Novobiocin hapten as well as synthesis method and application thereof

Abstract

The invention discloses a novobiocin hapten as well as synthesis and application thereof. The structure of the novobiocin hapten is as shown in formula I in the specification. The neomycin hapten provided by the invention can fully expose the antigenic determinant of the neomycin and maintain the spatial conformation of the neomycin, has good immunogenicity, and is mild in synthesis conditions, few in reaction steps, high in yield and capable of reducing the cost of raw materials. The novobiocin colloidal gold detection product provided by the invention has the characteristics of strong specificity and high sensitivity, can realize accurate and rapid detection of novobiocin in a sample, and has no cross reaction with other drugs. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly to a novobiocin hapten, a synthesis method thereof and an application thereof. Background Art

[0002] Novobiocin is an aminocoumarin antibiotic isolated from Streptomyces niveus, and is a potent inhibitor of bacterial DNA gyrase and eukaryotic DNA topoisomerase II. It is a Gram-positive bacterium that can specifically target the activity of the GyrB subunit involved in energy transfer, thereby interfering with DNA synthesis and ultimately affecting bacterial growth. Its antibacterial spectrum is similar to that of penicillin, and it is mainly used to treat infections caused by drug-resistant Staphylococcus aureus, such as pneumonia, sepsis, etc. However, it is easy to cause bacteria to develop drug resistance, so it is advisable to be used in combination with other antibacterial drugs.

[0003] With the rapid development of the national animal husbandry, novobiocin, as a drug with good antibacterial effect and simple production process, has been gradually widely used in veterinary clinics. On the other hand, its irregular use poses a potential hazard to food safety, thus threatening people's physical health. Some countries and regions have also stipulated the residue limit of novobiocin in animal-derived foods. Therefore, it is very important to establish a rapid, safe and stable detection method for novobiocin.

[0004] Currently, the main detection method for novobiocin is the high performance liquid chromatography-tandem mass spectrometry method established by Shen Lei et al. There has also been a method of using microbial filter paper strips to detect novobiocin. These methods are relatively complex in sample processing, have strict requirements for operation, and have a long detection time, and it is difficult to simultaneously process a large number of samples. In contrast, the colloidal gold immunochromatography method can make up for the above deficiencies. The test strip is convenient to carry, easy to operate, has low professional requirements for the testers, and has a short reaction time, and is suitable for rapid on-site detection of a large number of samples. It has now been widely used in the detection of food, biology, environment, drugs, clinical medicine and other fields. Therefore, the present invention provides a novobiocin hapten with stable structure, and couples it with a carrier protein to prepare a novobiocin complete antigen. The colloidal gold test strip prepared by this antigen can achieve accurate and rapid detection of novobiocin. Summary of the Invention

[0005] The purpose of the present invention is to provide a novobiocin hapten, a synthesis method thereof and an application thereof, to achieve accurate and rapid detection of novobiocin 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 objectives of the present invention is to provide a novobiocin hapten, and the structure of the novobiocin hapten is shown in Formula I: Formula I.

[0007] Another objective of the present invention is to provide a method for preparing the above novobiocin hapten, and the steps are as follows: S1. Accurately weigh the novobiocin raw material, completely dissolve it in anhydrous pyridine and methanol (1:1), protect it with nitrogen, then cool it to 0°C, add carboxymethoxyamine hemihydrochloride, heat it to 60°C, and stir and react for 8 hours to obtain reaction solution A; S2. Monitor the mixture A by TLC, confirm that the reaction is complete, spin-dry it, dropwise add purified water to quench it, extract it twice with ethyl acetate, combine the organic phases, wash it twice with saturated sodium chloride aqueous solution to obtain the crude novobiocin hapten; S3. Purification: Purify the product obtained in S2 with a reverse-phase FLASH column to obtain the purified novobiocin hapten.

[0008] Preferably, the detailed steps are as follows: S1. Accurately weigh 500 mg of novobiocin raw material, completely dissolve it in 20 mL of anhydrous pyridine and methanol (1:1), protect it with nitrogen, then cool it to 0°C, add 97 mg of carboxymethoxyamine hemihydrochloride, heat it to 60°C, and stir and react for 8 hours to obtain reaction solution A; S2. Monitor the mixture A by TLC, confirm that the reaction is complete, spin-dry it, dropwise add 10 mL of purified water to quench it, extract it twice with 15 mL of ethyl acetate, combine the organic phases, wash it twice with saturated sodium chloride aqueous solution, and spin-dry the organic phase to obtain the crude novobiocin hapten; S3. Purification: Purify the product obtained in S2 with a reverse-phase FLASH column to obtain the purified novobiocin hapten.

[0009] Another objective of the present invention is to provide a novobiocin complete antigen, which is obtained by conjugating the above novobiocin hapten with a carrier protein, and the structure of the novobiocin complete antigen is shown in Formula II: Formula II.

[0010] Among them, Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0011] Another objective of the present invention is to provide a method for preparing the above novobiocin complete antigen.

[0012] Specifically, the method is the EDC / NHS coupling technology. When the carrier protein is bovine serum albumin (BSA) or chicken ovalbumin (OVA), the preparation method of the novobiocin complete antigen is as follows: Dissolve the above novobiocin hapten in DMF (N,N-dimethylformamide), slowly add NHS and EDC, and stir at room temperature for 6 h to obtain an activated solution of novobiocin hapten; weigh BSA or OVA and dissolve it in PBS buffer (0.01 mol / L, pH 7.4), stir evenly, and slowly drop it into the activated hapten solution, and stir at room temperature for 16 h. Dialyze the obtained reaction solution with PBS buffer at 0.01 mol / L and pH 7.4, change the buffer every 12 h during dialysis, and dialyze for 72 h to obtain a complete novobiocin antigen with BSA as the carrier protein.

[0013] The fifth object of the present invention is to provide a novobiocin colloidal gold detection product, which is characterized in that the above-mentioned complete novobiocin antigen is coated on a nitrocellulose membrane.

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

[0015] 2. The novobiocin colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity and strong stability, can realize the accurate and rapid detection of novobiocin, 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 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, other implementation drawings can be obtained according to the provided drawings without creative work.

[0017] The structures, ratios, sizes, etc. shown 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 under which the present invention can be implemented. Therefore, they do not have technical substance. Any modification of the structure, change of the proportional relationship or adjustment of the size should still fall within the scope covered by the technical content disclosed in the present invention without affecting the efficacy and the purpose that the present invention can achieve.

[0018] Figure 1 It is the MS spectrum of novobiocin hapten; Figure 2 It is the synthetic structure diagram of novobiocin hapten; Figure 3Sensitivity test results of novobiocin in fresh milk Figure 4 Specificity test results of novobiocin colloidal gold test strip Specific implementation mode

[0019] The following specific embodiments illustrate the implementation mode of the present invention. Those skilled in the art 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, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present invention.

[0020] Example 1: Preparation of novobiocin hapten The structure of the novobiocin hapten is shown in Formula I: Formula I

[0021] The preparation method of the novobiocin hapten described in this example is as follows: S1: Accurately weigh the novobiocin raw material, completely dissolve it in anhydrous pyridine and methanol (1:1), protect it with nitrogen, then cool it to 0°C, add carboxymethoxyamine hemihydrochloride, heat it to 60°C, and stir and react for 8 hours to obtain reaction solution A; S2: Monitor the mixture A by TLC, confirm that the reaction is complete, spin-dry it, dropwise add purified water for quenching, extract it twice with ethyl acetate, combine the organic phases, wash it twice with saturated sodium chloride aqueous solution to obtain the crude novobiocin hapten; S3: Purification: Purify the product obtained in S2 with a reverse-phase FLASH column to obtain the purified novobiocin hapten.

[0022] Preferably, the detailed steps are as follows: S1: Accurately weigh 500 mg of novobiocin raw material, completely dissolve it in 20 mL of anhydrous pyridine and methanol (1:1), protect it with nitrogen, then cool it to 0°C, add 97 mg of carboxymethoxyamine hemihydrochloride, heat it to 60°C, and stir and react for 8 hours to obtain reaction solution A; S2: Monitor the mixture A by TLC, confirm that the reaction is complete, spin-dry it, dropwise add 10 mL of purified water for quenching, extract it twice with 15 mL of ethyl acetate, combine the organic phases, wash it twice with saturated sodium chloride aqueous solution, and spin-dry the organic phase to obtain the crude novobiocin hapten; S3: Purification: Purify the product obtained in S2 with a reverse-phase FLASH column to obtain the purified novobiocin hapten.

[0023] After column purification, a novobiocin carboxyl derivative with a higher purity is obtained. The novobiocin carboxyl derivative is detected by LC-MS. As shown by Figure 1 , the negative signal of the novobiocin hapten is 684.2. The molecular weight of the novobiocin hapten is 685.24, thus determining the hapten structure. The synthesis route of the novobiocin hapten is as shown by Figure 2 .

[0024] Example 2. Preparation of the novobiocin complete antigen 1. Use the EDC / NHS coupling technology to prepare the novobiocin complete antigen S1. Weigh 40 mg of the pure hapten prepared in Example 1, dissolve it in 1 mL of DMF (N,N-dimethylformamide), slowly add 13 mg of NHS and 23 mg of EDC, and stir at room temperature for 6 h to obtain the novobiocin hapten activation solution; S2. Weigh 120 mg of BSA or OVA, dissolve it in 10 mL of PBS buffer solution with a concentration of 0.01 mol / L and a pH of 7.4, stir evenly, slowly drop it into the activated hapten solution, and stir at room temperature for 16 h. Dialyze with PBS buffer solution with a pH of 7.2 and a concentration of 0.01 mol / L, change the buffer solution once every 12 h during dialysis, and dialyze for 72 h to obtain the novobiocin complete antigen with the carrier protein being BSA or OVA.

[0025] Its reaction route is as follows:

[0026] Among them, Protein represents the carrier protein, and the carrier protein is any one of bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0027] Example 3. Rapid detection of novobiocin residues in milk by colloidal gold immunochromatography I. Preparation of the novobiocin 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 1.5 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 grayish black and finally to orange red. After the color is stable, continue to heat for 5 min, cool to room temperature, and supplement ultrapure water to the original volume.

[0028] (2) Preparation of colloidal gold labeled with novobiocin monoclonal antibody Adjust the pH value of the colloidal gold solution to 8.0, and stir it evenly with a constant-speed stirrer. At the same time, add novobiocin monoclonal antibody drop by drop. The amount of labeled antibody is 1 μg / mL of colloidal gold. After 1 hour, add BSA equivalent to the amount of antibody, and continue to react for 30 min. Then add PEG equivalent to the amount of antibody. After adding, continue to stir for 30 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 novobiocin monoclonal antibody.

[0029] (3)Preparation of freeze-dried novobiocin monoclonal antibody-colloidal gold complex (hereinafter referred to as freeze-dried gold) Prepare the freeze-drying buffer, and use novobiocin colloidal gold and the freeze-drying buffer to prepare the freeze-dried gold solution. Stir and mix evenly for 20 min, and dispense it into the 96-well microplate at 60 μl / well; among them, the freeze-drying buffer consists of 0.05 M phosphate buffer, 0.5% BSA, and 3% trehalose; the ratio of novobiocin colloidal gold to the freeze-drying buffer in the freeze-dried gold solution is 1:5; put the coated 96-well microplate into a vacuum freeze dryer with a preset freeze-drying program for treatment. After the freeze-drying process is completed, take out the freeze-dried gold, cover it with a rubber cap, and put it into an aluminum foil bag with a desiccant for storage and standby.

[0030] (4)Preparation of novobiocin colloidal gold test strip Spray 0.5 mg / mL novobiocin-coated antigen solution on the nitrocellulose membrane to form the test line T line, and spray 0.3 mg / mL goat anti-mouse IgG to form the control line C line. The test line and the control line are parallel to each other, and the distance between the two is 0.65 cm. Then, on the bottom plate, in the same direction, sequentially lap and adhere the sample pad, the nitrocellulose membrane with the test line T line sprayed with novobiocin-coated antigen and the control line C line sprayed with goat anti-mouse IgG, and the absorbent paper.

[0031] Among them, the test line is close to the sample pad, and the control line is close to the absorbent paper. Cut the adhered test strip into test strips of equal width, combine with the freeze-dried gold, and assemble them into a test strip card. Seal and dry the test strip card for storage.

[0032] II. Sample detection Using the competitive immuno-chromatographic technique, qualitatively detect the novobiocin residue in the sample. Coat the novobiocin antigen on the test line (T line) of the nitrocellulose membrane, and label the corresponding novobiocin monoclonal antibody on the colloidal gold. During the detection, when there is no novobiocin in the test sample, the colloidal gold particles will bind to the novobiocin antigen coated on the membrane when chromatographing to the test line position, and the T line will show color. When there is novobiocin in the test sample, it will bind to the gold-labeled specific monoclonal antibody on the conjugate pad to form an immune complex. When the complex chromatographs to the test line position, it will not bind to the novobiocin-coated antigen on the membrane, and the T line will not show color or the color will become lighter.

[0033] (1) Detection steps: The method is as follows: Take 200 μL of fresh milk in a micro-well, slowly aspirate and mix well with the freeze-dried gold reagent in the micro-well, incubate at 20 - 25 °C for 5 min, insert the labeled test strip into the micro-well to fully immerse it in the solution, and after incubating at room temperature for 5 min, perform the result judgment. 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.

[0034] (2) Sensitivity detection of the novobiocin colloidal gold test strip card: Perform a spiking experiment on negative milk until the final concentration of novobiocin reaches 10, 15, 20 μg / kg. The results are shown in Table 1. Figure 3 As shown, the sensitivity of the novobiocin colloidal gold test strip card in the present invention can reach 20 μg / kg, and the CV value is less than 15%.

[0035] Table 1. Sensitivity of the novobiocin colloidal gold test strip

[0036] (3) Specificity detection of the novobiocin colloidal gold test strip card: Add 500 μg / kg of novobiocin, β-lactams, tetracyclines, fluoroquinolones, sulfonamides, erythromycin, and lincomycin to negative fresh milk respectively to verify the specificity of the test strip card. The specific detection results are shown in Figure 4 . The detection results are all negative except for novobiocin, indicating that the novobiocin colloidal gold test strip card has excellent specificity and no cross-reaction with the above added drugs.

[0037] (4) Product stability experiment The novobiocin colloidal gold products in the present invention were respectively placed under the conditions of 4°C and 37°C for accelerated tests. The novobiocin standard product at 20 µg / kg was detected at 0d, 7d, 14d, and 28d respectively to evaluate the stability performance of the products. The results are shown in Table 2. When aged to 28 days, the novobiocin colloidal gold detection products under the conditions of 4°C and 37°C still had good sensitivity when detecting the 20 μg / kg standard product, with no obvious difference from the detection results on the first day, and the CVs were all <10%, indicating that the test strip provided by the present invention has good stability.

[0038] Table 2 Stability of the novobiocin colloidal gold detection test strip

[0039] In summary, the colloidal gold detection test strip of the present invention has the characteristics of high sensitivity, strong specificity, and good stability, and can be applied to the rapid detection of novobiocin in fresh milk.

Claims

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

2. A method for preparing the biotin hapten according to claim 1, characterized in that: S1. Accurately weigh the raw material of neomycin, completely dissolve it in anhydrous pyridine and methanol (1:1), protect with nitrogen, then cool to 0°C, add carboxymethylhydroxylamine hemihydrochloride, heat to 60°C, stir and react for 8 hours to obtain reaction solution A; S2, monitoring the mixed solution A by TLC to confirm that the reaction is complete, spin-drying, adding purified water dropwise for quenching, extracting twice with ethyl acetate, combining the organic phases, and washing twice with a saturated sodium chloride aqueous solution to obtain a crude product of neomycin hapten; S3. Purification: Purify the product obtained in S2 using a reverse phase FLASH column to obtain the purified neomycin hapten.

3. A complete neomycin antigen, characterized in that: The novel mycin 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 complete neomycin antigen according to claim 3, characterized in that: When the carrier protein is bovine serum albumin or chicken ovalbumin, the method comprises: The neomycin hapten described in claim 1 is dissolved in DMF (N,N-dimethylformamide), and NHS and EDC are slowly added, and the mixture is stirred at room temperature for 5 hours to obtain a neomycin hapten activation solution; bovine serum albumin or chicken ovalbumin is weighed and dissolved in a 0.01 mol / L PBS buffer solution with a pH of 7.4, and the mixture is stirred evenly, and the mixture is slowly added dropwise to the activated hapten solution, and the mixture is stirred at room temperature for 16 hours. The obtained reaction solution is dialyzed using a 0.01 mol / L PBS buffer solution with a pH of 7.4, during which the buffer solution is changed every 12 hours, and the dialyzation is performed for 72 hours to obtain a neomycin complete antigen whose carrier protein is bovine serum albumin or chicken ovalbumin.

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

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