495g (9.583mol) sodium dihydrogen phosphate dihydrate, dissolve them in deionized water, and bring the volume to 5.0L to obtain 0.01M PBS buffer with pH 7.4.

[0071] ⑦ Weigh 127.5 mg of bovine serum albumin and dissolve it in 25.5 ml of PBS buffer to obtain bovine serum albumin solution.

[0072] ⑧ Under rapid stirring, the supernatant of ⑤ is slowly added dropwise to the bovine serum albumin solution of ⑦, with a volume ratio of supernatant to bovine serum albumin solution of 1:5. The resulting mixture is stored at 4°C overnight to obtain the artificial antigen mixture.

[0073] ⑨ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 7 times with the PBS buffer described in ⑥. After dialysis, centrifuge and collect the supernatant to obtain arecoline artificial antigen IV.

[0074] The synthetic route is shown below:

[0075]

[0076] Where DIPEA stands for N,N-diisopropylethylamine, MeCN stands for acetonitrile, TFA stands for trifluoroacetic acid, and DCM stands for dichloromethane, and so on.

[0077] Comparative Example 2

[0078] This embodiment provides a method for preparing arecoline artificial antigen V, including the following steps:

[0079] (1) Preparation of arecoline artificial hapten I:

[0080] ①-⑥ are the same as in Example 1.

[0081] (2) Preparation of arecoline artificial antigen V:

[0082] Bovine gamma globulin was used as a carrier and conjugated with arecoline artificial hapten I. The conjugation steps were the same as in Example 1 to obtain arecoline artificial antigen V.

[0083] The synthetic route is shown below:

[0084]

[0085] BGG stands for bovine gamma globulin, and the same applies below.

[0086] Comparative Example 3

[0087] This embodiment provides a method for preparing arecoline artificial antigen VI, including the following steps:

[0088] (1) Preparation of arecoline artificial hapten III:

[0089] ①-④ are the same as Comparative Example 1.

[0090] (2) Preparation of arecoline artificial antigen VI:

[0091] Bovine gamma globulin was used as a carrier and conjugated with arecoline hapten III. The conjugation steps were the same as in Comparative Example 1 to obtain arecoline artificial antigen VI.

[0092] The synthetic route is shown below:

[0093]

[0094] Comparative Example 4

[0095] This embodiment provides a method for preparing arecoline artificial antigen VII, including the following steps:

[0096] (1) Preparation of arecoline artificial hapten I:

[0097] ①-③ are the same as in Example 1.

[0098] (2) Preparation of arecoline artificial antigen VII:

[0099] ④ Weigh 150 mg (0.622 mmol) of arecoline artificial hapten I and place it in a 50 ml round-bottom flask. Dissolve it in 7.5 ml of DMF, then add 103 μl (0.746 mmol) of triethylamine and 96 μl (0.746 mmol) of isobutyl chloroformate. Stir the mixture at 20–25 °C for 18 h. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0100] ⑤-⑧ Same as in Example 1, arecoline artificial antigen VII was obtained.

[0101] The synthetic route is shown below:

[0102]

[0103] Et3N represents triethylamine, and the same applies below.

[0104] Comparative Example 5

[0105] This embodiment provides a method for preparing arecoline artificial antigen VIII, including the following steps:

[0106] (1) Preparation of arecoline artificial hapten I:

[0107] ①-③ are the same as in Example 1.

[0108] (2) Preparation of arecoline artificial antigen VIII:

[0109] ④ Same as Comparative Example 4, ⑤-⑧ Bovine gamma globulin was used as a carrier to conjugate with arecoline artificial hapten I. The conjugation steps were the same as in Example 1 to obtain arecoline artificial antigen VIII.

[0110] The synthetic route is shown below:

[0111]

[0112] Comparative Example 6

[0113] This embodiment provides a method for preparing arecoline artificial antigen IX, including the following steps:

[0114] (1) Preparation of arecoline artificial hapten III:

[0115] ①-④ are the same as Comparative Example 1.

[0116] (2) Preparation of arecoline artificial antigen IX:

[0117] ⑤ Weigh 126 mg (0.710 mmol) of artificial hapten into a 50 ml round-bottom flask, add 6.3 ml of DMF, then add 118 μl (0.852 mmol) of triethylamine and 110 μl (0.852 mmol) of isobutyl chloroformate. Stir and react at 20–25 °C for 18 h. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0118] ⑥-⑨ are the same as in Comparative Example 1; thus, arecoline artificial antigen IX is obtained.

[0119] The synthetic route is shown below:

[0120]

[0121] Comparative Example 7

[0122] This embodiment provides a method for preparing arecoline artificial antigen X, including the following steps:

[0123] (1) Preparation of arecoline artificial hapten III:

[0124] ①-④ are the same as Comparative Example 1.

[0125] (2) Preparation of arecoline artificial antigen X:

[0126] ⑤ Same as Comparative Example 6.

[0127] ⑥-⑨ Bovine gamma globulin was used as a carrier and conjugated with arecoline hapten III. The conjugation steps were the same as those in Comparative Example 1 to obtain arecoline artificial antigen X.

[0128] The synthetic route is shown below:

[0129]

[0130] Test Example 1: Performance Determination of Arecoline Artificial Antigen

[0131] (1) Identification of arecoline artificial antigen:

[0132] Molar absorptivity ε: Arecoline artificial hapten solutions with concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, and 40 μg / ml were prepared using PBS buffer. UV scanning revealed that the maximum absorption wavelength of the arecoline hapten was 388 nm. Absorbance values ​​were measured at 388 nm, with parallel samples prepared for each concentration. The molar absorptivity (i.e., molar absorption coefficient) is calculated as follows: ε = Absorbance value / Molar concentration.

[0133] Determination of conjugate protein concentration: Prepare 1 ml of bovine serum albumin solutions with concentrations of 0 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, and 120 μg / ml using PBS buffer. Add 3 ml of Coomassie brilliant blue staining solution, mix immediately, and incubate at 30°C for 5 minutes. Perform parallel tests for each concentration. Measure the absorbance at 655 nm and plot the relationship between protein concentration and absorbance. Dilute the artificial antigen solution (prepared with PBS buffer) at a certain ratio, measure the absorbance of the artificial antigen at 655 nm, and read the corresponding protein concentration value of the artificial antigen solution from the curve.

[0134] Conjugation ratio determination: Prepare a 100 μg / ml bovine gamma globulin PBS solution. Dilute the conjugate (i.e., arecoline artificial antigen) to 100 μg / ml with PBS. Measure the absorbance A1 at 368 nm. Measure the absorbance A2 using PBS as a blank. The conjugation ratio γ is then calculated as: γ = [(A1 - A2) / ε] / (100 × 10⁻⁶) / ε. -3 / 66400).

[0135] Where ε is the molar absorptivity (L / mol), 66400 is the molecular weight of bovine serum albumin, and 100 × 10⁻⁶ is the molecular weight of bovine serum albumin. -3 This refers to bovine serum albumin concentration (g / L).

[0136] When bovine gamma globulin is used as a carrier, the conjugation ratio is calculated as follows: γ=[(A1-A2) / ε] / (100×10 -3 / 150000); where 150000 is the molecular weight of bovine gamma globulin.

[0137] The test results are shown in Table 1.

[0138] Table 1. Coupling ratios and molar absorption coefficients of various arecoline artificial antigens.

[0139] serial number Artificial antigen Coupling ratio Coupling protein concentration molar absorption coefficient Example 1 I 28 3.357 mg / ml 5882.63 Comparative Example 1 IV 15 2.650mg / ml 5923.61 Comparative Example 2 V 22 3.062 mg / ml 5882.63 Comparative Example 3 VI 17 2.285mg / ml 5923.61 Comparative Example 4 VII 23 3.086 mg / ml 5882.63 Comparative Example 5 VIII 18 2.743 mg / ml 5882.63 Comparative Example 6 IX 21 3.036 mg / ml 5923.61 Comparative Example 7 X 2 0.836mg / ml 5923.61

[0140] As shown in Table 1, the structure of the artificial hapten, the activation method of the artificial hapten, and the type of carrier protein all affect the coupling ratio when the artificial hapten cross-links with the carrier protein.

[0141] (2) Animal Immunization

[0142] New Zealand white rabbits were immunized with the prepared arecoline artificial antigens, and the titers of the resulting immune serum were detected by ELISA. The results are shown in Table 2.

[0143] Table 2. Results of titer tests for various immune sera.

[0144] serial number Arecoline artificial antigen Immune serum titer Example 1 II 1：70000 Comparative Example 1 IV 1：33000 Comparative Example 2 V 1：7000 Comparative Example 3 VI 1：3000 Comparative Example 4 VII 1：36000 Comparative Example 5 VIII 1：5000 Comparative Example 6 IX 1：6000 Comparative Example 7 X 1：4000

[0145] As shown in Table 2, compared with Example 1, the immune sera obtained by immunizing animals using arecoline artificial antigens in each comparative ratio had lower titers and could not be used in immunoassays. However, the immune sera obtained by immunizing animals using arecoline artificial antigen II had a titer of 1:70,000, which is perfectly suitable for immunoassays and provides a more convenient, rapid, and accurate method for arecoline detection.

Claims

1. An artificial hapten of arecoline, characterized in that, Its molecular structure is shown in formula (Ⅰ): ； Equation (Ⅰ).

2. The method for preparing the arecoline artificial hapten according to claim 1, characterized in that, The preparation method includes the following steps: Step (1): Dilute concentrated ammonia to obtain DAS solution; Step (2): Dissolve demethylarecoline hydrochloride in deionized water, add the DAS solution obtained in step (1) to adjust the pH to alkaline, extract with dichloromethane multiple times, collect the dichloromethane phase, dry, filter, and dry to obtain colorless oily substance A. Step (3): The colorless oily substance A obtained in step (2) is mixed with succinic anhydride in a molar ratio of 1: (1.5~2.5) in a solvent. 4-Dimethylaminopyridine is added as a catalyst, and the mixture is stirred and refluxed at 100~110℃ for 20~21 h. After the reaction is complete, the arecoline artificial hapten is obtained by thin-layer chromatography.

3. The preparation method according to claim 2, characterized in that, In step (2), the pH value is adjusted to 9.

4. The preparation method according to claim 2, characterized in that, In step (3), the molar ratio of colorless oily substance A to succinic anhydride is 1:

2.

5. The preparation method according to claim 2, characterized in that, The solvent mentioned in step (3) is either pyridine or dichloromethane.

6. An artificial arecoline antigen, characterized in that, The arecoline hapten of claim 1 is purified after conjugation with bovine serum albumin, and its molecular structure is shown in formula (II): ； Formula (II) BSA stands for bovine serum albumin.

7. The method for preparing the arecoline artificial antigen according to claim 6, characterized in that, Includes the following steps: Step (1): Mix arecoline artificial hapten, N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in a molar ratio of 1:(1.35~1.5):(1.35~1.5) in N,N-dimethylformamide, stir and react at 20~30℃ for 18~20 hours, centrifuge after the reaction is completed, and take the supernatant. Step (2): Add the supernatant to the bovine serum albumin solution, place the resulting mixture at 3-5°C and let it stand overnight. After dialysis and centrifugation, take the supernatant to obtain the arecoline artificial antigen.

8. The preparation method according to claim 7, characterized in that, In step (2), the concentration of the bovine serum albumin solution is 5 mg / mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:

5.

9. The use of the arecoline artificial antigen according to claim 6 in the preparation of anti-arecoline antibodies.

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