Artificial hapten and artificial antigen of trihexylamine, and preparation method and application of artificial hapten and artificial antigen of trihexylamine

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a trihexylamine artificial hapten, an artificial antigen as well as a preparation method and application of the trihexylamine artificial hapten and the trihexylamine artificial antigen. The molecular structural formula of the trihexylamine artificial hapten is as shown in a formula (I), and the molecular structural formula of the trihexylamine artificial antigen is as shown in a formula (II). The trihexyle artificial antigen can be applied to the field of preparation of anti-trihexyle antibodies. According to the present invention, the trihexylamine artificial hapten maximally retains the characteristic structure of the trihexylamine, has the active group capable of being coupled with the carrier protein, and can be used as the antigenic determinant; the further prepared trihexylamine artificial antigen can be immunized to obtain an anti-trihexylamine antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit reaches up to 1: 256000, and the trihexylamine artificial antigen can be used for rapid and accurate immunodetection and immunoassay of trihexylamine.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to an artificial hapten of benzhexol, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Trihexyphenidyl, also known as Trihexyphenidyl and Antan, is a relatively common anticholinergic drug. Trihexyphenidyl can block choline and play a role in the central nervous system, maintaining the balance of the central nervous system and inhibiting muscle rigidity and tremor. It is mainly used in medicine to improve the discomfort symptoms caused by Parkinson's disease and Parkinson's syndrome. It can also be used for extrapyramidal diseases caused by drugs.

[0003] As the drug has become more popular, cases of overdose and abuse of trihexyphenidyl have occurred. Trihexyphenidyl overdose and abuse can cause a range of adverse reactions. Symptoms of overdose include dilated pupils, dry mucous membranes, redness of the face, intestinal and bladder weakness, and high temperature at high doses. Trihexyphenidyl can cause agitation, confusion, and hallucinations due to its effects on the central nervous system. Untreated overdose can result in death, especially in children, where respiratory depression and cardiac arrest may be precursors to death.

[0004] Patients who overdose on trihexyphenidyl may also experience dry mouth, anhidrosis, dilated pupils, nausea, vomiting, abnormal heart rate, high fever, decreased gastrointestinal motility, hesitant or retained urination, rash, confusion, restlessness, agitation, poor coordination, paranoia, psychosis, delirium, hallucinations, coma, respiratory failure, circulatory collapse, and death.

[0005] At present, the detection of benzhexol mainly relies on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance hydrogen spectrum (H NMR), nuclear magnetic resonance carbon spectrum (13C NMR), nuclear magnetic resonance fluorine spectrum (19F NMR), infrared spectroscopy (IR), thin layer chromatography (TLC), etc. However, all of them have the disadvantages of expensive instruments, time-consuming detection, and need professional technicians to operate, which makes it difficult to meet the requirements of modern detection for rapid and accurate detection. Therefore, it is very necessary to establish rapid, sensitive and accurate detection technology.

[0006] Immunochromatographic test strip rapid detection technology can make up for all the above shortcomings. This technology has the characteristics of strong specificity, high sensitivity, simplicity and economy. It does not require professional skills and expensive and complex instruments and equipment to achieve qualitative and semi-quantitative detection of various analytes such as antigens, haptens, antibodies, etc. It is widely used in the detection of hormones, tumor markers, drugs, bacteria, viruses and other substances. Antigens and antibodies are essential raw materials for this technology, so it is necessary to provide an effective preparation method of benzhexol antigen antibodies, which can be used for immunoassay of benzhexol.

[0007] Since most small molecule compounds (molecular weight less than 1000), including benzhexol, are not immunogenic, that is, they lack T cell epitopes and cannot directly induce the animal body to produce specific antibodies, small molecules are called haptens. Through appropriate chemical modification, a connecting arm with an active group at the end is added to a certain position of the hapten molecule structure, and then combined with a macromolecular carrier to generate a hapten-carrier conjugate (i.e., artificial antigen). The artificial antigen can indirectly induce the proliferation and differentiation of B cells with the help of T cell epitopes, and then produce specific antibodies, thereby preparing anti-benzhexol antibodies.

[0008] There is no related report on artificial hapten and artificial antigen of trihexyphenidyl in the prior art. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention provides a trihexyphenidyl artificial hapten, an artificial antigen and a preparation method thereof, and applies the trihexyphenidyl artificial antigen to the preparation of an anti-trihexyphenidyl antibody with high detection sensitivity and strong specificity.

[0010] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A trihexyphenidyl artificial hapten, the molecular structure of which is shown in formula (I):

[0011] The artificial hapten of benzhexol provided by the invention retains the characteristic structure of benzhexol to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant.

[0012] The method for preparing the above-mentioned trihexyphenidyl artificial hapten comprises the following steps: (S.1) diluting concentrated ammonia water with deionized water, then adding benzhexyphenidyl hydrochloride to obtain free benzhexyphenidyl, mixing the free benzhexyphenidyl with ethyl bromoacetate in N,N-dimethylformamide, adding sodium hydride, heating and stirring under reflux until the reaction is completed, drying, adding deionized water, extracting with ethyl acetate, collecting the organic phase, drying, filtering, drying, separating and purifying, and obtaining a white solid A; (S.2) dissolving the white solid A obtained in step (S.1) in methanol and tetrahydrofuran, adding sodium hydroxide solution, heating and stirring until the reaction is complete, adjusting the pH with hydrochloric acid, extracting with ethyl acetate, drying, separating and purifying, and obtaining the artificial hapten I of benzhexidine.

[0013] The present invention adopts N,N-dimethylformamide-sodium hydride system and uses sodium hydride as catalyst, which helps to better combine the connecting arm with the free-state benzhexol, thereby effectively reducing the generation of by-products, further improving the yield and saving costs. In addition, the present invention introduces a connecting arm on the hydroxyl group of benzhexol, and the characteristic structure of benzhexol can be retained to the greatest extent by introducing the connecting arm on the modification site. Compared with the general cyclic structure used as the connecting arm, the connecting arm used in the present invention is a linear structure, which helps to reduce the non-specific binding generated during immunoassay, and can also effectively reduce the immune response to the connecting arm generated during immunization, so that the specificity of the artificial hapten is enhanced, the conformation is kept stable in the solution, and the immune characteristics of the benzhexol artificial antigen II are improved.

[0014] Preferably, in step (S.1), the molar ratio of free benzhexol to ethyl bromoacetate is 1:2-10.

[0015] Preferably, the reaction temperature of the heating and stirring reflux process in step (S.1) is 60 to 70° C., and the reaction time is 12 to 20 hours; The reaction temperature of the heating and stirring process in step (S.2) is 0 to 40°C, and the reaction time is 10 to 20 hours.

[0016] Controlling the reaction temperature and reaction time can help reduce by-products to a minimum, thereby increasing yield and reducing costs.

[0017] Preferably, the separation and purification process in step (S.1) adopts a TLC separation and purification method, the developing agent used is ethyl acetate, and the solvent and eluent used are ethanol.

[0018] Using ethyl acetate as a developing agent helps to separate the product, facilitates subsequent operations, and saves costs.

[0019] A trihexyphenidyl artificial antigen, the molecular structure of which is shown in formula (II): Among them, BGG is bovine immunoglobulin G.

[0020] The method for preparing the above-mentioned trihexyphenidyl artificial antigen comprises the following steps: The artificial hapten I of benzhexol as claimed in claim 1 is combined with bovine immunoglobulin gamma by using the N-hydroxysuccinimide active ester method to obtain the artificial antigen II of benzhexol.

[0021] Preferably, the method for preparing the above-mentioned benzhexol artificial antigen comprises the following steps: (a) mixing the benzhexol artificial hapten I as claimed in claim 1 with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, heating and stirring for reaction, centrifuging after the reaction is completed, and taking the supernatant; (b) adding the supernatant obtained in step (a) dropwise to the bovine immunoglobulin solution and mixing evenly to obtain a mixed solution, and letting it stand. After the reaction is complete, dialyzing and centrifuging are performed, and the supernatant is taken to obtain the benzhexidine artificial antigen II.

[0022] Preferably, the molar ratio of the benzhexol artificial hapten I to cyclohexylcarbodiimide and N-hydroxysuccinimide added in step (a) is 1:(1.1-1.5):(1.1-1.5); The concentration of the bovine immunoglobulin G solution in step (b) is 4-6 mg / mL; the volume ratio of the supernatant obtained in step (a) added in the mixing process to the bovine immunoglobulin G solution is 1:8-12.

[0023] As further preferred, the method for preparing the bovine gamma globulin solution comprises the following steps: The bovine immunoglobulin G is dissolved in 0.01 M PBS buffer, wherein the pH of the PBS buffer is 7.2-7.4.

[0024] The present invention uses bovine immunoglobulin as a macromolecular carrier, which has the following advantages compared with traditional bovine serum albumin: (1) the bovine immunoglobulin of the present invention has strong immunogenicity, and can more easily obtain high-titer serum by immunizing New Zealand white rabbits; (2) From the perspective of antigen-antibody binding test, the artificial antigen obtained by using bovine immunoglobulin G as a carrier binds to the benzhexol antibody with stronger specificity, higher sensitivity, and better gradient, and is more suitable for immunodetection of benzhexol.

[0025] As a further preference, the molar ratio of the benzhexol artificial hapten I added in step (a) to cyclohexylcarbodiimide and N-hydroxysuccinimide is 1:(1.3-1.4):(1.3-1.4).

[0026] Use of the above-mentioned trihexyphenidyl artificial antigen in the preparation of anti-trihexyphenidyl antibodies.

[0027] The invention discloses an anti-benzhexyl antibody, which is a globulin obtained by immunizing animals with the above-mentioned benzhexyl artificial antigen and can generate specific immune reaction with benzhexyl and its analogs.

[0028] The invention has been found through repeated experiments that the titer of the immune serum obtained by immunizing New Zealand white rabbits with the artificial benzhexol antigen is 1:256000. This indicates that the artificial benzhexol antigen of the invention can be used to immunize and obtain anti-benzhexol antibodies with high affinity, high sensitivity and strong specificity, and the anti-benzhexol antibodies can be used for immunodetection and analysis of benzhexol.

[0029] Therefore, the present invention has the following beneficial effects: The artificial hapten of the benzhexol retains the characteristic structure of the benzhexol to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant. The artificial benzhexol antigen prepared further can be used for immunization to obtain anti-benzhexol antibodies 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 1:256000, and can be used for rapid and accurate immunodetection and immunoanalysis of benzhexol. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The flowchart is a preparation process of the benzhexol artificial antigens II and IV of Example 1 and Comparative Example 2 of the present invention.

[0031] Figure 2 It is a liquid chromatogram of the artificial hapten I of benzhexol of the present invention.

[0032] Figure 3 It is the mass spectrum of the artificial hapten I of benzhexol of the present invention.

[0033] Figure 4 These are UV scans of BGG, trihexyphenidyl artificial hapten I, and trihexyphenidyl artificial antigen II.

[0034] Figure 5 The flowchart of the preparation of the benzhexidine artificial antigens III and V in Comparative Examples 1 and 3 is shown.

[0035] Figure 6 This is a flow chart for the preparation of the benzhexidine artificial antigens VII and IX in Comparative Examples 4 and 6.

[0036] Figure 7 This is a flow chart for the preparation of the benzhexidine artificial antigens VIII and X in Comparative Examples 5 and 7. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0038] Example 1 This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0039] A method for preparing a trihexyphenidyl artificial antigen comprises the following steps: (1) Preparation of trihexyphenidyl artificial hapten I: (S.1) (S.11) Prepare DAS solution: dilute 5 mL of concentrated ammonia water to 13 mL with deionized water to obtain DAS solution; (S.12) Weigh 500 mg (1.483 mmol) of benzhexyphenidyl hydrochloride into a 50 mL centrifuge tube, add 15 mL of deionized water to dissolve, and adjust pH to 9 with the above DAS solution. A large amount of precipitation appears. Extract with 20 mL of dichloromethane each time, extract 3 times, collect the organic phase and dry it with anhydrous magnesium sulfate, filter, and dry to obtain 444 mg (1.475 mmol) of free benzhexyphenidyl; (S.13) Weigh 100 mg (0.332 mmol) of free benzhexol and 295 μL (2.658 mmol) of ethyl bromoacetate, mix and dissolve in 5 mL of N,N-dimethylformamide (DMF), add 53 mg (1.328 mmol) of 60% sodium hydride as a catalyst, and reflux and stir at 65°C for 16 h. TLC detection (developing solvent is ethyl acetate: concentrated ammonia water = 200:1, v / v) shows that the reaction is complete, and the product point Rf = 0.2. Directly transfer to dryness, add 20 mL of deionized water to dissolve, extract with 20 mL of ethyl acetate each time, extract twice, collect the organic phase, dry with anhydrous magnesium sulfate, filter, transfer to dryness, and obtain a light yellow solid. Separate and purify by TLC (developing solvent is ethyl acetate, solvent and eluent are ethanol), and obtain 115 mg (0.298 mmol) of white solid A; (S.2) (S.21) Prepare 1 mol / L sodium hydroxide aqueous solution: dissolve 8 g of sodium hydroxide solid in 200 mL of deionized water to obtain 1 mol / L sodium hydroxide aqueous solution; (S.22) 115 mg (0.298 mmol) of the white solid A obtained in step (S.1) was dissolved in a 50 mL single-necked flask with 3.2 mL of methanol (MeOH) and 2.7 mL of tetrahydrofuran (THF), and 13 mL of the 1 mol / L sodium hydroxide aqueous solution prepared in the above step (S.21) was added, and the mixture was stirred at 25°C for 16 h. The reaction was completed by TLC detection (developing solvent was dichloromethane: 95% ethanol: 1,4-dioxane: concentrated ammonia water = 10:8:1:1, v / v / v / v, hereinafter collectively referred to as MOP, and the solvent and eluent were anhydrous ethanol), with the raw material point Rf = 0.7 and the product point Rf = 0.3; (S.23) Prepare 1 mol / L hydrochloric acid: dilute 16.95 mL of 37% hydrochloric acid to 200 mL with deionized water to obtain 1 mol / L hydrochloric acid; (S.24) The reaction solution after stirring reaction in the above step (S.22) was adjusted to pH=3 with 1 mol / L hydrochloric acid prepared in the above step (S.23), and extracted with 20 mL of ethyl acetate each time, extracted 3 times, collected the organic phase, dried over anhydrous magnesium sulfate, filtered, and dried to obtain a white solid, which was separated and purified by TLC (developing agent: MOP, solvent and eluent: anhydrous ethanol) to obtain 67 mg (0.187 mmol) of benzhexol artificial hapten I.

[0040] The molecular structure of the artificial hapten I of benzhexol is shown in formula (I):

[0041] The preparation reaction process of the artificial hapten I of benzhexol in this example is shown in Figure 1 The liquid chromatogram of the artificial hapten I of trihexyphenidyl is shown in Figure 2 The mass spectrum of benzhexol hapten I is shown in Figure 3 .

[0042] from Figure 2 It can be seen that the purity of the purified benzhexol artificial hapten I reaches more than 95%. Figure 3 It can be seen that the characteristic peaks of the benzhexol artificial hapten I obtained in this example are 360.51, 382.49, 383.5, and 404.52, respectively corresponding to [M+H] + 、[M+Na] + 、[M+Na+H] + 、[M+HCOO] - , it can be basically determined that the final compound obtained in step (S.13) is the artificial hapten I of benzhexol designed by the present invention.

[0043] (2) Preparation of trihexyphenidyl artificial antigen II: (a) 67 mg (0.187 mmol) of the benzhexol artificial hapten I obtained in the above step (S.24) was placed in a 50 mL round-bottom flask, 3.35 mL of DMF was added, and 51 mg (0.252 mmol) of cyclohexylcarbodiimide (DCC) and 28 mg (0.252 mmol) of N-hydroxysuccinimide were added and mixed evenly, and the mixture was stirred at 25° C. for 16 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected for later use; (b) 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate were weighed and dissolved in deionized water to a volume of 5.0 L to obtain a 0.01 M PBS buffer solution, wherein the pH of the PBS buffer solution was 7.4; Weigh 167.5 mg of bovine gamma globulin (BGG) and dissolve it in 33.5 mL of the prepared PBS buffer to obtain a bovine gamma globulin solution with a concentration of 5 mg / mL; Under rapid stirring, slowly dropwise adding the supernatant obtained in step (a) into the bovine immunoglobulin solution to mix evenly, the volume ratio of the supernatant to the bovine immunoglobulin solution being 1:10, and the obtained mixed solution being stored at 4° C. overnight to obtain a trihexyphenidyl artificial antigen mixed solution; The benzhexol artificial antigen mixture was transferred into a dialysis bag and dialyzed 9 times with the prepared PBS buffer. After the dialysis, the mixture was centrifuged and the supernatant was taken to obtain the benzhexol artificial antigen II. That is, the benzhexol-bovine immunoglobulin conjugate (such as formula II). The ultraviolet scanning images of the benzhexol artificial antigen before and after preparation are shown in FIG. Figure 4 The molecular structure of the trihexyphenidyl artificial antigen II is shown in formula (II): Among them, BGG is bovine immunoglobulin G.

[0044] Figure 4 In the figure, curve a is the ultraviolet scanning diagram of the artificial hapten of benzhexol, curve b is the ultraviolet scanning diagram of the artificial antigen of benzhexol, and curve c is the ultraviolet scanning diagram of bovine gamma globulin. The maximum absorption wavelength of the artificial hapten I of benzhexol is 297nm, the maximum absorption wavelength of the artificial antigen II of benzhexol is 286nm, and the maximum absorption wavelength of bovine gamma globulin is 280nm. Compared with benzhexol hapten I and bovine gamma globulin, the maximum absorption wavelength of the artificial antigen II of benzhexol changes significantly. It can be explained that benzhexol and bovine gamma globulin are successfully coupled.

[0045] Example 2 The difference between this embodiment and embodiment 1 is that: This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0046] A method for preparing an artificial trihexyphenidyl antigen, wherein: (1) Preparation process of trihexyphenidyl artificial hapten I: In step (S.13), 100 mg (0.332 mmol) of free benzhexol and 368.47 μL (3.32 mmol) of ethyl bromoacetate were weighed and dissolved in 5 mL of N,N-dimethylformamide (DMF); the mixture was stirred under reflux at 60° C. for 12 h; in step (S.22), the mixture was stirred at 0° C. for 10 h; the rest was the same as in Example 1. Finally, artificial hapten I of benzhexol was obtained.

[0047] (2) Preparation process of trihexyphenidyl artificial antigen II: In step (a), 67 mg (0.187 mmol) of benzhexol artificial hapten I is placed in a 50 mL round-bottom flask, 3.35 mL of DMF is added, and then 41.63 mg (0.2057 mmol) of cyclohexylcarbodiimide and 22.85 mg (0.2057 mmol) of N-hydroxysuccinimide are added and mixed evenly; in step (b), 134 mg of bovine gamma globulin is weighed and dissolved in 33.5 mL of the above-prepared PBS buffer to obtain a bovine gamma globulin solution with a concentration of 4 mg / mL; the volume ratio of the supernatant to the bovine gamma globulin solution is 1:8. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, benzhexol artificial antigen II is obtained.

[0048] Example 3 The difference between this embodiment and embodiment 1 is that: This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0049] A method for preparing an artificial trihexyphenidyl antigen, wherein: (1) Preparation process of trihexyphenidyl artificial hapten I: In step (S.13), 100 mg (0.332 mmol) of free benzhexol and 73.69 μL (0.664 mmol) of ethyl bromoacetate were weighed and mixed and dissolved in 5 mL of N,N-dimethylformamide (DMF); the mixture was stirred under reflux at 70° C. for 20 h; in step (S.22), the mixture was stirred at 40° C. for 20 h; the rest was the same as in Example 1. Finally, the artificial hapten I of benzhexol was obtained.

[0050] (2) Preparation process of trihexyphenidyl artificial antigen II: In step (a), 67 mg (0.187 mmol) of benzhexol artificial hapten I is placed in a 50 mL round-bottom flask, 3.35 mL of DMF is added, and then 56.77 mg (0.2805 mmol) of cyclohexylcarbodiimide and 31.17 mg (0.2805 mmol) of N-hydroxysuccinimide are added and mixed evenly; in step (b), 201 mg of bovine gamma globulin is weighed and dissolved in 33.5 mL of the above-prepared PBS buffer to obtain a bovine gamma globulin solution with a concentration of 6 mg / mL; the volume ratio of the supernatant to the bovine gamma globulin solution is 1:12. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, benzhexol artificial antigen II is obtained.

[0051] Comparative Example 1 The difference between this comparative example and Example 1 is: This comparative example provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen III and a preparation method thereof.

[0052] A method for preparing an artificial benzhexol antigen, wherein: (1) the preparation process of the artificial benzhexol hapten I is the same as that in Example 1. (2) the preparation process of the artificial benzhexol antigen III: the bovine immunoglobulin (i.e., carrier protein) in step (b) is replaced with bovine serum albumin (BSA); the volume ratio of the supernatant to the bovine immunoglobulin solution of 1:10 is replaced with the volume ratio of the supernatant to the bovine serum albumin solution of 1:5. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, the artificial benzhexol antigen III is obtained. The preparation reaction process of the artificial benzhexol antigen III in this comparative example is as follows: Figure 5 shown.

[0053] Comparative Example 2 The difference between this comparative example and Example 1 is: This comparative example provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen IV and a preparation method thereof.

[0054] A method for preparing an artificial antigen IV of trihexyphenidyl, wherein: (1) the preparation process of the artificial hapten I of trihexyphenidyl is the same as that in Example 1. (2) the preparation process of the artificial antigen IV of trihexyphenidyl: the volume ratio of the supernatant to the bovine immunoglobulin solution in step (b) is 1:10, which is replaced by a volume ratio of the supernatant to the bovine immunoglobulin solution of 1:5. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, the artificial antigen IV of trihexyphenidyl is obtained. The preparation reaction process of the artificial antigen IV of trihexyphenidyl in this comparative example is as follows: Figure 1 shown.

[0055] Comparative Example 3 The difference between this comparative example and Example 1 is: This comparative example provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen V and a preparation method thereof.

[0056] A method for preparing an artificial antigen V of trihexyphenidyl, wherein: (1) the preparation process of the artificial hapten I of trihexyphenidyl is the same as that in Example 1. (2) the preparation process of the artificial antigen V of trihexyphenidyl: the bovine immunoglobulin (i.e., carrier protein) in step (b) is replaced with bovine serum albumin; the volume ratio of the supernatant to the bovine immunoglobulin solution of 1:10 is replaced with the volume ratio of the supernatant to the bovine serum albumin solution of 1:10. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, the artificial antigen V of trihexyphenidyl is obtained. The preparation reaction process of the artificial antigen V of trihexyphenidyl in this comparative example is as follows: Figure 5 shown.

[0057] Comparative Example 4 The difference between this comparative example and Example 1 is: This comparative example provides a trihexyphenidyl artificial hapten VI and a preparation method thereof, and a trihexyphenidyl artificial antigen VII and a preparation method thereof.

[0058] A method for preparing trihexyphenidyl artificial antigen VII comprises the following steps: (1) Preparation of artificial hapten VI of trihexyphenidyl: (S.1) (S.11) Prepare DAS solution: dilute 5 mL of concentrated ammonia water to 13 mL with deionized water to obtain DAS solution; (S.12) Weigh 500 mg (1.483 mmol) of benzhexyphenidyl hydrochloride into a 50 mL centrifuge tube, add 15 mL of deionized water to dissolve, and adjust pH to 9 with the above DAS solution. A large amount of precipitation appears. Extract with 20 mL of dichloromethane each time, extract 3 times, collect the organic phase and dry it with anhydrous magnesium sulfate, filter, and dry to obtain 444 mg (1.475 mmol) of free benzhexyphenidyl; (S.13) Take 100 mg (0.332 mmol) of the free benzhexol obtained in step (S.12) and add 10 mL of dichloromethane to a 50 mL round-bottom flask. Place in an ice bath and stir to cool to 0°C, then add 67 μL (0.481 mmol) of triethylamine. Dissolve 49 μL (0.398 mmol) of succinic acid monoester chloride in 2 mL of dichloromethane and slowly drop it into the system. White smoke is generated. After the addition is complete, the system is light brown. Transfer to 25°C and stir to react for 5.5 hours. TLC detection (developing solvent is ethyl acetate: ammonia water = 200:1, v / v) shows that the reaction is complete, and the product point Rf = 0.4. Dried to obtain 169 mg of brown solid B; (S.2) (S.21) Prepare 1 mol / L sodium hydroxide aqueous solution: dissolve 8 g of sodium hydroxide solid in 200 mL of deionized water to obtain 1 mol / L sodium hydroxide aqueous solution; (S.22) 169 mg of the brown solid B obtained in step (S.13) was dissolved in 4 mL of methanol and 3.4 mL of tetrahydrofuran in a 50 mL round-bottom flask, and 16 mL of the 1 mol / L sodium hydroxide aqueous solution prepared in step (S.21) was added, and the mixture was stirred at 25°C for 18 h. The reaction was complete after TLC detection (developing agent: MOP, solvent and eluent: anhydrous ethanol). Product point Rf = 0.3; (S.23) Prepare 1 mol / L hydrochloric acid: dilute 16.95 mL of 37% hydrochloric acid to 200 mL with deionized water to obtain 1 mol / L hydrochloric acid; (S.24) The reaction solution after stirring reaction in the above step (S.22) was adjusted to pH=3 with 1 mol / L hydrochloric acid prepared in step (S.23), and extracted with 20 mL of ethyl acetate each time, extracted 3 times, collected the organic phase, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 148 mg of light yellow solid. After TLC separation and purification (developing agent was MOP, and solvent and eluent were anhydrous ethanol), 110 mg (0.274 mmol) of benzhexol artificial hapten VI was obtained.

[0059] (2) Preparation of trihexyphenidyl artificial antigen VII: (a) 110 mg (0.274 mmol) of the benzhexol artificial hapten VI obtained in the above step (S.24) was placed in a 50 mL round-bottom flask, 5.5 mL of DMF was added, and then 76 mg (0.374 mmol) of cyclohexylcarbodiimide and 43 mg (0.373 mmol) of N-hydroxysuccinimide were added and mixed evenly, and the mixture was stirred at 25° C. for 16 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected for later use; (b) 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate were weighed and dissolved in deionized water to a volume of 5.0 L to obtain a 0.01 M PBS buffer solution, wherein the pH of the PBS buffer solution was 7.4; Weigh 137.5 mg of bovine serum albumin and dissolve it in 27.5 mL of the prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 5 mg / mL; Under rapid stirring, slowly dropwise adding the supernatant obtained in step (a) into the bovine serum albumin solution and mixing evenly, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is stored at 4° C. overnight to obtain a trihexyphenidyl artificial antigen mixed solution; The trihexyphenidyl artificial antigen mixture was transferred into a dialysis bag and dialyzed 9 times with the prepared PBS buffer solution. After the dialysis, the mixture was centrifuged and the supernatant was collected to obtain the trihexyphenidyl artificial antigen VII.

[0060] Comparative Example 5 The difference between this comparative example and comparative example 4 is that: This comparative example provides a trihexyphenidyl artificial hapten VI and a preparation method thereof, and a trihexyphenidyl artificial antigen VIII and a preparation method thereof.

[0061] A method for preparing an artificial benzhexol antigen VIII, wherein: (1) the preparation process of the artificial benzhexol hapten VI is the same as that in Comparative Example 4. (2) the preparation process of the artificial benzhexol antigen VIII: the bovine serum albumin (i.e., carrier protein) in step (b) is replaced with bovine immunoglobulin gamma, and the volume ratio of the supernatant to the bovine serum albumin solution of 1:5 is replaced with a volume ratio of the supernatant to the bovine immunoglobulin solution of 1:5. The same coupling steps as in Comparative Example 4 are adopted, and the rest are the same as in Comparative Example 4. Finally, the artificial benzhexol antigen VIII is obtained. The preparation reaction process of the artificial benzhexol antigen VIII in this comparative example is as follows: Figure 7 shown.

[0062] Comparative Example 6 The difference between this comparative example and comparative example 4 is that: This comparative example provides a trihexyphenidyl artificial hapten VI and a preparation method thereof, and a trihexyphenidyl artificial antigen IX and a preparation method thereof.

[0063] A method for preparing an artificial antigen IX of trihexyphenidyl, wherein: (1) the preparation process of the artificial hapten VI of trihexyphenidyl is the same as that in Comparative Example 4. (2) the preparation process of the artificial antigen IX of trihexyphenidyl: the volume ratio of the supernatant to the bovine serum albumin solution in step (b) is 1:5, which is replaced by a volume ratio of the supernatant to the bovine serum albumin solution of 1:10. The same coupling steps as in Comparative Example 4 are adopted, and the rest are the same as in Comparative Example 4. Finally, the artificial antigen IX of trihexyphenidyl is obtained. The preparation reaction process of the artificial antigen IX of trihexyphenidyl in this comparative example is as follows: Figure 6 shown.

[0064] Comparative Example 7 The difference between this comparative example and comparative example 4 is that: This comparative example provides a trihexyphenidyl artificial hapten VI and a preparation method thereof, and a trihexyphenidyl artificial antigen X and a preparation method thereof.

[0065] A method for preparing an artificial antigen X of trihexyphenidyl, wherein: (1) the preparation process of the artificial hapten VI of trihexyphenidyl is the same as that in Comparative Example 4. (2) the preparation process of the artificial antigen X of trihexyphenidyl: the bovine serum albumin (i.e., carrier protein) in step (b) is replaced with bovine immunoglobulin gamma, and the volume ratio of the supernatant to the bovine serum albumin solution of 1:5 is replaced with the volume ratio of the supernatant to the bovine immunoglobulin solution of 1:10. The same coupling steps as in Comparative Example 4 are adopted, and the rest are the same as in Comparative Example 4. Finally, the artificial antigen X of trihexyphenidyl is obtained. The preparation reaction process of the artificial antigen X of trihexyphenidyl in this comparative example is as follows: Figure 7 shown.

[0066]

Performance determination of trihexyphenidyl artificial antigen

[0067] Table 1: Calculation table of molar absorption coefficients of trihexyphenidyl artificial haptens I and VI

[0068] Determination of protein concentration of conjugate: Prepare 1 mL of bovine serum albumin solution 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 with PBS buffer, add 3 mL of Coomassie Brilliant Blue staining solution, mix immediately, warm in a 30°C water bath for 5 min, make parallel samples for each concentration, measure the absorbance at 655 nm, and draw a curve of the relationship between protein concentration and absorbance. Dilute the artificial antigen solution (prepared with PBS buffer) in a certain proportion, measure the absorbance of the artificial antigen at 655 nm, read the corresponding protein concentration value of the artificial antigen solution from the curve, and the calculated data results are shown in Table 2 below.

[0069] Coupling ratio determination: prepare 100 μg / mL bovine immunoglobulin PBS solution, dilute the conjugate (i.e., benzhexol artificial antigen) to 100 μg / mL with PBS, measure the absorbance value A1 at 297 nm, and measure the absorbance value A2 with PBS as blank. The coupling ratio γ is: γ=[(A1-A2) / ε] / (100×10 -3 / 43000), the calculation data results are shown in Table 2 below.

[0070] Where ε is the molar absorption coefficient (L / mol), 43000 is the molecular weight of bovine gamma globulin, and 100×10 -3 is the concentration of bovine immunoglobulin (g / L).

[0071] When bovine serum albumin is used as a carrier, the coupling ratio is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 66400); wherein 66400 is the molecular weight of bovine serum globulin, and the calculated data results are shown in Table 2 below.

[0072] Table 2: Calculation data of coupling ratio of each benzhexol artificial antigen and protein concentration of the coupling product

[0073] As can be seen from Table 2, the structure of the artificial hapten, the volume ratio between the artificial hapten and the carrier protein solution when coupled, and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten and the carrier protein are cross-linked.

[0074] (2) Animal immunization The prepared artificial antigens of benzhexol were used to immunize New Zealand white rabbits, and the titers of the immune sera obtained were tested by ELISA. The test results are shown in Table 3 below.

[0075] Table 3: Titer test results of each immune serum serial number Trihexyphenidyl artificial antigen Immune serum titer Example 1 II 256000 Comparative Example 1 III 128000 Comparative Example 2 IV 64000 Comparative Example 3 V / Comparative Example 4 VII 16000 Comparative Example 5 VIII / Comparative Example 6 IX 64000 Comparative Example 7 X 32000 .

[0076] As shown in Table 3, compared with Example 1, the immune serum obtained by animal immunization using the artificial benzhexol antigens of each comparative example has a low titer and is not suitable for immunoassay. The artificial benzhexol antigen V and artificial benzhexol antigen VIII obtained in comparative examples 3 and 5 are relatively turbid, and a large amount of precipitation occurs after freezing and storage, and the stability is very poor, so they cannot be used as immune antigens. The immune serum obtained by animal immunization using artificial benzhexol antigen II has a titer of 1:256000, which can be used in immunoassay completely, and can provide a more convenient, rapid and accurate approach for the detection of benzhexol.

[0077] In summary, the present invention discloses an artificial hapten of benzhexol, an artificial antigen, and a preparation method and application thereof. The molecular structural formula of the artificial hapten of benzhexol is shown in formula (I), and the molecular structural formula of the artificial antigen of benzhexol is shown in formula (II). The artificial antigen of benzhexol can be applied to the field of preparation of anti-benzhexol antibodies. The artificial hapten of benzhexol of the present invention retains the characteristic structure of benzhexol to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant; the artificial antigen of benzhexol obtained by further preparation can be immunized to obtain anti-benzhexol polyclonal antibodies with high affinity, high sensitivity and strong specificity, and the titer of the immune serum obtained by immunizing New Zealand white rabbits can reach 1:256000, which can be used for rapid and accurate immunodetection and immunoanalysis of benzhexol.

[0078] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A trihexyphenidyl artificial hapten, characterized in that: Its molecular structure is shown in formula (I): Formula (I).

2. The method for preparing a trihexyphenidyl artificial hapten as claimed in claim 1, characterized in that: The following steps are involved: (S.1) diluting concentrated ammonia water with deionized water, then adding benzhexyphenidyl hydrochloride to obtain free benzhexyphenidyl, mixing the free benzhexyphenidyl with ethyl bromoacetate in N,N-dimethylformamide, adding sodium hydride, heating and stirring under reflux until the reaction is completed, drying, adding deionized water, extracting with ethyl acetate, collecting the organic phase, drying, filtering, drying, separating and purifying, and obtaining a white solid A; (S.2) dissolving the white solid A obtained in step (S.1) in methanol and tetrahydrofuran, adding sodium hydroxide solution, heating and stirring until the reaction is complete, adjusting the pH with hydrochloric acid, extracting with ethyl acetate, drying, separating and purifying, and obtaining the artificial hapten I of benzhexidine.

3. The method for preparing a trihexyphenidyl artificial hapten according to claim 2, wherein: In step (S.1), the molar ratio of free benzhexol to ethyl bromoacetate is 1:2-10.

4. The method for preparing a trihexyphenidyl artificial hapten according to claim 2, wherein: The reaction temperature of the heating and stirring reflux process in step (S.1) is 60-70°C, and the reaction time is 12-20h; The reaction temperature of the heating and stirring process in step (S.2) is 0 to 40°C, and the reaction time is 10 to 20 hours.

5. A trihexyphenidyl artificial antigen, characterized in that: Its molecular structure is shown in formula (II): Formula (II); Among them, BGG is bovine immunoglobulin G.

6. The method for preparing a trihexyphenidyl artificial antigen as claimed in claim 5, characterized in that: The following steps are involved: The artificial hapten I of benzhexol as claimed in claim 1 is combined with bovine immunoglobulin gamma by using the N-hydroxysuccinimide active ester method to obtain the artificial antigen II of benzhexol.

7. The method for preparing a trihexyphenidyl artificial antigen according to claim 6, characterized in that: The following steps are involved: (a) mixing the benzhexol artificial hapten I as claimed in claim 1 with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, heating and stirring for reaction, centrifuging after the reaction is completed, and taking the supernatant; (b) adding the supernatant obtained in step (a) dropwise to the bovine immunoglobulin solution and mixing evenly to obtain a mixed solution, and letting it stand. After the reaction is complete, dialyzing and centrifuging are performed, and the supernatant is taken to obtain the benzhexidine artificial antigen II.

8. The method for preparing a trihexyphenidyl artificial antigen according to claim 7, characterized in that: The molar ratio of the benzhexol artificial hapten I added in step (a) to cyclohexylcarbodiimide and N-hydroxysuccinimide is 1:(1.1-1.5):(1.1-1.5); The concentration of the bovine immunoglobulin G solution in step (b) is 4-6 mg / mL; the volume ratio of the supernatant obtained in step (a) added in the mixing process to the bovine immunoglobulin G solution is 1:8-12.

9. Use of the trihexyphenidyl artificial antigen as claimed in claim 5 in the preparation of anti-trihexyphenidyl antibodies.

10. An anti-benzhexol antibody, characterized in that: The globulin is obtained by immunizing animals with the artificial benzhexol antigen as claimed in claim 5 and can generate specific immune reaction with benzhexol and its analogs.

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