A trihexyphenidyl artificial hapten, artificial antigen, and preparation method and application thereof

Abstract

The present invention belongs to the field of biochemical technology, and specifically relates to an artificial hapten and an artificial antigen for benzhexyphenidyl, as well as a preparation method and application thereof. The molecular structural formula of the artificial hapten for benzhexyphenidyl is shown in Formula (I), and the molecular structural formula of the artificial antigen for benzhexyphenidyl is shown in Formula (II). The artificial hapten for benzhexyphenidyl can be used in the field of preparing anti-benzhexyphenidyl antibodies. The artificial hapten for benzhexyphenidyl of the present invention retains the characteristic structure of benzhexyphenidyl to the greatest extent, and has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant. The artificial hapten for benzhexyphenidyl obtained by further preparation can be used to immunize and obtain anti-benzhexyphenidyl 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 1:256,000, and can be used for rapid and accurate immunodetection and immunoanalysis of benzhexyphenidyl.

Description

Technical Field

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

[0002] Trihexyphenidyl, also known as trihexyphenidyl and antan, is a common anticholinergic drug. It blocks choline and acts within the central nervous system, maintaining balance and inhibiting muscle rigidity and tremors. Medically, it is primarily used to alleviate the discomfort associated with Parkinson's disease and Parkinson's syndrome. It can also be used for drug-induced extrapyramidal disorders.

[0003] With the increasing popularity of trihexyphenidyl, cases of overdose and abuse have emerged. Trihexyphenidyl overdose and abuse can result in a range of adverse reactions. Symptoms of overdose include dilated pupils, dry mucous membranes, facial redness, bowel and bladder weakness, and, at high doses, a high temperature. Trihexyphenidyl can cause agitation, confusion, and hallucinations due to its effects on the central nervous system. Untreated overdose can be fatal, especially in children, with respiratory depression and cardiac arrest being possible 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] Currently, the detection of benzhexol mainly relies on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance hydrogen spectroscopy (H NMR), nuclear magnetic resonance carbon spectroscopy (13C NMR), nuclear magnetic resonance fluorine spectroscopy (19F NMR), infrared spectroscopy (IR), thin-layer chromatography (TLC), etc. However, all of these methods have the disadvantages of expensive instruments, time-consuming detection, and the need for professional technicians to operate, making it difficult to meet the modern requirements of rapid and accurate detection. Therefore, it is very necessary to establish a rapid, sensitive and accurate detection technology.

[0006] Immunochromatographic rapid detection technology can overcome all of these shortcomings. This technology boasts strong specificity, high sensitivity, simplicity, and cost-effectiveness. It can achieve qualitative and semi-quantitative detection of various analytes, including antigens, haptens, and antibodies, without requiring specialized expertise or expensive, complex instrumentation. It is widely used in the detection of substances such as hormones, tumor markers, drugs, bacteria, and viruses. Since antigens and antibodies are essential raw materials for this technology, it is necessary to provide an effective method for preparing trihexyphenidyl antigen-antibodies for use in trihexyphenidyl immunoassays.

[0007] Since most small molecule compounds (molecular weight less than 1000), including benzhexol, are non-immunogenic, meaning they lack T-cell epitopes and cannot directly induce the production of specific antibodies in animals, small molecules are called haptens. Through appropriate chemical modification, a linker with an active group at the end is added to a certain position of the hapten molecule. This is then combined with a macromolecular carrier to produce a hapten-carrier conjugate (i.e., an artificial antigen). This artificial antigen can indirectly induce the proliferation and differentiation of B cells through the use of T-cell epitopes, subsequently producing specific antibodies, thereby preparing anti-benzhexol antibodies.

[0008] There is no relevant report on trihexyphenidyl artificial hapten or artificial antigen 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 anti-trihexyphenidyl antibodies with high detection sensitivity and strong specificity.

[0010] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0011] A trihexyphenidyl artificial hapten, whose molecular structure is shown in formula (I):

[0012]

[0013] The artificial hapten of benzhexol provided by 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 serve as an antigenic determinant.

[0014] The method for preparing the above-mentioned trihexyphenidyl artificial hapten comprises the following steps:

[0015] (S.1) Concentrated aqueous ammonia is diluted with deionized water, and then benzhexyphenidyl hydrochloride is added to obtain free benzhexyphenidyl. The free benzhexyphenidyl is mixed with ethyl bromoacetate in N,N-dimethylformamide, sodium hydride is added, and the mixture is heated and stirred under reflux until the reaction is complete. The mixture is drained, deionized water is added, and the mixture is extracted with ethyl acetate. The organic phase is collected, dried, filtered, drained, separated, and purified to obtain a white solid A.

[0016] (S.2) The white solid A obtained in step (S.1) is dissolved in methanol and tetrahydrofuran, and sodium hydroxide solution is added. The mixture is heated and stirred until the reaction is complete. The pH is adjusted with hydrochloric acid, and the mixture is extracted with ethyl acetate. The mixture is dried, separated, and purified to obtain the artificial hapten I of benzhexol.

[0017] The present invention adopts an N,N-dimethylformamide-sodium hydride system and uses sodium hydride as a catalyst, which helps to better combine a connecting arm with 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 at the hydroxyl group of benzhexol. By introducing the connecting arm at this modification site, the characteristic structure of benzhexol can be retained to the greatest extent. Compared with the use of a general cyclic structure as a connecting arm, the connecting arm adopted by 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, thereby enhancing the specificity of the artificial hapten, maintaining conformational stability in solution, and improving the immune properties of the benzhexol artificial antigen II.

[0018] Preferably, in step (S.1), the molar ratio of free benzhexol to ethyl bromoacetate is 1:2-10.

[0019] Preferably, the reaction temperature during the heating and stirring reflux process in step (S.1) is 60-70° C., and the reaction time is 12-20 h;

[0020] The reaction temperature during the heating and stirring process in step (S.2) is 0-40°C, and the reaction time is 10-20h.

[0021] By controlling the reaction temperature and reaction time, it is helpful to reduce by-products to a minimum, thereby increasing the yield and reducing costs.

[0022] Preferably, the separation and purification process in step (S.1) adopts a TLC separation and purification method, the developing solvent used is ethyl acetate, and the solvent and eluent used are ethanol.

[0023] Using ethyl acetate as a developing agent helps to separate the product, facilitates subsequent operations, and saves costs.

[0024] A trihexyphenidyl artificial antigen, the molecular structure of which is shown in formula (II):

[0025]

[0026] Among them, BGG is bovine immunoglobulin G.

[0027] The method for preparing the above-mentioned trihexyphenidyl artificial antigen comprises the following steps:

[0028] The benzhexidine artificial hapten I as claimed in claim 1 is combined with bovine immunoglobulin gamma by using the N-hydroxysuccinimide active ester method to obtain the benzhexidine artificial antigen II.

[0029] Preferably, the method for preparing the above-mentioned benzhexol artificial antigen comprises the following steps:

[0030] (a) mixing the benzhexol artificial hapten I as claimed in claim 1 with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, heating and stirring to react, centrifuging after the reaction is completed, and collecting the supernatant;

[0031] (b) adding the supernatant obtained in step (a) dropwise to the bovine immunoglobulin solution and mixing evenly to obtain a mixed solution, and allowing the mixed solution to stand. After the reaction is complete, dialyzing and centrifugation are performed, and the supernatant is collected to obtain trihexyphenidyl artificial antigen II.

[0032] 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);

[0033] 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 during the mixing process to the bovine immunoglobulin G solution is 1:8-12.

[0034] As further preferred, the method for preparing the bovine gamma globulin solution comprises the following steps:

[0035] The bovine immunoglobulin G is dissolved in 0.01 M PBS buffer solution, wherein the pH of the PBS buffer solution is 7.2-7.4.

[0036] The present invention uses bovine immunoglobulin G as a macromolecular carrier, which has the following advantages compared to traditional bovine serum albumin: (1) the bovine immunoglobulin G has strong immunogenicity and can more easily obtain high-titer serum by immunizing New Zealand white rabbits;

[0037] (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.

[0038] As a further preference, the molar ratio of the benzhexol artificial hapten I to cyclohexylcarbodiimide and N-hydroxysuccinimide added in step (a) is 1:(1.3-1.4):(1.3-1.4).

[0039] Use of the above-mentioned benzhexol artificial antigen in preparing anti-benzhexol antibodies.

[0040] 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.

[0041] The present invention has found through repeated experiments that the titer of immune serum obtained by immunizing New Zealand white rabbits with the artificial benzhexol antigen is 1:256,000. This indicates that the artificial benzhexol antigen of the present invention can be used to immunize and obtain anti-benzhexol antibodies with high affinity, high sensitivity, and strong specificity. The anti-benzhexol antibodies can be used for immunodetection and analysis of benzhexol.

[0042] Therefore, the present invention has the following beneficial effects:

[0043] The artificial benzhexol hapten 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 serve as an antigenic determinant. The artificial benzhexol antigen further prepared 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 1:256,000, and the immune serum can be used for rapid and accurate immunodetection and immunoanalysis of benzhexol. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a flow chart for the preparation of the benzhexol artificial antigens II and IV of Example 1 and Comparative Example 2 of the present invention.

[0045] Figure 2 It is a liquid chromatogram of the artificial hapten I of benzhexol of the present invention.

[0046] Figure 3 It is the mass spectrum of the artificial hapten I of benzhexol of the present invention.

[0047] Figure 4 These are UV scans of BGG, trihexyphenidyl artificial hapten I, and trihexyphenidyl artificial antigen II.

[0048] Figure 5 This is a flow chart for the preparation of the trihexyphenidyl artificial antigens III and V in Comparative Examples 1 and 3.

[0049] Figure 6 This is a flow chart for the preparation of the trihexyphenidyl artificial antigens VII and IX in Comparative Examples 4 and 6.

[0050] Figure 7 This is a flow chart for the preparation of the benzhexol artificial antigens VIII and X in Comparative Examples 5 and 7. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0052] Example 1

[0053] This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0054] A method for preparing a trihexyphenidyl artificial antigen comprises the following steps:

[0055] (1) Preparation of trihexyphenidyl artificial hapten I:

[0056] (S.1)

[0057] (S.11) Prepare DAS solution: Dilute 5 mL of concentrated ammonia to 13 mL with deionized water to obtain DAS solution.

[0058] (S.12) Weigh 500 mg (1.483 mmol) of benzhexyphenidyl hydrochloride into a 50 mL centrifuge tube and dissolve in 15 mL of deionized water. Adjust the pH to 9 with the DAS solution described above. A large amount of precipitate will form. Extract the mixture three times with 20 mL of dichloromethane each time. Collect the organic phase, dry it over anhydrous magnesium sulfate, filter it, and transfer it to dryer to yield 444 mg (1.475 mmol) of free benzhexyphenidyl.

[0059] (S.13) 100 mg (0.332 mmol) of free benzhexol was mixed with 295 μL (2.658 mmol) of ethyl bromoacetate and dissolved in 5 mL of N,N-dimethylformamide (DMF). 53 mg (1.328 mmol) of 60% sodium hydride was added as a catalyst and the mixture was stirred under reflux at 65°C for 16 h. TLC analysis (developing solvent: ethyl acetate:concentrated aqueous ammonia = 200:1, v / v) showed that the reaction was complete, with a product Rf = 0.2. The mixture was directly transferred to dryness, dissolved in 20 mL of deionized water, and extracted twice with 20 mL of ethyl acetate each time. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and transferred to dryness to yield a pale yellow solid. Separation and purification by TLC (developing solvent: ethyl acetate, solvent and eluent: ethanol) yielded 115 mg (0.298 mmol) of white solid A.

[0060] (S.2)

[0061] (S.21) Prepare a 1 mol / L sodium hydroxide solution: Dissolve 8 g of solid sodium hydroxide in 200 mL of deionized water to obtain a 1 mol / L sodium hydroxide solution.

[0062] (S.22) 115 mg (0.298 mmol) of the white solid A obtained in step (S.1) was dissolved in 3.2 mL of methanol (MeOH) and 2.7 mL of tetrahydrofuran (THF) in a 50 mL single-necked flask. 13 mL of the 1 mol / L aqueous sodium hydroxide solution prepared in step (S.21) was added, and the mixture was stirred at 25°C for 16 h. TLC analysis (developing solvent: dichloromethane: 95% ethanol: 1,4-dioxane: concentrated aqueous ammonia = 10:8:1:1, v / v / v / v, hereinafter collectively referred to as MOP; the solvent and eluent were anhydrous ethanol) indicated that the reaction was complete, with Rf = 0.7 for the raw material and Rf = 0.3 for the product.

[0063] (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.

[0064] (S.24) The reaction solution after stirring and reacting 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, and extracted three times. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain a white solid. The solid was separated and purified by TLC (developing solvent: MOP, solvent and eluent: anhydrous ethanol) to obtain 67 mg (0.187 mmol) of benzhexol artificial hapten I.

[0065] The molecular structure of trihexyphenidyl artificial hapten I is shown in formula (I):

[0066]

[0067] The preparation reaction process of the artificial hapten I of benzhexol in this embodiment is shown in Figure 1 The liquid chromatography diagram of trihexyphenidyl artificial hapten I is shown in Figure 2 The mass spectrum of benzhexol hapten I is shown in Figure 3 .

[0068] 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 benzhexol artificial hapten I designed by the present invention.

[0069] (2) Preparation of trihexyphenidyl artificial antigen II:

[0070] (a) 67 mg (0.187 mmol) of the benzhexol artificial hapten I obtained in step (S.24) above was placed in a 50 mL round-bottom flask, 3.35 mL of DMF was added, and then 51 mg (0.252 mmol) of cyclohexylcarbodiimide (DCC) and 28 mg (0.252 mmol) of N-hydroxysuccinimide were added and mixed uniformly. The mixture was stirred at 25°C for 16 h. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected for later use.

[0071] (b) Weigh 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 and dissolve them in deionized water to 5.0 L to obtain 0.01 M PBS buffer solution. The pH of the PBS buffer solution was 7.4.

[0072] Weigh 167.5 mg of bovine gamma globulin (BGG) and dissolve it in 33.5 mL of the prepared PBS buffer to obtain a 5 mg / mL BGG solution.

[0073] Under rapid stirring, the supernatant obtained in step (a) is slowly added dropwise to the bovine immunoglobulin solution and mixed evenly, the volume ratio of the supernatant to the bovine immunoglobulin solution being 1:10, and the resulting mixture is stored at 4° C. overnight to obtain a trihexyphenidyl artificial antigen mixture;

[0074] 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 collected to obtain the benzhexol artificial antigen II. This is also the benzhexol-bovine immunoglobulin conjugate (Formula II). The UV 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):

[0075]

[0076] Among them, BGG is bovine immunoglobulin G.

[0077] Figure 4 In the figure, curve a is the UV scanning diagram of the artificial hapten of benzhexol, curve b is the UV scanning diagram of the artificial antigen of benzhexol, and curve c is the UV 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 significantly changes. It can be illustrated that benzhexol and bovine gamma globulin are successfully coupled thus.

[0078] Example 2

[0079] The difference between this embodiment and embodiment 1 is that:

[0080] This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0081] A method for preparing a trihexyphenidyl artificial antigen, wherein:

[0082] (1) Preparation process of trihexyphenidyl artificial hapten I:

[0083] In step (S.13), 100 mg (0.332 mmol) of free benzhexol was weighed and mixed with 368.47 μL (3.32 mmol) of ethyl bromoacetate, 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. All other steps were the same as in Example 1. This yielded the benzhexol artificial hapten I.

[0084] (2) Preparation process of trihexyphenidyl artificial antigen II:

[0085] In step (a), 67 mg (0.187 mmol) of benzhexyphenidyl artificial hapten I was placed in a 50 mL round-bottom flask, 3.35 mL of DMF was added, and then 41.63 mg (0.2057 mmol) of cyclohexylcarbodiimide and 22.85 mg (0.2057 mmol) of N-hydroxysuccinimide were added and mixed uniformly. In step (b), 134 mg of bovine immunoglobulin (BIG) was weighed and dissolved in 33.5 mL of the prepared PBS buffer to obtain a 4 mg / mL BIG solution. The volume ratio of the supernatant to the BIG solution was 1:8. The same coupling steps as in Example 1 were used, with all other steps being the same as in Example 1. Finally, benzhexyphenidyl artificial antigen II was obtained.

[0086] Example 3

[0087] The difference between this embodiment and embodiment 1 is that:

[0088] This embodiment provides a trihexyphenidyl artificial hapten I and a preparation method thereof, and a trihexyphenidyl artificial antigen II and a preparation method thereof.

[0089] A method for preparing a trihexyphenidyl artificial antigen, wherein:

[0090] (1) Preparation process of trihexyphenidyl artificial hapten I:

[0091] In step (S.13), 100 mg (0.332 mmol) of free benzhexol was weighed and mixed with 73.69 μL (0.664 mmol) of ethyl bromoacetate, 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. All other steps were the same as in Example 1. Finally, the benzhexol artificial hapten I was obtained.

[0092] (2) Preparation process of trihexyphenidyl artificial antigen II:

[0093] In step (a), 67 mg (0.187 mmol) of benzhexol artificial hapten I was placed in a 50 mL round-bottom flask, 3.35 mL of DMF was added, and then 56.77 mg (0.2805 mmol) of cyclohexylcarbodiimide and 31.17 mg (0.2805 mmol) of N-hydroxysuccinimide were added and mixed uniformly. In step (b), 201 mg of bovine immunoglobulin (BIG) was weighed and dissolved in 33.5 mL of the prepared PBS buffer to obtain a 6 mg / mL BIG solution. The volume ratio of the supernatant to the BIG solution was 1:12. The same coupling steps as in Example 1 were used, with all other steps being the same as in Example 1. Finally, benzhexol artificial antigen II was obtained.

[0094] Comparative Example 1

[0095] The difference between this comparative example and Example 1 is:

[0096] 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.

[0097] A method for preparing a benzhexol artificial antigen, wherein: (1) the preparation process of the benzhexol artificial hapten I is the same as that in Example 1. (2) the preparation process of the benzhexol artificial 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 a 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 benzhexol artificial antigen III is obtained. The preparation reaction process of the benzhexol artificial antigen III in this comparative example is as follows: Figure 5 shown.

[0098] Comparative Example 2

[0099] The difference between this comparative example and Example 1 is:

[0100] 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.

[0101] A method for preparing a benzhexol artificial antigen IV, wherein: (1) the preparation process of the benzhexol artificial hapten I is the same as that in Example 1. (2) the preparation process of the benzhexol artificial antigen IV: 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 benzhexol artificial antigen IV is obtained. The preparation reaction process of the benzhexol artificial antigen IV in this comparative example is as follows: Figure 1 shown.

[0102] Comparative Example 3

[0103] The difference between this comparative example and Example 1 is:

[0104] 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.

[0105] A method for preparing an artificial antigen V of benzhexol, wherein: (1) the preparation process of the artificial hapten I of benzhexol is the same as that in Example 1. (2) the preparation process of the artificial antigen V of benzhexol: 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 benzhexol is obtained. The preparation reaction process of the artificial antigen V of benzhexol in this comparative example is as follows: Figure 5 shown.

[0106] Comparative Example 4

[0107] The difference between this comparative example and Example 1 is:

[0108] 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.

[0109] A method for preparing trihexyphenidyl artificial antigen VII comprises the following steps:

[0110] (1) Preparation of trihexyphenidyl artificial hapten VI:

[0111] (S.1)

[0112] (S.11) Prepare DAS solution: Dilute 5 mL of concentrated ammonia to 13 mL with deionized water to obtain DAS solution.

[0113] (S.12) Weigh 500 mg (1.483 mmol) of benzhexyphenidyl hydrochloride into a 50 mL centrifuge tube and dissolve in 15 mL of deionized water. Adjust the pH to 9 with the DAS solution described above. A large amount of precipitate will form. Extract the mixture three times with 20 mL of dichloromethane each time. Collect the organic phase, dry it over anhydrous magnesium sulfate, filter it, and transfer it to dryer to yield 444 mg (1.475 mmol) of free benzhexyphenidyl.

[0114] (S.13) 100 mg (0.332 mmol) of the free benzhexol obtained in step (S.12) was added to a 50 mL round-bottom flask, 10 mL of dichloromethane was added, the mixture was placed in an ice bath and stirred to cool to 0°C, and 67 μL (0.481 mmol) of triethylamine was added. 49 μL (0.398 mmol) of succinic acid monoester chloride was dissolved in 2 mL of dichloromethane and slowly added dropwise to the system. White smoke was generated, and the system turned light brown after the addition was complete. The mixture was transferred to 25°C and stirred for 5.5 hours. TLC analysis (developing solvent: ethyl acetate: ammonia water = 200:1, v / v) showed that the reaction was complete, with a product spot Rf = 0.4. The mixture was dried to obtain 169 mg of brown solid B.

[0115] (S.2)

[0116] (S.21) Prepare a 1 mol / L sodium hydroxide solution: Dissolve 8 g of solid sodium hydroxide in 200 mL of deionized water to obtain a 1 mol / L sodium hydroxide solution.

[0117] (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. 16 mL of the 1 mol / L sodium hydroxide aqueous solution prepared in step (S.21) was added. The mixture was stirred at 25°C for 18 h. TLC (developing solvent: MOP, solvent and eluent: anhydrous ethanol) showed that the reaction was complete. The product point Rf = 0.3;

[0118] (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.

[0119] (S.24) The reaction solution after the stirring reaction in step (S.22) was adjusted to pH 3 with 1 mol / L hydrochloric acid prepared in step (S.23). The mixture was extracted three times with 20 mL of ethyl acetate each time. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and dried to obtain 148 mg of a light yellow solid. The solid was separated and purified by TLC (developing solvent: MOP, solvent and eluent: anhydrous ethanol) to obtain 110 mg (0.274 mmol) of trihexyphenidyl artificial hapten VI.

[0120] (2) Preparation of trihexyphenidyl artificial antigen VII:

[0121] (a) 110 mg (0.274 mmol) of the artificial hapten VI of benzhexol obtained in step (S.24) above 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 uniformly. The mixture was stirred at 25°C for 16 h. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected for later use.

[0122] (b) Weigh 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 and dissolve them in deionized water to 5.0 L to obtain 0.01 M PBS buffer solution. The pH of the PBS buffer solution was 7.4.

[0123] 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;

[0124] Under rapid stirring, the supernatant obtained in step (a) is slowly added dropwise to the bovine serum albumin solution and mixed evenly, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the resulting mixture is stored at 4°C overnight to obtain a trihexyphenidyl artificial antigen mixture;

[0125] The trihexyphenidyl artificial antigen mixture was transferred into a dialysis bag and dialyzed 9 times with the above-prepared PBS buffer. After the dialysis, the mixture was centrifuged and the supernatant was collected to obtain the trihexyphenidyl artificial antigen VII.

[0126] Comparative Example 5

[0127] The difference between this comparative example and comparative example 4 is:

[0128] 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.

[0129] A method for preparing a benzhexol artificial antigen VIII, wherein: (1) the preparation process of the benzhexol artificial hapten VI is the same as that in Comparative Example 4. (2) the preparation process of the benzhexol artificial antigen VIII: the bovine serum albumin (i.e., carrier protein) in step (b) is replaced with bovine immunoglobulin G, 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 benzhexol artificial antigen VIII is obtained. The preparation reaction process of the benzhexol artificial antigen VIII in this comparative example is as follows: Figure 7 shown.

[0130] Comparative Example 6

[0131] The difference between this comparative example and comparative example 4 is:

[0132] 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.

[0133] A method for preparing a trihexyphenidyl artificial antigen IX, wherein: (1) the preparation process of the trihexyphenidyl artificial hapten VI is the same as that in Comparative Example 4. (2) the preparation process of the trihexyphenidyl artificial antigen IX: the volume ratio of the supernatant to the bovine serum albumin solution in step (b) is replaced by a volume ratio of the supernatant to the bovine serum albumin solution of 1:5 to 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 trihexyphenidyl artificial antigen IX is obtained. The preparation reaction process of the trihexyphenidyl artificial antigen IX in this comparative example is as follows: Figure 6 shown.

[0134] Comparative Example 7

[0135] The difference between this comparative example and comparative example 4 is:

[0136] 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.

[0137] A method for preparing a benzhexol artificial antigen X, wherein: (1) the preparation process of the benzhexol artificial hapten VI is the same as that in Comparative Example 4. (2) the preparation process of the benzhexol artificial antigen X: the bovine serum albumin (i.e., carrier protein) in step (b) is replaced with bovine immunoglobulin G, 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 1:10. The same coupling steps as in Comparative Example 4 are adopted, and the rest are the same as in Comparative Example 4. Finally, the benzhexol artificial antigen X is obtained. The preparation reaction process of the benzhexol artificial antigen X in this comparative example is as follows: Figure 7 shown.

[0138]

Performance determination of trihexyphenidyl artificial antigen

[0139] (1) Identification of trihexyphenidyl artificial antigen:

[0140] Molar Absorption Coefficient ε: Buffer solutions of trihexyphenidyl artificial hapten were prepared at concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. UV scanning revealed that the maximum absorption wavelength of trihexyphenidyl artificial hapten I was 297 nm, and that of artificial hapten VI was 286 nm. The absorbance of the corresponding antigens was measured at 297 nm and 286 nm, respectively, with replicates prepared for each concentration. The molar absorption coefficient (i.e., molar absorption coefficient) was calculated as follows: ε = absorbance / molar concentration. The calculated data are shown in Table 1 below.

[0141] Table 1: Calculation table of molar absorption coefficients of trihexyphenidyl artificial haptens I and VI

[0142]

[0143] Determination of conjugate protein concentration: 1 mL of bovine serum albumin solution at 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 was prepared in PBS buffer. 3 mL of Coomassie Brilliant Blue staining solution was added and immediately mixed. The samples were warmed in a 30°C water bath for 5 minutes. Parallel samples were prepared for each concentration and the absorbance was measured at 655 nm. A curve was plotted showing the relationship between protein concentration and absorbance. The artificial antigen solution (prepared in PBS buffer) was diluted in a certain proportion and the absorbance of the artificial antigen was measured at 655 nm. The corresponding protein concentration of the artificial antigen solution was read from the curve. The calculated data are shown in Table 2 below.

[0144] 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), and the calculation data results are shown in Table 2 below.

[0145] Where ε is the molar absorption coefficient (L / mol), 43000 is the molecular weight of bovine gamma globulin, 100×10 -3 is the concentration of bovine immunoglobulin G (g / L).

[0146] 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.

[0147] Table 2: Calculation results of coupling ratios of various benzhexol artificial antigens and protein concentrations of the conjugates

[0148]

[0149]

[0150] 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.

[0151] (2) Animal immunization

[0152] Each prepared artificial antigen of trihexyphenidyl was used to immunize New Zealand white rabbits, and the titer of the obtained immune serum was tested by ELISA. The test results are shown in Table 3 below.

[0153] Table 3: Titer test results of each immune serum

[0154] 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 .

[0155] As shown in Table 3, compared with Example 1, the immune serum obtained by animal immunization using the artificial benzhexol antigens in each comparative example had a low titer and was not suitable for immunoassay. The artificial benzhexol antigen V and artificial benzhexol antigen VIII obtained in comparative examples 3 and 5 were relatively turbid, and a large amount of precipitation occurred after freezing and storage, and their stability was very poor, so they could not be used as immune antigens. The immune serum obtained by animal immunization using the artificial benzhexol antigen II had a titer of 1:256,000, which could be used in immunoassay fully and could provide a more convenient, rapid and accurate approach for the detection of benzhexol.

[0156] 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 hapten of benzhexol can be used in the field of preparing 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, which can serve as an antigenic determinant. The artificial hapten of benzhexol obtained by further preparation can be used to immunize and obtain anti-benzhexol polyclonal antibodies with high affinity, high sensitivity, and strong specificity. The titer of the immune serum obtained by immunizing New Zealand white rabbits can reach 1:256,000, which can be used for rapid and accurate immunodetection and immunoanalysis of benzhexol.

[0157] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, based on the ideas provided by the present invention, there may be changes in the specific implementation methods, and these changes should also be considered as the scope of protection 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, wherein: The following steps are involved: (S.1) Concentrated aqueous ammonia is diluted with deionized water, and then benzhexyphenidyl hydrochloride is added to obtain free benzhexyphenidyl. The free benzhexyphenidyl is mixed with ethyl bromoacetate in N,N-dimethylformamide, sodium hydride is added, and the mixture is heated and stirred under reflux until the reaction is complete. The mixture is drained, deionized water is added, and the mixture is extracted with ethyl acetate. The organic phase is collected, dried, filtered, drained, separated, and purified to obtain a white solid A. (S.2) The white solid A obtained in step (S.1) is dissolved in methanol and tetrahydrofuran, and sodium hydroxide solution is added. The mixture is heated and stirred until the reaction is complete. The pH is adjusted with hydrochloric acid, and the mixture is extracted with ethyl acetate. The mixture is dried, separated, and purified to obtain the artificial hapten I of benzhexol.

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 during the heating and stirring reflux process in step (S.1) is 60-70°C, and the reaction time is 12-20 hours; The reaction temperature during the heating and stirring process in step (S.2) is 0-40°C, and the reaction time is 10-20h.

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, wherein: The following steps are involved: The benzhexidine artificial hapten I as claimed in claim 1 is combined with bovine immunoglobulin gamma by using the N-hydroxysuccinimide active ester method to obtain the benzhexidine artificial antigen II.

7. The method for preparing a trihexyphenidyl artificial antigen according to claim 6, wherein: 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 to react, centrifuging after the reaction is completed, and collecting 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 allowing the mixed solution to stand. After the reaction is complete, dialyzing and centrifugation are performed, and the supernatant is collected to obtain trihexyphenidyl artificial antigen II.

8. The method for preparing a trihexyphenidyl artificial antigen according to claim 7, wherein: 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 during 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 preparing 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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