Medetomidine artificial hapten, artificial antigen and preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a medetomidine artificial hapten, an artificial antigen as well as a preparation method and application of the medetomidine artificial hapten and the artificial antigen. The medetomidine artificial hapten maximally retains the characteristic structure of medetomidine, has an active group capable of being coupled with carrier protein, and can be used as an antigenic determinant; the medetomidine artificial antigen obtained by further preparation can be immunized to obtain an anti-medetomidine polyclonal antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit is as high as 1: 135000, and the medetomidine artificial antigen can be used for rapid and accurate immunodetection and immunoassay of medetomidine.

Description

Technical Field

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

[0002] Medetomidine is a class of alpha-2 adrenergic receptor agonist sedatives, primarily used for sedation in intensive care units and as an adjunct to general anesthesia. Medetomidine produces its sedative effect by selectively stimulating alpha-2 adrenergic receptors in the central nervous system, characterized by providing effective sedation while maintaining the patient's arousal. This drug exhibits a dose-dependent sedative effect, producing mild sedation at low doses and achieving deep sedation with increasing doses.

[0003] Medetomidine has been found to be effective in various animal models associated with drug abuse, addiction, and dependence, and may represent a new potential treatment for these diseases. Currently, the detection of medetomidine mainly relies on high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), and mass spectrometry (MS), but these methods have drawbacks such as expensive equipment, time-consuming testing, and the need for specialized technicians, failing to meet the requirements of modern testing for speed and accuracy.

[0004] Immunoassay can overcome all the above shortcomings. It is an analytical method that uses the specific binding reaction between antigen and antibody to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to establishing immunoassay methods for small molecule compounds is the ability to produce medemidin antigen and antibodies with high affinity and specificity for medemidin. However, most small molecule compounds (molecular weight less than 1000), including medemidin, lack immunogenicity, meaning they lack T-cell epitopes and cannot directly induce the production of specific antibodies in animals. Therefore, small molecules are called haptens. Through appropriate chemical modification, a linker arm with an active group at its end is added to a certain position in the hapten molecule structure. This linker is then combined with a macromolecular carrier to generate a hapten-carrier conjugate (i.e., an artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells through T-cell epitopes, thereby producing specific antibodies. Therefore, the efficient synthesis of artificial antigens is a prerequisite and key to ensuring the success of immunoassay. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a medetomidine artificial hapten, an artificial antigen, and a method for preparing the same, and applies the medetomidine artificial antigen to the preparation of anti-medetomidine antibodies with high sensitivity and specificity.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: A medetomidine artificial hapten, characterized in that its molecular structure is shown in formula (I): ; Equation (Ⅰ).

[0007] The medemidin artificial hapten of the present invention retains the characteristic structure of medemidin to the greatest extent and has an active group that can be coupled with carrier proteins, and can be used as an antigenic determinant.

[0008] The method for preparing a medemididine artificial hapten as described above includes the following steps: (S.1) DAS solution was obtained by diluting concentrated ammonia with deionized water. Metopril hydrochloride was dissolved in deionized water and added to DAS solution to adjust the pH to alkaline. Then it was extracted with dichloromethane, the dichloromethane phase was collected, dried, filtered and dried to obtain colorless oily substance A. (S.2) Dissolve the colorless oily substance A obtained in step (S.1) in concentrated sulfuric acid, add potassium nitrate, and stir the reaction in an ice-water bath. After the reaction is complete, adjust the pH, dry the mixture directly, extract with anhydrous ethanol, filter, dry, and purify to obtain yellow bubbly solid B. (S.3) Add iron powder and acetic acid I to the reaction vessel and heat to obtain a mixed reaction solution. Dissolve the yellow bubbly solid B obtained in step (S.2) in acetic acid II and add it dropwise to the mixed reaction solution to carry out the reaction. After the reaction is completed, filter, dry directly, extract with anhydrous ethanol, filter, dry, and purify to obtain a pale yellow solid C. (S.4) Dissolve the pale yellow solid C obtained in step (S.3) in anhydrous acetone, add potassium carbonate and ethyl bromoacetate, and stir the reaction at room temperature under nitrogen protection. After the reaction is completed, filter, dry and purify to obtain pale yellow bubbly solid D. (S.5) Dissolve the pale yellow bubbly solid D obtained in step (S.4) in tetrahydrofuran and anhydrous methanol, add sodium hydroxide solution, stir at room temperature until the reaction is complete, adjust the pH with hydrochloric acid, directly dry, extract with anhydrous ethanol, filter, dry, and purify to obtain metoprimidine artificial hapten I.

[0009] This invention introduces a linker arm at the β-position of the benzene ring of medetomidine via a nitration reaction. Introducing a linker arm at this modification site can preserve the characteristic structure of medetomidine to the greatest extent, and the modification site is as far away as possible from other characteristic functional groups of medetomidine, so as to expose its characteristic parts to the outside as much as possible, thereby avoiding interference with specific antigenic determinants and maximizing recognition by the immune body.

[0010] Compared with the use of a ring-shaped linker arm, the linker arm used in this invention is chain-shaped. The linker arm is of appropriate length, which ensures that the medemidin hapten small molecule can be fully exposed on the surface of the artificial antigen, and minimizes the recognition of the linker arm by T cells during immunization. In this way, the antibodies obtained by immunization have stronger specificity and affinity for medemidin.

[0011] Preferably, in step (S.1), DAS solution is added to adjust the pH to 9.

[0012] Preferably, the sample is extracted three times with dichloromethane in step (S.1).

[0013] Under the above reaction conditions, the yield of colorless oily substance A is relatively high, the post-processing procedure is relatively simple, and it is relatively easy to purify.

[0014] Preferably, in step (S.2), the ratio of colorless oily substance A to concentrated sulfuric acid and potassium nitrate is 90~110:0.8~1.2:45~55mg / mL / mg.

[0015] Preferably, in step (S.2), the colorless oily substance A obtained in step (S.1) is dissolved in concentrated sulfuric acid at 0°C; the reaction is carried out by stirring in an ice-water bath at 0°C; the pH of the reaction system is adjusted to about 5-7 with sodium bicarbonate; and the purification method is thin-layer chromatography.

[0016] Under the above reaction conditions, the reaction in an ice-water bath at 0°C effectively controls the exothermic reaction of concentrated sulfuric acid. The subsequent processing of the residue by extraction with anhydrous ethanol is relatively simple, and the product is also easier to purify. Thin-layer chromatography (TLC) can maximize the extraction of the target product.

[0017] Preferably, in step (S.3), the ratio of iron powder, acetic acid I, yellow bubbly solid B to acetic acid II is 800~840:4.5~5.5:90~110:4.5~5.5 mg / mL / mg / mL.

[0018] Preferably, in step (S.4), the ratio of the pale yellow solid C, anhydrous acetone, potassium carbonate and ethyl bromoacetate is 90~110:15~25:450~550:106~126 mg / mL / mg / μL.

[0019] Under the above reaction conditions, the pale yellow solid C is readily soluble in anhydrous acetone, and the yield and purity of the pale yellow bubbly solid D are both high, with a relatively simple post-processing procedure. Under nitrogen protection, the generation of reaction byproducts can be controlled, thereby improving yield and purity.

[0020] Preferably, in step (S.5), the ratio of the pale yellow bubbly solid D, tetrahydrofuran, anhydrous methanol and sodium hydroxide solution is 90~110:1.25~1.75:1.5~2.1:7~9 mg / mL / mL.

[0021] Preferably, the reaction time for the stirring process in the ice-water bath in step (S.2) is 2 to 4 hours.

[0022] Preferably, the reaction temperature of the heating process in step (S.3) is 45~55℃ and the reaction time is 5~7h.

[0023] Preferably, the reaction time for the room temperature stirring process in step (S.4) is 16~24h.

[0024] Preferably, the reaction time for the room temperature stirring process in step (S.5) is 3 to 5 hours.

[0025] A medetomidine artificial antigen, the molecular structure of which is shown in formula (II): ; Formula (II); BSA stands for bovine serum albumin.

[0026] The method for preparing a medemididine artificial antigen as described above includes the following steps: Medetomidine artificial hapten I as described in claim 1 was bound to bovine serum albumin using the N-hydroxysuccinimide active ester method to obtain medetomidine artificial antigen II.

[0027] As a preferred embodiment, a method for preparing medemididine artificial antigen includes the following steps: (a) Mix metopridine artificial hapten I with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, heat and stir to react, and centrifuge after the reaction is completed to collect the supernatant; (b) The supernatant obtained in step (a) is added dropwise to the bovine serum albumin solution and mixed evenly to obtain a mixture. After the reaction is complete, the mixture is dialyzed and centrifuged to obtain the supernatant, which yields metoprimidine artificial antigen II.

[0028] Preferably, the molar ratio of metoprimidine artificial hapten I to cyclohexylcarbodiimide and N-hydroxysuccinimide added in step (a) is 1:(1.3~1.4):(1.3~1.4).

[0029] Preferably, the concentration of bovine serum albumin solution in step (b) is 4.5~6 mg / mL; the volume ratio of the supernatant obtained in step (a) to the bovine serum albumin solution added during the mixing process is 1:9.5~12.

[0030] As a further preferred embodiment, the method for preparing the bovine serum albumin solution includes the following steps: Bovine serum albumin was dissolved in 0.01M PBS buffer, the pH of which was 7.2-7.4.

[0031] This invention uses bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared with bovine gamma globulin (BGG): ① Bovine serum albumin has many lysine residues, which makes it more immunogenic. It also has numerous antigenic determinants, which can easily elicit a stronger immune response and has high coupling efficiency. ② Bovine serum albumin has a highly conserved structure and high stability, making it less susceptible to external factors, while bovine gamma globulin is more easily affected by external factors, prone to precipitation, and thus affecting the performance of the antigen. ③ The coupling of bovine serum albumin with haptens is highly efficient and makes it easier to improve the specificity of antibodies. The anti-medetomidine polyclonal antibody obtained by immunizing animals with medetomidine artificial antigen formed by binding bovine serum albumin with medetomidine artificial hapten has better specificity.

[0032] The application of the medetomidine artificial antigen as described above in the preparation of anti-medetomidine antibodies.

[0033] An anti-medetomidine antibody is a globulin obtained by animal immunization with the medetomidine artificial antigen as described above, and capable of generating a specific immune response to medetomidine and its analogues.

[0034] Through repeated experiments, this invention has found that immunizing New Zealand white rabbits with the medemididine artificial antigen yields an immune serum titer of 1:135000. This indicates that the medemididine artificial antigen of this invention can produce anti-medemididine polyclonal antibodies with high affinity, high sensitivity, and strong specificity, which can be used for the immunodetection and analysis of medemididine.

[0035] Therefore, the present invention has the following beneficial effects: The medemididine artificial hapten of the present invention retains the characteristic structure of medemididine to the greatest extent and has an active group that can couple with the carrier protein, and can serve as an antigenic determinant. The medemididine artificial antigen obtained by further preparation can be used to immunize and obtain anti-medemididine polyclonal antibodies with high affinity, high sensitivity and high specificity. The titer of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:135000, which can be used for rapid and accurate immunodetection and immunoanalysis of medemididine. Attached Figure Description

[0036] Figure 1 This is a flowchart illustrating the preparation process of metoprimidine artificial antigen II in Example 1.

[0037] Figure 2 This is a high-performance liquid chromatogram of metoprimidine artificial hapten I from Example 1.

[0038] Figure 3 This is a high-resolution mass spectrum of the medemidin artificial hapten I from Example 1.

[0039] Figure 4 The image shows the ultraviolet absorption spectra of bovine serum albumin, medemidin artificial hapten I, and medemidin artificial antigen II in Example 1.

[0040] Figure 5 The flowchart shows the preparation process of medetomidine artificial antigen IV in Comparative Example 1.

[0041] Figure 6 The flowchart shows the preparation process of medetomidine artificial antigen V in Comparative Example 2.

[0042] Figure 7 The flowchart shows the preparation process of metomididine artificial antigen VI in Comparative Example 3.

[0043] Figure 8 The flowchart shows the preparation process of metomididine artificial antigen VII in Comparative Example 4.

[0044] Figure 9 The flowchart shows the preparation process of medetomidine artificial antigen VIII in Comparative Example 5.

[0045] Figure 10 The flowchart shows the preparation process of medetomidine artificial antigen IX in Comparative Example 6.

[0046] Figure 11 The flowchart shows the preparation process of medetomidine artificial antigen X in Comparative Example 7. Detailed Implementation

[0047] The present invention will be further described below with reference to 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 some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained 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.

[0048] Example 1 This embodiment provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen II and its preparation method.

[0049] A method for preparing medemididine artificial antigen includes the following steps: (1) Preparation of medemidin artificial hapten I: (S.1) Dilute 5 mL of concentrated ammonia with deionized water to 13 mL to obtain a DAS solution. Weigh 200 mg (0.845 mmol) of metomididine hydrochloride, dissolve it in 10 mL of deionized water, and then adjust the pH to 9 with DAS solution. There was no significant change. Extract the aqueous phase with 15 mL of dichloromethane each time, and extract three times. Collect the dichloromethane phase, dry it with anhydrous magnesium sulfate, filter it, and transfer it to dryness to obtain 150 mg (0.749 mol) of colorless oily substance A. Perform TLC on the colorless oily substance A. The chromatographic solution was 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia = 8:1:10:1 (v / v). The product Rf = 0.8. (S.2) 150 mg (0.749 mmol) of the colorless oily substance A obtained in step (S.1) was slowly dissolved in 1.5 mL of concentrated sulfuric acid cooled to 0 °C. 75 mg of potassium nitrate was added, and the mixture was stirred in an ice-water bath at 0 °C for 3 h. After the reaction was completed, the pH of the reaction system was adjusted to approximately 5-7 with sodium bicarbonate. The residue was directly dried to obtain a yellow residue. The residue was extracted with 5 mL of anhydrous ethanol each time, for a total of 3 extractions. The residue was filtered, and the ethanol phase was dried to obtain 328 mg of yellow oily substance. 166 mg (0.677 mmol) of yellow bubbly solid B was purified by thin-layer chromatography. The yellow bubbly solid B was detected by TLC. The chromatographic solution was 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1 (v / v). The product Rf = 0.4. The solvent and eluent were anhydrous ethanol. (S.3) In a 50 mL round-bottom flask, add 1361 mg (24.3 mmol) of iron powder and 8.3 mL of acetic acid I, and heat to 50 °C to obtain a mixed reaction solution. Dissolve 166 mg (0.677 mmol) of the yellow bubbly solid B obtained in step (S.2) in 8.3 mL of acetic acid II, and slowly add it dropwise to the mixed reaction solution. React at 50 °C for 6 h. After the reaction is complete, filter to remove the remaining iron powder, wash the iron powder with an appropriate amount of acetic acid, and dry the filtrate directly to obtain a reddish-brown solid residue. Extract with 5 mL of anhydrous ethanol each time, for 3 extractions, filter and dry, and purify by thin-layer chromatography to obtain 68 mg (0.307 mmol) of pale yellow solid C. The pale yellow solid C was analyzed by TLC. The chromatographic solution was 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1 (v / v). The product Rf = 0.2. The solvent and eluent were anhydrous ethanol. (S.4) Dissolve 68 mg (0.307 mmol) of the pale yellow solid C obtained in step (S.3) in 13.6 mL of anhydrous acetone in a 50 mL round-bottom flask. Add 340 mg (2.46 mmol) of potassium carbonate and 78.9 μL (0.712 mmol) of ethyl bromoacetate. Under nitrogen protection, stir at room temperature for more than 17 h. After the reaction is complete, filter and dry to obtain a pale yellow bubbly solid. Purify by thin-layer chromatography to obtain 138 mg (0.458 mmol) of pale yellow bubbly solid D. Analyze the pale yellow bubbly solid by TLC. The chromatographic solution was ethyl acetate:25 wt% ammonia water = 10 mL:200 μL (v / v). The product Rf = 0.8. The solvent and eluent were anhydrous ethanol. (S.5) Dissolve 138 mg (0.458 mmol) of the pale yellow bubbly solid D obtained in step (S.4) in 2.07 mL of tetrahydrofuran and 2.48 mL of anhydrous methanol. Add 11 mL of 1N sodium hydroxide aqueous solution. The solution darkens and becomes turbid. Stir rapidly at room temperature for 4 h. Adjust the pH to approximately 4-5 with 1N hydrochloric acid. Dry the mixture to obtain a solid-oil mixture residue. Extract the residue three times with 10 mL of anhydrous ethanol each time. Filter, dry the mixture, and purify by thin-layer chromatography to obtain 50 mg (0.183 mmol) of medetomidine artificial hapten I. TLC detection of the medetomidine artificial hapten I was performed using a chromatographic buffer of 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia = 8:1:10:1 (v / v). The product Rf = 0.5. Anhydrous ethanol was used as both solvent and eluent.

[0050] The molecular structure of metomididine artificial hapten I is shown in formula (I): ; Equation (Ⅰ).

[0051] The high-performance liquid chromatogram of medetomidine artificial hapten I in this embodiment is as follows: Figure 2 As shown, the ultraviolet detector wavelength is 286 nm. The high-resolution mass spectrum of the medemididine artificial hapten I in this embodiment is shown below. Figure 3 As shown.

[0052] from Figure 2 It can be seen that the purity of the purified medemidin artificial hapten reaches over 98%. Figure 3 It can be seen that the characteristic peaks of medemidin artificial hapten I obtained in this embodiment are 274.25, 296.23, 297.24, and 318.26, corresponding to [M+H], respectively. + [M+Na] + [M+ Na+H] + [M+HCOO] -Given that the theoretical molecular weight of medetomidine artificial hapten I is 273.24, it can be basically determined that the final compound obtained in step (S.5) is the medetomidine artificial hapten designed in this invention.

[0053] (2) Preparation of medemidin artificial antigen II: (a) Place 50 mg (0.183 mmol) of metoprimidine artificial hapten I obtained in step (S.5) into a 50 mL round-bottom flask, add 2.5 mL of N,N-dimethylformamide (DMF), then add 28 mg (0.247 mmol) of N-hydroxysuccinimide (NHS) and 51 mg (0.247 mmol) of cyclohexylcarbodiimide (DCC), stir at 25 °C for 20 h, centrifuge at 8000 rpm for 5 min after the reaction is completed, and take the supernatant for later use; (b) Weigh 14.5 g (0.0405 mol) disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) sodium chloride, and 1.495 g (0.00958 mol) sodium dihydrogen phosphate dihydrate, dissolve in deionized water, and bring the volume to 5.0 L to obtain a 0.01 M PBS buffer with a pH of 7.4. Weigh 125 mg bovine serum albumin (BSA) and dissolve it in 25 mL of the prepared PBS buffer to obtain a BSA solution with a concentration of 5 mg / mL. Under rapid stirring, slowly add the supernatant obtained in step (a) to the BSA solution at a volume ratio of 1:10. The resulting mixture is stored at 4 °C overnight to obtain the artificial antigen mixture. Transfer the artificial antigen mixture to a dialysis bag and dialyze it 9 times with the prepared PBS buffer. After dialysis, centrifuge and collect the supernatant to obtain the medemididine artificial antigen II: medemididine-bovine serum albumin conjugate. The preparation flowchart of medetomidine artificial antigen II in this embodiment is as follows: Figure 1 As shown. The ultraviolet absorption spectra of bovine serum albumin, medemidin artificial hapten I, and medemidin artificial antigen II in this embodiment are as follows. Figure 4 As shown.

[0054] The molecular structure of metomididine artificial antigen II is shown in formula (II): ; Formula (II); BSA stands for bovine serum albumin.

[0055] Figure 4In the diagram, curve a represents the UV absorption curve of medemididine artificial hapten I, curve b represents the UV absorption curve of medemididine artificial antigen II, and curve c represents the UV absorption curve of bovine serum albumin (BSA). The maximum absorption wavelength of medemididine artificial hapten I is 286 nm, that of BSA is 295 nm, and that of medemididine artificial antigen II is 293 nm. Compared with medemididine hapten I and BSA, the maximum absorption wavelength of medemididine artificial antigen II shows a significant change, indicating that the conjugation of medemididine hapten I with BSA was successful.

[0056] Example 2 The difference between this embodiment and Embodiment 1 is that: This embodiment provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen II and its preparation method.

[0057] A method for preparing medetomidine artificial antigen, wherein, (1) Preparation process of medemidin artificial hapten I: In step (S.2), 150 mg (0.749 mmol) of colorless oily substance A was slowly dissolved in 1.33 mL of concentrated sulfuric acid cooled to 0 °C, and 75 mg (0.742 mmol) of potassium nitrate was added. The mixture was stirred in an ice-water bath at 0 °C for 2 h. In step (S.3), 1422 mg (25.39 mmol) of iron powder and 8 mL of acetic acid I were added and the mixture was heated to 45 °C to obtain a mixed reaction solution. 160 mg (0.653 mmol) of yellow bubbly solid B was dissolved in 8 mL of acetic acid II and slowly added dropwise to the mixed reaction solution. The reaction was carried out at 45 °C for 5 h. In step (S.4), 60 mg (0.271 mmol) of pale yellow solid C was dissolved in 10 mL of anhydrous acetone in a 50 mL round-bottom flask, and 300 mg (2.17 mmol) of potassium carbonate and 70.7 μL (0.638 mmol) of ethyl bromoacetate were added. The reaction was carried out under nitrogen protection and stirred at room temperature for 16 h. In step (S.5), 130 mg (0.431 mmol) of pale yellow bubbly solid D was dissolved in 1.81 mL of tetrahydrofuran and 2.17 mL of anhydrous methanol, and 10.1 mL of 1N sodium hydroxide aqueous solution was added. The solution darkened and became turbid. The reaction was carried out by rapid stirring at room temperature for 3 h. 50 mg of metoprimidine artificial hapten I was used for the next reaction.

[0058] (2) Preparation process of medemidin artificial antigen II: In step (b), 117 mg of bovine serum albumin was weighed and dissolved in 26 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 4.5 mg / mL; the volume ratio of the supernatant to the bovine serum albumin solution was 1:9.5. Everything else was the same as in Example 1.

[0059] Example 3 The difference between this embodiment and Embodiment 1 is that: This embodiment provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen II and its preparation method.

[0060] A method for preparing medetomidine artificial antigen, wherein, (1) Preparation process of medemidin artificial hapten I: In step (S.2), 150 mg (0.749 mmol) of colorless oily substance A was slowly dissolved in 1.64 mL of concentrated sulfuric acid cooled to 0 °C, and 75 mg (0.742 mmol) of potassium nitrate was added. The mixture was stirred in an ice-water bath at 0 °C for 4 h. In step (S.3), 1260 mg (22.5 mmol) of iron powder and 8.25 mL of acetic acid I were added and the mixture was heated to 55 °C to obtain a mixed reaction solution. 165 mg (0.673 mmol) of yellow bubbly solid B was dissolved in 8.25 mL of acetic acid II and slowly added dropwise to the mixed reaction solution. The reaction was carried out at 55 °C for 7 h. In step (S.4), 65 mg (0.294 mmol) of pale yellow solid C was dissolved in 14.78 mL of anhydrous acetone in a 50 mL round-bottom flask, and 325 mg (2.356 mmol) of potassium carbonate and 74.5 μL (0.672 mmol) of ethyl bromoacetate were added. The reaction was carried out under nitrogen protection and stirred at room temperature for 24 h. In step (S.5), 135 mg (0.448 mmol) of pale yellow bubbly solid D was dissolved in 2.15 mL of tetrahydrofuran and 2.58 mL of anhydrous methanol, and 11 mL of 1N sodium hydroxide aqueous solution was added. The solution darkened and became turbid. The reaction was carried out with rapid stirring at room temperature for 5 h. 50 mg of metoprimidine artificial hapten I was used for the next reaction.

[0061] (2) Preparation process of medemidin artificial antigen II: In step (b), 126 mg of bovine serum albumin was weighed and dissolved in 21 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 6 mg / mL; the volume ratio of the supernatant to the bovine serum albumin solution was 1:12. Everything else was the same as in Example 1.

[0062] Comparative Example 1 The difference between this comparative example and Example 1 is as follows: This comparative example provides a medetomidine artificial hapten III and its preparation method, and a medetomidine artificial antigen IV and its preparation method.

[0063] A method for preparing medetomidine artificial antigen, wherein, (1) Preparation process of medemidin artificial hapten III: In step (S.1), 180 mg (0.899 mmol) of colorless oily substance A was obtained; steps (S.2) to (S.5) were replaced by: dissolving 180 mg (0.899 mmol) of colorless oily substance A in 7.2 mL of anhydrous pyridine in a 50 mL round-bottom flask, adding 45 mg (0.368 mmol) of 4-dimethylaminopyridine (DMAP) and 181 mg (1.798 mmol) of succinic anhydride, refluxing at 100 °C for 17 h, and after the reaction was completed, directly drying to obtain 288 mg of light yellow oily substance, which was purified by thin-layer chromatography to obtain 96 mg (0.319 mmol) of metoprimidine artificial hapten III. The metomididine artificial hapten III was detected by TLC. The chromatography buffer was 95 vol% ethanol: 1,4-dioxane: dichloromethane: 25 wt% ammonia water = 8:1:10:1 (v / v). The product Rf = 0.5. The solvent and eluent were anhydrous ethanol.

[0064] Preparation process of medemidin artificial antigen IV: Step (a) is replaced by: placing 96 mg (0.319 mmol) of medemididine artificial hapten III into a 50 mL round-bottom flask, adding 4.8 mL of DMF, then adding 55 mg (0.479 mmol) of NHS and 98.7 mg (0.479 mmol) of DCC, stirring overnight at room temperature, centrifuging after the reaction, and collecting the supernatant for later use; in step (b), weighing 0.240 g of bovine serum albumin and dissolving it in 48 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 5 mg / mL. Everything else is the same as in Example 1. Finally, medemididine artificial antigen IV (i.e., medemididine-bovine serum albumin conjugate) is obtained. The preparation process of medemididine artificial antigen IV in this comparative example is as follows: Figure 5 As shown.

[0065] Comparative Example 2 The difference between this comparative example and Comparative Example 1 is: This comparative example provides a medetomidine artificial hapten III and its preparation method, and a medetomidine artificial antigen V and its preparation method.

[0066] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten III is the same as that in comparative example 1.

[0067] (2) Preparation process of medemididine artificial antigen V: In step (b), bovine gamma globulin (as a carrier) was used to replace bovine serum albumin and conjugated with medemididine artificial hapten III. The conjugation steps were the same as in Comparative Example 1, and all other steps were the same as in Comparative Example 1. Finally, medemididine artificial antigen V was obtained. The preparation process of medemididine artificial antigen V in this comparative example is as follows: Figure 6 As shown.

[0068] Comparative Example 3 The difference between this comparative example and Comparative Example 1 is: This comparative example provides a medetomidine artificial hapten III and its preparation method, and a medetomidine artificial antigen VI and its preparation method.

[0069] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten III is the same as that in comparative example 1.

[0070] (2) Preparation process of medetomidine artificial antigen VI: Step (a) is replaced by: Weighing 96 mg (0.319 mmol) of medetomidine artificial hapten III into a 50 mL round-bottom flask, adding 2.9 mL of DMF, then adding 44 μL (0.319 mmol) of triethylamine, stirring in an ice bath for 30 min, then adding 82.6 μL (0.638 mmol) of isobutyl chloroformate, continuing to stir in an ice bath for 2 h, centrifuging after the reaction, and taking the supernatant for later use. Everything else is the same as in Comparative Example 1. Finally, medetomidine artificial antigen VI is obtained. The preparation process of medetomidine artificial antigen VI in this comparative example is as follows: Figure 7 As shown.

[0071] Comparative Example 4 The difference between this comparative example and Comparative Example 3 is: This comparative example provides a medetomidine artificial hapten III and its preparation method, and a medetomidine artificial antigen VII and its preparation method.

[0072] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten III is the same as that in comparative example 1.

[0073] (2) Preparation process of medemididine artificial antigen VII: In step (b), bovine gamma globulin (as a carrier) was used to replace bovine serum albumin and conjugated with medemididine artificial hapten III. The conjugation steps were the same as in Comparative Example 3, and all other steps were the same as in Comparative Example 3. Finally, medemididine artificial antigen VII was obtained. The preparation process of medemididine artificial antigen VII in this comparative example is as follows: Figure 8 As shown.

[0074] Comparative Example 5 The difference between this comparative example and Example 1 is as follows: This comparative example provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen VIII and its preparation method.

[0075] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten I is the same as in Example 1.

[0076] (2) Preparation process of medetomidine artificial antigen VIII: Step (a) is replaced by: Weighing 50 mg (0.183 mmol) of medetomidine artificial hapten I into a 50 mL round-bottom flask, adding 2.1 mL of DMF, then adding 23 μL (0.183 mmol) of triethylamine, stirring in an ice bath for 30 min, then adding 47.3 μL (0.366 mmol) of isobutyl chloroformate, continuing to stir in an ice bath for 2 h, centrifuging after the reaction, and taking the supernatant for later use. Step (b) replaces deionized water with double-distilled water. Everything else is the same as in Example 1. Finally, medetomidine artificial antigen VIII is obtained. The preparation process of medetomidine artificial antigen VIII in this comparative example is as follows: Figure 9 As shown.

[0077] Comparative Example 6 The difference between this comparative example and Comparative Example 5 is: This comparative example provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen IX and its preparation method.

[0078] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten I is the same as in Example 1.

[0079] (2) Preparation process of medetomidine artificial antigen IX: In step (b), bovine gamma globulin (as a carrier) was used to replace bovine serum albumin and conjugated with medetomidine artificial hapten I. The conjugation steps were the same as in Comparative Example 5, and all other steps were the same as in Comparative Example 5. Finally, medetomidine artificial antigen IX was obtained. The preparation process of medetomidine artificial antigen IX in this comparative example is as follows: Figure 10 As shown.

[0080] Comparative Example 7 The difference between this comparative example and Example 1 is as follows: This comparative example provides a medetomidine artificial hapten I and its preparation method, and a medetomidine artificial antigen X and its preparation method.

[0081] A method for preparing medetomidine artificial antigen, wherein, (1) The preparation process of medetomidine artificial hapten I is the same as in Example 1.

[0082] (2) Preparation process of medetomidine artificial antigen X: In step (b), bovine gamma globulin (as a carrier) was used to replace bovine serum albumin and conjugated with medetomidine artificial hapten I. The conjugation steps were the same as in Example 1, and all other steps were the same as in Example 1. Finally, medetomidine artificial antigen X was obtained. The preparation process of medetomidine artificial antigen X in this comparative example is as follows: Figure 11 As shown.

[0083] [Performance Determination and Analysis of Metomidin Artificial Antigen] (1) Identification of metoprimidine artificial antigen: Molar absorptivity ε: Medetomidine artificial hapten I and medetomidine artificial hapten III were prepared according to the methods described in Example 1 and Comparative Example 1, respectively. Medetomidine artificial hapten solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL were then prepared using PBS buffer. UV scanning revealed that the maximum absorption wavelength of medetomidine artificial hapten I was 286 nm, and the maximum absorption wavelength of medetomidine artificial hapten III was 290 nm. The absorbance values ​​of the corresponding medetomidine artificial haptens were measured at 286 nm and 290 nm, with parallel samples prepared for each concentration. The molar absorptivity (i.e., molar absorption coefficient) was calculated using the formula: ε = absorbance value / molar concentration. The calculation results are shown in Table 1 below.

[0084] Table 1: Calculation of Molar Absorption Coefficients of Medemidin Artificial Hapten I and III .

[0085] Determination of conjugate protein concentration: Medetomidine artificial antigen was prepared according to the methods described in Example 1 and Comparative Examples 1-7. 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 were prepared using PBS buffer. 3 mL of Coomassie brilliant blue staining solution was added, and the mixture was immediately mixed and incubated at 30°C for 5 minutes. Parallel samples were prepared for each concentration, and absorbance was measured at 655 nm. A curve showing the relationship between protein concentration and absorbance was plotted. The medetomidine artificial antigen solution (prepared with PBS buffer) was diluted at a certain ratio, and the absorbance of the medetomidine artificial antigen was measured at 655 nm. The corresponding protein concentration values ​​of the medetomidine artificial antigen solution were read from the curve, and the calculated data are shown in Table 2 below.

[0086] Conjugation ratio determination: Prepare a 100 μg / mL bovine serum albumin PBS solution. Dilute the conjugate (i.e., medemidin artificial antigen II) to 100 μg / mL with PBS. Measure the absorbance A1 at 293 nm. Use bovine serum albumin PBS as a blank and measure the absorbance A2. The conjugation ratio γ is calculated using the formula: γ = [(A1 - A2) / ε] / (100 × 10⁻⁶). -3 / 66400), where ε is the molar absorptivity (L / mol), 66400 is the molecular weight of bovine serum albumin, 100 × 10⁻⁶. -3The value represents bovine serum albumin concentration (g / L). The calculated data are shown in Table 2 below.

[0087] When bovine gamma globulin is used as a carrier, the coupling ratio γ is calculated using the formula: γ = [(A1 - A2) / ε] / (100 × 10⁻⁶). -3 / 43000), where 43000 is the molecular weight of bovine gamma globulin, 100 × 10⁻⁶. -3 The value represents the concentration of bovine gamma globulin (g / L). The calculated data are shown in Table 2 below.

[0088] Table 2: Conjugation ratios and molar absorption coefficients of various medetomidine artificial antigens serial number Artificial antigen <![CDATA[A1]]> <![CDATA[A2]]> Coupling protein concentration (mg / mL) Coupling ratio molar absorption coefficient Example 1 II 0.205 0.025 3.55 90 1328 Comparative Example 1 Ⅳ 0.190 0.025 3.28 76 1442 Comparative Example 2 Ⅴ 0.038 0.021 0.94 5 1442 Comparative Example 3 Ⅵ 0.160 0.025 2.98 62 1442 Comparative Example 4 VII 0.071 0.021 1.86 15 1442 Comparative Example 5 VIII 0.141 0.025 2.54 58 1328 Comparative Example 6 IX 0.058 0.021 1.42 12 1328 Comparative Example 7 X 0.024 0.021 0.68 1 1328

[0089] As shown in Tables 1 and 2, the structure of the artificial hapten, the activation method of the artificial hapten, and the structure of the carrier protein all affect the coupling ratio when the artificial hapten cross-links with the carrier protein.

[0090] (2) Animal immunization Medetomidine artificial antigens were prepared according to the methods described in Example 1 and Comparative Examples 1-7, respectively. New Zealand white rabbits were immunized with each prepared medetomidine artificial antigen, and the titers of the resulting immune serum were detected by ELISA. The results are shown in Table 3.

[0091] Table 3: Titer Test Results of Various Immune Serums serial number Metopidine Artificial Antigen Immune serum titer Example 1 Ⅱ 1:135000 Comparative Example 1 Ⅳ 1:58000 Comparative Example 2 Ⅴ / Comparative Example 3 Ⅵ 1:41000 Comparative Example 4 Ⅶ 1:18000 Comparative Example 5 Ⅷ 1:41000 Comparative Example 6 Ⅸ 1:18000 Comparative Example 7 Ⅹ /

[0092] As shown in Table 3, compared with Example 1, the immune sera obtained by animal immunization using the medemididine artificial antigens in each comparative example had lower titers and could not be used for immunoassay. Specifically, medemididine artificial antigen V obtained in Comparative Example 2 and medemididine artificial antigen X obtained in Comparative Example 7 showed a large amount of precipitation immediately during dialysis, indicating very low conjugate protein concentration and poor conjugation effect; therefore, no titer determination was performed. Medemididine artificial antigen VII obtained in Comparative Example 4 and medemididine artificial antigen IX obtained in Comparative Example 6 showed a large amount of precipitation after cryopreservation, indicating poor stability; therefore, they could not be used as immunoantigens. However, the immune sera obtained by animal immunization using medemididine artificial antigen II (Example 1) had a titer of 1:135000 and was stable, making it perfectly suitable for immunoassay and providing a more convenient, rapid, and accurate method for medemididine detection.

[0093] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

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

2. The method for preparing a medemidin artificial hapten as described in claim 1, characterized in that, Includes the following steps: (S.1) DAS solution was obtained by diluting concentrated ammonia with deionized water. Metopril hydrochloride was dissolved in deionized water and added to DAS solution to adjust the pH to alkaline. Then it was extracted with dichloromethane, and the dichloromethane phase was collected, dried, filtered and dried to obtain colorless oily substance A. (S.2) Dissolve the colorless oily substance A obtained in step (S.1) in concentrated sulfuric acid, add potassium nitrate, and stir the reaction in an ice-water bath. After the reaction is complete, adjust the pH, dry the mixture directly, extract with anhydrous ethanol, filter, dry, and purify to obtain yellow bubbly solid B. (S.3) Add iron powder and acetic acid I to the reaction vessel and heat to obtain a mixed reaction solution. Dissolve the yellow bubbly solid B obtained in step (S.2) in acetic acid II and add it dropwise to the mixed reaction solution to carry out the reaction. After the reaction is completed, filter, dry directly, extract with anhydrous ethanol, filter, dry, and purify to obtain a pale yellow solid C. (S.4) Dissolve the pale yellow solid C obtained in step (S.3) in anhydrous acetone, add potassium carbonate and ethyl bromoacetate, and stir the reaction at room temperature under nitrogen protection. After the reaction is completed, filter, dry and purify to obtain pale yellow bubbly solid D. (S.5) Dissolve the pale yellow bubbly solid D obtained in step (S.4) in tetrahydrofuran and anhydrous methanol, add sodium hydroxide solution, stir at room temperature until the reaction is complete, adjust the pH with hydrochloric acid, directly dry, and extract with anhydrous ethanol. Filter, dry, and purify to obtain metoprimidine artificial hapten I.

3. The method for preparing a medemidin artificial hapten according to claim 2, characterized in that, In step (S.2), the ratio of colorless oily substance A to concentrated sulfuric acid and potassium nitrate is 90~110:0.8~1.2:45~55mg / mL / mg; In step (S.3), the ratio of iron powder, acetic acid I, yellow bubbly solid B to acetic acid II is 800~840:4.5~5.5:90~110:4.5~5.5 mg / mL / mg / mL; In step (S.4), the ratio of pale yellow solid C, anhydrous acetone, potassium carbonate and ethyl bromoacetate is 90~110:15~25:450~550:106~126 mg / mL / mg / μL. In step (S.5), the ratio of the pale yellow bubbly solid D, tetrahydrofuran, anhydrous methanol and sodium hydroxide solution is 90~110:1.25~1.75:1.5~2.1:7~9 mg / mL / mL.

4. The method for preparing a medemidin artificial hapten according to claim 2, characterized in that, The reaction time for the stirring process in the ice-water bath in step (S.2) is 2-4 hours; The reaction temperature in step (S.3) is 45~55℃ and the reaction time is 5~7h. The reaction time for the room temperature stirring process in step (S.4) is 16~24h; The reaction time for the room temperature stirring process in step (S.5) is 3~5 hours.

5. A medetomidine artificial antigen, characterized in that, Its molecular structure is shown in formula (II): ； Formula (II); BSA stands for bovine serum albumin.

6. The method for preparing a medemidin artificial antigen as described in claim 5, characterized in that, Includes the following steps: Medetomidine artificial hapten I as described in claim 1 was bound to bovine serum albumin using the N-hydroxysuccinimide active ester method to obtain medetomidine artificial antigen II.

7. The method for preparing a medemidin artificial antigen according to claim 6, characterized in that, Includes the following steps: (a) Mix metopridine artificial hapten I with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, heat and stir to react, and centrifuge after the reaction is completed to collect the supernatant; (b) The supernatant obtained in step (a) is added dropwise to the bovine serum albumin solution and mixed evenly to obtain a mixture. After the reaction is complete, the mixture is dialyzed and centrifuged to obtain the supernatant, which yields metoprimidine artificial antigen II.

8. The method for preparing a medemidin artificial antigen according to claim 7, characterized in that, The molar ratio of metoprimidine artificial hapten I to cyclohexylcarbodiimide and N-hydroxysuccinimide added in step (a) is 1:(1.3~1.4):(1.3~1.4). The concentration of bovine serum albumin solution in step (b) is 4.5~6 mg / mL; the volume ratio of the supernatant obtained in step (a) to the bovine serum albumin solution added during the mixing process is 1:9.5~12.

9. The use of the medetomidine artificial antigen as described in claim 5 in the preparation of anti-medetomidine antibodies.

10. An anti-metopidine antibody, characterized in that, It is a globulin obtained by animal immunization with the medemidin artificial antigen as described in claim 5, and can produce a specific immune response with medemidin and its analogues.

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