Nitrazene substance artificial hapten, artificial antigen and preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a nitrazene substance artificial hapten, an artificial antigen and a preparation method and application of the nitrazene substance artificial hapten and the artificial antigen. The artificial hapten of the nitrazene substance retains the characteristic structure of the nitrazene substance to the greatest extent, has an active group capable of being coupled with carrier protein, and can be used as an antigenic determinant; the further prepared artificial antigen of the nitrazene substance can be used for immunizing to obtain an anti-nitrazene substance antibody with high affinity, high sensitivity and strong specificity, and a polyclonal antibody obtained by immunizing a New Zealand white rabbit can be used for carrying out rapid and accurate immunodetection and immunoassay on the nitrazene substance.

Description

Technical Field

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

[0002] Nitrazenes are a class of emerging potent synthetic opioid psychoactive substances, which are composed of several different types of drugs. Their material structure is shown in the following formula (III), and the specific list is shown in Table 1 below. Nitazenes are a type of potent synthetic opioid drug that has a rapid sedative effect. Their potency may be stronger than fentanyl, hundreds of times that of heroin, and 1,000 times that of morphine. Compared with other opioid drugs, nitrazenes are more likely to cause the user's breathing to weaken or stop. Criminals mix them into "Mohe cigarettes" (Xinjiang local cigarettes, green or yellow granular tobacco) and sell them for smoking. The abuse methods are soaking them in drinks, smoking them with hot water, etc. After smoking, symptoms such as general weakness, nausea, foaming at the mouth, dry cough, vomiting, and paralysis appear, and severe cases can cause sudden death. Therefore, for regulatory purposes, it is necessary to develop a method for rapid detection of nitrazenes.

[0003] Formula (III); Wherein, R1 represents an alkyl group, including -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -(CH2)3CH3, etc.; R2 represents -NO2 or -H; R3 represents -N(CH2CH3)2, -NHCH2CH3, pyrrolyl, piperidinyl, etc.

[0004] Table 1: Partial list of Nitazenes

[0005] At present, the detection of nitrazoles mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), mass spectrometry (MS), etc. However, these methods have the disadvantages of expensive instruments, time-consuming detection, and the need for professional technicians to operate, and cannot meet the requirements of modern detection for rapidity and accuracy.

[0006] Immunoassay can make up for all the above shortcomings. Immunoassay is an analytical method that uses antigen-antibody specific binding reactions to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to establishing an immunoassay for small molecule compounds is to be able to produce antibodies with high affinity and high specificity for small molecule compounds. However, since most small molecule compounds (molecular weight less than 1000), including nitrazolidines, are not immunogenic, that is, they lack T cell epitopes and cannot directly induce the animal body to produce specific antibodies, small molecules are called haptens. Through appropriate chemical modification, a connecting arm with an active group at the end is added to a certain site of the hapten molecular structure, and then combined with a macromolecular carrier to generate a hapten-carrier conjugate (i.e., artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells with the help of T cell epitopes, and then produce specific antibodies. Therefore, the synthesis of efficient artificial antigens is the premise and key to ensure immunoassay.

[0007] There is no related report on artificial haptens and artificial antigens of nitrazole substances in the prior art. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides an artificial hapten and an artificial antigen of a nitrazolone substance and a preparation method thereof, and applies the artificial antigen of a nitrazolone substance to the preparation of an anti-nitrazolone substance antibody with high detection sensitivity and strong specificity.

[0009] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: An artificial hapten of a nitrazole substance, the molecular structure of which is shown in formula (I):

[0010] The present invention selects the most common site R1 of nitrazole substances as the coupling site, and introduces a connecting arm from the R1 site, which can retain the characteristic structure of nitrazole substances to the greatest extent, and has an active group that can be coupled with a carrier protein, which can be used as an antigenic determinant and can be used for immunodetection of nitrazole substances.

[0011] The method for preparing the artificial hapten of nitrazole substances as described above comprises the following steps: (S.1) Dissolve the free-state metonizine with hydrobromic acid, heat under reflux until the reaction is complete, directly transfer to dryness, add purified water, adjust the pH with concentrated ammonia, extract with ethyl acetate and collect the organic phase, dry, filter, and transfer to dryness to obtain a light yellow solid A; (S.2) dissolving the light yellow solid A obtained in step (S.1) in anhydrous acetone, adding potassium carbonate and ethyl bromoacetate, heating under reflux for reaction, cooling to room temperature after the reaction is completed, filtering, and drying to obtain a reddish brown oil B; (S.3) The reddish brown oil B obtained in step (S.2) is dissolved in tetrahydrofuran and anhydrous methanol, and an aqueous sodium hydroxide solution is added, and the mixture is rapidly stirred at room temperature. After the reaction is complete, the pH is adjusted with a hydrochloric acid solution, and the organic phase is extracted with dichloromethane and collected, dried, filtered, transferred to dryness, and purified to obtain an artificial hapten I of a nitrazole substance.

[0012] The present invention can retain the characteristic structure of the nitrazolene substance to the greatest extent by introducing a connecting arm at the R1 site of the nitrazolene substance, and the modification site is as far away from its characteristic functional group as possible, so that its characteristic part is exposed to the outside to the greatest extent, so as to avoid interference with the specific antigen determinant cluster and be recognized by the immune body to the greatest extent. Compared with the use of a ring-shaped connecting arm, the connecting arm used in the present invention is chain-shaped, and the length of the connecting arm is appropriate, so as to ensure that the nitro-nitrogen substance hapten small molecule can be fully exposed on the surface of the artificial antigen, and the recognition degree of the connecting arm by T cells during immunization is reduced as much as possible, thereby promoting the specificity and affinity of the antibodies obtained by immunization to the nitrazolene substance to be stronger.

[0013] Preferably, in step (S.1): The material-liquid ratio of the added free metonizine to hydrobromic acid is 35-45:1 mg / mL; The reaction temperature of the heating reflux reaction is 90-110°C, and the reaction time is 17-20h.

[0014] Preferably, in step (S.2): The solid-liquid ratio of the added light yellow solid A, anhydrous acetone, potassium carbonate and ethyl bromoacetate is 95-105:8-12:430-450:120-128 mg / mL / mg / μL; The reaction temperature of the heating reflux reaction is 65-75°C, and the reaction time is 3.5-5h.

[0015] Preferably, the solid-liquid ratio of the reddish brown oil B, tetrahydrofuran, anhydrous methanol and sodium hydroxide aqueous solution added in step (S.3) is 95-105:1.40-1.50:2.0-2.1:4-6 mg / mL / mL / mL.

[0016] An artificial antigen of nitrazole substances, the molecular structure of which is shown in formula (II): Among them, BSA is bovine serum albumin.

[0017] The method for preparing the above-mentioned artificial antigen of nitrazole substances comprises the following steps: The artificial hapten I of the nitrazolidine substance as claimed in claim 1 is combined with bovine serum albumin by using the N-hydroxysuccinimide active ester method to obtain the artificial antigen II of the nitrazolidine substance.

[0018] The present invention uses bovine serum albumin (BSA) as a macromolecular carrier, which has the following advantages compared with bovine gamma globulin (BGG): ① Bovine serum albumin contains many lysine residues, has stronger immunogenicity, has many antigenic determinants, is easy to cause a stronger immune response, and has high coupling efficiency; ② Bovine serum albumin has a highly conservative structure, high stability, and is not easily affected by external factors, while bovine immunoglobulin G is more susceptible to external factors and is prone to precipitation, which in turn affects the performance of the antigen; ③ The coupling efficiency of bovine serum albumin and hapten is high, and it is easier to improve the specificity of the antibody. The artificial antigen of nitrazolyl substances formed by the combination of bovine serum albumin and artificial hapten of nitrazolyl substances has better specificity of the anti-nitrazolyl substances antibody obtained by animal immunization; ④ Bovine serum albumin is relatively cheap and easy to obtain, which helps to reduce costs and increase product competitiveness.

[0019] Preferably, a method for preparing an artificial antigen of nitrazole substances comprises the following steps: (a) mixing the nitrazole artificial hapten I as claimed in claim 1 with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, stirring at room temperature, centrifuging after the reaction is completed, and collecting the supernatant; (b) adding the supernatant obtained in step (a) dropwise to the bovine serum albumin solution and mixing thoroughly to obtain a mixed solution, and letting the mixed solution stand. After the reaction is completed, dialyzing and centrifuging are performed, and the supernatant is taken to obtain the nitrazole artificial antigen II.

[0020] Preferably, the bovine serum albumin solution in step (b) is prepared by dissolving bovine serum albumin in 0.01 M PBS buffer (pH=7.2-7.4).

[0021] Preferably, the molar ratio of the nitrazole artificial hapten I to cyclohexylcarbodiimide and N-hydroxysuccinimide added in step (a) is 1:(1.3-1.4):(1.3-1.4); The concentration of the bovine serum albumin solution added in step (b) is 9-12 mg / mL; the volume ratio of the added supernatant to the bovine serum albumin solution is 0.8-1.2:9-12.

[0022] The use of the artificial nitrazolone antigen as described above in the preparation of anti-nitrazolone antibody.

[0023] An anti-nitrazolium antibody is a globulin obtained by immunizing animals with the artificial nitrazolium antigens described above and capable of generating a specific immune reaction with nitrazolium substances.

[0024] The present invention has been found through repeated experiments that the titer of the immune serum obtained by immunizing New Zealand white rabbits with artificial antigens of nitrazolene substances is 1:128000. This shows that the artificial antigens of nitrazolene substances of the present invention can be used to immunize and obtain anti-nitrazolene substances antibodies with high affinity, high sensitivity and strong specificity, and the anti-nitrazolene substances antibodies can be used for immunological detection and analysis of nitrazolene substances.

[0025] Therefore, the present invention has the following beneficial effects: The artificial nitroazine hapten of the present invention retains the characteristic structure of nitroazine substances to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant cluster; the artificial nitroazine antigen prepared further can be immunized to obtain anti-nitroazine antibodies with high affinity, high sensitivity and strong specificity, and the polyclonal antibodies obtained by immunizing New Zealand white rabbits can be used for rapid and accurate immunodetection and immunoanalysis of nitroazine substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a reaction process diagram for the preparation of artificial antigen II of nitrazole substances in Example 1.

[0027] Figure 2 This is the liquid chromatogram of the artificial hapten I of the nitrazole substance in Example 1.

[0028] Figure 3 This is the mass spectrum of the artificial hapten I of the nitrazole substance in Example 1.

[0029] Figure 4 This is the UV scanning image before and after the preparation of the artificial antigen II of the nitrazole substance in Example 1.

[0030] Figure 5 This is a reaction process diagram for the preparation of artificial antigen IV of nitrazole substances in Comparative Example 1.

[0031] Figure 6 This is a reaction process diagram for the preparation of artificial antigen V of nitrazole substances in Comparative Example 2.

[0032] Figure 7 This is a reaction process diagram for the preparation of artificial antigen VI of nitrazole substances in Comparative Example 3.

[0033] Figure 8 This is a reaction process diagram for the preparation of artificial antigen VII of nitrazole substances in Comparative Example 4.

[0034] Fig. 9This is a reaction process diagram for the preparation of artificial antigen VIII of nitrazole substances in Comparative Example 5.

[0035] Fig.10 This is a reaction process diagram for the preparation of artificial antigen IX of nitrazole substances in Comparative Example 6.

[0036] Fig.11 This is a reaction process diagram for the preparation of artificial antigen X of nitrazole substances in Comparative Example 7. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0038] Example 1 This embodiment provides an artificial hapten I of a nitrazole substance and a preparation method thereof, and an artificial antigen II of a nitrazole substance and a preparation method thereof.

[0039] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: (S.1) Dissolve 200 mg (0.523 mmol) of free metonizine in 5 mL of 48% HBr and place in a 50 mL round-bottom flask. Reflux at 100°C for 18 h. After the reaction is complete, transfer directly to dryness. Add 15 mL of purified water and adjust the pH to 9 with concentrated ammonia water. A large amount of white precipitate is produced. Extract with 20 mL of ethyl acetate each time, extract twice, collect the organic phase, dry the organic phase with anhydrous sodium sulfate, filter, and transfer to dryness to obtain 180 mg (0.489 mmol) of light yellow solid A. The light yellow solid A was subjected to TLC detection, and the chromatographic solution was ethyl acetate: ammonia water = 10 mL: 200 μL, and the product Rf = 0.2; (S.2) Dissolve 180 mg (0.489 mmol) of the light yellow solid A obtained in step (S.1) in 18 mL of anhydrous acetone in a 50 mL round-bottom flask, add 792 mg of potassium carbonate and 223 μL of ethyl bromoacetate, and stir under reflux at 70°C for reaction. After 2 hours, the solution changes from light yellow to orange-red, and then to wine red. The reaction ends after 4 hours. Stop the reaction, cool to room temperature, filter, dry, and turn to dryness to obtain 210 mg (0.462 mmol) of a reddish brown oil B. The reddish brown oil B is subjected to TLC detection, and the chromatographic liquid is ethyl acetate:ethanol = 5:1 (v / v), and the product Rf = 0.8; (S.3) 210 mg (0.462 mmol) of the reddish brown oil B obtained in step (S.2) was dissolved in 3.05 mL of tetrahydrofuran (THF) and 4.35 mL of anhydrous methanol (MeOH), placed in a 50 mL round-bottom flask, and 10.5 mL of 1 mol / L sodium hydroxide aqueous solution was added. The solution color deepened and became turbid, and the reaction was stirred rapidly at room temperature for 4 hours. After the reaction was completed, the pH was adjusted to 4 with a 1 mol / L hydrochloric acid aqueous solution, and 20 mL of dichloromethane was used each time for extraction, and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate, filtered, and dried to obtain 190 mg of a light yellow oil, which was purified by thin layer chromatography to obtain 156 mg (0.366 mmol) of nitrazole artificial hapten I.

[0040] The molecular structure of the artificial hapten I of the nitrazole substance is shown in formula (I):

[0041] The artificial hapten I of the nitrazole substance was detected by TLC, the chromatographic solution was 95% ethanol:1,4-dioxane:dichloromethane:ammonia water=8:1:10:1 (v / v / v / v), and the product Rf=0.3.

[0042] The chromatographic solution used in the thin layer chromatography method was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1 (v / v / v / v), the product Rf = 0.3, and the solvent and eluent were anhydrous ethanol.

[0043] The liquid chromatogram of the artificial hapten Ⅰ of nitrazole substances is shown in Figure 2 (UV detector, wavelength 290nm) as shown. The mass spectrum of artificial hapten I of nitrazole is shown in Figure 3 shown.

[0044] from Figure 2 It can be seen that the purity of the artificial hapten I of the nitrazole substance obtained after purification reaches more than 98%. Figure 3It can be seen that the characteristic peaks of the artificial hapten I of the nitrazole substance obtained in this example are 427.2, 449.18, 450.19, and 471.21, respectively corresponding to [M+H] + 、[M+Na] + 、[M+Na+H] + 、[M+HCOO] - The theoretical molecular weight of the artificial hapten I of the nitrazole olefins is 426.19, so it can be basically determined that the final compound obtained in step (S.3) is the artificial hapten I of the nitrazole olefins designed by the present invention.

[0045] (2) Preparation of artificial antigen II of nitrazole substances: (a) 156 mg (0.366 mmol) of the nitrazole artificial hapten I obtained in the above step (S.3) was placed in a 50 mL round-bottom flask, 7.8 mL of N,N-dimethylformamide (DMF) was added, and then 57 mg (0.494 mmol) of N-hydroxysuccinimide (NHS) and 102 mg (0.494 mmol) of cyclohexylcarbodiimide (DCC) were added, and the mixture was stirred at 25°C for 16 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected for later use; (b) (b1) Weigh 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate and dissolve them in deionized water to a volume of 5.0 L to obtain a 0.01 M PBS buffer solution with a pH of 7.4; (b2) weighing 780 mg of bovine serum albumin and dissolving it in 78 mL of the PBS buffer obtained in step (b1) to obtain a bovine serum albumin solution with a concentration of 10 mg / mL; (b3) slowly adding the supernatant obtained in step (a) to the bovine serum albumin solution obtained in step (b2) under rapid stirring, wherein the volume ratio of the added supernatant to the bovine serum albumin solution is 1:10, and the obtained mixture is stored at 4° C. overnight to obtain an artificial antigen mixture; (b4) The artificial antigen mixture obtained in step (b3) is transferred into a dialysis bag, and dialyzed 9 times with the PBS buffer obtained in step (b1). After the dialysis is completed, the mixture is centrifuged and the supernatant is taken to obtain the nitrazole artificial antigen II (i.e., nitrazole-bovine serum protein conjugate). The preparation reaction process of the nitrazole artificial antigen II in this embodiment is shown in Figure 1 The UV scanning images of the artificial antigen II of nitrazole substances before and after preparation are shown in Figure 4 The molecular structure of the artificial antigen II of nitrazole is shown in formula (II): Among them, BSA is bovine serum albumin.

[0046] Figure 4 In the figure, curve a is the UV scanning graph of artificial hapten Ⅰ of nitrazolidine substances, curve b is the UV scanning graph of artificial antigen Ⅱ of nitrazolidine substances, and curve c is the UV scanning graph of bovine serum albumin. Compared with artificial hapten Ⅰ of nitrazolidine substances and bovine serum albumin, the maximum absorption wavelength of artificial antigen Ⅱ of nitrazolidine substances changes significantly, indicating that artificial hapten Ⅰ of nitrazolidine substances is successfully coupled with bovine serum albumin.

[0047] Example 2 The difference between this embodiment and embodiment 1 is that: A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: Wherein, in step (S.1), 200 mg (0.523 mmol) of free metonizine was dissolved in 5.7 mL of 48% HBr; refluxed at 90°C for 17 h; in step (S.2), 175 mg (0.475 mmol) of light yellow solid A obtained in step (S.1) was dissolved in 14.7 mL of anhydrous acetone in a 50 mL round-bottom flask, 769.5 mg of potassium carbonate and 221 μL of ethyl bromoacetate were added, stirred and refluxed at 65°C, and the reaction was completed after 3.5 h; in step (S.3), 206 mg (0.454 mmol) of reddish brown oil B obtained in step (S.2) was dissolved in 3.04 mL of tetrahydrofuran (THF) and 4.34 mL of anhydrous methanol (MeOH), placed in a 50 mL round-bottom flask, and 8.67 mL of 1 mol / L sodium hydroxide aqueous solution was added. The rest is the same as in Example 1.

[0048] (2) Preparation of artificial antigen II of nitrazole substances: Wherein, in step (a), 140 mg (0.328 mmol) of the artificial hapten I of the nitrazole olefin substance obtained in the above step (S.3) is placed in a 50 mL round-bottom flask; 49 mg (0.426 mmol) of N-hydroxysuccinimide (NHS) and 87.8 mg (0.426 mmol) of cyclohexylcarbodiimide (DCC) are added; in step (b2), 945 mg of bovine serum albumin is weighed and dissolved in 105 mL of the PBS buffer obtained in step (b1) to obtain a bovine serum albumin solution with a concentration of 9 mg / mL; in step (b3), the volume ratio of the supernatant added to the bovine serum albumin solution is 0.8:12. The rest is the same as in Example 1.

[0049] Example 3 The difference between this embodiment and embodiment 1 is that: A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: Wherein, in step (S.1), 200 mg (0.523 mmol) of free metonizine was dissolved in 4.44 mL of 48% HBr; refluxed at 110°C for 20 h; in step (S.2), 170 mg (0.481 mmol) of light yellow solid A obtained in step (S.1) was dissolved in 19.4 mL of anhydrous acetone in a 50 mL round-bottom flask, 728.6 mg of potassium carbonate and 207 μL of ethyl bromoacetate were added, stirred and refluxed at 75°C, and the reaction was completed after 5 h; in step (S.3), 198 mg (0.436 mmol) of reddish brown oil B obtained in step (S.2) was dissolved in 2.83 mL of tetrahydrofuran (THF) and 3.96 mL of anhydrous methanol (MeOH), placed in a 50 mL round-bottom flask, and 11.3 mL of 1 mol / L sodium hydroxide aqueous solution was added. The rest is the same as in Example 1.

[0050] (2) Preparation of artificial antigen II of nitrazole substances: Wherein, in step (a), 138 mg (0.324 mmol) of the artificial hapten I of the nitrazole olefin substance obtained in the above step (S.3) is placed in a 50 mL round-bottom flask; 52.2 mg (0.454 mmol) of N-hydroxysuccinimide (NHS) and 94 mg (0.454 mmol) of cyclohexylcarbodiimide (DCC) are added; in step (b2), 624 mg of bovine serum albumin is weighed and dissolved in 52 mL of the PBS buffer obtained in step (b1) to obtain a bovine serum albumin solution with a concentration of 12 mg / mL; in step (b3), the volume ratio of the supernatant added to the bovine serum albumin solution is 1.2:9. The rest is the same as in Example 1.

[0051] Comparative Example 1 The difference between this comparative example and Example 1 is: This comparative example provides an artificial hapten III of a nitrazole substance and a preparation method thereof, and an artificial antigen IV of a nitrazole substance and a preparation method thereof.

[0052] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten III of nitrazole substances: (S.1) 200 mg (0.523 mmol) of free metonizine was dissolved in 20 mL of anhydrous ethanol, 53 mg of 10% target carbon was added, hydrogen was introduced, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, the mixture was filtered and dried to obtain 168 mg (0.477 mmol) of a yellow oil C. The yellow oil C was subjected to TLC detection, and the chromatographic solution was ethyl acetate: ammonia water = 10 mL: 200 μL, and the product Rf = 0.2; (S.2) Take 168 mg (0.477 mmol) of the yellow oil C obtained in step (S.1), dissolve it in 10 mL of pyridine, place it in a 50 mL round-bottom flask, add 143.1 mg (1.431 mmol) of succinic anhydride, and reflux at 100 ° C for 20 h. After the reaction is completed, turn it to dryness directly to obtain a yellow oil, add 20 mL of dichloromethane and 20 mL of purified water, and collect the aqueous phase. The aqueous phase is turned to dryness, extracted with 10 mL of ethanol each time, extracted 3 times, the organic phase is dried, filtered, and turned to dryness to obtain a light yellow oil, which is purified by thin layer chromatography to obtain 93 mg (0.206 mmol) of nitrazole artificial hapten III, and the solvent and eluent are anhydrous ethanol. TLC detection of artificial hapten III of nitrazole substances was performed, and the chromatographic liquid was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1 (v / v / v / v), and the product Rf = 0.4. The chromatographic liquid used in the thin layer chromatography method was 95% ethanol: 1,4-dioxane: dichloromethane: ammonia water = 8: 1: 10: 1 (v / v / v / v), and the product Rf = 0.4.

[0053] (2) Preparation of artificial antigen IV of nitrazole substances: (a) 93 mg (0.206 mmol) of the nitro-olefin artificial hapten III obtained in the above step (S.2) was placed in a 50 mL round-bottom flask, 4.65 mL of DMF was added, and then 32 mg (0.278 mmol) of NHS and 57 mg (0.278 mmol) of DCC were added, and the mixture was stirred at room temperature for overnight reaction. After the reaction was completed, the supernatant was centrifuged and set aside; Wherein, deionized water is replaced with double distilled water in step (b1); in step (b2), 0.465 g of bovine serum albumin is weighed and dissolved in 46.5 mL of PBS buffer obtained in step (b1). The rest is the same as in Example 1. Finally, the nitrazole artificial antigen IV is obtained. The preparation reaction process of the nitrazole artificial antigen IV in this comparative example is as follows Figure 5 shown.

[0054] Comparative Example 2 The difference between this comparative example and comparative example 1 is: This comparative example provides an artificial hapten III of a nitrazole substance and a preparation method thereof, and an artificial antigen V of a nitrazole substance and a preparation method thereof.

[0055] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten III of nitrazole substances: Among them, steps (S.1) to (S.2) are the same as those in Comparative Example 1.

[0056] (2) Preparation of artificial antigen V of nitrazole substances: Wherein, the bovine serum albumin (i.e., carrier protein) in steps (b2) to (b3) is replaced with bovine gamma globulin. The same coupling steps as in Comparative Example 1 are adopted, and the rest are the same as in Comparative Example 1. Finally, the nitrazole artificial antigen V is obtained. The preparation reaction process of the nitrazole artificial antigen V in this comparative example is as follows: Figure 6 shown.

[0057] Comparative Example 3 The difference between this comparative example and comparative example 1 is: This comparative example provides an artificial hapten III of a nitrazole substance and a preparation method thereof, and an artificial antigen VI of a nitrazole substance and a preparation method thereof.

[0058] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten III of nitrazole substances: Among them, steps (S.1) to (S.2) are the same as those in Comparative Example 1.

[0059] (2) Preparation of artificial antigen VI of nitrazole substances: Wherein, step (a) is replaced by: 93 mg (0.206 mmol) of the artificial hapten III of the nitrazole olefin substance obtained in the above step (S.2) is placed in a 50 mL round-bottom flask, 4.65 mL of DMF is added, and then 28.5 μL (0.206 mmol) of triethylamine is added, and the mixture is stirred in an ice bath for 30 min, followed by the addition of 53.3 μL (0.412 mmol) of isobutyl chloroformate, and the mixture is stirred in an ice bath for 2 h. After the reaction is completed, the mixture is centrifuged, and the supernatant is taken for later use. Steps (b1) to (b4) are the same as those in comparative example 1. Finally, the artificial antigen VI of the nitrazole olefin substance is obtained. The preparation reaction process of the artificial antigen VI of the nitrazole olefin substance in this comparative example is as follows: Figure 7 shown.

[0060] Comparative Example 4 The difference between this comparative example and comparative example 1 is: This comparative example provides an artificial hapten III of a nitrazole substance and a preparation method thereof, and an artificial antigen VII of a nitrazole substance and a preparation method thereof.

[0061] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten III of nitrazole substances: Among them, steps (S.1) to (S.2) are the same as those in Comparative Example 1.

[0062] (2) Preparation of artificial antigen VII of nitrazole substances: The bovine serum albumin (i.e., carrier protein) in steps (b2) to (b3) was replaced with bovine gamma globulin. The same coupling steps as in Comparative Example 3 were adopted, and the rest was the same as in Comparative Example 3. Finally, the nitrazole artificial antigen VII was obtained. The preparation reaction process of the nitrazole artificial antigen VII in this comparative example is as follows: Figure 8 shown.

[0063] Comparative Example 5 The difference between this comparative example and Example 1 is: This comparative example provides an artificial hapten I of a nitrazole substance and a preparation method thereof, and an artificial antigen VIII of a nitrazole substance and a preparation method thereof.

[0064] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: Among them, steps (S.1) to (S.3) are the same as those in Example 1.

[0065] (2) Preparation of artificial antigen VIII of nitrazole substances: Wherein, step (a) is replaced by: weigh the artificial hapten I of the nitrazole substance obtained in step (S.3) and place it in a 50mL round-bottom flask, add 5mL DMF, then add 32.5μL (0.235mmol) triethylamine, stir in an ice bath for 30min, then add 60.8μL (0.47mmol) isobutyl chloroformate, continue stirring and reacting in an ice bath for 2h, centrifuge after the reaction is completed, take the supernatant and set aside. In step (b1), deionized water is replaced with double distilled water; in step (b2), 0.500g bovine serum albumin is weighed and dissolved in 50mL PBS buffer obtained in step (b1). The rest is the same as in Example 1. Finally, the artificial antigen VIII of the nitrazole substance is obtained. The preparation reaction process of the artificial antigen VIII of the nitrazole substance in this comparative example is as follows Fig. 9 shown.

[0066] Comparative Example 6 This comparative example provides an artificial hapten I of a nitrazole substance and a preparation method thereof, and an artificial antigen IX of a nitrazole substance and a preparation method thereof.

[0067] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: Among them, steps (S.1) to (S.3) are the same as those in Example 1.

[0068] (2) Preparation of artificial antigen IX of nitrazole substances: Wherein, the bovine serum albumin (i.e., carrier protein) in steps (b2) to (b3) is replaced with bovine gamma globulin. The same coupling steps as in Comparative Example 5 are adopted, and the rest are the same as in Comparative Example 5. Finally, the nitrazole artificial antigen IX is obtained. The preparation reaction process of the nitrazole artificial antigen IX in this comparative example is as follows: Fig.10 shown.

[0069] Comparative Example 7 The difference between this comparative example and Example 1 is: This comparative example provides a nitrazole artificial hapten I and a preparation method thereof, and a nitrazole artificial antigen X and a preparation method thereof.

[0070] A method for preparing an artificial antigen of a nitrazole substance comprises the following steps: (1) Preparation of artificial hapten I of nitrazole substances: Among them, steps (S.1) to (S.3) are the same as those in Example 1.

[0071] (2) Preparation of artificial antigen X of nitrazole substances: Wherein, the bovine serum albumin (i.e., carrier protein) in steps (b2) to (b3) is replaced by bovine gamma globulin. The same coupling steps as in Example 1 are adopted, and the rest are the same as in Example 1. Finally, the nitrazole artificial antigen X is obtained. The preparation reaction process of the nitrazole artificial antigen X in this comparative example is as follows: Fig.11 shown.

[0072]

Performance determination of artificial antigens of nitrazole substances

[0073] Table 1:

[0074] Determination of protein concentration of conjugate: Prepare 1 mL of bovine serum albumin 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, 100 μg / mL, and 120 μg / mL with PBS buffer, add 3 mL of Coomassie Brilliant Blue staining solution, mix immediately, warm in a 30°C water bath for 5 min, make parallel samples for each concentration, measure the absorbance at 655 nm, and draw a curve of the relationship between protein concentration and absorbance. Dilute the artificial antigen solution of nitrazolidine substances (prepared with PBS buffer) in a certain proportion, measure the absorbance of the artificial antigen of nitrazolidine substances at 655 nm, and read the corresponding protein concentration value of the artificial antigen solution of nitrazolidine substances from the curve.

[0075] Coupling ratio determination: prepare 100 μg / mL bovine serum albumin PBS solution, dilute the conjugate (i.e., nitrazole artificial antigen II) to 100 μg / mL with PBS, measure the absorbance value A1 at 290 nm, and measure the absorbance value A2 with bovine serum albumin PBS as blank. The coupling ratio γ is: γ=[(A1-A2) / ε] / (100×10 -3 / 66400). Where ε is the molar absorption coefficient (L / mol), 66400 is the molecular weight of bovine serum albumin, and 100×10 -3 is the concentration of bovine serum albumin (g / L).

[0076] When bovine immunoglobulin G is used as the carrier, the coupling ratio is calculated as follows: γ = [(A1-A2) / ε] / (100×10 -3 / 43000). Wherein, 43000 is the molecular weight of bovine gamma globulin. The calculation results of the coupling ratio and molar absorption coefficient of each nitrazole artificial antigen are shown in Table 2 below.

[0077] Table 2:

[0078] As shown in Tables 1 and 2, the structure of the artificial hapten of the nitrazine substance, the activation method of the artificial hapten of the nitrazine substance and the structure of the carrier protein all have an influence on the coupling ratio when the artificial hapten of the nitrazine substance is cross-linked with the carrier protein.

[0079] (2) Animal immunization Artificial antigens of various nitrazole substances were prepared according to the methods in Example 1 and Comparative Examples 1 to 7, and New Zealand white rabbits were immunized with the prepared artificial antigens of various nitrazole substances. The titer of the obtained immune serum was tested by ELISA method. The test results are shown in Table 3 below.

[0080] Table 3: Titer test results of each immune serum serial number Artificial antigens of nitrazoles Immune serum titer Example 1 Ⅱ 1:128000 Comparative Example 1 Ⅳ 1:64000 Comparative Example 2 Ⅴ 1:16000 Comparative Example 3 Ⅵ 1:32000 Comparative Example 4 Ⅶ / Comparative Example 5 Ⅷ 1:64000 Comparative Example 6 Ⅸ / Comparative Example 7 Ⅹ 1:32000 .

[0081] It can be seen from Table 3 that, compared with Example 1, the titers of the immune sera obtained by immunizing animals with the artificial antigens of nitrazolene substances in each comparative example are all low, and cannot be used in immunoassays. Among them, a large amount of precipitation immediately appeared during the dialysis process of the artificial antigen IX of nitrazolene substances obtained in Comparative Example 6, and the protein concentration of the conjugate was very low, and the coupling effect was not good. The coupling effect of the artificial antigen VII of nitrazolene substances obtained in Comparative Example 4 was also poor, and a large amount of precipitation appeared after frozen storage, and the stability was very poor, so it could not be used as an immune antigen. The immune serum obtained by immunizing animals with artificial antigen II of nitrazolene substances has a titer of 1:128000, and its properties are stable, which can be fully used in immunoassays, and can provide a more convenient, rapid and accurate way for the detection of nitrazolene substances.

[0082] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. An artificial hapten of nitrazole substances, characterized in that: Its molecular structure is shown in formula (I):

2. The method for preparing an artificial hapten of a nitrazole substance as claimed in claim 1, characterized in that: The following steps are involved: (S.1) Dissolve the free-state metonizine with hydrobromic acid, heat under reflux until the reaction is complete, directly transfer to dryness, add purified water, adjust the pH with concentrated ammonia, extract with ethyl acetate and collect the organic phase, dry, filter, and transfer to dryness to obtain a light yellow solid A; (S.2) dissolving the light yellow solid A obtained in step (S.1) in anhydrous acetone, adding potassium carbonate and ethyl bromoacetate, heating under reflux for reaction, cooling to room temperature after the reaction is completed, filtering, and drying to obtain a reddish brown oil B; (S.3) The reddish brown oil B obtained in step (S.2) is dissolved in tetrahydrofuran and anhydrous methanol, and an aqueous sodium hydroxide solution is added, and the mixture is rapidly stirred at room temperature. After the reaction is complete, the pH is adjusted with a hydrochloric acid solution, and the organic phase is extracted with dichloromethane and collected, dried, filtered, transferred to dryness, and purified to obtain an artificial hapten I of a nitrazole substance.

3. The method for preparing an artificial hapten of nitrazole substances according to claim 2, characterized in that: In step (S.1): The material-liquid ratio of the added free metonizine to hydrobromic acid is 35-45:1 mg / mL; The reaction temperature of the heating reflux reaction is 90-110°C, and the reaction time is 17-20h.

4. The method for preparing an artificial hapten of nitrazole substances according to claim 2, characterized in that: In step (S.2): The solid-liquid ratio of the added light yellow solid A, anhydrous acetone, potassium carbonate and ethyl bromoacetate is 95-105:8-12:430-450:120-128 mg / mL / mg / μL; The reaction temperature of the heating reflux reaction is 65-75°C, and the reaction time is 3.5-5h.

5. The method for preparing an artificial hapten of nitrazole substances according to claim 2, characterized in that: The solid-liquid ratio of the reddish brown oil B, tetrahydrofuran, anhydrous methanol and sodium hydroxide aqueous solution added in step (S.3) is 95-105:1.40-1.50:2.0-2.1:4-6 mg / mL / mL / mL.

6. An artificial antigen of nitrazole substances, characterized in that: Its molecular structure is shown in formula (II): Among them, BSA is bovine serum albumin.

7. The method for preparing an artificial antigen of nitrazole substances according to claim 6, characterized in that: The following steps are involved: The artificial hapten I of the nitrazolidine substance as claimed in claim 1 is combined with bovine serum albumin by using the N-hydroxysuccinimide active ester method to obtain the artificial antigen II of the nitrazolidine substance.

8. The method for preparing an artificial antigen of nitrazole substances according to claim 7, characterized in that: The following steps are involved: (a) mixing the nitrazole artificial hapten I as claimed in claim 1 with cyclohexylcarbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, stirring at room temperature, centrifuging after the reaction is completed, and collecting the supernatant; (b) adding the supernatant obtained in step (a) dropwise to the bovine serum albumin solution and mixing thoroughly to obtain a mixed solution, and letting the mixed solution stand. After the reaction is completed, dialyzing and centrifuging are performed, and the supernatant is taken to obtain the nitrazole artificial antigen II.

9. The method for preparing an artificial antigen of nitrazole substances according to claim 8, characterized in that: The molar ratio of the nitrazole artificial hapten I added in step (a) to cyclohexylcarbodiimide and N-hydroxysuccinimide is 1:(1.3-1.4):(1.3-1.4); The concentration of the bovine serum albumin solution added in step (b) is 9-12 mg / mL; the volume ratio of the added supernatant to the bovine serum albumin solution is 0.8-1.2:9-12.

10. Use of the artificial nitrazolone antigen according to claim 6 in preparing anti-nitrazolone antibodies.

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