A diclazuril antigen and its synthesis method and application

Abstract

The present invention discloses a diclazuril antigen and its synthesis and application. The structure of the diclazuril hapten is shown in Formula I. The diclazuril complete antigen obtained by coupling the hapten with a protein can fully expose the antigenic determinant of diclazuril and maintain the spatial conformation of diclazuril, and has good immunogenicity. Furthermore, the diclazuril colloidal gold detection product prepared by using the diclazuril complete antigen has the characteristics of high sensitivity, good specificity and strong stability, and can realize the rapid detection of diclazuril in a sample.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a diclazuril antigen and a synthesis method and application thereof. Background Art

[0002] Diclazuril (DIC) is a new broad-spectrum triazine anticoccidial drug. Its chemical name is chlorazine benzyl cyanide. It can effectively control chicken cecal coccidia and destroy coccidia oocysts at low doses. It is widely used in poultry farming. DIC has good effects on common types of chicken coccidia, such as tender, pile-type, toxic, Brucella, giant and malaise. The anticoccidial effect varies slightly depending on the species of coccidia. The results of the study showed that its anticoccidial effect is better than anticoccidial drugs such as monensin, amprolium, and nicarbazin. It has the characteristics of high efficiency and low toxicity. It is currently an anticoccidial drug with the lowest concentration in mixed feed. The large-scale use of anticoccidial drugs may cause the drug prototype or metabolites to accumulate in edible animal products (tissues, milk, eggs, etc.), thereby entering the food chain and threatening human health. Eggs are one of the common high-nutrition foods in life. Failure to strictly abide by the withdrawal period and drug exposure in bedding or drinking water will lead to drug residues in eggs, posing a potential risk to food safety. At the same time, there are literature reports that DIC residues occur in milk. The clinical dosage of DIC is small, and it is slowly eliminated in cattle after continuous use. Even if it contains only 5% residue, it takes up to 15 days to degrade. The Ministry of Agriculture and Rural Affairs stipulates in the "National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food" (GB31650-2019) that DIC is prohibited in milk. However, in the actual clinical application process, due to the failure to observe the administration time in time, cross-infection of bedding, feed and water sources, etc., DIC residues may occur in milk. In view of this, a rapid, sensitive and efficient detection method is currently needed to achieve rapid detection of diclazuril.

[0003] Enzyme-linked immunosorbent assay has the advantages of high sensitivity, strong specificity, low instrument and equipment requirements, and relatively simple sample pre-treatment, and is suitable for market monitoring and on-site monitoring. The immunoassay based on antigen-antibody specific binding is mainly based on monoclonal antibodies and polyclonal antibodies, and has the characteristics of simple sample pre-treatment, simple operation, rapid, sensitive and high throughput. It is praised as the most competitive and challenging rapid detection technology in the 21st century, and has broad application prospects in the field of food safety. However, for immunoassay, antibodies are core raw materials, and the effect of antibodies depends largely on the antigen structure that causes the corresponding animals to have an immune response. Therefore, the present invention provides a structurally stable antigen, and the colloidal gold test strip prepared by the antigen can achieve accurate and rapid detection of diclazuril. Summary of the invention

[0004] The purpose of the present invention is to provide a diclazuril antigen and a synthesis method and application thereof, so as to achieve accurate and rapid detection of diclazuril without cross-reaction with other drugs.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] One of the purposes of the present invention is to provide a diclazuril hapten, the structure of which is shown in Formula I:

[0007] Formula I.

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned diclazuril hapten, the steps are as follows:

[0009] S1. Accurately weigh the diclazuril raw material, dissolve it in anhydrous 1,4-dioxane, cool it to 0°C, add mercaptopropionic acid, Pd(OAc)2, BINAP, slowly drop triethylamine, and keep the high temperature at 90°C for reflux reaction;

[0010] S2. After about 8 hours, the mixed solution obtained in step S1 is monitored by TLC. After confirming that the reaction is complete, the mixture is cooled to 0°C, purified water is added dropwise for quenching, 3M hydrochloric acid is used to adjust the system (pH = 3), and further extracted with ethyl acetate to obtain a crude diclazuril hapten;

[0011] S3. Purification: The crude product obtained in step S2 is purified in a reverse phase FLASH-C18 column to obtain the diclazuril hapten.

[0012] Preferably, the steps are as follows:

[0013] S1. Accurately weigh 500 mg of diclazuril raw material, dissolve it in anhydrous 1,4-dioxane, cool it to 0°C, add 136 mg of mercaptopropionic acid, 25 mg of Pd(OAc)2, 425 mg of BINAP, slowly drop 186 mg of triethylamine, and keep the high temperature at 90°C for reflux reaction;

[0014] S2. After about 8 hours, the progress of the reaction was monitored by TLC. After the reaction was completed, the temperature was lowered to 0°C, purified water was added dropwise for quenching, 3M hydrochloric acid was used to adjust the system (pH = 3), and 25 mL of ethyl acetate was further added for extraction. The mixture was stirred for 5 minutes, and the mixture was allowed to stand for separation. The organic phase was retained and washed once with 20 mL of purified water and once with 20 mL of saturated sodium chloride aqueous solution. The organic phase was retained and further spin-dried to obtain 650 mg of crude diclazuril hapten.

[0015] S3. Purification: 650 mg of the crude diclazuril hapten obtained in step S2 was purified on a reverse phase FLASH-C18 column to obtain 340 mg of diclazuril hapten with high purity.

[0016] The third object of the present invention is to provide a complete diclazuril antigen, which is obtained by coupling the above-mentioned diclazuril hapten with a carrier protein, and the structure of the complete diclazuril antigen is shown in Formula II:

[0017] Formula II.

[0018] Herein, Protein represents a carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0019] A fourth object of the present invention is to provide a method for preparing the above-mentioned diclazuril complete antigen.

[0020] Specifically, when the carrier protein is bovine serum albumin, the preparation method of diclazuril complete antigen is as follows:

[0021] The above-mentioned diclazuril hapten was dissolved in DMF, NHS and EDC were slowly added, and the mixture was stirred at room temperature for 6 hours to obtain the diclazuril hapten activation solution; bovine serum albumin was weighed and dissolved in PBS buffer (0.01 mol / L, pH 7.4), and then the diclazuril hapten activation solution was slowly added dropwise, and the mixture was stirred at room temperature for 16 hours. The obtained reaction solution was dialyzed with PBS buffer (0.01 mol / L, pH 7.4), and the buffer was changed every 12 hours for 72 hours to obtain the diclazuril complete antigen with bovine serum albumin as the carrier protein.

[0022] Specifically, when the carrier protein is chicken ovalbumin, the preparation method of diclazuril complete antigen is as follows:

[0023] Dissolve the above diclazuril hapten in anhydrous methanol, add tri-n-butylamine dropwise, stir at 4°C for 30 minutes, then add isopropyl chloroformate and stir for 1 hour. Weigh chicken ovalbumin and add it to the reaction solution for 4 hours. Dialyze the resulting reaction solution with PBS buffer (0.01 mol / L, pH 7.4), replace the buffer every two hours, and dialyze for 48 hours to obtain the diclazuril complete antigen with chicken ovalbumin as the carrier protein.

[0024] The fifth object of the present invention is to provide a diclazuril colloidal gold detection product, characterized in that the above-mentioned diclazuril complete antigen is coated on the nitrocellulose membrane, and the detection results can be obtained quickly and efficiently.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention provides a connecting arm with a specific length and containing an active group -COOH derived from the diclazuril structure. After the hapten is connected to the carrier protein, the resulting complete antigen can fully expose the antigenic determinant of diclazuril and maintain the spatial conformation of diclazuril, thus having good immunogenicity.

[0027] 2. The diclazuril colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity and strong stability, can realize rapid and accurate detection of diclazuril, and has no cross-reaction with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0029] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0030] Figure 1 This is the synthetic structure diagram of diclazuril hapten;

[0031] Figure 2 is the MS spectrum of diclazuril hapten;

[0032] Figure 3 The results are for the detection sensitivity of diclazuril in fresh milk samples;

[0033] Figure 4 This is the specific test result of diclazuril colloidal gold test strip. DETAILED DESCRIPTION

[0034] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1. Preparation of diclazuril hapten

[0036] The structure of the diclazuril hapten is shown in Formula I:

[0037]

[0038] The preparation method of the diclazuril hapten described in this embodiment, its specific operation process is as follows:

[0039] S1. Accurately weigh 500 mg of diclazuril raw material, dissolve it in 20 mL of anhydrous 1,4-dioxane, cool it to 0°C, add 136 mg of mercaptopropionic acid, 25 mg of Pd(OAc)2, 425 mg of BINAP, slowly drop 186 mg of triethylamine, and keep the high temperature at 90°C for reflux reaction;

[0040] S2. Monitor the progress of the reaction by TLC. After the reaction is completed, cool to 0°C, add purified water dropwise for quenching, use 3M hydrochloric acid to adjust the system (pH=3), then add 25 mL of ethyl acetate for extraction, stir for 5 minutes, stand for separation, retain the organic phase, wash once with 20 mL of purified water, wash once with 20 mL of saturated sodium chloride aqueous solution, retain the organic phase and further spin-dry to obtain 650 mg of crude diclazuril hapten;

[0041] S3, purification: 650 mg of the crude diclazuril hapten obtained in step S2 was purified on a reverse phase FLASH-C18 column to obtain 340 mg of diclazuril hapten with high purity. Figure 1 Further, Figure 2 It can be seen that the molecular weight of the hapten is 478, and the purity of the hapten is as high as 95.3%.

[0042] Example 2: Preparation of diclazuril complete antigen

[0043] 1. Preparation of diclazuril hapten-bovine serum albumin conjugate (diclazuril-BSA)

[0044] S1, weigh 10 mg of the pure hapten in Example 1, dissolve it in 1 mL of DMF, slowly add 15 mg of NHS and 22 mg of EDC, stir at room temperature for 6 h, and obtain a diclazuril hapten activation solution;

[0045] S2. Weigh 22 mg BSA, dissolve it in 2 mL PBS buffer (0.01 mol / L, pH 7.4), stir evenly, slowly add it to the activated hapten solution, and stir at room temperature for 16 hours. Dialyze with 0.01 mol / L, pH 7.4 PBS buffer, change the buffer solution every 12 hours, and dialyze for 72 hours to obtain the complete antigen of diclazuril with bovine serum albumin as the carrier protein.

[0046] The reaction route is as follows:

[0047]

[0048] 2. Preparation of diclazuril hapten-chicken ovalbumin conjugate (diclazuril-OVA)

[0049] S1. Weigh 5 mg of the pure hapten of diclazuril in Example 1, dissolve it in 500 μL of anhydrous methanol, add 30 μL of tri-n-butylamine dropwise, and stir at 4° C. for 30 min.

[0050] S2, add 60 μL isopropyl chloroformate and stir for 1 h;

[0051] S3, weigh 20 mg of OVA, slowly add it to the reaction solution obtained in step S2, and react for 4 hours;

[0052] S4. Dialyze with 0.01 mol / L PBS buffer solution, pH 7.4, changing the buffer solution every 2 hours. Dialyze for 48 hours to obtain the complete antigen of diclazuril with chicken ovalbumin (OVA) as the carrier protein.

[0053] The reaction route is as follows:

[0054]

[0055] Example 3: Rapid detection of diclazuril residues in fresh milk samples by colloidal gold immunochromatography

[0056] 1. Preparation of diclazuril colloidal gold detection reagent

[0057] (1) Preparation of colloidal gold

[0058] Take 1 mL of 1% (mass percentage) chloroauric acid solution, add 99 mL of ultrapure water to make a final concentration of 0.01% (mass percentage) chloroauric acid solution, heat to boiling, then take 1.6 mL of 1% (mass percentage) trisodium citrate and quickly add it to the boiling chloroauric acid solution at one time, continue heating until the solution changes from light yellow to blue-black and finally to bright red, continue heating for 5 minutes after the color stabilizes, cool to room temperature, and add pure water to the original volume.

[0059] (2) Preparation of colloidal gold-labeled diclazuril monoclonal antibody

[0060] Adjust the pH value of the colloidal gold solution to 8.0, stir evenly with a constant speed stirrer, and add diclazuril monoclonal antibody (purchased from Power Biotechnology (Shenzhen) Co., Ltd.) dropwise at the same time. The amount of antibody added is 2 μg / mL colloidal gold. After 1 hour, add PEG equivalent to the antibody. After fully reacting for 30 minutes, add BSA equivalent to the antibody. After adding, continue stirring for 30 minutes. Centrifuge at 9000rpm for 30 minutes to obtain a uniform gold-labeled antibody precipitate, then resuspend in PBS for use, and obtain colloidal gold-labeled diclazuril monoclonal antibody.

[0061] (3) Preparation of diclazuril colloidal gold test strips

[0062] A diclazuril-coated antigen solution (0.1 mg / mL) was sprayed on the nitrocellulose membrane to form a detection line T, and goat anti-mouse IgG (0.2 mg / mL) was sprayed on the nitrocellulose membrane to form a control line C. The detection line and the control line were parallel to each other, and the distance between them was 0.65 cm. Then, on the bottom plate, the sample pad, the detection line T sprayed with diclazuril-coated antigen, and the control line C sprayed with goat anti-mouse IgG were overlapped and bonded in sequence on the nitrocellulose membrane and absorbent paper in the same direction.

[0063] The detection line is close to the sample pad, the control line is close to the absorbent paper, the stuck detection test paper is cut into test strips of equal width; colloidal gold-labeled diclazuril monoclonal antibody is added into the micropores and freeze-dried; the micropores and test strips are sealed and dried for storage.

[0064] 2. Sample Testing

[0065] (1) Testing steps:

[0066] The method is as follows: take 200 μL of fresh milk sample into the microwell, slowly aspirate and mix with the colloidal gold-labeled diclazuril monoclonal antibody in the microwell, incubate at 40°C for 3 minutes, then insert the test strip and incubate at 40°C for 5 minutes. Take out the test strip, gently scrape off the sponge pad of the test strip, and read the result. If the T line and the C line show purple-red bands at the same time and the T line is stronger than the C line, the result is negative; if the T line is lighter than the C line or the C line is colored but the T line is not colored, the result is positive; if both the C line and the T line are not colored, the detection reagent has expired.

[0067] (2) Sensitivity test

[0068] Fresh milk samples were spiked to final concentrations of 0, 2, 5, 10, and 20 μg / kg. Figure 3As shown in the figure, with the increase of the added concentration of diclazuril, the color of the T line gradually faded. When the mass fraction was 5 μg / kg, the color of the T line was significantly weaker than that of the C line, indicating a positive result. It was thus determined that the sensitivity of the diclazuril colloidal gold detection device of the present invention could reach 5 μg / kg.

[0069] (3) Specificity test

[0070] In the negative fresh milk samples, 500 μg / kg of acetochlor, chlorpyrifos, carbaryl, and carbofuran were added to verify the specificity of the test strips. The specificity test results are shown in Figure 4 The test results were all negative, indicating that the diclazuril colloidal gold product has good specificity and has no cross-reaction with the above-mentioned added drugs.

[0071] (4) Product stability test

[0072] The diclazuril colloidal gold product was placed at 4°C and 37°C for accelerated testing, and the diclazuril standard 5 μg / kg was detected at 0d, 7d, 14d, and 30d, respectively, to evaluate the stability performance of the product. The test results are shown in Table 1, and the CV value is calculated. When aged to the 30th day, the diclazuril colloidal gold product under 4°C and 37°C conditions still has good sensitivity in detecting the 5 μg / kg standard, and there is no significant difference from the test results on the first day, and the CV is <15%, indicating that the test strip provided by the present invention has good stability.

[0073] Table 1 Stability of diclazuril colloidal gold test strips

[0074]

[0075] In summary, the colloidal gold test strip of the present invention has the characteristics of high sensitivity, strong specificity and good stability, and can be applied to the colloidal gold immunochromatography method for rapid detection of diclazuril residues in fresh milk samples.

Claims

1. A diclazuril hapten, characterized in that: The structure of the diclazuril hapten is shown in Formula I, Formula I.

2. A method for preparing the diclazuril hapten according to claim 1, characterized in that: S1. Accurately weigh the diclazuril raw material, dissolve it in anhydrous 1,4-dioxane, cool it to 0°C, add mercaptopropionic acid, Pd(OAc)2, BINAP, slowly drop triethylamine, and reflux at 90°C for reaction; S2. After 8 hours, the mixed solution obtained in step S1 was monitored by TLC. After confirming that the reaction was complete, the mixture was cooled to 0°C, purified water was added dropwise for quenching, the system was adjusted with 3M hydrochloric acid at a pH of 3, and extracted with ethyl acetate to obtain a crude diclazuril hapten; S3. Purification: The crude product obtained in step S2 is purified in a reverse phase FLASH-C18 column to obtain the diclazuril hapten.

3. A complete diclazuril antigen, characterized in that: The diclazuril hapten according to claim 1 is coupled with a carrier protein, wherein the carrier protein is either bovine serum albumin or chicken ovalbumin.

4. A method for preparing the diclazuril complete antigen according to claim 3, characterized in that: When the carrier protein is bovine serum albumin, the method comprises: The diclazuril hapten described in claim 1 is dissolved in DMF, NHS and EDC are slowly added, and the mixture is stirred at room temperature for 6 hours to obtain a diclazuril hapten activation solution; bovine serum albumin is weighed and dissolved in a 0.01 mol / L PBS buffer solution with a pH of 7.4, and then the diclazuril hapten activation solution is slowly added dropwise, and the mixture is stirred at room temperature for 16 hours. The resulting reaction solution is dialyzed with a 0.01 mol / L PBS buffer solution with a pH of 7.4, and the buffer solution is replaced every 12 hours for 72 hours to obtain a diclazuril complete antigen with bovine serum albumin as the carrier protein.

5. A method for preparing the diclazuril complete antigen according to claim 3, characterized in that: When the carrier protein is chicken ovalbumin, the method comprises: The diclazuril hapten according to claim 1 is dissolved in anhydrous methanol, tri-n-butylamine is added dropwise, stirred at 4°C for 30 minutes, and then isopropyl chloroformate is added, stirred for 1 hour, chicken ovalbumin is weighed, added to the reaction solution, reacted for 4 hours, and the obtained reaction solution is dialyzed with 0.01 mol / L PBS buffer with a pH of 7.4, the buffer is replaced every two hours, and the dialyzation is performed for 48 hours to obtain the diclazuril complete antigen with chicken ovalbumin as the carrier protein.

6. A diclazuril colloidal gold detection product, characterized in that The nitrocellulose membrane is coated with the diclazuril complete antigen prepared according to claim 4 or claim 5.

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