Etiazepine derivative hapten and preparation method thereof, Etiazepine derivative antigen and preparation method thereof, and kit

Abstract

The invention relates to the technical field of biochemical engineering, in particular to an etiazepine derivative hapten and a preparation method thereof, an etiazepine derivative antigen and a preparation method thereof and a kit. The structural formula of the etiazepine derivative hapten is as follows: # imgabs0 #, wherein m is any integer between 1 and 4. An etiazepine derivative antigen formed by the etiazepine derivative hapten is high in immune titer and good in antibody affinity, and etiazepine can be rapidly detected when the etiazepine derivative hapten is applied to a kit product.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to an etizidine derivative hapten and a preparation method thereof, an etizidine derivative antigen and a preparation method thereof, and a kit. Background Art

[0002] In order to control drug abuse, fentanyl analogs are strictly controlled, and many non-fentanyl-related opioids have gradually appeared in the recreational opioid market, such as etizidine.

[0003] Etizidine (N,N-diethyl-2-[(5-nitro-2-{[4-(propan-2-yl)-oxy]-phenyl}methyl)-benzimidazol-1-yl]ethylenediamine, scientific name iso or toni), molecular formula: C 23 H 30 N4O3; molecular weight: 410.51; English name: isotonitazene, usually a white or off-white powder, its structural formula is as follows:

[0004]

[0005] In order to strictly control the application of etizidine, it is necessary to be able to detect etizidine in a timely and rapid manner. At present, the analysis and detection of etizidine mainly rely on large-scale analytical instruments such as high performance liquid chromatography (HPLC), gas chromatography (GC), and mass spectrometry (MS). However, the above-mentioned analytical methods have the disadvantages of expensive instruments, time-consuming detection, the need for professional technicians to operate, and the need for complicated processing of the samples to be tested. Immunoassay can overcome the defects of the above-mentioned methods. It has the advantages of strong specificity, simple operation, short detection time, no need for professional operation, and suitability for large-scale detection. It has been widely used in the rapid detection of various diseases, drug abuse, and food safety. Therefore, it is urgent to develop an etizidine antigen, immunize to produce antibodies, and apply it to rapid diagnostic reagents at the same time, so that drug abuse can be detected more quickly and conveniently.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide etizidine derivative haptens and preparation methods thereof, etizidine derivative antigens and preparation methods thereof and kits. The etizidine derivative antigens formed by the etizidine derivative haptens provided in the embodiments of the present invention have high immune titers and good antibody affinity, and can be applied to kit products to quickly detect etizidine.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides an etizidine derivative hapten, the structural formula of which is shown below:

[0010] Wherein m is any integer between 1 and 4.

[0011] In an alternative embodiment, m is 2, 3 or 4.

[0012] In an optional embodiment, the structural formula is as follows:

[0013]

[0014] In a second aspect, the present invention provides a method for preparing the etizidine derivative hapten described in the aforementioned embodiment, which is synthesized according to the following synthesis route:

[0015]

[0016] In an optional embodiment, the step of forming compound F comprises: mixing compound E with tert-butyl bromide, alkaline material and iodine salt for reaction;

[0017] Preferably, the molar ratio of the compound E to the tert-butyl bromide is 1:(2.2-2.6);

[0018] The molar ratio of the compound E to the alkaline material is 1:(1.2-1.5);

[0019] The molar ratio of the compound E to the iodine salt is 1:(0.9-1.1);

[0020] Preferably, the conditions for forming the compound F include: a reaction temperature of 50-75° C. and a reaction time of 10-16 hours;

[0021] Preferably, the hydrolysis reaction step comprises: mixing compound F with a solvent, cooling the mixture, and then mixing the mixture with an acidic substance, and then heating the mixture to react;

[0022] Preferably, the temperature is lowered to 0-4°C and the temperature is raised to 20-30°C.

[0023] In an optional embodiment, compound E is synthesized according to the following synthetic route:

[0024]

[0025] In an optional embodiment, the step of forming compound A comprises: reacting 1-chloro-2,4-dinitrobenzene and Mix and react at 80-100°C;

[0026] Wherein, the 1-chloro-2,4-dinitrobenzene and The molar ratio is 1:(1.1-1.3);

[0027] Preferably, the step of forming compound B comprises: mixing compound A and (NH4)2S and reacting them at 50-95°C;

[0028] Preferably, the molar ratio of the compound A to the (NH4)2S is 1:(2.5-3.5);

[0029] Preferably, the step of forming compound C comprises: mixing compound B and 4-methoxyphenylacetic acid to react at 20-40° C.;

[0030] Preferably, the molar ratio of the compound B to the 4-methoxyphenylacetic acid is 1:(1.1-1.3);

[0031] Preferably, the step of forming compound D comprises: mixing compound C and a catalyst to react at 70-90° C.;

[0032] Preferably, the molar ratio of the compound C to the catalyst is 1:(1.8-2.2):

[0033] Preferably, the step of forming compound E comprises: mixing compound D and Lewis acid to react at 20-30°C;

[0034] Preferably, the molar ratio of the compound D to the Lewis acid is 1:(3.5-4.5).

[0035] In a third aspect, the present invention provides an etizidine derivative antigen, the structural formula of which is shown below:

[0036] Wherein m is any integer between 1 and 4.

[0037] In a fourth aspect, the present invention provides a method for preparing an etizide derivative antigen, comprising: activating the etizide derivative hapten described in the aforementioned embodiment, and then coupling with a protein.

[0038] In a fifth aspect, the present invention provides a kit comprising the etizide derivative antigen described in the aforementioned embodiment.

[0039] The present invention has the following beneficial effects: the etizidine derivative antigen formed by the etizidine derivative hapten provided in the embodiment of the present invention has high immune titer and good antibody affinity. The etizidine artificial antigen provided in the embodiment of the present invention can be used for animal immunization to obtain the corresponding etizidine antibody, which is used for the research of various etizidine immunoassays and as a key raw material for the production of etizidine immunochromatography kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 The mass spectrum of the etizidine derivative hapten provided in Example 1 of the present invention;

[0042] Figure 2 This is a high performance liquid chromatogram of the etizidine derivative hapten provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] In a first aspect, the present invention provides an etizidine derivative hapten, the structural formula of which is shown below:

[0045] Wherein m is any integer between 1 and 4. For example, m is any value between 1, 2, 3 and 4. For example, when m is 2, the structural formula of the etizidine derivative hapten is as follows:

[0046] In a second aspect, the present invention provides a method for preparing the etizidine derivative hapten described in the aforementioned embodiment, which is synthesized according to the following synthesis route:

[0047]

[0048] The specific process is as follows:

[0049] The step of forming compound A comprises: reacting 1-chloro-2,4-dinitrobenzene and Mix at room temperature, and then heat to 80-100°C for reaction; wherein the molar ratio of the two is 1:(1.1-1.3).

[0050] The step of forming compound B includes: mixing compound A and (NH4)2S and reacting them at 50-95°C; wherein the molar ratio of compound A to (NH4)2S is 1:2.5-3.5, for example, 1:3.

[0051] The step of forming compound C comprises: mixing compound B and 4-methoxyphenylacetic acid to react at 20-40° C.; wherein the molar ratio of compound B to 4-methoxyphenylacetic acid is 1:(1.1-1.3).

[0052] The step of forming compound D comprises: mixing compound C and a catalyst to react at 70-90° C.; wherein the molar ratio of compound C to the catalyst is 1:(1.8-2.2): the catalyst includes but is not limited to phosphorus pentachloride.

[0053] The step of forming compound E comprises: mixing compound D and Lewis acid for reaction at 20-30° C., wherein the molar ratio of compound D to Lewis acid is 1:(3.5-4.5). Lewis acid includes but is not limited to boron tribromide.

[0054] The step of forming compound F comprises: mixing compound E with tert-butyl bromide, alkaline material and iodine salt for reaction; wherein the molar ratio of compound E to tert-butyl bromide is 1:(2.2-2.6); the molar ratio of compound E to alkaline material is 1:(1.2-1.5); the molar ratio of compound E to iodine salt is 1:(0.9-1.1); the reaction temperature is 50-75°C and the reaction time is 10-16 hours. Alkaline material includes but is not limited to potassium tert-butoxide and sodium methoxide. Iodine salt includes but is not limited to potassium iodide.

[0055] The step of forming compound G, i.e., etizidine derivative hapten, comprises: mixing compound F with a solvent, cooling the mixture, and then mixing the mixture with an acidic substance, and then heating the mixture for reaction; wherein the temperature is lowered to 0-4° C. and the temperature is raised to 20-30° C. The acidic substance includes, but is not limited to, trifluoroacetic acid.

[0056] It should be noted that in each step of the above reaction, a reaction solvent is added according to the situation. The solvent includes but is not limited to dichloromethane, pyridine, DMF, chloroform, ethanol and water. The amount and type of the added solvent are selected and adjusted according to the actual situation, and the embodiments of the present invention will not be described in detail.

[0057] After each step of the reaction, post-treatment is performed. The post-treatment is all existing technology. For example, the post-treatment includes but is not limited to column, pH adjustment, drying, baking, washing, extraction and solvent removal, etc., which will not be described in detail in the embodiments of the present invention.

[0058] In a third aspect, the present invention provides an etizidine derivative antigen, the structural formula of which is shown below:

[0059] Wherein m is any integer between 1 and 4.

[0060] The protein selection carrier protein includes, but is not limited to, any one of BGG (bovine gamma globulin), BTG (bovine thyroglobulin) and KLH (keyhole limpet hemocyanin).

[0061] In a fourth aspect, the present invention provides a method for preparing an etizide derivative antigen, comprising: activating the etizide derivative hapten described in the aforementioned embodiment, for example, activating the etizide derivative hapten with NHS and / or EDC, and then coupling with a protein.

[0062] In a fifth aspect, the present invention provides a kit comprising the etizide derivative antigen described in the aforementioned embodiment.

[0063] The etizidine derivative antigen provided by the present invention has high immune titer and good antibody affinity. The reagents and drugs used in the preparation method provided by the embodiment of the present invention are economical, environmentally friendly and low in cost; and the preparation method is simple to operate, has efficient synthesis, and stable products, and has the potential for large-scale production in the actual production process.

[0064] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0065] Example 1

[0066] The embodiment of the present invention provides a method for preparing an etizidine derivative hapten (hapten G), comprising:

[0067] The synthesis was carried out according to the following synthesis route:

[0068]

[0069] Specifically:

[0070] Synthesis of compound A: Weigh 1-chloro-2,4-dinitrobenzene (4.05 g, 20 mmol) and add it to a 100 ml single-necked bottle. At room temperature, add 35 ml of ethanol and stir magnetically to dissolve the raw material. Then add (2.78 g, 24 mmol) was heated to 90°C and reacted overnight.

[0071] Post-treatment: After the reaction was cooled to room temperature, 40 ml of deionized water was added to dilute the reaction, and the pH of the system was adjusted to about 10 with concentrated ammonia water. A large amount of precipitation appeared, and the solution was evacuated under vacuum. The filter cake was collected and dried to obtain 5 g of yellow solid product A with a yield of 89%.

[0072] Synthesis of compound B: Take compound A (2.82 g, 10 mmol) and add it into a 100 ml single-mouth bottle, add 30 ml of ethanol and 10 ml of deionized water, add 10.2 ml of (NH4)2S (20%) under magnetic stirring, after adding, heat to 60 ° C and react for half an hour, then heat to 90 ° C and react for about 20 hours.

[0073] Post-treatment: cool in an ice-water bath, add 2N hydrochloric acid to adjust the pH of the reaction solution to about 1, concentrate in a water bath at 50°C under reduced pressure to remove most of the ethanol, adjust the pH to alkaline with concentrated ammonia water, add a large amount of water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, filter, add silica gel to the organic phase, mix the sample and purify by column, use DCM:MeOH (50:1-5:1) as the eluent to pass through the column in a gradient, and obtain 1.2 g of yellow solid compound B after purification, with a yield of 47.6%.

[0074] Synthesis of compound C: Under N2 protection, compound B (1.2 g, 4.76 mmol) was added to a 100 ml single-necked bottle, and 40 ml of dry DCM was added, followed by 4-methoxyphenylacetic acid (870 mg, 5.24 mmol) and EEDQ (1.3 g, 5.24 mmol). The temperature was raised to 35°C and the reaction was stopped after 6-7 h.

[0075] Post-treatment: After the reaction was cooled to room temperature, 40 ml of deionized water was added to dilute the reaction, and the pH of the system was adjusted to about 11 with concentrated ammonia water. DCM was used for extraction three times, and the organic phase was collected. After washing with saturated sodium chloride, anhydrous sodium sulfate was added to dry, filtered, and silica gel was added to the organic phase for column purification. The eluent was DCM:MeOH (50:1-20:1) gradient column. After purification, 920 mg of yellow-brown oily compound C was obtained with a yield of 48.3%.

[0076] Synthesis of compound D: Under N2 protection, compound C (426 mg, 1.06 mmol) was added to a 100 ml two-necked bottle, 15 ml of dry chloroform was added to dissolve, and then phosphorus pentachloride (444 mg, 2.13 mmol) was added, and the mixture was heated to 80°C for reaction overnight.

[0077] Post-treatment: add 40 ml of water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase and purify by column, use DCM:MeOH (30:1-10:1) as eluent to obtain 250 mg of compound D as a yellow oil with a yield of 61%.

[0078] Synthesis of compound E: Under N2 protection, compound D (123 mg, 0.32 mmol) was added to a 50 ml single-necked bottle, 3 ml of dry dichloromethane solution was added, the mixture was cooled to 0°C in an ice-water bath, boron tribromide (321 mg, 1.28 mmol) was added dropwise, and after the addition was completed, the temperature was naturally raised to react overnight.

[0079] Post-treatment: water was slowly added dropwise in a 0°C water bath to quench the reaction, and then 30 ml of water was added. The mixture was extracted five times with DCM. The organic phase was collected, washed with saturated sodium chloride, and dried with anhydrous sodium sulfate. The organic phase was concentrated and passed through a column with an eluent ratio of DCM:MeOH = 20:1. 100 mg of compound E was obtained with a yield of 85%.

[0080] Synthesis of compound F: Compound E (55 mg, 0.15 mmol) was added into a 50 ml single-necked bottle, tert-butyl bromobutyrate (83 mg, 0.37 mmol), Cs2CO3 (71 mg, 0.22 mmol), and then 3 ml of DMF solution was added. After the addition was complete, the temperature was raised to 60°C and the reaction was allowed to proceed overnight.

[0081] Post-treatment: add 40 ml of water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase and purify by column, use DCM:MeOH (30:1-10:1) as eluent to obtain 39 mg of yellow oily compound F with a yield of 51%.

[0082] Synthesis of hapten G: Compound F (39 mg, 0.76 mmol) was added to a 50 ml single-necked bottle, 2 ml of dichloromethane solution was added, the mixture was cooled to 0°C in an ice-water bath, 0.5 ml of trifluoroacetic acid was added dropwise, and after the addition was completed, the temperature was naturally raised to react for 1 hour.

[0083] Post-treatment: add 40 ml of saturated sodium bicarbonate, adjust the pH of the system to about 6 with dilute hydrochloric acid, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase, mix the sample and purify it by column, use DCM:MeOH (10:1-3:1) as eluent to pass through the column, and obtain 29 mg of yellow solid hapten G with a yield of 83.5%.

[0084] The embodiment of the present invention also provides the synthesis of the artificial antigen G-BGG: 50 mg of the etizidine derivative hapten G is dissolved in 2 ml of DMF, and then 30 mg of NHS and 50 mg of EDC are added, and the reaction is carried out at room temperature overnight. Then 500 mg of BGG is weighed and dissolved in 50 ml of PBS, and the solution is dissolved evenly. Then the reaction solution is added to the prepared protein solution, and the reaction is allowed to stand at 4 degrees for 8 hours. After the reaction is completed, centrifugation is performed at 10,000 r / min for 10 min to obtain a supernatant, and the solubility is measured by the Lorwy method to obtain the final synthetic antigen, the hapten G-BGG.

[0085] Example 2-3

[0086] Examples 2-3 respectively provide a method for preparing an etizidine derivative antigen, which is basically the same as the method for preparing an etizidine derivative antigen (i.e., hapten G-BGG) provided in Example 1, except that different proteins are used. Specifically, Example 2 synthesizes hapten G-BTG; Example 3 synthesizes hapten G-KLH.

[0087] Comparative Example 1

[0088] This comparative example provides a method for preparing hapten H, comprising: synthesizing according to the following synthesis path:

[0089]

[0090] Synthesis of hapten H: Compound E (37 mg, 0.1 mmol) was added into a 50 ml single-necked bottle, 3 ml of pyridine was added to dissolve, and 15 mg of succinic anhydride was added, and the temperature was raised to 90°C to react overnight.

[0091] Post-treatment: cool to room temperature, remove pyridine under pressure, add 30 ml of water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase, mix the sample and purify by column, use DCM:MeOH (10:1-5:1) as eluent to pass through the column, and obtain 20 mg of yellow solid hapten H with a yield of 42.7%.

[0092] This comparative example provides a hapten H-BGG: 50 mg of hapten H is dissolved in 2 ml of DMF, and then 30 mg of NHS and 50 mg of EDC are added, and the mixture is reacted at room temperature overnight. Then 500 mg of BGG is weighed and dissolved in 50 ml of PBS, and the mixture is dissolved evenly. The reaction solution is then added to the prepared protein solution, and the mixture is allowed to react at 4° C. for 8 hours. After the reaction is completed, the mixture is centrifuged at 10,000 r / min for 10 min to obtain a supernatant, and the solubility is measured by the Lorwy method to obtain the final synthetic antigen, hapten H-BGG.

[0093] Comparative Examples 2-3

[0094] Comparative Examples 2-3 respectively provide a method for preparing an etizidine derivative antigen, which is basically the same as the method for preparing the antigen (i.e., hapten H-BGG) provided in Comparative Example 1, except that different proteins are used. Specifically, Comparative Example 2 synthesizes hapten H-BTG; Comparative Example 3 synthesizes hapten H-KLH.

[0095] Comparative Example 4

[0096] This comparative example provides a method for preparing hapten L, comprising: synthesizing according to the following synthesis path:

[0097]

[0098] The details are as follows:

[0099] Synthesis of compound I: Under N2 protection, compound B (1.2 g, 4.76 mmol) was added to a 100 ml single-necked bottle, 40 ml of dry DCM was added, and 4-isopropoxyphenylacetic acid (1.01 g, 5.24 mmol) and EEDQ (1.3 g, 5.24 mmol) were added in sequence. The temperature was raised to 35°C and the reaction was stopped after 6-7 h.

[0100] Post-treatment: After the reaction was cooled to room temperature, 40 ml of deionized water was added to dilute the reaction, and the pH of the system was adjusted to about 11 with concentrated ammonia water. DCM was used for extraction three times, and the organic phase was collected. After washing with saturated sodium chloride, anhydrous sodium sulfate was added to dry, filtered, and silica gel was added to the organic phase for column purification. The eluent was DCM:MeOH (50:1-20:1) gradient column. After purification, 850 mg of yellow-brown oily compound I was obtained with a yield of 48.3%.

[0101] Synthesis of compound J: Under N2 protection, compound I (409 mg, 1 mmol) was added into a 100 ml two-necked bottle, 15 ml of dry chloroform was added to dissolve, and then phosphorus pentachloride (416 mg, 2 mmol) was added, and the mixture was heated to 80°C for reaction overnight.

[0102] Post-treatment: add 40 ml of water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase and purify by column, use DCM:MeOH (30:1-10:1) as eluent to obtain 220 mg of yellow oily compound J, with a yield of 56%.

[0103] Synthesis of compound K: Compound J (90 mg, 0.23 mmol) was added into a 50 ml single-necked bottle, 3 ml of dichloromethane and 1 ml of acetic acid solution were added, and then zinc powder (123 mg, 1.88 mmol) was added, and the mixture was reacted at room temperature for 12 h.

[0104] Post-treatment: add 40 ml of water, adjust the pH to weak alkalinity with ammonia water, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, spin dry the organic phase, add silica gel to mix the sample and purify it by column, use DCM:MeOH (30:1-10:1) as eluent to pass through the column in a gradient to obtain 60 mg of yellow oily compound K with a yield of 76%.

[0105] Synthesis of hapten L: Compound K (60 mg, 0.17 mmol) was added to a 50 ml single-necked bottle, 3 ml of pyridine was added, and then succinic anhydride (20 mg, 0.20 mmol) was added, and the mixture was heated to 80°C for overnight reaction.

[0106] Post-treatment: add 40 ml of water, adjust the pH to neutral, extract three times with DCM, collect the organic phase, wash with saturated sodium chloride, add anhydrous sodium sulfate to dry, add silica gel to the organic phase and purify by column, use DCM:MeOH (10:1-3:1) as eluent to pass through the column, and obtain 44 mg of yellow oily hapten L with a yield of 57%.

[0107] This comparative example provides a hapten L-BGG: 50 mg of hapten L is dissolved in 2 ml of DMF, 30 mg of NHS and 50 mg of EDC are added, and the mixture is reacted at room temperature overnight. Then 500 mg of BGG is weighed and dissolved in 50 ml of PBS, and the mixture is dissolved evenly. The reaction solution is then added to the prepared protein solution, and the mixture is allowed to react at 4° C. for 8 hours. After the reaction is completed, the mixture is centrifuged at 10,000 r / min for 10 min to obtain a supernatant, and the solubility is measured by the Lorwy method to obtain the final synthetic antigen, hapten L-BGG.

[0108] Comparative Examples 5-6

[0109] Comparative Examples 5-6 respectively provide a method for preparing an etizidine derivative antigen, which is basically the same as the method for preparing the antigen (i.e., hapten L-BGG) provided in Comparative Example 4, except that different proteins are used. Specifically, Comparative Example 5 synthesizes hapten L-BTG; Comparative Example 6 synthesizes hapten L-KLH.

[0110] Characterization

[0111] The hapten G of the etizidine derivative prepared in Example 1 was detected by high performance liquid chromatography, and the detection conditions were as follows: detection wavelength: 214 nm; mobile phase: acetonitrile / water, wherein, gradient elution (time program: 0.01-5 min acetonitrile / water 10%-100%, 5.01-12 min acetonitrile 100%); the chromatographic column was C18.

[0112] The etizidine derivative hapten G prepared in Example 1 was subjected to mass spectrometry detection.

[0113] Results Figure 1 and Figure 2 .according to Figure 1 It can be seen that the measured value of m / z[M+H] is 455.4, which is consistent with the theoretical value. Figure 2 It can be seen that the peak position is at 7.15 min and the purity is above 95%, indicating that the compound synthesized in Example 1 of the present invention is the etizidine derivative hapten G with the desired structure.

[0114] Test 1

[0115] Determination of coupling ratio of etizide artificial antigen:

[0116] Molar absorption coefficient ε: PBS buffer was used to prepare etizide artificial hapten solutions with concentrations of 0ug / ml, 5ug / ml, 10ug / ml, 20ug / ml, 30ug / ml, and 40ug / ml. The UV scanning graph showed that the maximum absorption wavelength of etizide hapten was 242nm. PBS was used as a control sample to measure the absorbance at 242nm, and parallel samples were made for each concentration. The calculation formula for the molar absorption coefficient (i.e., molar absorption coefficient) is: ε = absorbance / molar concentration.

[0117] Determination of protein concentration of conjugate: Prepare 1 ml of bovine serum albumin solution with concentrations of 0ug / ml, 10ug / ml, 20ug / ml, 30ug / ml, 40ug / ml, 60ug / ml, 80ug / ml, and 100ug / ml with PBS buffer, add 3 ml of Coomassie Brilliant Blue staining solution, mix immediately, warm in a 30°C water bath for 5 minutes, make parallel samples for each concentration, measure the absorbance at 655nm, and draw a curve of the relationship between protein concentration and absorbance. Dilute the artificial antigen solution (prepared with PBS buffer) in a certain proportion, measure the absorbance of the artificial antigen at 655nm, and read the corresponding protein concentration value of the artificial antigen solution from the curve.

[0118] Coupling ratio determination: prepare 100ug / ml bovine serum albumin PBS solution, dilute the conjugate (i.e., etizidine artificial antigen) to 100ug / ml with PBS, use PBS as blank control, and measure the absorbance values ​​A2 and A1 at 255nm respectively.

[0119] The coupling ratio γ is: γ = [(A1-A2) / ε] / (100×10 3 / 68000).

[0120] Where ε is the molar absorption coefficient (L / mol), 68000 is the molecular weight of bovine serum albumin, and 100×10 3 is the concentration of bovine serum albumin (g / L).

[0121] The results are shown in Table 1.

[0122] Table 1 Coupling ratio

[0123] Artificial Antigen Coupling ratio Conjugate protein concentration Molar Absorption Coefficient Hapten G-BGG 15 5.7mg / ml 5628.5 Hapten G-BTG 22 9.2mg / ml 6258.9 Hapten G-KLH 16 2.9mg / ml 4430.3 Hapten H-BGG 8 7.8mg / ml 5643.2 Hapten H-BTG 6 6.5mg / ml 4861.2 Hapten H-KLH 8 3.6mg / ml 5981.3 Hapten L-BGG 12 8.3mg / ml 5238.6 Hapten L-BTG 9 6.2mg / ml 5264.3 Hapten L-KLH 5 4.4mg / ml 5366.5

[0124] As can be seen from Table 1, each hapten small molecule is coupled to a carrier protein to form a synthetic antigen.

[0125] Test 2

[0126] Animal immunization: The etizidine derivative antigens of Examples 1-3 and Comparative Examples 1-6 were added to adjuvants and injected into mice for immunization. A booster shot was given seven days later, and a third shot was given on the 20th day. Blood was collected one month later to test serum titers. The results are shown in Table 2.

[0127] Table 2 Immune serum titer

[0128]

[0129]

[0130] According to Table 2, (1) the hapten G-BTG has the best immune effect and the highest immune titer; further cell fusion, ELISA screening, and purification are performed to finally obtain suitable antibodies and matching antigens, which can be used in the immunoassay of etizide, providing a faster and more accurate way for the detection of etizide.

[0131] (2) The antigen formed by the etizidine derivative hapten provided in the embodiment of the present invention has good immune titer. If the structure of the etizidine derivative hapten is changed, the titer of the antigen formed is low and cannot meet the requirements of practical application.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An etizidine derivative hapten, characterized in that: Its structural formula is as follows: Wherein m is any integer between 1 and 4.

2. The etizidine derivative hapten according to claim 1, characterized in that m is 2, 3 or 4.

3. The etizidine derivative hapten according to claim 1, characterized in that Its structural formula is as follows:

4. A method for preparing the hapten of the etizide derivative according to claim 1, characterized in that: The synthesis was carried out according to the following synthesis route:

5. The preparation method according to claim 4, characterized in that: The step of forming compound F comprises: mixing compound E with tert-butyl bromide, alkaline material and iodine salt for reaction; Preferably, the molar ratio of the compound E to the tert-butyl bromide is 1:(2.2-2.6); The molar ratio of the compound E to the alkaline material is 1:(1.2-1.5); The molar ratio of the compound E to the iodine salt is 1:(0.9-1.1); Preferably, the conditions for forming the compound F include: a reaction temperature of 50-75° C. and a reaction time of 10-16 hours; Preferably, the hydrolysis reaction step comprises: mixing compound F with a solvent, cooling the mixture, and then mixing the mixture with an acidic substance, and then heating the mixture to react; Preferably, the temperature is lowered to 0-4°C and the temperature is raised to 20-30°C.

6. The preparation method according to claim 4, characterized in that: Compound E was synthesized according to the following synthetic route:

7. The preparation method according to claim 6, characterized in that: The step of forming compound A comprises: reacting 1-chloro-2,4-dinitrobenzene and Mix and react at 80-100°C; Wherein, the 1-chloro-2,4-dinitrobenzene and The molar ratio is 1:(1.1-1.3); Preferably, the step of forming compound B comprises: mixing compound A and (NH4)2S and reacting them at 50-95°C; Preferably, the molar ratio of the compound A to the (NH4)2S is 1:(2.5-3.5); Preferably, the step of forming compound C comprises: mixing compound B and 4-methoxyphenylacetic acid to react at 20-40° C.; Preferably, the molar ratio of the compound B to the 4-methoxyphenylacetic acid is 1:(1.1-1.3); Preferably, the step of forming compound D comprises: mixing compound C and a catalyst to react at 70-90° C.; Preferably, the molar ratio of the compound C to the catalyst is 1:(1.8-2.2): Preferably, the step of forming compound E comprises: mixing compound D and Lewis acid to react at 20-30°C; Preferably, the molar ratio of the compound D to the Lewis acid is 1:(3.5-4.5).

8. An etizidine derivative antigen, characterized in that Its structural formula is as follows: Wherein m is any integer between 1 and 4.

9. A method for preparing an etizide derivative antigen, characterized in that: include: The hapten of the etizide derivative according to claim 1 is activated and then coupled to a protein.

10. A kit, characterized in that: It is characterized in that It comprises the etizide derivative antigen as claimed in claim 8.

Images