Niazine artificial hapten as well as preparation method and application thereof

Abstract

The invention belongs to the field of chemical detection, and particularly relates to a niazine artificial hapten as well as a preparation method and application thereof. The invention provides a niazine artificial hapten, which retains the characteristic structure of niazine to the greatest extent, has an active group capable of being coupled with carrier protein, can be used as an antigenic determinant, and has high sensitivity when being subjected to immunoassay; the invention provides a niazine artificial antigen based on the niazine artificial hapten, a niazine antibody is obtained by using the artificial antigen to immunize an animal, and the niazine artificial antigen and the niazine antibody can be applied to colloidal gold immunochromatography detection of niazine. The method has the advantages of high sensitivity, high detection gradient and good specificity.

Description

Technical Field

[0001] The invention belongs to the field of chemical detection, and specifically relates to a nicdazole-type artificial hapten and a preparation method and application thereof. Background Art

[0002] Opioids are a large class of painkillers that act on brain cells. Low doses can relieve pain, but overdose can cause breathing difficulties and lead to death. Taking opioids can bring a sense of pleasure or excitement and can easily lead to addiction.

[0003] Nitazenes are a new type of synthetic opioids belonging to benzimidazole derivatives, including ethinizine, metonizine, and edonizine. It has strong μ-opioid receptor activity and is more potent than nitrazines, making it a potent opioid. The purity of nitrazines can reach more than 95%, but their toxic concentration level is very low, which means that even at extremely small doses, it may pose a serious threat to human health. Therefore, for regulatory purposes, it is necessary to develop a method for rapid, trace detection of nitrazines.

[0004] For other opioid drugs, such as morphine, patent application CN112480193A discloses an artificial hapten, artificial antigen, polyclonal antibody and their applications for opioid drugs, in which a D-glucuronic acid arm is derived from the hydroxyl group on the phenol ring of the morphine molecule, and a carboxyl group is successfully introduced into the morphine molecule to obtain an artificial hapten of a morphine derivative, which is then coupled with a carrier protein to obtain an artificial antigen of a morphine derivative with better antigenic properties. However, there are no related reports on artificial haptens and artificial antigens of the nicotinic acid class in the prior art. Summary of the invention

[0005] Based on the demand for rapid detection of nicotinic acid products and related technologies in the prior art, the present invention provides a nicotinic acid artificial hapten and a preparation method and application thereof. The specific technical scheme is as follows: In a first aspect, the present invention provides a class of nicadrenal artificial haptens, the general structural formula of the nicadrenal artificial haptens is shown in (X): (X) Where R1 is -C 2 H 5 , -H; R2 is -NO 2 、-NH 2 , -H; n = 2~5.

[0006] Furthermore, the structure of the nicrazine-based artificial hapten is shown in formula (I):

[0007] (I).

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned nicrazine-based artificial hapten, comprising the following steps: S1: 2,4-Dinitrofluorobenzene and diethylethylenediamine undergo coupling reaction, and after separation and purification, an intermediate product A as shown in formula A is obtained; (A) S2: The intermediate product A undergoes a reduction reaction with ammonium sulfide, and after separation and purification, an intermediate product B as shown in formula B is obtained; (B) S3: The intermediate product B undergoes a coupling reaction with p-hydroxyphenylacetic acid, and after separation and purification, an intermediate product C as shown in Formula III is obtained; (C) S4: The intermediate product C undergoes a nitrogen heterocyclization reaction under acidic conditions, and after separation and purification, an intermediate product IV as shown in formula D is obtained; (D) S5: The intermediate product IV reacts with ethyl bromobutyrate in the presence of a base to undergo an etherification reaction, and after separation and purification, an intermediate product E as shown in formula E is obtained; (E) S6: The intermediate product E is hydrolyzed under alkaline conditions, and after separation and purification, a nicrazine-type artificial hapten as shown in formula (I) is obtained.

[0009] The present application prepares a nicazine compound with a long arm from a simple molecule through a series of reactions. The nicazine compound retains the characteristic structure of the nicazine compound and has a carboxyl group at the end of the long arm, which can be coupled with a carrier protein as an active group. The nicazine compound can be used as a hapten and antigenic determinant for the subsequent preparation of artificial antigens.

[0010] Furthermore, in the above-mentioned preparation method S1 of the nicrazine-type artificial hapten, the specific steps are: dissolving 2,4-dinitrofluorobenzene in toluene, adding diethylethylenediamine, stirring at a constant temperature of 70°C, reacting for 12 hours, adding ethyl acetate and water, extracting by separation, drying over anhydrous sodium sulfate, filtering, and concentrating the organic phase to obtain an intermediate product A.

[0011] Furthermore, the mass ratio of the 2,4-dinitrofluorobenzene to diethylethylenediamine is 5:3-4.

[0012] Furthermore, the mass ratio of the 2,4-dinitrofluorobenzene to diethylethylenediamine is 5:3.4.

[0013] Furthermore, in the above-mentioned preparation method S2 of the nicdine-type artificial hapten, the specific steps are: dissolving the intermediate product A in tetrahydrofuran, adding a 50% aqueous solution of ammonium sulfide, stirring at room temperature for 12 hours, adding ethyl acetate and water, extracting, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase, adding an appropriate amount of acetonitrile to dissolve, adding silica gel, spin drying, and purifying by column chromatography: the eluent PE / EA 1 / 5 obtains the intermediate product B.

[0014] Furthermore, the mass ratio of the intermediate product A to the 50% aqueous solution of ammonium sulfide is 10:6-8.

[0015] Furthermore, the mass ratio of the intermediate product A to the 50% aqueous solution of ammonium sulfide is 11:15.9.

[0016] Furthermore, in the preparation method S3 of the above-mentioned nicdazole-type artificial hapten, the specific steps are: dissolving the intermediate product B in dichloromethane, adding p-hydroxyphenylacetic acid and dicyclohexylcarbodiimide (DCC), stirring at room temperature for 4 hours, adding ethyl acetate and water, extracting, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase, adding an appropriate amount of acetonitrile to dissolve, adding silica gel, spinning dry, and purifying by column chromatography: the eluent PE / EA 1 / 2 obtains the intermediate product C.

[0017] Furthermore, the mass ratio of the intermediate product B to p-hydroxyphenylacetic acid is 8:4-5.

[0018] Furthermore, the mass ratio of the intermediate product B to p-hydroxyphenylacetic acid is 8:4.5.

[0019] Furthermore, dicyclohexylcarbodiimide is used as a dehydrating agent to promote the esterification reaction, and the molar ratio of the intermediate product B to dicyclohexylcarbodiimide is 1.5-2.

[0020] Furthermore, in the above-mentioned preparation method S4 of the nicrazine-type artificial hapten, the specific steps are: dissolving the intermediate product C in concentrated hydrochloric acid, stirring at a constant temperature of 70°C for 12 hours, adjusting the pH to 7-8 with a 10% sodium hydroxide solution, adding ethyl acetate and water, extracting the mixture, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase, and spinning to obtain the intermediate product D.

[0021] Furthermore, in the above-mentioned preparation method S5 of the nicrazine-type artificial hapten, the specific steps are: dissolving the intermediate product D in N,N-dimethylformamide (DMF), adding ethyl 4-bromobutyrate and anhydrous potassium carbonate, stirring at room temperature for 12 hours, adding ethyl acetate and water, extracting by separation, drying with anhydrous sodium sulfate, filtering, concentrating the organic phase, adding an appropriate amount of acetonitrile to dissolve, adding silica gel, spinning dry, and purifying by column chromatography: the eluent PE / EA 1 / 4 obtains the intermediate product E.

[0022] Furthermore, the mass ratio of the intermediate product D to ethyl 4-bromobutyrate is 7:5.6.

[0023] Furthermore, in the above-mentioned preparation method S6 of the nicdinis artificial hapten, the specific steps are: dissolving the intermediate product E in ethanol, adding sodium hydroxide (10%) solution, stirring at room temperature for 12 hours, adding ethyl acetate and water, extracting, drying over anhydrous sodium sulfate, filtering, concentrating the organic phase, adding an appropriate amount of acetonitrile to dissolve, adding silica gel, spinning dry, and purifying by column chromatography: the eluent EA is 100% to obtain the nicdinis artificial hapten (I).

[0024] In a third aspect, the present invention provides a nicadine-based artificial antigen, which is obtained by coupling the above-mentioned nicadine-based artificial hapten with a carrier protein.

[0025] Furthermore, the carrier protein is bovine serum albumin.

[0026] Furthermore, the structure of the nicrazine-based artificial antigen is shown in Formula VII; (II).

[0027] Furthermore, the coupling ratio of the nicotinic acid artificial antigen is greater than 25.

[0028] In a fourth aspect, the present invention provides a method for preparing the above-mentioned nicadine artificial antigen, wherein the nicadine artificial antigen is obtained by combining the above-mentioned nicadine artificial hapten with a carrier protein through an activated ester method.

[0029] Further, the following steps are included: S1: reacting the above-mentioned nicrazine-based artificial hapten with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a solvent to obtain a reaction solution I; S2: dissolving the carrier protein to obtain reaction solution II; S3: Mix the reaction solution I and the reaction solution II. After the reaction is completed, filter and centrifuge, and take the supernatant to obtain the nicrazine artificial antigen.

[0030] Furthermore, in the above-mentioned preparation method S1 of the nicrazine artificial antigen, the specific steps are: take the nicrazine hapten, dissolve it in N,N-dimethylformamide (DMF), then add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stir the reaction at room temperature, and centrifuge to obtain the supernatant after the reaction is completed.

[0031] Furthermore, the mass ratio of the nicrazine artificial hapten to N-hydroxysuccinimide is 1:0.5~1.

[0032] Furthermore, the mass ratio of the nicrazine artificial hapten to N-hydroxysuccinimide is 1:0.9.

[0033] Furthermore, in the above-mentioned method S2 for preparing the nicardipine artificial antigen, the specific steps are: dissolving the carrier protein in PBS buffer to obtain a carrier protein solution.

[0034] Furthermore, the concentration of the carrier protein solution is 5 mg / mL.

[0035] Furthermore, in the above-mentioned method S3 for preparing the nicardipine artificial antigen, the specific steps are: under rapid stirring, slowly adding the supernatant obtained in S1 to the carrier protein solution to obtain an artificial antigen mixture, transferring the artificial antigen mixture into a dialysis bag, dialyzing with PBS buffer, and after the dialysis is completed, centrifuging and taking the supernatant to obtain the artificial antigen.

[0036] Furthermore, in the above-mentioned method S3 for preparing the isonitrile artificial antigen, the supernatant obtained in S1 is mixed with the carrier protein solution in a volume ratio of 1:5.

[0037] Furthermore, in S3, the mass ratio of the nicrazine artificial hapten in S1 used in the mixing process to the carrier protein in S2 used is 1:1~1.5.

[0038] Furthermore, during the mixing process of S3, the mass ratio of the nicrazine artificial hapten used in S1 to the carrier protein used in S2 was 1:1.25.

[0039] Furthermore, the coupling ratio of the nicotinic acid artificial antigen prepared in S3 is greater than 25.

[0040] In a fifth aspect, the present invention provides a nicotinoid antibody, which is prepared by immunizing experimental animals with the above-mentioned nicotinoid artificial antigen.

[0041] In a sixth aspect, the present invention provides a method for preparing the above-mentioned nicardipine antibody, comprising the following steps: (1) mixing a 1 mg / mL PBS solution of a nicardipine artificial antigen with Freund's complete adjuvant to obtain a mixed solution; (2) injecting the mixed solution obtained in step (1) into the immunized animal; (3) Blood is collected from the immunized animal, and immune serum containing nicardipine antibodies is separated from the blood.

[0042] Furthermore, in the above-mentioned method for preparing the nicadine antibody, in step (1), the volume ratio of the nicadine artificial antigen PBS solution to Freund's complete adjuvant is 1:1.

[0043] Furthermore, in the above-mentioned method for preparing the nicadrenal antibody, in step (2), the mixed solution prepared in step (1) is used to inject the immunized animal, and 21 days later, the mixed solution of nicadrenal artificial antigen PBS solution and Freund's complete adjuvant is again injected into the New Zealand white rabbit, and then the injection is performed every 14 days, for a total of 5 injections.

[0044] In a seventh aspect, the present invention provides the use of the above-mentioned nicadine artificial hapten, nicadine artificial antigen, or nicadine antibody in the immunodetection of nicadine compounds and the preparation of immunodetection reagents for nicadine compounds.

[0045] Furthermore, the reagent for detecting nicrazine compounds is colloidal gold immunochromatographic test paper.

[0046] The artificial antigen of nicazines provided by the present invention can be applied to colloidal gold immunochromatography detection of nicazines, and can quickly and conveniently realize the qualitative detection of nicazines, with a sensitivity of up to 100 ng / mL.

[0047] In an eighth aspect, the present invention provides a composition for detecting nicardipines, comprising a coating source and an antibody; The coating is originally the above-mentioned nicrazine artificial antigen; The antibody is prepared by immunizing an animal with a nicardipine immunogen.

[0048] Furthermore, the nicrazine immunogen is a nicrazine compound or the above-mentioned nicrazine artificial antigen.

[0049] In a ninth aspect, the present invention provides a product for detecting nicardipines, comprising the above-mentioned composition.

[0050] Furthermore, the product is a colloidal gold immunochromatographic test paper.

[0051] In a tenth aspect, the present invention provides a colloidal gold immunochromatographic test paper for detecting nicardipine compounds, comprising the following steps: (1) Preparation of colloidal gold solution using trisodium citrate reduction method; (2) Add colloidal gold solution to K 2 CO 3 The solution is evenly dispersed, and then a nicazine antibody solution is added for labeling reaction. After the reaction is completed, a skim milk powder solution is added for blocking, and the precipitate is collected by centrifugation. The precipitate is re-dissolved with a re-solution and then sprayed onto the glass fiber to obtain a nicazine antibody gold label pad; (3) coating the nicotinoid artificial antigen solution on the detection line on the nitrocellulose membrane, coating the goat anti-mouse IgG antibody solution on the quality control line on the nitrocellulose membrane, and drying; (4) Assemble the sample absorption pad, nitrocellulose membrane, nicotinic acid antibody gold label pad, and water absorption pad into a colloidal gold test strip.

[0052] Furthermore, the formula of the reconstituted solution is: 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 chelating agent, 0.05% sodium azide, and the pH value is adjusted to 7.4 with HCl.

[0053] Furthermore, the preparation process of the colloidal gold solution is as follows: measure 80 mL of pure water, put it into 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 it at 5°C away from light for later use.

[0054] Furthermore, the specific steps for preparing the nicrazine antibody gold label pad are: Take 3 mL of the prepared colloidal gold solution and add 1.0% K 2 CO 3 After adjusting the pH value to 8.2, pipette 1.5 μL of 1 mg / mL nicotinic acid antibody, slowly add it to the colloidal gold solution, shake well and let it stand for 35 min. Then take 10 μL of 10% skim milk powder and add it dropwise to the solution, shake well and let it stand for 35 min. Then put it into the centrifuge and centrifuge it at 5°C for 25 minutes. After completion, aspirate the supernatant and discard it. Dissolve the precipitate with a reconstitution solution according to 65% of the volume of the colloidal gold solution used for the original initial labeling, mix well, and obtain the reconstituted colloidal gold marker; spray 1.5 mL of the reconstituted colloidal gold marker on a 0.5 cm × 25 cm glass fiber and dry it at 35°C for 6 hours.

[0055] Furthermore, the sample absorption pad is immersed in a phosphate buffer solution containing 0.5% BSA, pH=7.2, and 0.1 mol / L, and dried at 37°C.

[0056] Furthermore, the step of coating the nitrocellulose membrane with the nitrocellulose artificial antigen and the goat anti-mouse IgG antibody is as follows: diluting the nitrocellulose artificial antigen with a phosphate buffer, coating it on the detection line T on the nitrocellulose membrane with a biodot film spotting instrument, diluting the goat anti-mouse IgG antibody with a pH=7.2, 0.01 mol / L PBS buffer, coating it on the quality control line C on the nitrocellulose membrane with a biodot film spotting instrument, and drying the coated reaction membrane.

[0057] Furthermore, in the colloidal gold test strip, the sample absorption pad, the nitrocellulose membrane, the nicdazole antibody gold label pad, and the water absorbent pad are sequentially adhered to 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 nicdazole antibody gold label pad, the end of the nicdazole antibody gold label pad is connected to the beginning of the water absorbent pad, the beginning of the sample absorption pad is aligned with the beginning of the bottom plate, and the end of the water absorbent pad is aligned with the end of the bottom plate.

[0058] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an artificial hapten of the nisazine class, which retains the characteristic structure of the nisazine class to the greatest extent, has an active group that can be coupled with a carrier protein, can be used as an antigenic determinant, and has high sensitivity during immunoassay. The present invention provides an artificial nisazine class antigen based on the artificial hapten of the nisazine class, which can be used in colloidal gold immunochromatography detection of the nisazine class, and has high sensitivity, detection gradient and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figure is a schematic diagram of the reaction formula for preparing the nicrazine artificial antigen II of the present invention; wherein THF represents tetrahydrofuran, DCC represents dicyclohexylcarbodiimide, 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 2 The mass spectrum of the hapten intermediate A in Example 1 of the present invention is shown in FIG. 1 , wherein Rel. Abundance (%) indicates relative abundance, and m / z indicates charge-to-mass ratio.

[0061] Figure 3 The mass spectrum of the hapten intermediate B in Example 1 of the present invention is shown in Figure 1; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0062] Figure 4 The mass spectrum of the hapten intermediate C in Example 1 of the present invention is shown in Figure 1; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0063] Figure 5 The mass spectrum of the hapten intermediate D in Example 1 of the present invention is shown in FIG. 1 , wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0064] Figure 6The mass spectrum of the hapten intermediate E in Example 1 of the present invention is shown in FIG. 1 , wherein Rel. Abundance (%) indicates relative abundance, and m / z indicates charge-to-mass ratio.

[0065] Figure 7 This is a liquid chromatogram of hapten I in Example 1 of the present invention; wherein, Response (mAU) represents the response value, and the unit is mAU.

[0066] Figure 8 It is the mass spectrum of hapten I in Example 1 of the present invention; wherein Rel. Abundance (%) represents relative abundance, and m / z represents charge-to-mass ratio.

[0067] Fig. 9 The figure is a schematic diagram of the reaction formula for preparing artificial antigen IV of comparative example 1 of the present invention; wherein THF represents tetrahydrofuran, DCC represents dicyclohexylcarbodiimide, DCM represents dichloromethane, EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0068] Fig.10 The figure is a schematic diagram of the reaction formula for preparing the artificial antigen V of Comparative Example 2 of the present invention; wherein THF represents tetrahydrofuran, DCC represents dicyclohexylcarbodiimide, DCM represents dichloromethane, EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BGG represents bovine gamma globulin.

[0069] Fig.11 The figure is a schematic diagram of the reaction formula for preparing the artificial antigen VI of comparative example 3 of the present invention; wherein THF represents tetrahydrofuran, DCC represents dicyclohexylcarbodiimide, DCM represents dichloromethane, NHS represents N-hydroxysuccinimide, DMF represents N,N-dimethylformamide, and BSA represents bovine serum albumin.

[0070] Fig.12 This is the absorption spectrum of hapten I in Example 1 of the present invention; wherein Rel. Response (%) represents the relative response value, and Wavelength represents the wavelength, and the unit is nm. DETAILED DESCRIPTION

[0071] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0072] 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.

[0073] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill 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 specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0074] The main materials and reagents used in this application: 2,4-Dinitrofluorobenzene, CAS No. 70-34-8; Diethylethylenediamine, CAS No. 100-36-7; p-Hydroxyphenylacetic acid, CAS No. 156-38-7; Dicyclohexylcarbodiimide (DCC), CAS No. 538-75-0; Ethyl 4-bromobutyrate, CAS No. 2969-81-5; 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 gamma globulin, CAS No. 9007-83-4.

[0075] Example 1 Preparation of Nisidine Artificial Haptens and Nisidine Artificial Antigens A method for preparing a nicotine-based artificial antigen, wherein the reaction formula is as follows: Figure 1 As shown, the specific steps include: (1) Preparation of artificial haptens of nicardipines The specific preparation steps are as follows: 1) Dissolve 10 g of 2,4-dinitrofluorobenzene in 100 mL of toluene, add 6.8 g of diethylethylenediamine, stir at 70 °C, react for 12 hours, add 200 mL of ethyl acetate and 200 mL of water, extract by separation, dry over anhydrous sodium sulfate, filter, and concentrate the organic phase to obtain 13.5 g of yellow solid with a yield of 91%. Perform mass spectrometry analysis and detection. The intermediate A is shown in the figure Figure 2 The mass-to-charge ratio (m / z) of the molecular ion peak was 283.3, which was consistent with its theoretical molecular weight of 282.1, confirming that intermediate product A was obtained; (A) 2) Dissolve 11 g of yellow solid A in 110 mL of tetrahydrofuran, add 15.9 g of 50% aqueous solution of ammonium sulfide, stir at room temperature for 12 hours, add 200 mL of ethyl acetate and 200 mL of water, extract by separation, dry over anhydrous sodium sulfate, filter, concentrate the organic phase, add appropriate amount of acetonitrile to dissolve, add 30 g of silica gel, spin dry, and purify by column chromatography: eluent PE / EA 1 / 5 to obtain 8.6 g of white solid, yield: 88%, perform mass spectrometry analysis, the mass spectrum of intermediate B is shown in Figure 3 The mass-to-charge ratio (m / z) of the molecular ion peak was 253.3, which was consistent with its theoretical molecular weight of 252.1, confirming that intermediate product B was obtained; (B) 3) 8 g of white solid B was dissolved in 80 mL of dichloromethane, 4.2 g of p-hydroxyphenylacetic acid and 3.5 g of DCC were added, and the mixture was stirred at room temperature for 4 hours. 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 spin-dried. The mixture was purified by column chromatography: the eluent was PE / EA 1 / 2 to obtain 10 g of white solid with a yield of 82%. The mass spectrum of the intermediate C was shown in FIG. Figure 4 The mass-to-charge ratio (m / z) of the molecular ion peak is 387.3, which is consistent with its theoretical molecular weight of 386.2, confirming that the intermediate product C is obtained; (C) 4) 8.5 g of white solid C was dissolved in 20 mL of concentrated hydrochloric acid, stirred at 70 °C for 12 hours, adjusted to pH 7-8 with 10% sodium hydroxide solution, added with 200 mL of ethyl acetate and 200 mL of water, separated and extracted, dried over anhydrous sodium sulfate, filtered, concentrated the organic phase, and dried to obtain 7.3 g of light yellow solid, yield: 90%. Mass spectrometry analysis was performed, and the mass spectrum of intermediate D is shown in Figure 5 The mass-to-charge ratio (m / z) of the molecular ion peak was 369.2, which was consistent with its theoretical molecular weight of 368.2, confirming that intermediate product D was obtained.

[0076] (D) 5) Dissolve 7 g of light yellow solid D in 70 mL of DMF, add 5.6 g of ethyl 4-bromobutyrate and 3.9 g of anhydrous potassium carbonate, stir at room temperature for 12 hours, add 200 mL of ethyl acetate and 200 mL of water, extract by separation, dry over anhydrous sodium sulfate, filter, concentrate the organic phase, add appropriate amount of acetonitrile to dissolve, add 20 g of silica gel, spin dry, and purify by column chromatography: eluent PE / EA 1 / 4 to obtain 7.8 g of light yellow oil, yield: 85%, perform mass spectrometry analysis, the mass spectrum of intermediate A is shown in Figure 6The mass-to-charge ratio (m / z) of the molecular ion peak was 483.4, which was consistent with its theoretical molecular weight of 482.3, confirming that intermediate product E was obtained.

[0077] (E) 6) 7.8 g of light yellow oil E was dissolved in 80 mL of ethanol, 30 mL of sodium hydroxide (10%) solution was added, and the mixture was stirred at room temperature for 12 hours. 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 for dissolution. 25 g of silica gel was added, and the mixture was spin-dried. Column chromatography was used for purification: the eluent EA was 100% to obtain 6.5 g of light yellow oil, with a yield of 88%. LC-MS detection was performed to confirm that the nicrazine artificial hapten (I) shown in formula (I) was obtained.

[0078] The liquid chromatogram of the nicardiline artificial hapten I is shown in Figure 7 , mass spectrum see Figure 8 .from Figure 7 It can be seen that the purity of the purified nicotinic acid artificial hapten reaches more than 97%. Figure 8 It can be seen that the molecular ion peak of the artificial hapten obtained in this example is 455.4, which is consistent with its theoretical molecular weight of 454.3. It is determined that the final compound prepared is the nicdazine artificial hapten I designed by the present invention.

[0079] (I).

[0080] (2) Preparation of artificial antigens of nicardipines 1) Take 100 mg of nicardipine hapten I in a 50 mL round-bottom flask, add 5 mL N,N-dimethylformamide (DMF), then add 90 mg N-hydroxysuccinimide (NHS) and 150 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), stir at room temperature for overnight reaction, centrifuge after the reaction, and take the supernatant for later use; 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 make up to 5.0 L to obtain 0.01 M PBS buffer with a pH of 7.4; 3) Weigh 0.125 g of bovine serum albumin and dissolve it in 25 mL of PBS buffer prepared in step 2) to obtain a bovine serum albumin solution with a concentration of 5 mg / ml; 4) Under rapid stirring, slowly dropwise add the supernatant obtained in step 1) to the bovine serum albumin solution, the volume ratio of the supernatant to the bovine serum albumin solution being 1:5, and the obtained mixture is stored at 4° C. overnight to obtain an artificial antigen mixture; 5) The artificial antigen mixture is transferred into a dialysis bag and dialyzed with PBS buffer for 4 times. After the dialysis, the supernatant is centrifuged to obtain the nicardipine artificial antigen (II).

[0081] Example 2 Preparation of Nisazine Antibodies The prepared nicardipine artificial antigen was used to immunize New Zealand white rabbits, and the specific steps were as follows: (1) diluting the synthesized nicardipine artificial antigen to 1 mg / mL with PBS to obtain an antigen solution, and then mixing 0.5 mL of the antigen solution with 0.5 mL of Freund's complete adjuvant and injecting it into New Zealand white rabbits; (2) 21 days later, inject the New Zealand white rabbits with a mixture of 0.5 mL of antigen solution and 0.5 mL of Freund's complete adjuvant, and then inject them every 14 days for a total of 5 injections; (3) Blood was collected from New Zealand white rabbits, immune serum was separated, and nisazine antibodies were purified.

[0082] Example 3 Preparation of Nizine Colloidal Gold Immunochromatographic Test Strips (1) Preparation of colloidal gold: Measure 80 mL of pure water, put it into 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 away from light for later use.

[0083] (2) Preparation of Nisazine Antibody Gold Label Pad: 1) The required raw materials are: the nicotinic acid antibody prepared in Example 2; the colloidal gold solution prepared in step (1); 1.0% K 2 CO 3 ; 10% skimmed milk powder; reconstituted solution; glass fiber.

[0084] The reconstitution solution formula is 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, 0.01mol / L Tris, 0.2% hydrolyzed chelating agent, 0.05% sodium azide, and the pH value is adjusted to 7.4 with HCl.

[0085] 2) Colloidal gold-nisazine antibody labeling process: Take 3 mL of the prepared colloidal gold solution and add 1.0% K 2 CO 3After adjusting the pH value to 8.2, pipette 1.5 μL of 1 mg / mL nicotinic acid antibody and slowly add it to the colloidal gold solution. Shake well and let it stand for 35 minutes. Then take 10 μL of 10% skim milk powder and add it dropwise to the solution. Shake well and let it stand for 35 minutes. Then put it into the centrifuge and centrifuge it at 5°C for 25 minutes. After completion, aspirate the supernatant and discard it. Dissolve the precipitate with a reconstitution solution at 65% of the volume of the colloidal gold solution used for the original initial labeling, mix well, and obtain the reconstituted colloidal gold marker.

[0086] 3) Solidification of colloidal gold marker: Spray 1.5 mL of the reconstituted colloidal gold marker onto a 0.5 cm × 25 cm glass fiber and dry at 35°C for 6 hours. Seal the aluminum foil bag and store at room temperature for later use.

[0087] (3) Preparation of sample absorption pad The sample absorption pad was immersed in a phosphate buffer solution containing 0.5% BSA, pH=7.2, 0.1 mol / L, and dried at 37°C to obtain a sample absorption pad; (4) Preparation of nitrocellulose membrane Coating process: dilute the nicrazine artificial antigen with phosphate buffer, and coat it on the detection line T on the nitrocellulose membrane with a biodot film spotter; 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 with a biodot film spotter; dry the coated reaction membrane to obtain a nitrocellulose membrane; (5) Assembly of test strips The sample absorption pad, nitrocellulose membrane, nicdazole 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 nicdazole antibody gold-labeled pad, the end of the nicdazole 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.

[0088] Comparative Example 1 Preparation of Nisidine Artificial Haptens and Nisidine Artificial Antigens Using Different Condensing Agents Preparation method of nicardipine artificial antigen (III) (reaction formula as shown in Fig. 9 ), including the following steps: (1) Preparation of nicotinic acid artificial hapten (III): Steps 1) to 4) are the same as those for the preparation of hapten in Example.

[0089] 5) Dissolve 5 g of light yellow solid D in 50 mL of pyridine, add 2.4 g of succinic anhydride, stir at room temperature for 12 hours, add 200 mL of ethyl acetate and 200 mL of water, separate and extract, dry over anhydrous sodium sulfate, filter, concentrate the organic phase, add appropriate amount of acetonitrile to dissolve, add 20 g of silica gel, spin dry, and purify by column chromatography: eluent PE / EA 1 / 3 to obtain 5.4 g of light yellow oil E, yield: 85%, LC-MS detection, confirming that artificial hapten (III) is obtained.

[0090] (2) Preparation of artificial antigens of the nicotinic acid class (IV): BSA was used as a carrier protein and coupled with hapten, and the coupling steps 1) to 5) were the same as those in the example.

[0091] Comparative Example 2 Preparation of Nisazine Artificial Antigens Using Bovine Gamma Globulin Preparation method of nicardipine artificial antigen (V) (reaction formula as shown in Fig.10 ), including the following steps: (1) Preparation of nicotinic acid artificial hapten (I): Steps 1) to 5) are the same as those in comparative example 1 for preparing hapten.

[0092] (2) Preparation of artificial haptens of nicotinic acid class: Bovine gamma globulin (BGG) was used as a carrier protein for coupling with hapten, and the coupling steps 1)-2) were the same as those in Example 1.

[0093] 3) Weigh 0.125 g of bovine immunoglobulin G and dissolve it in 25 mL of PBS buffer prepared in step 2) to obtain a bovine serum albumin solution with a concentration of 5 mg / ml; 4) under rapid stirring, slowly adding the supernatant obtained in step 1) to the bovine immunoglobulin G solution in a volume ratio of 1:5, and the obtained mixture is stored at 4° C. overnight to obtain an artificial antigen mixture; 5) The artificial antigen mixture is transferred into a dialysis bag and dialyzed with PBS buffer for 4 times. After the dialysis, the supernatant is centrifuged to obtain the nicardipine artificial antigen (V).

[0094] Comparative Example 3 Preparation of Nisazine Artificial Antigens Using Different Activation Methods Preparation method of nicardipine artificial antigen (VI) (reaction formula as shown in Fig.11 ), including the following steps: (1) Preparation of artificial haptens of nicotinic acid class: Steps 1) to 5) are the same as those in Example 1 for hapten preparation (I).

[0095] (2) Preparation of artificial haptens of nicotinic acid class: BSA was used as carrier protein and hapten coupling, where: 1) Take 100 mg of nicardipine hapten (I) in a 50 mL round-bottom flask, add 5 mL of dichloromethane (DCM), then add 90 mg of triethylamine and 120 mg of isobutyl chloroformate, stir at room temperature for overnight reaction, centrifuge after the reaction, and take the supernatant for later use; 2)-5) The antigen preparation was the same as in Example 1 to obtain the nicrazine artificial antigen (VI).

[0096] Test Example 1 Performance test of nicardipine artificial antigens (1) Determination of molar absorption coefficient PBS buffer was used to prepare 0μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, and 40μg / mL nicrazine artificial hapten solutions. The ultraviolet absorption spectra showed that Fig.12 It can be seen that the maximum absorption wavelength of the nisazine hapten is 240 nm. The absorbance is measured at 240 nm, and parallel samples are made for each concentration. The calculation formula for the molar absorption coefficient (i.e., molar absorption coefficient) is: ε = absorbance / molar concentration.

[0097] (2) Determination of the concentration of artificial antigen protein of nicardipine The antigen concentration was measured by the lowry method, and 1 mL of bovine serum protein solution with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL was prepared with PBS buffer, and parallel samples were made for each concentration. The absorbance was detected at 650 nm using an ultraviolet spectrophotometer, and a curve of the relationship between protein concentration and absorbance was drawn. The nicdazole artificial antigens prepared in Example 1 and Comparative Examples 1-3 were dissolved in PBS buffer, diluted at a ratio of 1 / 8, and the absorbance of the nicdazole artificial antigens was measured at 650 nm. The corresponding protein concentration value of the nicdazole artificial antigen solution was read from the curve to calculate the protein concentration of the nicdazole artificial antigen.

[0098] 3) Determination of coupling ratio of nicardipine artificial antigens Prepare 100 μg / mL bovine serum albumin PBS solution, dilute the nicardipine artificial antigen to 100 μg / mL with PBS, and measure the absorbance value A at 240 nm. 1 , using PBS solution as blank to measure the absorbance value A 2 The coupling ratio γ is calculated as follows: γ = [(A 1 -A 2 ) / ε] / (100×10 -3 / 66000); 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), When bovine gamma globulin (BGG) is used as a carrier, γ = [(A 1 -A 2 ) / 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).

[0099] The coupling ratios of various nicardipine artificial antigens and the corresponding molar absorption coefficients of haptens are shown in Table 1 Table 1 Performance test of nicardipine artificial antigens II, IV, V, VI

[0100] 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.

[0101] Test Example 2 Activity Detection of Nisazine Artificial Antigens The titer of the immune serum prepared in Example 2 was tested by ELISA method. The test results are shown in Table 2.

[0102] Table 2 Immune serum titer

[0103] Table 2 shows the titer test results of each immune serum. Compared with Example 1, the titers of the immune serum obtained by immunizing animals with artificial nicadine antigens in each comparative example are all low and cannot be used in immunoassays. However, the titer of the immune serum obtained by immunizing animals with artificial nicadine antigen II is 1:90,000, which can be used in immunoassays and can provide a convenient, fast and accurate way for the detection of nicadines.

[0104] Test Example 3 Performance Determination of Urine Colloidal Gold Immunochromatographic Detection Reagent Strip Functional test of the colloidal gold immunochromatographic detection reagent strip prepared in Example 3 The specific operation is as follows: collect fresh clinical negative urine, add nicardipine standard to prepare samples of 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, 300 ng / mL. Cross-interference substance solution: use negative urine to add different drugs, common medicines, etc. to prepare a 100μg / mL concentration solution sample.

[0105] 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 of the reagent strip are colored, the test result is negative (-); if the quality control line is colored and the test line is not colored, the test result is positive (+); if the quality control line is not colored, the test line is colored, or both the quality control line and the test line are not colored (X), the test result is invalid.

[0106] The results of the sensitivity test of this test case are shown in Table 3. The minimum detection limit of the colloidal gold immunochromatographic test strip for nicardipines is 100 ng / mL. All tests above 100 ng / mL are positive, and all tests below 100 ng / mL are negative. The drug cross-interference experiment is shown in Table 4. The samples of 100 μg / mL concentration of 30 common drugs were prepared using negative urine, and the results were all negative.

[0107] Table 3 Sensitivity test results of colloidal gold immunochromatographic test strips for nicardipines

[0108] Table 4 Results of drug cross-interference test of colloidal gold immunochromatographic test strips of nicardipines

Claims

1. A nicotinic acid-based artificial hapten, characterized in that: The general structural formula of the nicrazine-based artificial hapten is shown in (X): （X） Among them, R1 is one of -C2H5 and -H; R2 is one of -NO2, -NH2, and -H; n = 2~5.

2. The nicrazine-based artificial hapten according to claim 1, characterized in that: The structure of the nicrazine-based artificial hapten is shown in formula (I): （Ⅰ）。 3. The method for preparing the nicrazine-based artificial hapten according to claim 2, characterized in that: The following steps are involved: S1: 2,4-Dinitrofluorobenzene and diethylethylenediamine undergo coupling reaction, and after separation and purification, an intermediate product A as shown in formula A is obtained; （A） S2: The intermediate product A undergoes a reduction reaction with ammonium sulfide, and after separation and purification, an intermediate product B as shown in formula B is obtained; （B） S3: The intermediate product B undergoes a coupling reaction with p-hydroxyphenylacetic acid, and after separation and purification, an intermediate product C as shown in formula C is obtained; （C） S4: The intermediate product III undergoes a nitrogen heterocyclization reaction under acidic conditions, and after separation and purification, an intermediate product IV as shown in formula D is obtained; （D） S5: The intermediate product D reacts with ethyl 4-bromobutyrate in the presence of a base to undergo an etherification reaction, and after separation and purification, an intermediate product E as shown in Formula E is obtained; （E） S6: The intermediate product E is hydrolyzed under alkaline conditions, and after separation and purification, the nicrazine-type artificial hapten shown in formula (I) is obtained.

4. A nicotinic acid artificial antigen, characterized in that: The nicadrenal artificial antigen is obtained by coupling the nicadrenal artificial hapten as claimed in claim 1 with a carrier protein.

5. The method for preparing the nicrazine artificial antigen according to claim 3, characterized in that: The following steps are involved: S1: reacting the nicrazine-based artificial hapten as claimed in claim 1 with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a solvent to obtain a reaction solution I; S2: dissolving the carrier protein to obtain reaction solution II; S3: Mix the reaction solution I and the reaction solution II. After the reaction is completed, filter and centrifuge, and take the supernatant to obtain the nicrazine artificial antigen.

6. A nicardipine antibody, characterized in that: The method is prepared by immunizing experimental animals with the artificial nicardipine antigen as described in any one of claims 4 or 5.

7. The method for preparing a nicardipine antibody according to claim 6, characterized in that: The following steps are involved: (1) mixing a 1 mg / mL PBS solution of a nicardipine artificial antigen with Freund's complete adjuvant to obtain a mixed solution; (2) injecting the mixed solution obtained in step (1) into the immunized animal; (3) Blood is collected from the immunized animal, immune serum is separated from the blood, and nicardipine antibodies are separated from the blood.

8. Use of the artificial hapten of the nicadine class as described in any one of claims 1 or 2, the artificial antigen of the nicadine class as described in any one of claims 4, or the antibody of the nicadine class as described in claim 7 in the preparation of a reagent for detecting nicadine class compounds.

9. The use according to claim 8, characterized in that: The reagent for detecting nicrazine compounds is colloidal gold immunochromatographic test paper.

10. A composition for detecting nicrazines, characterized in that: Including coating source and antibody; The coating is the nicrazine artificial antigen as described in claim 3; The antibody is prepared by immunizing an animal with a nicardipine immunogen.

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