Decoquinate hapten and synthesis and application thereof

Abstract

The invention discloses a decoquinate hapten as well as synthesis and application thereof. The structure of the decoquinate hapten is shown as a formula I. Compared with 3-6 carbon chains, the synthetic method of the decoquinate hapten provided by the invention has the characteristics of mild reaction conditions, high yield, few reaction steps and capability of reducing the cost of raw materials. The decoquinate colloidal gold detection product prepared from the decoquinate complete antigen provided by the invention has the characteristics of strong specificity, high sensitivity and the like, can realize accurate detection of decoquinate in a sample, and has no cross reaction with other drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a decoquinate hapten, its synthesis and application. Background Art

[0002] Decoquinate belongs to quinoline anti-coccidial drugs and is used for the prevention and treatment of coccidiosis. This type of anti-coccidial drug has the advantages of low toxicity, good tolerance, and fast metabolism, and is widely used. Decoquinate is the only chemically-based anti-coccidial drug approved for use by the governments of Japan, the European Union, the United States, and China in the world, and has no compatibility taboos with veterinary drug feed additives currently permitted in China. Although the toxicity of this type of anti-coccidial drug is relatively low, the use of anti-coccidial drugs is likely to cause coccidia to develop drug resistance, and there is a tendency to increase the dosage in livestock production. To ensure the safety of animal-derived foods, the maximum residue limit of decoquinate in chicken target tissues is clearly defined in the National Food Safety Standard - Maximum Residue Limits of Veterinary Drugs in Foods (GB 31650-2019) issued in China in 2019.

[0003] As the main anti-coccidial drug on the current market, decoquinate has a long medication cycle, and there is a greater possibility of its residue caused by careless operation, with a potential risk of in vivo accumulation, which may lead to the residue of this drug in the edible tissues of animals.

[0004] Currently, the detection methods of decoquinate mainly include gas chromatography, high-performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and surface-enhanced Raman spectroscopy. These methods mainly rely on precise data acquisition and analysis systems and can perform quantitative analysis, but they require expensive instrument equipment and trained professionals, and are not suitable for on-site and rapid detection of a large number of samples. In recent years, there have been more and more methods for detecting residues using bioanalysis techniques. Enzyme-linked immunosorbent assay is one of the most commonly used immunoassay methods, with the characteristics of high specificity and rapid detection, but multiple washing of the plate and spectrophotometric determination during the operation process are also not suitable for field and on-site detection, and cannot meet the urgent requirements of emergency handling such as at import and export ports and over-standard incidents. In contrast, colloidal gold immunochromatography can make up for the above deficiencies. The test strip is convenient to carry, easy to operate, has low professional requirements for testers, and has a short reaction time, and is suitable for on-site rapid detection of a large number of samples, and has now been widely used in the detection of fields such as food, biology, environment, medicine, and clinical medicine. Summary of the Invention

[0005] The purpose of the present invention is to provide a decoquinate hapten, its synthesis method and application, to achieve the precise detection of decoquinate and have no cross-reaction with other drugs.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions: One of the purposes of the present invention is to provide a decoquinate hapten, and the structure of the decoquinate hapten is shown in Formula I: Formula I.

[0007] Another purpose of the present invention is to provide a method for preparing the above-mentioned decoquinate hapten, and the steps are as follows: S1. Accurately weigh the decoquinate raw material, dissolve it in N,N-dimethylformamide, protect it with nitrogen, then cool it to 0 °C, add ethyl 4-bromobutyrate, slowly add potassium carbonate, and slowly add a catalytic amount of tetrabutylammonium iodide, keep the temperature at 60 °C, and stir for 12 hours; S2. After 12 h, monitor the mixture obtained in step S1 by TLC, confirm that the reaction is complete, cool it to 0 °C, dropwise add purified water to quench, extract with ethyl acetate, retain the organic phase, wash once with 10 mL of purified water and once with 10 mL of saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain the crude product of decoquinate; S3. Take the crude product obtained in S2, dissolve it in methanol, slowly dropwise add 3M lithium hydroxide aqueous solution for ester hydrolysis, stir at room temperature for 6 hours, detect by TLC, after complete reaction, dropwise add purified water to quench, free with 3M hydrochloric acid until the pH of the system is equal to 5, a large amount of solid precipitates, and filter to obtain the crude decoquinate hapten; S4. Purification: Dissolve the crude product obtained in step S3 with 1M sodium hydroxide aqueous solution, wash twice with ethyl acetate, retain the aqueous phase, free with 3M hydrochloric acid until the pH is equal to 5, filter after solid precipitation to obtain the decoquinate hapten.

[0008] Preferably, the detailed steps are as follows: S1. Accurately weigh 2.0 g of the decoquinate raw material, dissolve it in 20 mL of N,N-dimethylformamide, protect it with nitrogen, then cool it to 0 °C, add 980 mg of ethyl 4-bromobutyrate, slowly add 790 mg of potassium carbonate, and slowly add 80 mg of a catalytic amount of tetrabutylammonium iodide, keep the temperature at 60 °C, and stir and react for 12 hours; S2. After 12 h, monitor the mixture obtained in step S1 by TLC, confirm that the reaction is complete, cool it to 0 °C, dropwise add 20 mL of purified water to quench, extract with 20 mL of ethyl acetate, retain the organic phase, wash once with 10 mL of purified water and once with 10 mL of saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain 2.3 g of the crude product obtained in S1; S3. Take 2.3 g of the crude product obtained in S2, dissolve it in 20 mL of methanol, slowly add 3M lithium hydroxide aqueous solution dropwise for ester hydrolysis, stir at room temperature for 6 hours, detect by TLC, when the reaction is complete, add 15 mL of purified water dropwise to quench, and use 3M hydrochloric acid for dissociation until the pH of the system is equal to 5, a large amount of solid precipitates, and filter to obtain 2.0 g of crude decoquinate hapten; S4. Purification: Dissolve 2.0 g of the crude product obtained in step S3 in 15 mL of 1M sodium hydroxide aqueous solution, wash twice with 15 mL of ethyl acetate, retain the aqueous phase, use 3M hydrochloric acid for dissociation until the pH is equal to 5, filter after solid precipitation, and 1.6 g of decoquinate hapten can be obtained.

[0009] The third object of the present invention is to provide a complete decoquinate antigen, which is obtained by conjugating the above-mentioned decoquinate hapten with a carrier protein, and the structure of the complete decoquinate antigen is shown in formula II: Formula II.

[0010] The conjugation method is the EDC / NHS conjugation technique, where Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) or chicken ovalbumin (OVA).

[0011] The fourth object of the present invention is to provide a method for preparing the above-mentioned complete decoquinate antigen.

[0012] Specifically, the method is the EDC / NHS conjugation technique. When the carrier protein is BSA or OVA, the preparation method of the complete decoquinate antigen is as follows: Dissolve the above-mentioned decoquinate hapten in DMF, slowly add NHS and EDC, stir at room temperature for 6 h to obtain a decoquinate hapten activation solution; weigh BSA or OVA and dissolve it in CB buffer (0.2 mol / L, pH 9.0), stir evenly, and slowly add it dropwise to the activated hapten solution, stir at room temperature for 16 h. Dialyze the obtained reaction solution with PBS buffer (0.01 mol / L, pH 7.4), change the buffer once every 12 h during dialysis, and dialyze for 72 h to obtain the complete decoquinate antigen with BSA or OVA as the carrier protein.

[0013] The fifth object of the present invention is to provide a decoquinate colloidal gold detection product, which is characterized in that the above-mentioned complete decoquinate antigen is coated on the nitrocellulose membrane, and the detection result can be obtained quickly and efficiently.

[0014] The present invention has the following beneficial effects: 1. The present invention provides a linker arm derived from the structure of decoquinate, having a specific length and containing an active group -COOH. After the hapten is linked to a carrier protein, the resulting complete antigen can fully expose the antigenic determinant of decoquinate and maintain the spatial conformation of decoquinate, having good immunogenicity.

[0015] 2. The decoquinate colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity, and strong stability, can achieve rapid and accurate detection of decoquinate, and has no cross-reaction with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained according to the provided drawings.

[0017] The structures, ratios, sizes, etc. illustrated in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limited conditions for the implementation of the present invention. Therefore, they do not have technical substance significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy and the purpose that the present invention can achieve, should still fall within the scope covered by the technical content disclosed in the present invention.

[0018] Figure 1 It is the MS spectrum of the decoquinate hapten; Figure 2 It is the synthetic structure diagram of the decoquinate hapten; Figure 3 It is the sensitivity detection result of decoquinate in chicken tissue samples; Figure 4 It is the specificity detection result of the decoquinate colloidal gold test strip. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The following specific embodiments illustrate the embodiments of the present invention. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention.

[0020] Example 1. Preparation of the Decoquinate Hapten The structure of the decoquinate hapten is shown in Formula I: Formula I.

[0021] The preparation method of the decoquinate hapten described in this example is as follows: S1. Accurately weigh 2.0 g of the decoquinate raw material, dissolve it in 20 mL of N,N-dimethylformamide, protect it with nitrogen, then cool it to 0 °C, add 980 mg of ethyl 4-bromobutyrate, slowly add 790 mg of potassium carbonate, and slowly add 80 mg of catalytic amount of 4-butylammonium iodide. Keep the temperature at 60 °C and stir for 12 hours; S2. After 12 h, monitor the mixture obtained in step S1 by TLC to confirm the completion of the reaction. Cool it to 0 °C, dropwise add 20 mL of purified water for quenching, extract with 20 mL of ethyl acetate, retain the organic phase, wash once with 10 mL of purified water and once with 10 mL of saturated sodium chloride aqueous solution, and dry with anhydrous sodium sulfate to obtain 2.3 g of the crude decoquinate product; S3. Take 2.3 g of the crude product obtained in S2, dissolve it in 20 mL of methanol, slowly dropwise add 3M lithium hydroxide aqueous solution for ester hydrolysis, stir at room temperature for 6 hours, detect by TLC. After the complete reaction, dropwise add 15 mL of purified water for quenching, and use 3M hydrochloric acid for dissociation until the pH of the system is equal to 5, and a large amount of solid precipitates. Filter to obtain 2.0 g of the crude decoquinate hapten; S4. Purification: Dissolve 2.0 g of the crude product obtained in step S3 in 15 mL of 1M sodium hydroxide aqueous solution, wash twice with 15 mL of ethyl acetate, retain the aqueous phase, use 3M hydrochloric acid for dissociation until the pH is equal to 5, filter after the solid precipitates to obtain 1.6 g of the decoquinate hapten.

[0022] After column purification, a decoquinate derivative with higher purity is obtained. Detect the decoquinate derivative by LC-MS. From Figure 1 it can be seen that the signal of the decoquinate hapten is 504.3 and the molecular weight of the decoquinate hapten is 503, thus determining the hapten structure. The synthesis route of the decoquinate hapten is as Figure 2 shown.

[0023] Example 2. Preparation of the complete decoquinate antigen 1. Use the EDC / NHS coupling technology to prepare the complete decoquinate antigen S1. Weigh 100 mg of the pure hapten prepared in Example 1, dissolve it in 2 mL of DMF, slowly add 25 mg of NHS and 46 mg of EDC, and stir at room temperature for 6 h to obtain the activated solution of the decoquinate hapten; S2. Weigh 120 mg of BSA or 90 mg of OVA, dissolve it in 20 mL of CB buffer (0.2 mol / L, pH 9.0), stir evenly, and slowly add it dropwise to the activated hapten solution. Stir at room temperature for 16 h. Dialyze with 0.2 mol / L CB buffer at pH 9.0, change the buffer every 12 h during dialysis, and dialyze for 72 h to obtain the complete antigen of decoquinate with bovine serum albumin or chicken ovalbumin as the carrier protein.

[0024] The reaction route is as follows:

[0025] Example 3. Rapid detection of decoquinate residues in chicken tissues by colloidal gold immunochromatography I. Preparation of decoquinate colloidal gold detection reagent (1) Preparation of colloidal gold Take 1 mL of 1% (mass percentage) chloroauric acid solution, add 99 mL of ultrapure water to prepare a chloroauric acid solution with a final concentration of 0.01% (mass percentage). After heating to boiling, take 1 mL of 1% (mass percentage) trisodium citrate and quickly add it to the boiling chloroauric acid solution at one time. Continue to heat until the solution changes from light yellow to blue-black and finally to wine red. After the color is stable, continue to heat for 5 min, cool to room temperature, and supplement ultrapure water to the original volume.

[0026] (2) Preparation of colloidal gold-labeled decoquinate monoclonal antibody Adjust the pH value of the colloidal gold solution to 8.0, stir evenly with a constant-speed stirrer, and add the decoquinate monoclonal antibody drop by drop to make the amount of labeled antibody 2 μg / mL. After 1 hour, add BSA equivalent to the amount of antibody, fully react for 30 min, then add PEG20000 equivalent to the amount of antibody. After adding, continue to stir for 10 min. Centrifuge at 9000 rpm for 10 min to obtain a homogeneous gold-labeled antibody precipitate, and then resuspend it with PBS for standby to obtain a colloidal gold solution labeled with decoquinate monoclonal antibody. Dilute this colloidal gold solution by 2 times and spray it on the glass fiber at a spraying amount of 5 μL / cm, and dry it at 40 °C for storage for standby. This is the colloidal gold conjugate pad.

[0027] (3) Preparation of decoquinate colloidal gold test strip Attach a nitrocellulose membrane to the PVC bottom plate and equilibrate it at 40% humidity for 30 min; spray a decoquinate-coated antigen solution at 1.5 mg / mL on the nitrocellulose membrane to form a test line T line, and spray goat anti-mouse IgG at 0.5 mg / mL to form a control line C line. The test line and the control line are parallel to each other, and the distance between the two is 0.5 cm. Then, on the bottom plate, in the same direction, sequentially lap and adhere a sample pad, a colloidal gold conjugate pad, the nitrocellulose membrane with the test line T line sprayed with decoquinate-coated antigen and the control line C line sprayed with goat anti-mouse IgG, and a blotting paper in turn.

[0028] Among them, the test line is close to the colloidal gold conjugate pad and the sample pad, and the control line is close to the blotting paper. Cut the adhered test strip into test strips of equal width and assemble them into a test card. Seal and store the test card in a dry place.

[0029] II. Sample Detection Adopt the competitive immuno-chromatographic technique to qualitatively detect the residue of decoquinate in the sample. During detection, when there is no decoquinate in the sample to be detected, the colloidal gold particles are chromatographed to the position of the test line and bind to the decoquinate antigen coated on the membrane, and the T line shows color. When there is decoquinate in the sample to be detected, it will bind to the gold-labeled specific monoclonal antibody in the conjugate pad to form an immune complex. When the complex is chromatographed to the position of the test line, it will not bind to the decoquinate-coated antigen coated on the membrane, and the T line does not show color or the color becomes lighter.

[0030] (1) Detection steps: The method is as follows: Weigh 1 g of chicken tissue sample, add 3 mL of anhydrous methanol solution, mix well by shaking, centrifuge, take the supernatant and add it to the PBS solution to mix well. Take 100 μL of the mixed solution and add it to the sample adding hole of the test card, incubate at 20 - 25 °C for 10 minutes, and then judge the result. If both the T line and the C line show purple-red bands and the color of the T line is stronger than that of the C line, the result is negative; if the color of the T line is lighter than that of the C line or the C line shows color while the T line does not show color, the result is positive; if neither the C line nor the T line shows color, the test reagent has expired.

[0031] (2) Sensitivity detection of the decoquinate colloidal gold test card: Perform spiking experiments on chicken tissue samples respectively to make their final concentrations 800, 1000, 1500, 2000 μg / kg. The results are as Figure 3 shown. The sensitivity of the decoquinate colloidal gold test card in the present invention can reach 1000 μg / kg, and the CV value is less than 15%.

[0032] (3) Specificity detection of the decoquinate colloidal gold test card: In the negative chicken tissue samples, drugs such as decoquinate, triadimenol, ofloxacin, sulfadiazine, gentamicin, and penicillin were added at 5 mg / kg respectively to verify the specificity of the test strip card. The specific detection results are shown in Figure 4 . The detection results were negative except for decoquinate, indicating that there was no cross-reaction between decoquinate and the above-added drugs, and the specificity was good.

[0033] (4)Product stability experiment The decoquinate colloidal gold products in the present invention were subjected to an accelerated test at 4°C and 37°C respectively. The decoquinate standard at 1000 µg / kg was detected at 0 d, 7 d, 14 d, and 30 d respectively to evaluate the stability performance of the product. The results are shown in Table 1. When aged to 30 days, the decoquinate colloidal gold test products at 4°C and 37°C still had good sensitivity when detecting the 1000 μg / kg standard, with no obvious difference from the detection results on the first day, and the CVs were all <10%, indicating that the test strip card provided by the present invention had good stability.

[0034] Table 1 Stability of the decoquinate colloidal gold test strip card

[0035] In summary, the colloidal gold test strip card of the present invention has the characteristics of high sensitivity, strong specificity, and good stability, and can be applied to the rapid detection of decoquinate in chicken tissue.

Claims

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

2. A synthesis method for preparing the decoquinate hapten according to claim 1, characterized in that: S1. Accurately weigh the decoquinate raw material, dissolve it in N,N-dimethylformamide, protect it with nitrogen, then cool it to 0 °C, add ethyl 4-bromobutyrate, slowly add potassium carbonate, and slowly add a catalytic amount of tetrabutylammonium iodide. Keep the temperature at 60 °C and stir for 12 hours. S2. After 12 hours, monitor the mixture obtained in step S1 by TLC to confirm the completion of the reaction. Cool it to 0 °C, dropwise add purified water to quench it, extract with ethyl acetate, retain the organic phase, wash it once with purified water and once with saturated sodium chloride aqueous solution, and dry it with anhydrous sodium sulfate to obtain the crude decoquinate product. S3. Take the crude product of S2, dissolve it in methanol, slowly dropwise add 3M lithium hydroxide aqueous solution for ester hydrolysis, stir at room temperature for 6 hours, detect by TLC. After complete reaction, dropwise add purified water to quench it, use 3M hydrochloric acid for dissociation until the pH of the system is equal to 5, and a large amount of solid precipitates. Filter to obtain the crude decoquinate hapten. S4. Purification: Dissolve the crude product obtained in step S3 in 1M sodium hydroxide aqueous solution, wash it twice with ethyl acetate, retain the aqueous phase, use 3M hydrochloric acid for dissociation until the pH of the system is equal to 5. After solid precipitation, filter to obtain the decoquinate hapten.

3. A decoquinate complete antigen, characterized in that It is prepared by coupling the decoquinate hapten described in claim 1 with a carrier protein. The coupling method is the EDC / NHS coupling technique, and the carrier protein is any one of bovine serum albumin or chicken ovalbumin.

4. A method for preparing the decoquinate complete antigen according to claim 3, characterized in that When the method is the EDC / NHS coupling technique and the carrier protein is bovine serum albumin (BSA) or chicken ovalbumin (OVA), the method includes: Dissolve the decoquinate hapten described in claim 1 in DMF, slowly add NHS and EDC, and stir at room temperature for 6 hours to obtain the activated decoquinate hapten solution. Weigh BSA or OVA and dissolve it in CB buffer (0.2 mol / L, pH 9.0), stir evenly, slowly dropwise add it to the activated hapten solution, stir at room temperature for 16 hours, dialyze the obtained reaction solution with 0.01 mol / L PBS buffer with pH 7.4, change the buffer once every 12 hours during dialysis, and dialyze for 72 hours to obtain the complete decoquinate antigen with BSA or OVA as the carrier protein.

5. Provide a decoquinate colloidal gold detection product, characterized in that The nitrocellulose membrane is coated with the complete decoquinate antigen described in claim 3 or claim 4.

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