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

Abstract

The present invention discloses an artificial hapten and an artificial antigen of tiletamine, as well as a preparation method and application thereof. The molecular structural formula of the artificial hapten of tiletamine is shown in Formula I, and the molecular structural formula of the artificial antigen of tiletamine is shown in Formula II. The present invention also discloses the application of the artificial hapten of tiletamine in the preparation of anti-tiletamine antibodies. The artificial hapten of tiletamine of the present invention retains the characteristic structure of tiletamine 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 tiletamine antigen prepared further can be used to immunize to obtain anti-tiletamine 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 1:85,000, and can be used for rapid and accurate immunodetection and immunoanalysis of tiletamine.

Description

Technical Field

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

[0002] Tiletamine, a white powdery substance, is a dissociative anesthetic primarily used for block and general anesthesia, and can also be used as a veterinary anesthetic. Tiletamine is quite popular in veterinary practice and is effective for anesthesia care in cats and dogs. It can complement zolazepam, rapidly relaxing muscles and causing analgesia of superficial and visceral pain without suppressing the respiratory center.

[0003] According to existing data, tiletamine is a phencyclidine drug with anxiolytic, sedative, anticonvulsant, and muscle relaxant effects. Although tiletamine is easy to use, it may also cause a number of adverse reactions. The main adverse reactions are psychomotor reactions such as hallucinations and nightmares during the awakening period. Some patients may experience diplopia, visual distortion, and even blindness. The abuse of tiletamine can cause great harm to people. In the short term, it can cause slurred speech and hallucinations, depression, sedation, hypnosis, and even drowsiness. Long-term abuse can cause memory loss and cognitive impairment, and lead to damage to heart function. It can also cause physical and psychological dependence and even addiction. For regulatory purposes, it is necessary to develop a method for rapid detection of tiletamine.

[0004] Currently, the detection of tiletamine mainly relies on high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), mass spectrometry (MS), etc. However, these methods have disadvantages such as expensive instruments, time-consuming detection, and the need for professional technicians to operate, which cannot meet the modern requirements of rapid and accurate detection.

[0005] Immunoassays can overcome all of these shortcomings. Immunoassays are analytical methods that utilize the specific binding reaction between antigens and antibodies to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to developing immunoassays for small molecule compounds is the ability to produce antibodies with high affinity and specificity for these compounds. However, since most small molecule compounds (molecular weight less than 1000), including tiletamine, are non-immunogenic, meaning they lack T-cell epitopes and cannot directly induce the production of specific antibodies in animals, small molecules are therefore called haptens. Through appropriate chemical modification, a linker with an active group at a specific position in the hapten molecule is added. This is then combined with a macromolecular carrier to produce a hapten-carrier conjugate (i.e., an artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells through the T-cell epitopes, leading to the production of specific antibodies. Therefore, the efficient synthesis of artificial antigens is a prerequisite and key to ensuring the effectiveness of immunoassays. Summary of the Invention

[0006] The present invention provides an artificial hapten of tiletamine, which retains the characteristic structure of tiletamine to the greatest extent, has an active group that can be coupled with a carrier protein, and can serve as an antigenic determinant.

[0007] To achieve the above technical objectives, the present invention is implemented through the following technical solutions:

[0008] The first object of the present invention is to provide an artificial hapten of tiletamine, the molecular structure of which is shown in formula I:

[0009]

[0010] A second object of the present invention is to provide a method for preparing the tiletamine artificial hapten, the method comprising the following steps:

[0011] (1) performing a substitution reaction between tiletamine and ethyl 5-bromovalerate to prepare a compound represented by formula A;

[0012] (2) hydrolyzing the compound represented by Formula A and adjusting the pH to be acidic to obtain the tiletamine artificial hapten represented by Formula I;

[0013] The reaction formula of the method is shown below:

[0014]

[0015] Furthermore, the method is preferably carried out according to the following steps:

[0016] (1) Tiletamine is dissolved in N,N-dimethylformamide (DMF), and ethyl 5-bromovalerate is added under the action of NaH. The mixture is heated to 60-70°C to carry out a substitution reaction. After the reaction is completed, the resulting reaction solution is post-treated to obtain the compound shown in Formula A;

[0017] The molar ratio of tiletamine to sodium hydride is 1:3-4;

[0018] The molar ratio of tiletamine to ethyl 5-bromovalerate is 1:1-2, preferably 1:1.5.

[0019] The volume dosage of the N,N-dimethylformamide is generally 3 to 10 mL / mmol based on the amount of tiletamine.

[0020] The reaction time of the substitution reaction is generally 10 to 30 hours, preferably 15 to 20 hours.

[0021] The post-treatment steps of the reaction solution in step (1) are generally as follows: after the reaction is completed, the reaction solution is evaporated to remove the solvent, the residue is extracted with ethyl acetate, the organic phase is dried, filtered, and the solvent is evaporated, and the obtained oil is purified by thin layer chromatography to obtain the compound shown in formula A.

[0022] The chromatographic solution for thin layer chromatography was a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1:4, and the product Rf = 0.3;

[0023] Under the reaction conditions, tiletamine is relatively soluble in N,N-dimethylformamide, and sodium hydride as a catalyst can accelerate the reaction process. The yield and purity of the light yellow oil A are both high, and the post-processing procedure is relatively simple.

[0024] (2) The compound represented by Formula A is dissolved in tetrahydrofuran and anhydrous methanol, and a 1N aqueous sodium hydroxide solution is added. The mixture is rapidly stirred at room temperature to carry out a hydrolysis reaction. After the reaction is completed, the pH is adjusted to 4-5. The resulting reaction solution is post-treated to prepare the tiletamine artificial hapten represented by Formula I;

[0025] The reaction time of the hydrolysis reaction is preferably 4 to 6 hours.

[0026] The molar ratio of the compound represented by formula A to NaOH in a 1N sodium hydroxide aqueous solution is generally 1:10 to 50;

[0027] The volume of anhydrous methanol is generally 10 to 20 mL / g based on the mass of the compound represented by formula A;

[0028] The volume amount of tetrahydrofuran is generally 8 to 20 mL / g based on the mass of the compound represented by formula D;

[0029] After the reaction is completed, 1N hydrochloric acid is generally used to adjust the pH to 4-5.

[0030] The post-treatment steps of the reaction solution in step (1) are generally as follows: the reaction solution is extracted with dichloromethane, the organic phases are combined, dried, filtered, the filtrate is evaporated to remove the solvent, and the obtained oil is purified by thin layer chromatography to obtain the tiletamine artificial hapten shown in formula I.

[0031] The chromatographic solution used in the thin layer chromatography method is a mixed solvent of 95% ethanol, 1,4-dioxane, dichloromethane, and ammonia water in a volume ratio of 8:1:10:1, and the product Rf is 0.5.

[0032] The mass fraction of ammonia water is 25-28%.

[0033] Under the reaction conditions, the light yellow oil A is easily hydrolyzed, the subsequent treatment procedure is relatively simple, and it is easier to purify.

[0034] By the above method, a linker is introduced into the hydrocarbon chain imino group of tiletamine. The introduction of the linker at this modification site can retain the characteristic structure of tiletamine to the greatest extent, and the modification site is as far away from the characteristic functional group of tiletamine as possible, so that its characteristic part is exposed to the outside to the greatest extent, thereby avoiding interference with specific antigenic determinants and maximizing recognition by the immune body.

[0035] Compared with a circular connecting arm, the connecting arm used in the present invention is chain-shaped and has an appropriate length, which ensures that the tiletamine hapten small molecule can be fully exposed on the carrier surface and reduces the recognition of the connecting arm by T cells during immunization as much as possible. In this way, the antibodies obtained by immunization have stronger specificity and affinity for tiletamine.

[0036] The third object of the present invention is to provide an artificial antigen of tiletamine, whose molecular structure is shown in II:

[0037]

[0038] In formula II, BSA is bovine serum albumin.

[0039] The fourth object of the present invention is to provide a method for preparing the tiletamine artificial antigen, which comprises combining the tiletamine artificial hapten shown in formula I with bovine serum albumin through a mixed anhydride method to obtain the tiletamine artificial antigen shown in formula II.

[0040] Specifically, when the mixed anhydride method is used to prepare the tiletamine artificial antigen, the method comprises the following steps:

[0041] (a) The tiletamine artificial hapten of Formula I, isobutyl chloroformate, and triethylamine are stirred in N,N-dimethylformamide (DMF) solvent at 0-5°C (generally in an ice bath) for 2-4 hours. After the reaction, the supernatant is collected by centrifugation.

[0042] The molar ratio of the tiletamine artificial hapten represented by formula I, isobutyl chloroformate, and triethylamine is 1:1.5-2.5:1-1.5, preferably 1:2:1.

[0043] The volume usage of the DMF solvent is 30-80 mL / g, preferably 50 mL / g, based on the mass of the tiletamine artificial hapten shown in Formula I.

[0044] (b) adding the supernatant dropwise to a bovine serum albumin solution, and allowing the resulting mixture to stand at 3-5° C. overnight (usually for 10-24 hours), dialyzing, and centrifuging to obtain the supernatant to obtain the tiletamine artificial antigen represented by formula II.

[0045] Unless otherwise specified, the bovine serum albumin solution in the present invention is prepared by dissolving bovine serum albumin in 0.01 M PBS buffer solution with a pH of 7.2 to 7.4.

[0046] In step (b), the concentration of the bovine serum albumin solution is 5 mg / mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5-6.

[0047] The present invention uses bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared to bovine gamma albumin (BGG): ① BSA is the most commonly used carrier protein, has stronger immunogenicity, possesses numerous antigenic determinants, and is likely to induce a stronger immune response; ② BSA molecules contain a large amount of lysine, have more free amino acids, and can maintain a higher solubility at different pH values ​​and ionic strengths. When dissolved in an organic solvent (such as dimethylformamide), its active groups remain soluble, and are therefore less likely to cause protein precipitation and the like; ③ BSA has many lysine residues and contains hundreds of primary amine and carboxyl groups, which can serve as targets for cross-linking with cross-linking reagents such as glutaraldehyde, N-hydroxysuccinimide ester, and EDC. It is easy to couple with haptens with high coupling efficiency, is less likely to produce polyclonal antibodies, and is more likely to improve antibody specificity. Anti-tiletamine antibodies obtained by animal immunization using tiletamine artificial antigens formed by combining bovine serum albumin with tiletamine artificial haptens have better specificity.

[0048] The present invention also provides the use of the tiletamine artificial antigen in preparing anti-tiletamine antibodies.

[0049] The fifth object of the present invention is to provide the use of the tiletamine artificial hapten or tiletamine artificial antigen in the preparation of anti-tiletamine antibodies.

[0050] The sixth object of the present invention is to provide an anti-tiletamine antibody, which is a globulin obtained by immunizing animals with the tiletamine artificial antigen and can produce a specific immune reaction with tiletamine.

[0051] The seventh object of the present invention is to provide the use of the anti-tiletamine antibody in detecting tiletamine.

[0052] The eighth object of the present invention is to provide a reagent for detecting tiletamine, wherein the reagent comprises the anti-tiletamine antibody.

[0053] The ninth object of the present invention is to provide a kit for detecting tiletamine, wherein the kit comprises the anti-tiletamine antibody.

[0054] Experiments have shown that the titer of the immune serum obtained by immunizing New Zealand white rabbits with the tiletamine artificial antigen was 1:85,000. This indicates that the tiletamine artificial antigen of the present invention can be used to immunize anti-tiletamine antibodies with high affinity, high sensitivity, and strong specificity, and that the anti-tiletamine antibodies can be used for immunodetection and analysis of tiletamine.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The tiletamine artificial hapten of the present invention retains the characteristic structure of tiletamine 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 tiletamine artificial antigen further prepared can be used for immunization to obtain anti-tiletamine 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 1:85,000, and can be used for rapid and accurate immunodetection and immunoanalysis of tiletamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The present invention is a preparation reaction formula of the tiletamine artificial antigen II.

[0058] Wherein, NaH represents sodium hydride, DMF represents N,N-dimethylformamide, THF represents tetrahydrofuran, MeOH represents anhydrous methanol, RT represents room temperature, BSA represents bovine serum albumin, and Et3N represents triethylamine, and the same applies hereinafter.

[0059] Figure 2 This is a liquid chromatogram of the tiletamine artificial hapten I prepared in Example 1 of the present invention.

[0060] The horizontal axis of the spectrum is time, unit is min; the vertical axis is the response value, unit is mAU.

[0061] Figure 3 This is the mass spectrum of the tiletamine artificial hapten I prepared in Example 1 of the present invention.

[0062] Wherein, Relative Abundance represents relative abundance; m / z represents charge-to-mass ratio.

[0063] Figure 4 These are UV scans of tiletamine artificial hapten I, tiletamine artificial antigen II, and bovine serum albumin.

[0064] Wherein, Abs represents the UV-visible absorption spectrum, and WL (nm) represents the wavelength (nm).

[0065] Figure 5 This is the reaction formula for preparing tiletamine artificial antigen IV in comparative example 1.

[0066] Wherein, Pyridine represents pyridine, DCC represents N,N'-dicyclohexylcarbodiimide, and the same applies hereinafter.

[0067] Figure 6 This is the reaction formula for preparing tiletamine artificial antigen V in comparative example 2.

[0068] Here, BGG stands for bovine gamma-gamma protein.

[0069] Figure 7 This is the reaction formula for the preparation of tiletamine artificial antigen VI in comparative example 3.

[0070] Figure 8 This is the reaction formula for the preparation of tiletamine artificial antigen VII in comparative example 4.

[0071] Figure 9 This is the reaction formula for the preparation of tiletamine artificial antigen VIII in comparative example 5.

[0072] Figure 10 This is the reaction formula for preparing tiletamine artificial antigen IX in comparative example 6.

[0073] Figure 11 This is the reaction formula for preparing tiletamine artificial antigen X in comparative example 7. DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings, specific embodiments and comparative examples, but the protection scope of the present invention is not limited thereto.

[0075] Example 1

[0076] This invention provides a method for preparing tiletamine artificial antigen II (reaction formula as shown in FIG. Figure 1 ), including the following steps:

[0077] (1) Preparation of artificial hapten I:

[0078] ① Dissolve 200 mg (0.896 mmol) of tiletamine in 5 ml of N,N-dimethylformamide and place in a 50 ml round-bottom flask. Add 108 mg (2.688 mmol) of 60% sodium hydride and stir at room temperature for 0.5 hour. Then add 213 μL (1.344 mmol) of ethyl 5-bromovalerate and heat to 65°C for 17 hours under reflux. After the reaction, transfer directly to dryness and extract the residue three times with 20 ml of ethyl acetate. The organic phases are combined, dried, filtered, and transferred to dryness to obtain 268 mg of a yellow oil. This is further purified by thin-layer chromatography to obtain 196 mg of a light yellow oil, Substance A. The solvent and eluent are anhydrous ethanol.

[0079] The light yellow oil A was subjected to TLC detection. The chromatographic solvent was ethyl acetate: petroleum ether in a volume ratio of 1:4. The product Rf was 0.3.

[0080] The chromatographic solution used in the thin layer chromatography method was ethyl acetate: petroleum ether in a volume ratio of 1:4, and the product Rf was 0.3.

[0081] ② Dissolve 196 mg (0.558 mmol) of pale yellow oil A in 2.35 ml of tetrahydrofuran and 2.94 ml of anhydrous methanol, add 19.6 ml of 1N aqueous sodium hydroxide solution, and react rapidly with stirring at room temperature for 5 hours. Adjust the pH to 4-5 with 1N aqueous hydrochloric acid solution, and extract three times with 30 ml of dichloromethane. The organic phases are combined, dried, filtered, and transferred to dryness. Purify by thin-layer chromatography to obtain 168 mg of tiletamine artificial hapten I. The solvent and eluent are anhydrous ethanol.

[0082] The tiletamine artificial hapten I was subjected to TLC detection, the chromatography solution was 95 vol% ethanol:1,4-dioxane:dichloromethane:25 wt% ammonia water in a volume ratio of 8:1:10:1, and the product Rf was 0.5;

[0083] The chromatographic solution used in the thin layer chromatography method was 95 vol% ethanol:1,4-dioxane:dichloromethane:25 wt% ammonia water in a volume ratio of 8:1:10:1, and the product Rf was 0.5;

[0084] The liquid chromatogram of tiletamine artificial hapten Ⅰ is shown in Figure 2 (UV detector, wavelength 285nm), the mass spectrum of tiletamine artificial hapten I is shown in Figure 3 .

[0085] from Figure 2 It can be seen that the purity of the purified tiletamine artificial hapten reaches more than 99.9%. Figure 3It can be seen that the mass-to-charge ratio (m / z) of the M+H peak of the tiletamine artificial hapten obtained in this example is 324.45, which is consistent with its theoretical relative molecular weight of 323. The mass-to-charge ratios (m / z) of its other three main fragment ion peaks are 294.18 and 310.14, respectively, which are consistent with the theoretical molecular weights of its two main fragments of 293 and 309. Based on the above data, it can be preliminarily determined that the final compound obtained in step ② is the tiletamine artificial hapten I designed by the present invention.

[0086] (2) Preparation of tiletamine artificial antigen II:

[0087] ③ Place 168 mg (0.520 mmol) of tiletamine artificial hapten I in a 50 ml round-bottom flask, add 8.4 ml of N,N-dimethylformamide (DMF), then add 72.2 μL (0.520 mmol) of triethylamine, place in an ice bath and stir for 30 min, then add 134.9 μL (1.04 mmol) of isobutyl chloroformate, stir in an ice bath for 2 h, centrifuge after the reaction is completed, take the supernatant and set aside.

[0088] ④ 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 double distilled water to 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0089] ⑤ Weigh 0.21 g of bovine serum albumin and dissolve it in 42 ml of the PBS buffer prepared in step ④ to obtain a bovine serum albumin solution with a concentration of 5 mg / ml.

[0090] ⑥ Under rapid stirring, the supernatant of step ③ was slowly added dropwise to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution was 1:5, and the resulting mixture was stored at 4°C overnight to obtain an artificial antigen mixture.

[0091] ⑦ Transfer the artificial antigen mixture into a dialysis bag and dialyze 9 times with the PBS buffer in step ④. After the dialysis, centrifuge and collect the supernatant to obtain artificial antigen II: tiletamine-bovine serum albumin conjugate. The UV scanning images of tiletamine artificial antigen II before and after preparation are shown in Figure 4 .

[0092] Figure 4In the figure, curve a is the UV scan of tiletamine artificial hapten I, curve b is the UV scan of tiletamine artificial antigen II, and curve c is the UV scan of bovine serum albumin. The maximum absorption wavelength of tiletamine artificial hapten I is 285 nm, the maximum absorption wavelength of tiletamine artificial antigen II is 273 nm, and the maximum absorption wavelength of bovine serum albumin is 280 nm. Compared with tiletamine artificial hapten I and bovine serum albumin, the maximum absorption wavelength of tiletamine artificial antigen II shows a significant shift, indicating that tiletamine artificial hapten I and bovine serum albumin are successfully coupled.

[0093] Comparative Example 1

[0094] This invention implements a preparation method of tiletamine artificial antigen IV (reaction formula as shown in Figure 5 ), including the following steps:

[0095] (1) Preparation of tiletamine artificial hapten III:

[0096] ① Dissolve 200 mg (0.896 mmol) of tiletamine in 20 ml of pyridine and place in a 50 ml round-bottom flask. Add 134.3 mg (1.343 mmol) of succinic anhydride and reflux at 100°C for 17 hours. After the reaction is complete, transfer directly to dryness to obtain 288 mg of a light yellow oil. Purify by thin-layer chromatography to obtain 190 mg of tiletamine artificial hapten III. The solvent and eluent are anhydrous ethanol.

[0097] TLC was performed on the tiletamine artificial hapten III. The chromatographic solution was 95 vol% ethanol:1,4-dioxane:dichloromethane:25 wt% ammonia water in a volume ratio of 8:1:10:1. The product Rf was 0.5.

[0098] The chromatographic solution used in the thin layer chromatography method was 95 vol% ethanol:1,4-dioxane:dichloromethane:25 wt% ammonia water in a volume ratio of 8:1:10:1, and the product Rf was 0.5.

[0099] (2) Preparation of tiletamine artificial antigen IV:

[0100] ② Place 190 mg (0.588 mmol) of tiletamine artificial hapten III in a 50 ml round-bottom flask, add 9.5 ml of N,N-dimethylformamide (DMF), then add 101 mg (0.882 mmol) of N-hydroxysuccinimide (NHS) and 182 mg (0.882 mmol) of cyclohexylcarbodiimide (DCC), stir at room temperature and react overnight. After the reaction is complete, centrifuge and collect the supernatant for later use.

[0101] ③ 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 double distilled water to a volume of 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0102] ④ Weigh 0.24 g of bovine serum albumin and dissolve it in 48 ml of the PBS buffer prepared in step ⑥ to obtain a bovine serum albumin solution with a concentration of 5 mg / ml.

[0103] ⑤ Under rapid stirring, the supernatant of step ② was slowly added dropwise to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution was 1:5, and the resulting mixture was stored at 4°C overnight to obtain an artificial antigen mixture.

[0104] ⑥ Transfer the artificial antigen mixture into a dialysis bag and dialyze 9 times with the PBS buffer in step ③. After the dialysis is completed, centrifuge and take the supernatant to obtain artificial antigen IV: tiletamine-bovine serum albumin conjugate.

[0105] Comparative Example 2

[0106] This invention provides a method for preparing tiletamine artificial antigen V (reaction formula as shown in FIG. Figure 6 ), including the following steps:

[0107] (1) Preparation of tiletamine artificial hapten III:

[0108] ①Same as Comparative Example 1.

[0109] (2) Preparation of tiletamine artificial antigen V:

[0110] Bovine immunoglobulin G was used as a carrier to couple with tiletamine artificial hapten III. The coupling steps ② to ⑥ were the same as those in comparative example 1 to obtain tiletamine artificial antigen V.

[0111] Comparative Example 3

[0112] This invention provides a method for preparing a tiletamine artificial antigen VI (reaction formula as shown in FIG. Figure 7 ), including the following steps:

[0113] (1) Preparation of tiletamine artificial hapten III:

[0114] ①Same as Comparative Example 1.

[0115] (2) Preparation of tiletamine artificial antigen VI:

[0116] ② Weigh 190 mg (0.588 mmol) of tiletamine artificial hapten III and place it in a 50 ml round-bottom flask. Add 9.5 ml of N,N-dimethylformamide (DMF) and then add 81.7 μL (0.588 mmol) of triethylamine. Stir on ice for 30 min. Then add 152.6 μL (1.176 mmol) of isobutyl chloroformate. Continue stirring on ice for 2 h. Centrifuge after the reaction is completed and take the supernatant for later use.

[0117] ③-⑥ were the same as in Comparative Example 1; and tiletamine artificial antigen VI was obtained.

[0118] Comparative Example 4

[0119] This invention provides a method for preparing tiletamine artificial antigen VII (reaction formula as shown in FIG. Figure 8 ), including the following steps:

[0120] (1) Preparation of tiletamine artificial hapten III:

[0121] ①Same as Comparative Example 1.

[0122] (2) Preparation of tiletamine artificial antigen VII:

[0123] Bovine immunoglobulin G was used as a carrier to couple with tiletamine artificial hapten III. The coupling steps ② to ⑥ were the same as those in comparative example 3 to obtain tiletamine artificial antigen VII.

[0124] Comparative Example 5

[0125] This invention provides a method for preparing tiletamine artificial antigen VIII (reaction formula as shown in FIG. Figure 9 ), including the following steps:

[0126] (1) Preparation of tiletamine artificial hapten I:

[0127] ①-② are the same as in Example 1.

[0128] (2) Preparation of tiletamine artificial antigen VIII:

[0129] ③ Weigh 168 mg (0.520 mmol) of tiletamine artificial hapten I into a 50 ml round-bottom flask, add 8.4 ml of N,N-dimethylformamide (DMF), then add 90 mg (0.78 mmol) of N-hydroxysuccinimide (NHS) and 161 mg (0.78 mmol) of cyclohexylcarbodiimide (DCC), stir at room temperature and react overnight. After the reaction is completed, centrifuge and collect the supernatant for later use.

[0130] ④ Weigh 14.5 g of disodium hydrogen phosphate dodecahydrate, 43.875 g of sodium chloride, and 1.495 g of sodium dihydrogen phosphate dihydrate and dissolve them in double-distilled water to a volume of 5.0 L to obtain 0.01 M PBS buffer solution with a pH of 7.4.

[0131] ⑤ Weigh 0.210 g of bovine serum albumin and dissolve it in 42 ml of PBS buffer to obtain a 5 mg / ml bovine serum albumin solution.

[0132] ⑥ Under rapid stirring, slowly add the supernatant to the bovine serum albumin solution. The volume ratio of the supernatant to the bovine serum albumin solution is 1:5. The resulting mixture is stored at 4°C overnight to obtain an artificial antigen mixture.

[0133] ⑦ Transfer the artificial antigen mixture into a dialysis bag and dialyze it 9 times with the above-mentioned PBS buffer. Centrifuge after dialysis and take the supernatant to obtain the tiletamine artificial antigen VIII.

[0134] Comparative Example 6

[0135] This invention implements a preparation method of tiletamine artificial antigen IX (reaction formula as shown in Figure 10 ), including the following steps:

[0136] (1) Preparation of tiletamine artificial hapten I:

[0137] ①-② are the same as in Example 1.

[0138] (2) Preparation of tiletamine artificial antigen IX:

[0139] Bovine immunoglobulin G was used as a carrier to couple with tiletamine artificial hapten I. The coupling steps ③-⑦ were the same as those in Comparative Example 5 to obtain tiletamine artificial antigen IX.

[0140] Comparative Example 7

[0141] This invention provides a method for preparing tiletamine artificial antigen X (reaction formula as shown in FIG. Figure 11 ), including the following steps:

[0142] (1) Preparation of tiletamine artificial hapten I:

[0143] ①-② are the same as in Example 1.

[0144] (2) Preparation of tiletamine artificial antigen X:

[0145] Bovine immunoglobulin G was used as a carrier to couple with the tiletamine artificial hapten I. The coupling steps ③-⑦ were the same as those in Example 1 to obtain the tiletamine artificial antigen X.

[0146] Performance determination of tiletamine artificial antigen

[0147] (1) Identification of tiletamine artificial antigen:

[0148] Molar Absorption Coefficient ε: Prepare tiletamine hapten solutions at concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, and 40 μg / ml in PBS buffer. UV scanning indicates that the maximum absorption wavelength of tiletamine hapten is 285 nm. Measure absorbance at 285 nm. Duplicate samples are prepared for each concentration. The molar absorption coefficient (i.e., molar absorption coefficient) is calculated as: ε = absorbance / molar concentration.

[0149] Determination of conjugate protein concentration: Prepare 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 in PBS buffer. Add 3 ml of Coomassie Brilliant Blue staining solution and mix immediately. Warm in a 30°C water bath for 5 minutes. Prepare replicates for each concentration and measure absorbance at 655 nm. Plot a curve showing the relationship between protein concentration and absorbance. Dilute the tiletamine artificial antigen solution (prepared in PBS buffer) in a specific ratio and measure the absorbance of the tiletamine artificial antigen at 655 nm. Read the corresponding protein concentration of the tiletamine artificial antigen solution from the curve.

[0150] Coupling ratio determination: prepare 100 μg / ml bovine serum albumin PBS solution, dilute the conjugate (i.e. tiletamine artificial antigen) to 100 μg / ml with PBS, measure the absorbance value A1 at 285 nm, and measure the absorbance value A2 with PBS as blank. The coupling ratio γ is: γ=[(A1-A2) / ε] / (100×10 -3 / 66000).

[0151] Where ε is the molar absorption coefficient (L / mol), 66000 is the molecular weight of bovine serum albumin, 100×10 -3 is the concentration of bovine serum albumin (g / L).

[0152] When bovine immunoglobulin G is used as a carrier, the coupling ratio is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 43000); wherein 43000 is the molecular weight of bovine immunoglobulin G.

[0153] Table 1 Coupling ratio and molar absorption coefficient of each tiletamine artificial antigen

[0154] serial number Artificial antigens Coupling ratio Conjugate protein concentration Molar absorption coefficient Example 1 Ⅱ 32 3.688mg / ml 6088.12 Comparative Example 1 Ⅳ 14 3.324mg / ml 6116.28 Comparative Example 2 Ⅴ 6 1.687mg / ml 6116.28 Comparative Example 3 Ⅵ 12 2.654mg / ml 6116.28 Comparative Example 4 Ⅶ 16 0.452mg / ml 6116.28 Comparative Example 5 Ⅷ 4 3.188mg / ml 6088.12 Comparative Example 6 Ⅸ 20 3.618mg / ml 6088.12 Comparative Example 7 Ⅹ 9 1.066mg / ml 6088.12

[0155] As can be seen from Table 1, the structure of the artificial hapten, the activation method of the artificial hapten and the structure of the carrier protein all have an impact on the binding ratio when the artificial hapten is cross-linked with the carrier protein.

[0156] (2) Animal immunization

[0157] New Zealand white rabbits were immunized with each of the prepared tiletamine artificial antigens, and the titers of the obtained immune sera were tested by ELISA. The test results are shown in Table 2.

[0158] Table 2 Titer test results of each immune serum

[0159] serial number tiletamine artificial antigen Immune serum titer Example 1 Ⅱ 1:85000 Comparative Example 1 Ⅳ 1:10000 Comparative Example 2 Ⅴ 1:6800 Comparative Example 3 Ⅵ 1:18000 Comparative Example 4 Ⅶ / Comparative Example 5 Ⅷ 1:16000 Comparative Example 6 Ⅸ 1:18000 Comparative Example 7 Ⅹ 1:14000

[0160] As shown in Table 2, compared with Example 1, the titers of the immune sera obtained by immunizing animals with the tiletamine artificial antigens in each comparative example were all low, making them unusable in immunoassays. The tiletamine artificial antigen VII obtained in Comparative Example 4 immediately showed a large amount of precipitation during dialysis, while the tiletamine artificial antigen V obtained in Comparative Example 2 was relatively turbid and showed a large amount of precipitation after cryopreservation, indicating poor stability and thus unsuitable for use as an immune antigen. However, the immune serum obtained by immunizing animals with the tiletamine artificial antigen II in Example 1 had a titer of 1:85,000 and was fully usable in immunoassays, providing a more convenient, rapid, and accurate method for detecting tiletamine.

Claims

1. A tiletamine artificial hapten, characterized in that: Its molecular structure is shown in Ⅰ:

2. The method for preparing the tiletamine artificial hapten according to claim 1, wherein The method comprises the following steps: (1) performing a substitution reaction between tiletamine and ethyl 5-bromovalerate to prepare a compound represented by formula A; (2) hydrolyzing the compound represented by Formula A and adjusting the pH to be acidic to obtain the tiletamine artificial hapten represented by Formula I; The reaction formula of the method is shown below:

3. The method according to claim 2, wherein The method is carried out according to the following steps: (1) Tiletamine is dissolved in N,N-dimethylformamide, and ethyl 5-bromovalerate is added under the action of NaH. The mixture is heated to 60-70°C to carry out a substitution reaction. After the reaction is completed, the resulting reaction solution is post-treated to obtain the compound shown in Formula A; (2) The compound represented by formula A is dissolved in tetrahydrofuran and anhydrous methanol, and a 1N sodium hydroxide aqueous solution is added. The mixture is rapidly stirred at room temperature to carry out a hydrolysis reaction. After the reaction is completed, the pH is adjusted to 4-5. The resulting reaction solution is post-treated to obtain the tiletamine artificial hapten represented by formula I.

4. A tiletamine artificial antigen, characterized in that Its molecular structure is shown in II: In formula II, BSA is bovine serum albumin.

5. The method for preparing the tiletamine artificial antigen according to claim 4, wherein: The method comprises the following steps: combining the tiletamine artificial hapten represented by formula I in claim 1 with bovine serum albumin through a mixed acid anhydride method to prepare the tiletamine artificial antigen represented by formula II.

6. The method for preparing the tiletamine artificial antigen according to claim 5, wherein: The method comprises the following steps: (a) The tiletamine artificial hapten represented by Formula I, isobutyl chloroformate, and triethylamine are stirred in N,N-dimethylformamide solvent at 0-5° C. for 2-4 hours, and the supernatant is collected by centrifugation after the reaction is completed; The molar ratio of the tiletamine artificial hapten represented by formula I, isobutyl chloroformate, and triethylamine is 1:1.5-2.5:1-1.5; (b) adding the supernatant dropwise to a bovine serum albumin solution, and allowing the resulting mixture to stand at 3-5° C. overnight. The supernatant was collected by dialysis and centrifugation to obtain the tiletamine artificial antigen represented by formula II.

7. Use of the tiletamine hapten according to claim 1 or the tiletamine artificial antigen according to claim 4 in the preparation of anti-tiletamine antibodies.

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