阅读说明：本技术 咪鲜胺半抗原、人工抗原和抗体及其制备方法和应用 (Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof ) 是由 万宇平 崔廷婷 崔海峰 张瑜 何方洋 马玉华 崔娜 赵正苗 于 2019-09-23 设计创作，主要内容包括：本发明公开了咪鲜胺半抗原、人工抗原和抗体及其制备方法和应用,本发明提供的咪鲜胺半抗原既最大程度保留了咪鲜胺的特征结构,使得咪鲜胺半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基；用咪鲜胺半抗原与载体蛋白偶联后得到的咪鲜胺免疫抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,经检测咪鲜胺抗体的灵敏度可达0.1μg/L,与其他农药的交叉反应率低,为后续建立咪鲜胺的各种免疫分析方法提供了基础。(The invention discloses a prochloraz hapten, an artificial antigen and an antibody as well as a preparation method and application thereof, the prochloraz hapten provided by the invention not only retains the characteristic structure of the prochloraz to the greatest extent, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz hapten is coupled with carrier protein to obtain the prochloraz immunizing antigen for immunizing animals, so that the prochloraz immunizing antigen is more favorable for stimulating the immune response of animals to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the detected prochloraz antibody can reach 0.1 mu g/L, the cross reaction rate with other pesticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of the prochloraz.)

1. A prochloraz hapten, which has the following structural formula:

2. the method for preparing prochloraz hapten as claimed in claim 1, comprising the following steps:

1) reacting 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid with 1, 2-dichloroethane to obtain an intermediate 1, wherein the intermediate 1 has a structural formula

2) Reacting the intermediate 1 with n-propylamine to obtain an intermediate 2, wherein the intermediate 2 has a structural formula

3) And reacting the intermediate 2 with bis (trichloromethyl) carbonate and imidazole to obtain the prochloraz hapten.

3. The method for preparing prochloraz hapten according to claim 2, wherein the step 1) comprises the following steps: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

4. The method for preparing prochloraz hapten according to claim 2, wherein the step 2) comprises the following steps: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

5. The method for preparing prochloraz hapten according to claim 2, wherein the step 3) comprises the following steps: dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz hapten.

6. A prochloraz artificial antigen which is a conjugate obtained by coupling a carrier protein and the prochloraz hapten as claimed in claim 1.

7. The prochloraz artificial antigen of claim 6, wherein said carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

8. The method for preparing prochloraz artificial antigen as claimed in claim 6, comprising the steps of:

dissolving the prochloraz hapten in an organic solvent, adding carbodiimide and N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4 hours to obtain solution A, wherein the mass ratio of the prochloraz hapten to the carbodiimide to the N-hydroxysuccinimide is (1), (2-3) to (2-3);

dissolving carrier protein in 0.05mol/L PB buffer solution to obtain solution B; and

and dropwise adding the solution A into the solution B, reacting for 24h at 4 ℃, and purifying to obtain the prochloraz artificial antigen.

9. A prochloraz antibody obtained by immunizing an animal with the prochloraz artificial antigen of claim 6, wherein the prochloraz antibody is specifically immunoreactive with prochloraz.

10. Use of the prochloraz antibody of claim 9 for detecting prochloraz residues.

Technical Field

The invention belongs to the field of food safety detection. More particularly, the invention relates to a prochloraz hapten, an artificial antigen and an antibody, and a preparation method and application thereof.

Background

Prochloraz (Prochloraz) is a broad-spectrum bactericide, and has control effects on various diseases of field crops, fruits and vegetables, turf and ornamental plants; in addition, prochloraz can be used for preserving the harvested fruits such as oranges and bananas, and has a wide application range. The dosage of the prochloraz is strictly regulated in various countries, for example, CAC, European Union and Japan all regulate the MRL value of the prochloraz in raw milk to be 0.05, 0.4 and 0.05mg/kg respectively, and national standard GB 2763-.

At present, the detection of prochloraz at home and abroad mainly comprises analysis methods such as gas chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. The instrument detection method has the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, so the instrument detection method cannot be widely applied in China and does not meet the requirements of on-site detection on accurately detecting and screening a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the research of the prochloraz residue.

When establishing an immunological detection method and applying the detection method to detect the residual quantity of the prochloraz, the key technology is to obtain an antibody with strong specificity and high sensitivity, and the aim is to realize the aim under the precondition that a proper prochloraz hapten is synthesized and prepared. However, at present, no related report aiming at the prochloraz hapten exists in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the hapten which can furthest reserve the characteristic structure of the prochloraz and has a connecting arm with a certain length and the preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.

The technical problem to be solved by the invention is realized by the following technical scheme:

the first purpose of the invention is to provide a prochloraz hapten which has the following structural formula:

the second purpose of the invention is to provide a preparation method of prochloraz hapten, which comprises the following steps:

1) reacting 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid with 1, 2-dichloroethane to obtain an intermediate 1, wherein the intermediate 1 has a structural formula

2) Reacting the intermediate 1 with n-propylamine to obtain an intermediate 2, wherein the intermediate 2 has a structural formula

3) And reacting the intermediate 2 with bis (trichloromethyl) carbonate and imidazole to obtain the prochloraz hapten.

Further, the step 1) comprises the following steps: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

Further, the step 2) comprises the following steps: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

Further, the step 3) comprises the following steps: dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz hapten.

The third purpose of the invention is to provide a prochloraz artificial antigen which is a conjugate obtained by coupling a carrier protein and the prochloraz hapten.

Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

The fourth purpose of the invention is to provide a preparation method of the prochloraz artificial antigen, which comprises the following steps:

dissolving the prochloraz hapten in an organic solvent, adding carbodiimide and N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4 hours to obtain solution A, wherein the mass ratio of the prochloraz hapten to the carbodiimide to the N-hydroxysuccinimide is (1), (2-3) to (2-3);

dissolving carrier protein in 0.05mol/L PB buffer solution to obtain solution B; and

and dropwise adding the solution A into the solution B, reacting for 24h at 4 ℃, and purifying to obtain the prochloraz artificial antigen.

The fifth purpose of the invention is to provide a prochloraz antibody which is obtained by immunizing animals with the prochloraz artificial antigen and can generate specific immunoreaction with the prochloraz.

Further, the prochloraz antibody is a monoclonal antibody or a polyclonal antibody.

The sixth purpose of the invention is to provide the application of the prochloraz antibody in detecting prochloraz residue.

The invention has the following beneficial effects:

the prochloraz hapten provided by the invention not only retains the characteristic structure of the prochloraz to the greatest extent, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz artificial antigen obtained by coupling the prochloraz hapten and the carrier protein is used for immunizing animals, so that the method is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and provides a basis for subsequently establishing various immunoassay methods of the prochloraz.

The preparation method of the prochloraz hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and high purity and yield of the prepared prochloraz hapten.

The prochloraz antibody obtained by adopting the prochloraz artificial antigen has good titer, specificity and affinity, the sensitivity can reach 0.1 mu g/L, and the cross reaction rate with other pesticides is low.

Drawings

FIG. 1 is the synthesis route of the prochloraz hapten

Detailed Description

In a first aspect, the present invention provides a prochloraz hapten having the following structural formula:

the prochloraz hapten provided by the invention not only retains the characteristic structure of the prochloraz to the greatest extent, so that the immunogenicity of the prochloraz hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the prochloraz artificial antigen obtained by coupling the prochloraz hapten and the carrier protein is used for immunizing animals, and is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity. The prochloraz hapten makes up the blank of the technical field of the domestic prochloraz immunological detection method, and lays a foundation for the further development of the prochloraz immunological detection method.

In a second aspect, the present invention provides a method for preparing the above-mentioned prochloraz hapten, which comprises the following steps:

1) reacting 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid with 1, 2-dichloroethane to obtain an intermediate 1, wherein the intermediate 1 has a structural formula

2) Reacting the intermediate 1 with n-propylamine to obtain an intermediate 2, wherein the intermediate 2 has a structural formula

3) And reacting the intermediate 2 with bis (trichloromethyl) carbonate and imidazole to obtain the prochloraz hapten.

Preferably, the step 1) includes the steps of: dissolving 2.69g of 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid in 60mL of 1, 2-dichloroethane, adding 0.57g of KOH, reacting at 60 ℃ for 6h, stopping the reaction, adding water for washing, and evaporating to dryness to obtain an intermediate 1.

Preferably, the step 2) includes the steps of: adding 40mL of n-propylamine, 0.31g of anhydrous sodium iodide and 1.38g of potassium carbonate into the intermediate 1, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the n-propylamine, adding water, shaking, extracting with ethyl acetate, collecting an organic phase, and recrystallizing the organic phase with n-hexane-dichloromethane to obtain an intermediate 2.

Preferably, the step 3) includes the steps of: dissolving 21.2 g of the intermediate in 80mL of dichloromethane, adding 1.1g of bis (trichloromethyl) carbonate and 0.3mL of triethylamine, stirring at room temperature for 3h, adding 0.27g of imidazole, continuing stirring for 7h, stopping the reaction, adding water, extracting, washing, evaporating to dryness to obtain red oily matter, loading on a silica gel column, eluting with petroleum ether-ethyl acetate, separating and purifying to obtain the prochloraz hapten.

The preparation method of the hapten mainly comprises the following steps: (1) generating corresponding functional groups by using the existing structure or intermediate of an object to be detected through oxidation, reduction, substitution, hydrolysis and other reactions; (2) transforming the metabolite or structural analogue of the hapten into a required hapten by using the metabolite or structural analogue as a primer; (3) raw materials and reagents are used for re-synthesis, and small molecules with functional groups are introduced at proper positions. The invention uses 3- (3-hydroxy-2, 4, 6-trichloro) -propionic acid as the starting material, synthesizes prochloraz hapten with carboxyl through 3 steps of reaction, has higher difficulty compared with the former two methods, but has the advantages of easy establishment of the optimal coupling site and easy establishment of reasonable spacing arms, and increases the possibility of generating antibodies aiming at characteristic structures.

The preparation method of the prochloraz hapten is reasonably designed according to the structural characteristics of the prochloraz, the used raw materials are easy to obtain, the reaction operation is simple, the reaction conditions are easy to control, and the prepared prochloraz hapten is high in purity and yield.

In a third aspect, the invention also provides a prochloraz artificial antigen which is a conjugate obtained by coupling carrier protein and the prochloraz hapten.

Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

The prochloraz hapten molecule only has immunoreactivity and does not have immunogenicity. Therefore, in order to confer immunogenicity on the prochloraz hapten molecules, the prochloraz hapten molecules are coupled and combined with suitable carrier protein molecules, thereby generating the prochloraz artificial antigen with both immunoreactivity and immunogenicity.

In a fourth aspect, the invention also provides a preparation method of the prochloraz artificial antigen, which comprises the following steps:

dissolving the prochloraz hapten in an organic solvent, adding carbodiimide and N-hydroxysuccinimide, fully and uniformly mixing, and reacting for 4 hours to obtain solution A, wherein the mass ratio of the prochloraz hapten to the carbodiimide to the N-hydroxysuccinimide is (1), (2-3) to (2-3);

dissolving carrier protein in 0.05mol/L PB buffer solution to obtain solution B; and

and dropwise adding the solution A into the solution B, reacting for 24h at 4 ℃, and purifying to obtain the prochloraz artificial antigen.

In the fifth aspect, the invention also provides a prochloraz antibody which is obtained by immunizing animals with the prochloraz artificial antigen and can generate specific immunoreaction with the prochloraz.

The prochloraz antibody can be a monoclonal antibody or a polyclonal antibody. In addition, the prochloraz antibody can be prepared by a method conventional in the art.

In a specific embodiment, the prochloraz antibody is a murine monoclonal antibody specific for a prochloraz artificial antigen of the above-mentioned prochloraz hapten.

The prochloraz antibody obtained by adopting the prochloraz artificial antigen has better titer, specificity and affinity, and has low cross reaction rate with other pesticides.

In a sixth aspect, the invention also provides an application of the prochloraz antibody in detection of prochloraz residues.

The invention induces immune animals to generate antibodies through the prochloraz artificial antigen, thereby being used in the prochloraz immunodetection analysis.

The prochloraz immunoassay comprises but is not limited to a prochloraz ELISA kit and a prochloraz colloidal gold test strip.

The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.