CN-115925637-B - Oxfendazole hapten and artificial antigen as well as preparation methods and application thereof

Abstract

The invention discloses an oxfendazole hapten, an artificial antigen, a preparation method and application thereof. The artificial antigen of the oxfendazole provided by the invention is an antigen obtained by coupling the oxfendazole hapten shown in the formula I with carrier protein. The method for synthesizing the artificial antigen of the oxfendazole is simple, has high purity and high yield, and has great value for preparation of the oxfendazole antibody and detection of oxfendazole drug residues.

Inventors

• MA LICAI
• Lv Shunquan
• LI RONGRONG
• QIN DANFENG
• XING WEIWEI
• JIA LIANGXI

Assignees

• 北京维德维康生物技术有限公司

Dates

Publication Date

20260512

Application Date

20220816

Claims (10)

1. 1. A compound has a structure shown in formula I: Formula I.
2. 2. A process for preparing the compound of claim 1, comprising the steps of: Adding N, N-dimethylformamide into 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-bromosuccinimide, stirring, adding 4- (aminomethyl) benzoic acid, reacting, extracting a reaction liquid, spin-drying, chromatography and spin-drying again to obtain solid 1, wherein the ratio of the 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, the N, N-dimethylformamide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-bromosuccinimide and the 4- (aminomethyl) benzoic acid is 500 mg/5 ml/448 mg/265 mg/446 mg.
3. 3. An oxfendazole antigen, which is an antigen obtained by coupling a compound according to claim 1 to a carrier protein.
4. 4. The oxfendazole antigen according to claim 3, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin, hemocyanin, murine serum protein, thyroxine or rabbit serum protein.
5. 5. The preparation method of the oxfendazole antigen according to claim 3 or 4 comprises the step of coupling the compound according to claim 1 with a carrier protein through an amide bond to obtain the oxfendazole antigen.
6. 6. The method for preparing an oxfendazole antigen according to claim 5, wherein the method comprises the steps of: (1) Dissolving the compound in N, N-dimethylformamide, then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and magnetically stirring at 20-25 ℃ for reaction for 2-3 hours to obtain a solution I; Wherein the ratio of the compound to the N, N-dimethylformamide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 13.73mg to 1.5ml to 21.5mg to 13mg; (2) Placing the carrier protein in 0.1M sodium bicarbonate buffer solution, stirring at 200rpm for 10min, and dissolving completely to obtain solution II, wherein the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer solution is 33.6-50mg:3.5ml; Wherein, if the carrier protein is bovine serum albumin, the ratio of the bovine serum albumin to the 0.1M sodium bicarbonate buffer solution is 50mg:3.5ml, and if the carrier protein is ovalbumin, the ratio of the ovalbumin to the 0.1M sodium bicarbonate buffer solution is 33.6mg:3.5ml; (3) Mixing the solution I and the solution II, specifically, dropwise adding the solution I into the solution II under the stirring of 1000rpm at the temperature of 0-4 ℃ and stirring and reacting for 24 hours at 500rpm to obtain a solution III; (4) The solution III was dialyzed with 0.01M phosphate buffer pH7.2 at 4℃for 3 days with stirring to give the oxfendazole antigen.
7. 7. The use of the compound of claim 1 or the oxfendazole antigen of claim 3, characterized in that a reagent for qualitatively or quantitatively detecting oxfendazole is prepared and an oxfendazole antibody is prepared.
8. 8. An antibody prepared by using the oxfendazole antigen according to claim 3, which is characterized in that the antibody is an anti-oxfendazole monoclonal antibody, the amino acid sequence of the heavy chain variable region of the anti-oxfendazole monoclonal antibody is shown as a sequence 1 in a sequence table, and the amino acid sequence of the light chain variable region of the anti-oxfendazole monoclonal antibody is shown as a sequence 2 in the sequence table.
9. 9. Use of an oxfendazole antigen according to claim 3 for the preparation of an enzyme linked immunosorbent assay kit and a colloidal gold assay card reagent.
10. 10. The use of the oxfendazole antigen according to claim 9, characterized in that the enzyme-linked immunosorbent assay kit and the colloidal gold detection card detect samples as dairy products and tissues, and the detection limit of the oxfendazole in the samples is 5 mug/kg, and the sensitivity is 0.20 mug/l.

Description

Oxfendazole hapten and artificial antigen as well as preparation methods and application thereof Technical Field The invention belongs to the technical field of food safety detection, and particularly relates to an oxfendazole hapten and an artificial antigen as well as a preparation method and application thereof. Background Oxfendazole is a novel, highly effective, broad-spectrum, low-toxic benzimidazole carbamate anthelmintic, which has been widely used in veterinary medicine, such as the prevention of gastrointestinal nematodes, pulmonary nematodes, liver flukes, etc. As oxfordazole is shown to have teratogenicity and mutability in animal safety evaluation tests, most countries or regions such as china, the united states, the european union and the like take oxfordazole as an important monitoring object of food safety, and the highest residual limit is formulated. At present, the method for measuring the oxfendazole residue mainly comprises a gas chromatography mass spectrometry (GC-MS), a High Performance Liquid Chromatography (HPLC), a liquid chromatography tandem mass spectrometry (LC-MS/MS) and the like, and the HPLC method has the advantages of accurate quantification and the like, but has the defects of relatively complex pretreatment during detection, low method sensitivity and the like in practical application. The GC-MS method can perform confirmatory detection and has high sensitivity, but the pretreatment process is complex, and derivatization treatment is needed. The LC-MS/MS method has the advantages of high sensitivity, good selectivity, accurate qualitative and quantitative, strong anti-interference capability and the like, but the method also has the defects of complex pretreatment process, long time consumption, high cost and the like. The immunochemistry analysis method has unique advantages in qualitative and quantitative aspects of antigen and antibody, and has the advantages of simple and rapid operation, low cost, higher sensitivity and large analysis sample size, and overcomes the defects of physicochemical analysis. Therefore, it is urgently needed to develop a rapid and accurate rapid detection method for screening mass animal foods to improve the current situation, fill the deficiency of domestic detection means, promote and standardize the technical level of the whole detection industry, and enhance the competitiveness of animal foods in the international market in China. Disclosure of Invention The invention aims to provide an oxfendazole hapten, an artificial antigen, a preparation method and application thereof. The artificial antigen of the oxfendazole provided by the invention is an antigen constructed on the basis of the hapten of the oxfendazole. The oxfendazole hapten belongs to the protection scope of the invention, and the structure of the oxfendazole hapten is shown as a formula I. The method for preparing the oxfendazole hapten provided by the invention specifically comprises the following steps: Adding dimethyl formamide (DMF) into 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, stirring, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-bromosuccinimide (NBS), stirring, adding 4- (aminomethyl) benzoic acid, reacting, extracting reaction liquid, spin-drying, chromatography and spin-drying again to obtain solid 1. The ratio of the 2-methoxycarbonylamino-3H-benzimidazole-5-carboxylic acid, dimethylformamide (DMF), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-bromosuccinimide (NBS) and 4- (aminomethyl) benzoic acid is 500mg:5 ml:447 mg:265 mg:4476 mg. The oxfendazole antigen constructed on the basis of the oxfendazole hapten also belongs to the protection scope of the invention. The oxfendazole antigen is an antigen obtained by coupling the oxfendazole hapten (formula I) with carrier protein. In one embodiment of the invention, the carrier protein is in particular Bovine Serum Albumin (BSA) or Ovalbumin (OVA). The preparation method of the oxfendazole antigen also belongs to the protection scope of the invention. The preparation method of the oxfendazole antigen specifically comprises the following steps of coupling the oxfendazole hapten (formula I) with carrier protein through an amide bond to obtain the oxfendazole antigen. In the invention, the oxfendazole antigen is prepared and obtained specifically according to a method comprising the following steps: (1) Dissolving the oxfendazole hapten (formula I) in Dimethylformamide (DMF), and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and magnetically stirring and reacting for 2-3 hours at 20-25 ℃ to obtain a solution I; Wherein the ratio of the oxfendazole hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 13.73mg:1.5ml:21.5mg:13mg. (2) Placing the carrier protein in 0.1M sodium