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

Abstract

The invention provides a medetomidine artificial hapten, an artificial antigen as well as a preparation method and application of the medetomidine artificial hapten and the artificial antigen. Specifically, the invention provides a medetomidine artificial hapten of formula (X), the artificial hapten maximally retains the characteristic structure of medetomidine, has an active group capable of being coupled with carrier protein, can be used as an antigenic determinant, and has high sensitivity during immunoassay; furthermore, the invention provides a medetomidine artificial antigen based on the medetomidine artificial hapten, and the medetomidine artificial antigen can be applied to colloidal gold immunochromatography detection of medetomidine and has relatively high sensitivity, detection gradient and relatively good specificity.

Description

Technical Field

[0001] The present invention belongs to the field of small molecule chemical detection, and specifically relates to a medetomidine artificial hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Medetomidine is a non-opioid veterinary anesthetic not approved for human use. Because it is an emerging adulterant, the Centers for Disease Control and Prevention is closely monitoring its use. Medetomidine is more potent than dextromethorphan. It may produce a longer-lasting sedative effect, is approximately 200 times more potent, and is approximately 10 times more selective than dextromethorphan. The combination of fentanyl and medetomidine has triggered a wave of new overdose poisonings, and with the increasing number of medetomidine-induced seizures in the United States, this has become a pressing public health concern.

[0003] There is no relevant report on medetomidine artificial hapten or medetomidine artificial antigen in the prior art. Summary of the Invention

[0004] Based on the demand for rapid detection of medetomidine products and related technologies in the prior art, the present invention provides a medetomidine artificial hapten, artificial antigen, and preparation method and application thereof. The specific technical solutions are as follows:

[0005] In one aspect, the present invention provides a medetomidine artificial hapten having a structure shown in the following formula (X):

[0006]

[0007] Where n=1~5.

[0008] In one aspect, the medetomidine artificial hapten of the present invention has a structure shown in the following formula (I):

[0009]

[0010] In another aspect, the present invention provides a method for synthesizing the artificial hapten of medetomidine of formula (I), comprising the following steps:

[0011] S1: Medetomidine reacts with ethyl 6-bromohexanoate to produce a nucleophilic reaction, and an intermediate product represented by formula (A) is obtained after separation and purification;

[0012]

[0013] S2: The intermediate product (A) is hydrolyzed under alkaline conditions, and after separation and purification, the artificial hapten of medetomidine as shown in formula (I) is obtained.

[0014] In one aspect, the method for synthesizing the artificial hapten of medetomidine of formula (I) of the present invention comprises the following steps:

[0015] S1: dissolving medetomidine in an organic solvent, adding ethyl 6-bromohexanoate, reacting, and separating and purifying to obtain an intermediate product represented by formula (A);

[0016] S2: dissolving the intermediate product (A) in alcohol, adding alkaline solution, reacting, separating and purifying to obtain the artificial hapten of medetomidine as shown in formula (I).

[0017] In one aspect, the organic solvent is selected from DMF, acetone or acetonitrile. Preferably, the organic solvent is DMF.

[0018] In one aspect, the alcohol is selected from ethanol, methanol or n-butanol. Preferably, the alcohol is ethanol.

[0019] In one aspect, the alkaline solution is selected from sodium hydroxide, potassium hydroxide or lithium hydroxide solution. Preferably, the alkaline solution is sodium hydroxide solution. More preferably, the alkaline solution is a 10% by mass sodium hydroxide solution.

[0020] In one aspect, the method for synthesizing the artificial hapten of medetomidine of formula (I) of the present invention comprises the following steps:

[0021] S1: dissolving medetomidine in DMF, adding ethyl 6-bromohexanoate, reacting, and separating and purifying to obtain an intermediate product represented by formula (A);

[0022] S2: dissolving the intermediate product (A) in ethanol, adding sodium hydroxide solution, reacting, separating and purifying to obtain the medetomidine artificial hapten as shown in formula (I).

[0023] In one aspect, the method for synthesizing the artificial hapten of medetomidine of formula (I) of the present invention comprises the following steps:

[0024] S1: Dissolve medetomidine in DMF, add ethyl 6-bromohexanoate, stir at 70±5°C, react for 12±2 hours, and separate and purify to obtain the intermediate product represented by formula (A);

[0025] S2: dissolving the intermediate product (A) in ethanol, adding sodium hydroxide solution, stirring at 20±5° C. for 12±2 hours, and obtaining the medetomidine artificial hapten represented by formula (I) after separation and purification.

[0026] In another aspect, the present invention provides an artificial medetomidine antigen, which is obtained by coupling the artificial medetomidine hapten described in the present invention with a carrier protein.

[0027] In one aspect, the synthesis of the medetomidine artificial antigen of the present invention comprises the following steps:

[0028] (1) dissolving the medetomidine artificial hapten described in the present invention in DMF, adding NHS and EDC, reacting, and centrifuging to obtain the supernatant;

[0029] (2) Dissolving the carrier protein with a buffer solution, and adding the supernatant of step (1) to the carrier protein solution to obtain the medetomidine artificial antigen.

[0030] In another aspect, the synthesis of the medetomidine artificial antigen of the present invention comprises the following steps:

[0031] (1) Dissolve 100 mg of the medetomidine artificial hapten described in the present invention in 5 mL of DMF, add 90 mg of NHS and 150 mg of EDC, stir at 20±5°C for 12±2 hours, and centrifuge to obtain the supernatant;

[0032] (2) 0.125 g of carrier protein was dissolved in 25 mL of 0.01 M PBS buffer solution with a pH of 7.4, and the supernatant of step (1) was added dropwise to the carrier protein solution at a volume ratio of 1:5 to obtain the medetomidine artificial antigen.

[0033] In one aspect, the carrier protein is bovine serum albumin. Preferably, the molecular weight of the bovine serum albumin is 66,000.

[0034] In another aspect, the present invention provides an artificial medetomidine antigen, which is obtained by coupling the artificial medetomidine hapten described in the present invention with bovine serum albumin.

[0035] In another aspect, the present invention provides a medetomidine antibody, characterized in that it is prepared by immunizing experimental animals with the medetomidine artificial antigen described in the present invention.

[0036] In one aspect, the experimental animal is selected from New Zealand rabbits, mice, and sheep. Preferably, the experimental animal is a New Zealand rabbit.

[0037] In one aspect, the medetomidine antibody of the present invention is obtained by subjecting New Zealand rabbits to one primary immunization and four booster immunizations with the medetomidine artificial antigen of the present invention.

[0038] In one aspect, the present invention provides use of the medetomidine artificial hapten of the present invention, the medetomidine artificial antigen of the present invention and / or the medetomidine antibody of the present invention in the preparation of a reagent for detecting medetomidine.

[0039] In one aspect, the present invention provides a medetomidine colloidal gold rapid detection device, comprising a test strip and a reaction pad, the test strip containing a detection line and a quality control line, the detection line being coated with the medetomidine artificial antigen described in the present invention, the quality control line being coated with a substance for detecting the medetomidine antibody, the reaction pad containing a colloidal gold-labeled medetomidine antibody, and the medetomidine antibody being the medetomidine antibody described in the present invention.

[0040] In one aspect, the test paper of the present invention contains a nitrocellulose membrane, the nitrocellulose membrane contains the detection line and the quality control line, the detection line is coated with the medetomidine artificial antigen of the present invention, the quality control line is coated with a substance for detecting the medetomidine antibody, the reaction pad contains colloidal gold-labeled medetomidine antibody, and the medetomidine antibody is the medetomidine antibody of the present invention.

[0041] In one aspect, the medetomidine colloidal gold rapid detection device of the present invention is a medetomidine colloidal gold immunochromatographic test strip.

[0042] In one aspect, the present invention provides a medetomidine colloidal gold immunochromatographic test strip, comprising: a sample absorption pad, a nitrocellulose membrane, a medetomidine antibody gold-labeled pad, and a water-absorbing pad are sequentially adhered to a bottom plate, the end of the sample absorption pad is connected to the beginning of the nitrocellulose membrane, the end of the nitrocellulose membrane is connected to the beginning of the medetomidine antibody gold-labeled pad, and the end of the medetomidine antibody gold-labeled pad is connected to the beginning of the water-absorbing pad, to assemble the colloidal gold test strip;

[0043] The nitrocellulose membrane comprises a detection line and a quality control line, the detection line is coated with the medetomidine artificial antigen of the present invention, and the quality control line is coated with a substance for detecting the medetomidine antibody;

[0044] The medetomidine antibody gold label pad contains colloidal gold-labeled medetomidine antibody, and the medetomidine antibody is the medetomidine antibody described in the present invention.

[0045] In one aspect, the substance used to detect the medetomidine antibody in the present invention is a second antibody against the medetomidine antibody; preferably, the second antibody is a goat anti-mouse IgG antibody.

[0046] In one aspect, the sample absorption pad of the present invention has been soaked in a buffer solution containing BSA.

[0047] In one aspect, the sample absorption pad of the present invention has been soaked in a buffer solution containing 0.5% BSA, pH=7.2, 0.1 mol / L, and then dried.

[0048] In one aspect, on the nitrocellulose membrane of the present invention, the detection line is coated with the medetomidine artificial antigen of the present invention, and the quality control line is coated with goat anti-mouse IgG antibody.

[0049] In one aspect, on the medetomidine antibody gold labeling pad described in the present invention, the medetomidine antibody labeled with colloidal gold is sprayed onto the glass fiber, and the medetomidine antibody is the medetomidine antibody described in the present invention.

[0050] In one aspect, the present invention provides a method for detecting medetomidine, characterized in that the method comprises using the medetomidine colloidal gold rapid detection device of the present invention for detection.

[0051] In one aspect, the sample of the present invention is a body fluid. Preferably, the body fluid is selected from blood, serum, saliva, sweat, and urine. More preferably, the body fluid is urine.

[0052] In one aspect, the sample of the present invention is urine.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention provides an artificial medetomidine hapten, which retains the characteristic structure of medetomidine to the greatest extent, has an active group that can be coupled to a carrier protein, can serve as an antigenic determinant, and has high sensitivity in immunoassay. Furthermore, the present invention provides an artificial medetomidine antigen based on the artificial medetomidine hapten, which can be used in colloidal gold immunochromatographic detection of medetomidine and has high sensitivity, detection gradient, and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the reaction formula for preparing the medetomidine artificial antigen II of the present invention; wherein EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0056] Figure 2 This is the mass spectrum of hapten intermediate A in Example 1 of the present invention.

[0057] Figure 3 This is the liquid chromatogram of hapten (I) in Example 1 of the present invention.

[0058] Figure 4 This is the mass spectrum of hapten (I) in Example 1 of the present invention.

[0059] Figure 5The figure is a schematic diagram of the reaction formula for preparing the artificial antigen (IV) of Comparative Example 1 of the present invention; wherein EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0060] Figure 6 This is the mass spectrum of hapten intermediate B in Comparative Example 1 of the present invention.

[0061] Figure 7 This is the mass spectrum of hapten (III) in comparative example 1 of the present invention.

[0062] Figure 8 Schematic diagram of the reaction formula for preparing the artificial antigen (V) of Comparative Example 2 of the present invention; wherein EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BGG represents bovine immunoglobulin gamma.

[0063] Figure 9 Schematic diagram of the reaction formula for preparing the artificial antigen (VI) in Comparative Example 3 of the present invention; wherein DCM represents dichloromethane, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0064] Figure 10 This is the absorption spectrum of hapten (I) in Example 1 of the present invention. DETAILED DESCRIPTION

[0065] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0066] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0067] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0068] The main materials and reagents used in this application:

[0069] Medetomidine, CAS No. 86347-15-1; ethyl 6-bromohexanoate, CAS No. 25542-62-5; ethyl 2-bromoacetate, CAS No. 105-36-2; N-hydroxysuccinimide (NHS), CAS No. 6066-82-6; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), CAS No. 25952-53-8; bovine serum albumin, CAS No. 9048-46-8; bovine immunoglobulin G, CAS No. 9007-83-4.

[0070] Example 1 Preparation of Medetomidine Artificial Hapten and Medetomidine Artificial Antigen

[0071] A method for preparing a medetomidine artificial antigen, wherein the reaction formula is as follows: Figure 1 As shown, the specific steps include:

[0072] (1) Preparation of artificial hapten of medetomidine

[0073] The specific preparation steps are as follows:

[0074] 1) 5 g of medetomidine was dissolved in 50 mL of DMF, 8.3 g of ethyl 6-bromohexanoate was added, the mixture was stirred at 70° C., the reaction was continued for 12 hours, 200 mL of ethyl acetate and 200 mL of water were added, the mixture was separated and extracted, the mixture was dried over anhydrous sodium sulfate, filtered, the organic phase was concentrated, an appropriate amount of acetonitrile was added and dissolved, 30 g of silica gel was added, the mixture was spin-dried, and the mixture was purified by column chromatography: the eluent was PE / EA (2 / 3), and 7.2 g of a light colorless oil was obtained, with a yield of 84%. The mixture was analyzed by mass spectrometry, as shown in FIG. Figure 2 The mass-to-charge ratio (m / z) of the molecular ion peak was 343.2, which was consistent with its theoretical molecular weight of 342.2, confirming that intermediate A was obtained;

[0075]

[0076] 2) 7.2 g of colorless oil A was dissolved in 80 mL of ethanol, 20 mL of sodium hydroxide (10%) solution was added, and the mixture was stirred at room temperature for 12 hours. The pH was adjusted to 2-3 with hydrochloric acid (2N), 200 mL of ethyl acetate and 200 mL of water were added, and the mixture was separated and extracted. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated and dissolved in an appropriate amount of acetonitrile. 25 g of silica gel was added, and the mixture was spin-dried. The mixture was purified by column chromatography: the eluent was EA100%, and 6.1 g of a colorless oil was obtained. The yield was 92%.

[0077] The liquid chromatography of artificial hapten (I) of medetomidine is shown in Figure 3 , mass spectrum see Figure 4 .from Figure 3 It can be seen that the purity of the purified artificial hapten of medetomidine reaches more than 96%. Figure 4It can be seen that the molecular ion peak of the artificial hapten obtained in this example is 315.1, which is consistent with its theoretical molecular weight of 314.2. It is determined that the final compound prepared is the medetomidine artificial hapten (I) designed by the present invention.

[0078]

[0079] (2) Preparation of artificial medetomidine antigen

[0080] 1) 100 mg of medetomidine hapten (I) was placed in a 50 mL round-bottom flask, 5 mL of N,N-dimethylformamide (DMF) was added, followed by 90 mg of N-hydroxysuccinimide (NHS) and 150 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and the mixture was stirred at room temperature overnight. After the reaction, the supernatant was centrifuged and set aside.

[0081] 2) Weigh 14.5 g of disodium hydrogen phosphate dodecahydrate, 43.8 g of sodium chloride, and 1.5 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 with a pH of 7.4.

[0082] 3) Weigh 0.125 g of bovine serum albumin and dissolve it in 25 mL of the PBS buffer prepared in step 2) to obtain a bovine serum albumin solution with a concentration of 5 mg / ml;

[0083] 4) slowly adding the supernatant obtained in step 1) dropwise to the bovine serum albumin solution under rapid stirring, with the volume ratio of the supernatant to the bovine serum albumin solution being 1:5, and the resulting mixture being stored at 4° C. overnight to obtain an artificial antigen mixture;

[0084] 5) The artificial antigen mixture was transferred into a dialysis bag and dialyzed four times with PBS buffer. After the dialysis, the supernatant was centrifuged to obtain the medetomidine artificial antigen (II).

[0085] Example 2 Preparation of Medetomidine Antibodies

[0086] The prepared artificial medetomidine antigen (II) was used to immunize New Zealand white rabbits. The specific steps are as follows:

[0087] (1) The synthesized medetomidine artificial antigen (II) was diluted to 1 mg / mL with PBS to obtain an antigen solution, and then 0.5 mL of the antigen solution was mixed with 0.5 mL of Freund's complete adjuvant and injected into New Zealand white rabbits;

[0088] (2) After 21 days, the New Zealand white rabbits were injected with a mixture of 0.5 mL of antigen solution and 0.5 mL of Freund's complete adjuvant, and then injected every 14 days for a total of 5 injections;

[0089] (3) Blood was collected from New Zealand white rabbits, immune serum was separated, and medetomidine antibodies were purified.

[0090] Example 3 Preparation of Medetomidine Colloidal Gold Immunochromatographic Test Strips

[0091] (1) Preparation of colloidal gold: Measure 80 mL of pure water, place it in a flask, heat and stir until boiling. Add 1.5 mL of 1% chloroauric acid solution to the boiling water, continue heating and stirring. When the solution boils again, accurately measure 1.5 mL of 1.0% trisodium citrate solution and quickly add it to the solution, and continue boiling. Observe the color of the solution. When the solution changes from yellow to purple-black and finally to wine red and stabilizes, continue heating for 15 minutes. After the solution temperature returns to room temperature, stop stirring, restore the volume to 80 mL with purified water, and store at 5°C in the dark for later use.

[0092] (2) Preparation of medetomidine antibody gold label pad:

[0093] 1) The required raw materials are: the medetomidine antibody prepared in Example 2; the colloidal gold solution prepared in step (1); 1.0% K2CO3; 10% skim milk powder; the reconstitution solution; and glass fiber.

[0094] The reconstitution solution was prepared according to the following final concentrations: 0.1% sodium chloride, 2% trehalose, 2% Triton X-100, 0.2% β-cyclodextrin, 2% BSA, 0.5% sodium citrate, 0.5% PEG20000, 0.01 mol / L Tris, 0.2% hydrolyzed chelate, and 0.05% sodium azide. The pH was adjusted to 7.4 with HCl.

[0095] 2) Colloidal gold-medetomidine antibody labeling process: Take 3 mL of the prepared colloidal gold solution, adjust the pH to 8.2 with 1.0% K₂CO₃, then pipette 1.5 μL of 1 mg / mL medetomidine antibody and slowly add it to the colloidal gold solution. Shake well and let it stand for 35 minutes. Then, add 10 μL of 10% skim milk powder dropwise to the solution, shake well, and let it stand for 35 minutes. Centrifuge at 5°C for 25 minutes. After completion, aspirate and discard the supernatant, and reconstitute the precipitate with a reconstitution solution at 65% the volume of the colloidal gold solution used for the initial labeling. Mix thoroughly to obtain the reconstituted colloidal gold label.

[0096] 3) Immobilization of colloidal gold label: Spray 1.5 mL of the reconstituted colloidal gold label onto a 0.5 cm × 25 cm glass fiber and dry at 35°C for 6 h. Seal the package in an aluminum foil bag and store at room temperature until ready for use.

[0097] (3) Preparation of sample absorption pad

[0098] The sample absorption pad was immersed in phosphate buffer containing 0.5% BSA, pH=7.2, 0.1 mol / L, and dried at 37°C to obtain the sample absorption pad;

[0099] (4) Preparation of nitrocellulose membrane

[0100] Coating process: Dilute the medetomidine artificial antigen with phosphate buffer and coat it on the test line T on the nitrocellulose membrane using a biodot coating instrument. Dilute the goat anti-mouse IgG antibody with pH 7.2, 0.01 mol / L PBS buffer and coat it on the quality control line C on the nitrocellulose membrane using a biodot coating instrument. Dry the coated reaction membrane to obtain a nitrocellulose membrane.

[0101] (5) Assembly of test strips

[0102] The sample absorption pad, nitrocellulose membrane, medetomidine antibody gold-labeled pad, and water-absorbing pad are sequentially pasted on the bottom plate, the end of the sample absorption pad is connected to the beginning of the nitrocellulose membrane, the end of the nitrocellulose membrane is connected to the beginning of the medetomidine antibody gold-labeled pad, the end of the medetomidine antibody gold-labeled pad is connected to the beginning of the water-absorbing pad, the beginning of the sample absorption pad is aligned with the beginning of the bottom plate, and the end of the water-absorbing pad is aligned with the end of the bottom plate to assemble a colloidal gold test strip.

[0103] Comparative Example 1 Preparation of Medetomidine Artificial Hapten and Medetomidine Artificial Antigen Using Different Connector Arms

[0104] Preparation method of artificial medetomidine antigen (Ⅳ) (reaction formula as shown Figure 5 ), including the following steps:

[0105] (1) Preparation of artificial hapten (III) of medetomidine:

[0106] The specific preparation steps are as follows:

[0107] 1) 6 g of medetomidine was dissolved in 50 mL of DMF, 6.3 g of ethyl 2-bromoacetate was added, the mixture was stirred at room temperature, and the reaction was carried out for 12 hours. 200 mL of ethyl acetate and 200 mL of water were added, the mixture was separated and extracted, and the mixture was dried over anhydrous sodium sulfate. The organic phase was concentrated and dissolved in an appropriate amount of acetonitrile. 30 g of silica gel was added, the mixture was spin-dried, and the mixture was purified by column chromatography: the eluent was PE / EA (2 / 3) to obtain 6.1 g of a light colorless oil, with a yield of 85%. The mixture was subjected to mass spectrometry analysis, as shown in FIG. Figure 6 The mass-to-charge ratio (m / z) of the molecular ion peak was 287.2, which was consistent with its theoretical molecular weight of 286.2, confirming that intermediate B was obtained;

[0108]

[0109] 2) 7.2 g of colorless oil A was dissolved in 80 mL of ethanol, 20 mL of sodium hydroxide (10%) solution was added, and the mixture was stirred at room temperature for 12 hours. The pH was adjusted to 2-3 with hydrochloric acid (2N), 200 mL of ethyl acetate and 200 mL of water were added, and the mixture was separated and extracted. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated. An appropriate amount of acetonitrile was added to dissolve the mixture, 25 g of silica gel was added, and the mixture was dried and purified by column chromatography: the eluent EA100% gave 4.9 g of colorless oil, with a yield of 90%. The mass spectrometry analysis was performed, as shown. Figure 7 The mass-to-charge ratio (m / z) of the molecular ion peak was 259.1, which was consistent with its theoretical molecular weight of 258.1, and was confirmed to be the artificial hapten (III) of medetomidine.

[0110]

[0111] (2) Preparation of Medetomidine Artificial Antigen (IV):

[0112] BSA was used as a carrier protein for coupling with hapten, and the coupling steps were basically the same as the corresponding coupling steps 1) to 5) in Example 1, except that the medetomidine artificial hapten (III) was used instead of the medetomidine artificial hapten (I).

[0113] Comparative Example 2 Preparation of Medetomidine Artificial Antigen Using Bovine Gamma Globulin

[0114] Preparation method of artificial medetomidine antigen (V) (reaction formula as shown Figure 8 ), including the following steps:

[0115] (1) Preparation of Medetomidine Artificial Hapten (I):

[0116] The preparation steps of the medetomidine artificial hapten (I) are the same as those in Example 1.

[0117] (2) Preparation of artificial medetomidine antigen:

[0118] Bovine gamma globulin (BGG) was used as a carrier protein for coupling with the hapten, and the coupling steps were the same as the corresponding coupling steps 1)-2) in Example 1.

[0119] 3) Weigh 0.125 g of bovine immunoglobulin G and dissolve it in 25 mL of the PBS buffer prepared in step 2) to obtain a 5 mg / ml bovine serum albumin solution;

[0120] 4) slowly adding the supernatant obtained in step 1) dropwise to the bovine immunoglobulin G solution under rapid stirring, with the volume ratio of the supernatant to the bovine immunoglobulin G solution being 1:5, and the resulting mixture being stored at 4° C. overnight to obtain an artificial antigen mixture;

[0121] 5) The artificial antigen mixture was transferred into a dialysis bag and dialyzed four times with PBS buffer. After the dialysis, the supernatant was centrifuged to obtain the medetomidine artificial antigen (V).

[0122] Comparative Example 3 Preparation of Medetomidine Artificial Antigens Using Different Activation Methods

[0123] Preparation method of artificial medetomidine antigen (VI) (reaction formula as shown Figure 9 ), including the following steps:

[0124] (1) Preparation of artificial hapten of medetomidine:

[0125] The preparation method of medetomidine artificial hapten (I) is the same as that in Example 1.

[0126] (2) Preparation of artificial medetomidine antigen:

[0127] BSA is used as a carrier protein to couple with hapten, wherein:

[0128] Step 1) 100 mg of medetomidine hapten (I) was placed in a 50 mL round-bottom flask, 5 mL of dichloromethane (DCM) was added, and then 90 mg of triethylamine and 120 mg of isobutyl chloroformate were added. The mixture was stirred at room temperature overnight. After the reaction was completed, the supernatant was centrifuged and set aside.

[0129] Steps 2)-5) are the same as the corresponding steps 2)-5) of the antigen preparation in Example 1, to obtain the medetomidine artificial antigen (VI).

[0130] Test Example 1 Performance Testing of Medetomidine Artificial Antigen

[0131] (1) Determination of molar absorption coefficient

[0132] Medetomidine artificial hapten solutions with concentrations of 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL were prepared with PBS buffer, and the UV absorption spectra were shown. Figure 10 The maximum absorption wavelength of medetomidine hapten is 198 nm. The absorbance was measured at 198 nm, with replicates made for each concentration. The molar absorptivity is calculated as: ε = absorbance / molar concentration.

[0133] (2) Determination of the concentration of medetomidine artificial antigen protein

[0134] Antigen concentration was measured using the Lowry method. 1 mL of bovine serum albumin solution was prepared in PBS buffer at concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. Duplicate samples were prepared for each concentration. The absorbance was measured at 650 nm using a UV spectrophotometer, and a curve plotting the relationship between protein concentration and absorbance was drawn. The medetomidine artificial antigen prepared in Example 1 and Comparative Examples 1-3 was dissolved in PBS buffer and diluted at a ratio of 1 / 8. The absorbance of the medetomidine artificial antigen was measured at 650 nm. The corresponding protein concentration of the medetomidine artificial antigen solution was read from the curve to calculate the protein concentration of the medetomidine artificial antigen.

[0135] 3) Determination of the coupling ratio of medetomidine artificial antigen

[0136] Prepare 100 μg / mL bovine serum albumin PBS solution, dilute the medetomidine artificial antigen to 100 μg / mL with PBS, measure the absorbance value A1 at 198 nm, and measure the absorbance value A2 with PBS solution as blank. The coupling ratio γ is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 66000);

[0137] Wherein, ε is the molar absorption coefficient (L / mol) measured in this example, 66000 is the molecular weight of bovine serum albumin, 100×10 -3 is the concentration of bovine serum albumin (g / L),

[0138] When bovine gamma globulin (BGG) is used as a carrier, γ = [(A1-A2) / ε] / (100×10 -3 / 150000), the molecular weight of bovine immunoglobulin G is 1500000, 100×10 -3 is the concentration of bovine serum albumin (g / L).

[0139] The coupling ratios of the medetomidine artificial antigens and the molar absorption coefficients of the corresponding haptens are shown in Table 1.

[0140] Table 1 Performance test of medetomidine artificial antigens II, IV, V, VI

[0141] serial number Artificial antigens Coupling ratio Antigen concentration Molar absorption coefficient Example 1 Ⅱ 23 2.98 mg / mL 5081.3 Comparative Example 1 Ⅳ 21 3.01 mg / mL 5062.7 Comparative Example 2 Ⅴ 22 2.88 mg / mL 5150.4 Comparative Example 3 Ⅵ 18 2.94 mg / mL 5112.7

[0142] 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 have a certain influence on the coupling ratio when the hapten and the carrier protein are combined.

[0143] Test Example 2 Activity Detection of Medetomidine Artificial Antigen

[0144] The titer of the immune serum prepared in Example 2 was tested by ELISA. The test results are shown in Table 2.

[0145] Table 2 Immune serum titer

[0146] serial number Medetomidine artificial antigen Immune serum titer Example 1 Ⅱ 1:85000 Comparative Example 1 Ⅳ 1:51000 Comparative Example 2 Ⅴ 1:18000 Comparative Example 3 Ⅵ 1:58000

[0147] Table 2 shows the titer test results for each immune serum. Compared with Example 1, the titers of the immune sera obtained from animals immunized with the artificial medetomidine antigen in each comparative example were all low and unusable in immunoassays. However, the titer of the immune sera obtained from animals immunized with the artificial medetomidine antigen II reached 1:85,000, making them suitable for immunoassays and providing a convenient, rapid, and accurate method for the detection of medetomidine.

[0148] Test Example 3 Performance Determination of Urine Colloidal Gold Immunochromatographic Detection Reagent Strip

[0149] Functional testing of the medetomidine colloidal gold immunochromatographic detection reagent strip prepared in Example 3

[0150] The specific procedure is as follows: Collect fresh clinically negative urine and add medetomidine standard to prepare samples at different concentrations: 0 ng / mL, 25 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 125 ng / mL, 150 ng / mL, 175 ng / mL, 200 ng / mL, and 300 ng / mL. Cross-interference substance solution: Use negative urine to add various drugs and common pharmaceuticals to prepare a solution sample at a concentration of 100 μg / mL.

[0151] When the above sample is added to the sample addition area of ​​the reagent strip, if both the test line and the quality control line show color, the test result is negative (-); if the quality control line shows color and the test line does not show color, the test result is positive (+); if the quality control line does not show color, the test line shows color, or both the quality control line and the test line do not show color (X), the test result is invalid.

[0152] The sensitivity test results for this test example are shown in Table 3. The minimum detection limit for the colloidal gold immunochromatographic test strip for medetomidine in urine is 100 ng / mL. Tests above 100 ng / mL are positive, while tests below 100 ng / mL are negative. The drug cross-interference test is shown in Table 4. Samples prepared at a concentration of 100 μg / mL using negative urine for 30 common drugs all returned negative results.

[0153] Table 3 Sensitivity test results of medetomidine urine colloidal gold immunochromatographic test strips

[0154] Concentration (ng / mL) result 0 -,-,-,-,- 25 -,-,-,-,- 50 -,-,-,-,- 75 -,-,-,-,- 100 -,+,+,+,- 125 +,+,+,+,+ 150 +,+,+,+,+ 175 +,+,+,+,+ 200 +,+,+,+,+ 300 +,+,+,+,+

[0155] Table 4 Drug cross-interference test results of medetomidine colloidal gold immunochromatographic test strips

[0156] drug result drug result drug result Dextromethorphan - Amphetamines - methaqualone - Fentanyl - Barbiturates - Methadone - Heroin - Buprenorphine - Morphine / opium - Zolpidem - Benzodiazepines - Oxycodone - Zopiclone - caffeine - Phencyclidine - Carbamazepine - Cathinone - Pregabalin - Psilocybin - Carfentanil - Dextropropoxyphene - Olanzapine - Clonazepam - tricyclic antidepressants - Zaleplon - cocaine - marijuana - Diazepam - Cotinine - Tramadol -

Claims

1. A medetomidine artificial hapten, characterized in that The medetomidine artificial hapten has a structure shown in the following formula (X): Where n=1~5.

2. The artificial hapten of medetomidine according to claim 1, characterized in that The medetomidine artificial hapten has a structure shown in the following formula (I):

3. A method for synthesizing the artificial hapten of medetomidine according to claim 2, characterized in that: The following steps are involved: S1: Medetomidine reacts with ethyl 6-bromohexanoate to produce a nucleophilic reaction, and an intermediate product represented by formula (A) is obtained after separation and purification; S2: The intermediate product (A) is hydrolyzed under alkaline conditions, and after separation and purification, the artificial hapten of medetomidine as shown in formula (I) is obtained.

4. A medetomidine artificial antigen, characterized in that The medetomidine artificial antigen is obtained by coupling the medetomidine artificial hapten according to claim 1 or 2 with a carrier protein.

5. The medetomidine artificial antigen according to claim 4, characterized in that The carrier protein is bovine serum albumin.

6. A medetomidine antibody, characterized in that The method is prepared by immunizing experimental animals with the medetomidine artificial antigen according to claim 4 or 5.

7. Use of the medetomidine artificial hapten according to claim 1 or 2, the medetomidine artificial antigen according to claim 4 or 5, and / or the medetomidine antibody according to claim 6 in the preparation of a reagent for detecting medetomidine.

8. A medetomidine colloidal gold rapid detection device, characterized in that: The detection device comprises a test strip and a reaction pad, the test strip comprises a detection line and a quality control line, the detection line is coated with the medetomidine artificial antigen according to claim 4 or 5, the quality control line is coated with a substance for detecting the medetomidine antibody, the reaction pad comprises a colloidal gold-labeled medetomidine antibody, and the medetomidine antibody is the medetomidine antibody according to claim 6.

9. The rapid detection device according to claim 8, characterized in that: The substance used to detect the medetomidine antibody is a secondary antibody against the medetomidine antibody; preferably, the secondary antibody against the medetomidine antibody is a goat anti-mouse IgG antibody.

10. A method for detecting medetomidine in a sample, characterized in that: The method comprises performing detection using the medetomidine colloidal gold rapid detection device according to claim 8 or 9.

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