阅读说明：本技术 一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用 (Preparation method and application of hapten and complete antigen for diethoxy thiophosphate organophosphorus pesticide ) 是由 何丽珊 张娅 顾明华 曾东强 李雪生 何惠欢 何冰 梁琼月 易国强 温绍聪 陈能 于 2020-07-22 设计创作，主要内容包括：本发明提供一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用。以1份5-羟基-2-硝基苯甲酸甲酯溶解在3-4份乙腈中,加入2-3份无水碳酸钠和1.2份氯硫代磷酸二乙酯制得中间体；将中间体溶于3-4份四氢呋喃,加入等体积的6mol/L盐酸,制得化合物,即二乙氧基硫代磷酸酯类有机磷农药半抗原。将半抗原与载体蛋白偶联,制备得到完全抗原。本发明的制备方法简单易行,制备得到的二乙氧基硫代磷酸酯类有机磷农药半抗原、二乙氧基硫代磷酸酯类有机磷农完全抗原,可有效地刺激被免疫动物产生灵敏度高、广谱性强的抗体,可用于免疫分析中,满足国内对部分二乙氧基硫代磷酸酯类有机磷农药残留的检测。(The invention provides a preparation method and application of a hapten and a complete antigen aiming at diethoxy thiophosphate organophosphorus pesticides. Dissolving 1 part of methyl 5-hydroxy-2-nitrobenzoate in 3-4 parts of acetonitrile, and adding 2-3 parts of anhydrous sodium carbonate and 1.2 parts of diethyl chlorothiophosphate to prepare an intermediate; and dissolving the intermediate in 3-4 parts of tetrahydrofuran, and adding 6mol/L hydrochloric acid with the same volume to prepare a compound, namely diethoxy thiophosphate organophosphorus pesticide hapten. Coupling hapten and carrier protein to prepare complete antigen. The preparation method is simple and feasible, and the prepared diethoxy thiophosphate organophosphorus pesticide hapten and the prepared diethoxy thiophosphate organophosphorus pesticide complete antigen can effectively stimulate immunized animals to generate antibodies with high sensitivity and strong broad spectrum, can be used in immunoassay, and can meet the requirement of domestic detection of partial diethoxy thiophosphate organophosphorus pesticide residues.)

1. A diethoxy thiophosphate organophosphorus pesticide hapten has a molecular formula as follows:

2. a process for preparing the diethoxy phosphorothioate organophosphorus pesticide hapten as claimed in claim 1, which comprises the following steps:

1) reacting methyl 5-hydroxy-2-nitrobenzoate with diethyl chlorothiophosphate to obtain an intermediate methyl 5- ((diethoxyphosphonothio) oxy) -2-nitrobenzoate;

2) hydrolyzing the intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester under an acidic condition to obtain 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid, namely diethoxy thiophosphate organophosphorus pesticide hapten.

3. The method of claim 2, wherein:

in the step 1), the reaction is carried out in the presence of anhydrous potassium carbonate;

the molar ratio of the 5-hydroxy-2-nitrobenzoic acid methyl ester to the anhydrous potassium carbonate is as follows: 1: 2-3;

the molar ratio of the 5-hydroxy-2-nitrobenzoic acid methyl ester to the diethyl chlorothiophosphate is as follows: 1: 1-1.5;

the reaction is carried out under the condition of heating reflux, the temperature of the reaction is 80 ℃, and the time is 2-3 hours;

the reaction is carried out in an organic solvent, and the organic solvent can be acetonitrile;

and after the reaction is finished, cooling the reaction system to room temperature, adding water to quench the reaction system, adding dichloromethane to extract and stratify, collecting an organic layer, drying and concentrating the organic layer, and performing column chromatography to separate to obtain an intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester.

4. A method according to claim 2 or 3, characterized in that: in the step 2), the hydrolysis is carried out in an organic solvent, and the organic solvent can be tetrahydrofuran specifically;

the hydrolysis is carried out at room temperature, and the hydrolysis time is 4-12 hours;

the hydrolysis operation comprises the following steps: dissolving 1 part by mole of intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester in 3-4 parts of tetrahydrofuran, adding equal-volume 6mol/L hydrochloric acid, stirring the reaction system at room temperature for 12 hours, adding 20 parts of water to quench the reaction, adding 20 parts of ethyl acetate to extract and stratify, collecting an organic layer, drying and concentrating, and then performing column chromatography to obtain a compound: 5- ((diethoxy phosphorothioyl) oxy) -2-nitrobenzoic acid, namely diethoxy thiophosphate organophosphorus pesticide hapten.

5. The diethoxy thiophosphate organophosphorus pesticide complete antigen has the following structural formula:

pr represents a carrier protein;

the diethoxy thiophosphate organophosphorus pesticide complete antigen is obtained by coupling the diethoxy thiophosphate organophosphorus pesticide hapten and carrier protein.

6. A process for preparing the complete antigen of the diethoxy thiophosphate organophosphorus pesticide of claim 5, comprising the steps of:

1) dissolving diethoxy thiophosphate organophosphorus pesticide hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature to obtain solution A;

2) dissolving carrier protein in carbonate buffer solution to obtain solution B;

3) dripping the solution A into the solution B, and stirring to obtain solution C;

4) putting the solution C into a dialysis bag, dialyzing the solution C in a phosphate buffer solution, and collecting the solution in the dialysis bag to obtain the diethoxy thiophosphate organophosphorus pesticide complete antigen;

specifically, in the step 1), the dosage ratio of the diethoxy thiophosphate organophosphorus pesticide hapten, the dimethylformamide, the dicyclohexylcarbodiimide and the N-hydroxysuccinimide sequentially can be as follows: 20 mu mol: 1-2 mL: 60-120 mu mol; the stirring time at room temperature is as follows: 18-24 hours;

in the step 2), the carrier protein is bovine serum albumin BSA, and a complete immune antigen is prepared;

the carrier protein is egg serum albumin OVA, and a complete coating antigen is prepared;

the dosage ratio of the carrier protein to the carbonate buffer solution is as follows: 0.4 mu mol: 3-6 mL;

the molar ratio of the diethoxy thiophosphate organophosphorus pesticide hapten to the carrier protein is as follows: 20:1-40:1, specifically 20: 1;

in the step 3), stirring for 10-12 hours at the temperature of 4-25 ℃;

in the step 4), in the dialysis process, the liquid is changed once every 3 hours, and multiple times of dialysis can be carried out, specifically 6 times of dialysis can be carried out.

7. Use of the hapten of the diethoxy thiophosphate organophosphorus pesticide described in claim 1 or the complete antigen of the diethoxy thiophosphate organophosphorus pesticide described in claim 5 for preparing a broad-spectrum antibody of the diethoxy thiophosphate organophosphorus pesticide.

8. A broad-spectrum antibody prepared from the complete antigen of the diethoxy thiophosphate organophosphorus pesticide as defined in claim 5.

9. The use of the broad spectrum antibody of claim 8 for any one of:

1) the application in detecting the diethoxy thiophosphate organophosphorus pesticide;

2) the application in preparing a kit for ELISA detection of diethoxy thiophosphate organophosphorus pesticides;

3) the application in preparing colloidal gold test paper for detecting the diethoxy thiophosphate organophosphorus pesticide.

10. A diethoxy phosphorothioate organophosphorus pesticide detection reagent or kit prepared from the broad-spectrum antibody of claim 8.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a hapten and a complete antigen of diethoxy thiophosphate organophosphorus pesticide.

Background

Diethoxy thiophosphate organophosphorus pesticides (DDPs) are pesticides which are used for killing insects by inhibiting the activity of acetylcholinesterase and interfering the nerve impulse of insects, have the characteristics of high efficiency, broad spectrum and low cost and are widely used in agricultural production. Although some highly toxic products such as parathion, methyl parathion and the like are forbidden to use the pesticides at present, and pesticides such as phorate, sulfotep and coumaphos cannot be used or limited on vegetables, fruit trees, tea leaves and Chinese herbal medicines, the high-toxicity pesticides still remain the diethoxy thiophosphate organophosphorus pesticides in the environment due to long-term use in the past and prevalence of low-toxicity pesticides at present. Therefore, establishing a rapid detection method for the pesticide has important significance for monitoring the environment and detecting the residual diethoxy thiophosphate organophosphorus pesticides in edible vegetables, fruits, grains and Chinese herbal medicines.

At present, the detection method of the diethoxy thiophosphate organophosphorus pesticide mainly comprises Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC) which are provided with different detectors, but the chromatographic method has complex operation, time consumption and high cost, usually needs to carry out complicated sample pretreatment, is difficult to meet the requirements of quick and economic field real-time detection and sample large-scale screening detection, the enzyme inhibition detection method is a quick detection method commonly used for detecting diethoxy thiophosphate organophosphorus pesticides in the market, but the accuracy of the method is about 30-40 percent, the enzyme-linked immunoassay is an immunoassay based on antigen-antibody specific reaction, has the characteristics of strong specificity, high sensitivity, no need of complex sample pretreatment, simple and convenient operation and low cost, and simultaneously, the colloidal gold test strip developed based on the method can be used for on-site rapid screening detection. Farmers use pesticides in a mixing way, so that a plurality of pesticides may remain in one sample, and therefore, the antibody with high cross reaction rate is prepared, and broad-spectrum specific immunodetection of a plurality of pesticides can be realized. The hapten and the antigen with excellent quality can excite the immunized animal to generate an antibody with high sensitivity and strong recognition capability, so the invention designs the hapten and the complete antigen aiming at the diethoxy thiophosphate organophosphorus pesticide, provides the preparation method and the corresponding application thereof, and aims to lay a foundation for rapidly detecting the diethoxy thiophosphate organophosphorus pesticide in the environment and food.

Disclosure of Invention

The invention aims to provide a hapten and a complete antigen aiming at diethoxy thiophosphate organophosphorus pesticide, which can effectively stimulate immunized animals to generate antibodies with high sensitivity and broad spectrum.

The invention also aims to provide a preparation method of the diethoxy thiophosphate organophosphorus pesticide hapten and complete antigen, which is simple and easy to implement.

The hapten and the complete antigen of the diethoxy thiophosphate organophosphorus pesticide prepared by the invention can be used for screening monoclonal antibodies for identifying the diethoxy thiophosphate organophosphorus pesticide in a broad spectrum manner, and the prepared antibodies can be used for establishing an enzyme-linked immunoassay method of the diethoxy thiophosphate organophosphorus pesticide, and can be applied to the rapid detection of the diethoxy thiophosphate organophosphorus pesticide possibly remaining in food.

The molecular formula of the diethoxy thiophosphate organophosphorus pesticide hapten provided by the invention is as follows:

the hapten is prepared by a method comprising the following steps:

1) reacting methyl 5-hydroxy-2-nitrobenzoate with diethyl chlorothiophosphate to obtain an intermediate methyl 5- ((diethoxyphosphonothio) oxy) -2-nitrobenzoate;

2) hydrolyzing the intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester under an acidic condition to obtain 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid, namely diethoxy thiophosphate organophosphorus pesticide hapten.

In step 1) of the above method, the reaction may be carried out in the presence of anhydrous potassium carbonate;

the molar ratio of the methyl 5-hydroxy-2-nitrobenzoate to the anhydrous potassium carbonate can be: 1: 2-3;

the molar ratio of methyl 5-hydroxy-2-nitrobenzoate to diethyl chlorothiophosphate may be: 1: 1-1.5, specifically 1: 1.2;

the reaction is carried out under the condition of heating reflux, the temperature of the reaction can be 80 ℃, and the time can be 2-3 hours;

the reaction is carried out in an organic solvent, and the organic solvent can be acetonitrile;

and after the reaction is finished, cooling the reaction system to room temperature, adding water to quench the reaction system, adding dichloromethane to extract and stratify, collecting an organic layer, drying and concentrating the organic layer, and performing column chromatography to separate to obtain an intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester.

In step 2), the hydrolysis is performed in an organic solvent, which may be tetrahydrofuran;

the hydrolysis is carried out at room temperature, and the hydrolysis time can be 4-12 hours, specifically 12 hours;

the hydrolysis is specifically carried out as follows: dissolving 1 part (mole part) of intermediate 5- ((diethoxyphosphorylthio) oxy) -2-nitrobenzoic acid methyl ester in 3-4 parts of tetrahydrofuran, adding equal-volume 6mol/L hydrochloric acid, stirring the reaction system at room temperature for 12 hours, adding 20 parts of water to quench the reaction, adding 20 parts of ethyl acetate to extract and layer, collecting an organic layer, drying and concentrating, and performing column chromatography to obtain a compound: 5- ((diethoxy phosphorothioyl) oxy) -2-nitrobenzoic acid, namely diethoxy thiophosphate organophosphorus pesticide hapten.

The structural formula of the diethoxy thiophosphate organophosphorus pesticide complete antigen provided by the invention is as follows:

pr represents a conjugated carrier protein.

The diethoxy thiophosphate organophosphorus pesticide complete antigen is obtained by coupling the diethoxy thiophosphate organophosphorus pesticide hapten and carrier protein.

The invention also provides a preparation method of the complete antigen, namely, the diethoxy thiophosphate organophosphorus pesticide hapten is coupled with carrier protein by a carbodiimide or mixed anhydride method to prepare the complete antigen.

Specifically, the carrier protein is coupled to the carboxyl group of the hapten by an activated ester method.

The specific operation for preparing the complete antigen is as follows:

1) dissolving diethoxy thiophosphate organophosphorus pesticide hapten in dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stirring at room temperature to obtain solution A;

2) dissolving carrier protein in carbonate buffer solution to obtain solution B;

3) dripping the solution A into the solution B, and stirring to obtain solution C;

4) and (3) putting the solution C into a dialysis bag, dialyzing the solution C in a phosphate buffer solution, and collecting the solution in the dialysis bag, namely the diethoxy thiophosphate organophosphorus pesticide complete antigen.

In the step 1) of the method, the dosage ratio of the diethoxy thiophosphate organophosphorus pesticide hapten, the dimethylformamide, the dicyclohexylcarbodiimide and the N-hydroxysuccinimide can be as follows in sequence: 20 mu mol: 1-2 mL: 60-120 mu mol; the method specifically comprises the following steps: 20 mu mol: 1 mL: 60 mu mol;

the room temperature stirring time may be: 18-24 hours;

in the step 2), the carrier protein can be bovine serum albumin BSA to prepare a complete immune antigen;

the carrier protein is egg serum albumin OVA, and a complete coating antigen is prepared;

the ratio of the carrier protein to the carbonate buffer may be: 0.4 mu mol: 3-6mL, which can be specifically: 0.4 mu mol: 3 mL;

the molar ratio of the diethoxy thiophosphate organophosphorus pesticide hapten to the carrier protein can be: 20:1-40:1, specifically 20: 1;

in the step 3), stirring for 10-12 hours (specifically, 10 hours) at a temperature of 4-25 ℃ (specifically, 4 ℃);

in the step 4), in the dialysis process, the liquid is changed once every 3 hours, and multiple times of dialysis can be carried out, specifically 6 times of dialysis can be carried out.

The invention also provides application of the diethoxy thiophosphate organophosphorus pesticide hapten or the diethoxy thiophosphate organophosphorus pesticide complete antigen in preparation of a broad-spectrum antibody of the diethoxy thiophosphate organophosphorus pesticide.

It is still another object of the present invention to provide a broad-spectrum antibody prepared from the above-mentioned diethoxy phosphorothioate complete antigen of organophosphorus pesticide.

The broad spectrum antibodies include polyclonal antibodies, monoclonal antibodies, heavy chain antibodies, and recombinant antibodies.

Any one of the following uses of the broad spectrum antibody:

1) the application in detecting the diethoxy thiophosphate organophosphorus pesticide;

2) the application in preparing a kit for ELISA detection of diethoxy thiophosphate organophosphorus pesticides;

3) the application in preparing colloidal gold test paper for detecting the diethoxy thiophosphate organophosphorus pesticide.

The invention also provides a diethoxy thiophosphate organophosphorus pesticide detection reagent or kit prepared from the broad-spectrum antibody.

The invention adopts the technical scheme that 1 part of methyl 5-hydroxy-2-nitrobenzoate is dissolved in 3-4 parts of acetonitrile, 2-3 parts of anhydrous sodium carbonate and 1.2 parts of diethyl chlorothiophosphate are added to prepare an intermediate: methyl 5- ((diethoxyphosphonothio) oxy) -2-nitrobenzoate. Dissolving the intermediate in 3-4 parts of tetrahydrofuran, and adding equal volume of 6mol/L hydrochloric acid to prepare a compound: 5- ((diethoxy phosphorothioyl) oxy) -2-nitrobenzoic acid, namely diethoxy thiophosphate organophosphorus pesticide hapten. Coupling hapten and carrier protein to prepare complete antigen. The preparation method is simple and feasible, and the prepared diethoxy thiophosphate organophosphorus pesticide hapten and the prepared diethoxy thiophosphate organophosphorus pesticide complete antigen can effectively stimulate immunized animals to generate antibodies with high sensitivity and strong broad spectrum, can be used in immunoassay, and can meet the requirement of domestic detection of diethoxy thiophosphate organophosphorus pesticide residues.

Drawings

FIG. 1 shows the UV absorption spectrum of complete antigens PA-BSA (left) and PA-OVA (right) prepared by the present invention.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.