CN100392405C - Pesticide carbaryl artificial antigen and antibody and its preparation method and application - Google Patents

Abstract

Pesticide carbaryl artificial antigen and antibody and its preparation method and application, relating to the preparation of carbamate pesticide carbamate small molecule compound artificial hapten, antigen and antibody with 6-aminocaproic acid molecular structure and its use in the establishment of immune system Application of analytical methods. The invention overcomes the problems of cumbersome and complicated traditional physical and chemical analysis methods, high cost and slow analysis speed, and provides a simple, fast, sensitive and accurate immune analysis technique. It uses 1-(5-carboxypentyl)-3-(1-naphthyl)urea and N-(2-naphthoyl)-6-aminocaproic acid as haptens, respectively with carrier proteins such as hemocyanin, Horseradish peroxidase linked synthetic artificial antigen and enzyme-labeled antigen. The artificial antigen is then immunized in animals, blood is taken, antiserum is separated, and antibodies are purified to obtain antibodies. The antibody is stable, has good specificity and sensitivity, and has a simple synthesis method, can be used for rapid immunodetection of the pesticide carbaryl, and has good application prospects.

Description

Pesticide carbaryl artificial antigen and antibody and its preparation method and application

technical field

The invention belongs to the technical field of immunochemistry and residue analysis of small-molecule pesticide compounds (molecular weight less than 1000 Daltons); relates to organic synthesis, immunochemistry, biochemistry and physicochemical testing techniques, etc. Design and synthesis of artificial haptens and specific antigens of carbamate pesticide small molecule compound carbamate, and preparation of specific antibodies for immunized animals and their application in the establishment of immunoassay methods.

Background technique

Carbaryl (also known as carbaryl) is a representative product of carbamate pesticides. In my country, carbaryl can be used for 141 kinds of crops and control 565 kinds of pests. It is currently the most widely registered insecticide. Carbaryl is widely used in vegetables, cotton, and field crops. Carbaryl is used as a flower thinning agent on fruit trees, and it is used to control harmful organisms in fish ponds in aquaculture. Because carbaryl is widely used in my country, carbaryl residues exist in many agricultural products in our country. With the continuous expansion of the scale of pesticide use, the impact of pesticide residues on the environment and the chronic and long-term effects on human health have attracted increasing attention and concern. For this reason, the restrictions on pesticide residues are becoming more and more stringent, and new requirements and higher standards are put forward for the analysis and measurement objects, types, quantities, ranges, indicators and other aspects. my country has stricter testing standards for carbaryl residues in agricultural products. According to GB 5009.21 regulations on carbaryl residues in grain, oil and vegetables: grain ≤ 5.0mg/kg, vegetables ≤ 2.0mg/kg, fruits ≤ 2.5mg/kg, edible oil ≤ 0.5mg/kg, tobacco ≤ 1.0 mg/kg.

However, due to the small molecule toxic chemicals with a molecular weight less than 100dolton (Dalton), such as pesticides and their metabolites, the traditional residual analysis methods mainly rely on gas chromatography (GC), liquid chromatography (HPLC) or mass spectrometry, etc. means of physical analysis. The traditional physical and chemical analysis method is cumbersome and complicated, with high cost and slow analysis speed, and it is difficult to meet the needs of actual analysis. Therefore, it is urgent to develop simple, fast and sensitive analysis techniques.

Different from macromolecules, immunoassays for small molecule compounds have their own characteristics:

(1) Small molecule compounds [MW ≤ 1000dolton] are generally not immunogenic, and cannot directly immunize animals to produce specific antibodies. They must synthesize haptens that highlight the specific parts of the three-dimensional structure of the molecule, and link them with macromolecular carriers to form conjugates. In order to immunize animals to produce specific antibodies against this target small molecule compound. The combination of this hapten and macromolecule carrier is called artificial antigen. The preparation of artificial antigen is not arbitrary, including the binding site, binding mode, carrier type, and any structural differences between the hapten and the target analyte, such as size, shape, composition, configuration, conformation, polarity, electron cloud density, etc. Various factors, such as, may greatly affect the properties of the corresponding antibodies, so they are the key to determine the production of specific antibodies and the establishment of immunoassay methods.

(2) Although the small molecular compound is not immunogenic, it has reactogenicity, that is, it has the ability to react immunologically with the corresponding antibody, and can be quantified in vitro, following the law of mass action.

(3) Analytical techniques for detecting small molecular compounds based on antigen-antibody immune reaction, currently, enzyme-linked immunoassay (ELISA) is mostly used. The use of enzymatic reactions to display the quantitative combination of antigens and antibodies is simple to operate and has considerable sensitivity. It has developed rapidly in recent years. The pesticide ELISA and simple enzyme immunology EIA technology developed since the 1980s have enabled the analysis of pesticide residues to gain greater vitality in the method; it is quite suitable for the rapid analysis of sample matrices that are too complex and difficult to analyze with ordinary physical and chemical methods. Value.

Immunoassay technology has been introduced into the field of pesticide residue analysis, and has become one of the most promising quantitative analysis techniques for development and application, and has received extensive attention. The first report on the immunoassay of pesticides was the antiserum of the pesticide malathion prepared by Centen et al. in 1967. In 1980, after Hammock and Mumma further explored the theory and technology of pesticide immunoassay methods, the ELISA pesticide analysis technology entered a new stage.

Pesticide small molecule compounds (molecular weight less than 1000 Daltons), rely on the basic principles of immunology, immunochemistry and biotechnology means. The key to this technical research is the molecular design and synthesis of haptens and the preparation of artificial whole antigens and antibodies. Therefore, the molecular immunological properties of target analytes and how to highlight and utilize these properties through chemical or biochemical techniques are important research contents in this field. This technology has become a new field of microanalysis research, and it can be regarded as a new analysis method alongside traditional analysis methods. The artificial antigen and specific antibody of carbaryl and the establishment of immunoassay method based on it have not been reported yet.

Contents of the invention

issues that need resolving:

In view of the above situation, there is an urgent need for a simple, fast and effective method for the detection of the pesticide carbaryl. The purpose of the present invention is by designing and synthesizing the hapten and the artificial antigen of the carbamate pesticide carbamate with 6-aminocaproic acid molecular structure; Unique feature is to highlight this type of pesticide molecular specific antigenic determinant, overcome again Overcoming the difficulty of chemical synthesis, the immunized animals induced specific antibodies with high affinity; and based on this, the ELISA method, that is, the rapid immunoassay method, was established to accurately detect the carbamate pesticide carbaryl.

Technical solutions:

The present invention designs and synthesizes small molecule target analyte hapten, and couples with carrier protein to prepare effective artificial antigen, immunizes animals to prepare small molecule analyte-specific antibody, utilizes the specific immunological reaction of antigen and antibody and is easily detected Detect the amplification effect of the identified marker, so as to qualitatively and quantitatively detect the ultra-trace small molecule target analyte in the sample, which can be used for sample determination. Its selectivity depends on the specificity of the immunological response, and its sensitivity depends on the affinity of the antibody and the detectability of the marker. Therefore, the residual amount of carbaryl with 1-naphthyl-N-carbamate structure in the sample can be analyzed and detected rapidly and accurately. The key to this technical research is the molecular design and synthesis of haptens and the preparation of artificial whole antigens and antibodies.

The structure of carbaryl is shown in the figure below:

The molecular structure of carbaryl can be divided into two structural units: one part is naphthyl, and the other part is carbamate. From the perspective of immunology, naphthyl has a complex spatial structure and a large molecular weight, which can be used as an antigenic determinant, while carbamate can be regarded as a linear long-chain molecule in the overall structure, which can be used as an arm connected to a carrier protein. . Therefore, when synthesizing a hapten, the naphthyl structure must be retained, and the carbamate chain can be appropriately modified to form an arm that can be linked to a carrier protein. Therefore, when designing and synthesizing the carbaryl hapten in this patent, a naphthyl-containing compound with active groups such as formyl groups is used to react with aminocaproic acid, and a synthetic method of introducing active side chains with different structures is used to synthesize the carbaryl hapten In this way, the structural similarity between the carbaryl hapten and carbaryl is maintained, and the hapten molecule has a suitable structure connected with the carrier protein.

The design and synthesis of hapten is the key to the success of this method. For highlighting the specific antigenic determinant of this type of pesticide molecule, the present invention selects 1-naphthyl-N-carbamate (b) structure

It is the basic structure of hapten, and its molecular weight MW is 300.

The present invention uses 1-(5-carboxypentyl)-3-(1-naphthyl)urea as a hapten, which is connected with a carrier protein and has a synthetic molecular formula of

or

compounds are artificial antigens.

The present invention uses N-(2-naphthoyl)-6-aminocaproic acid (c) as a hapten

Linked with horseradish peroxidase as an enzyme-labeled antigen, its molecular weight MW is 285.

The above-mentioned artificial antigens are then immunized by animals, blood is collected, anti-whole serum is separated, and specific antibodies are obtained by purification.

The preparation method of pesticide carbaryl (carbaryl) specific antibody:

(1) Hapten design and synthesis

Using a naphthyl-containing compound with a formyl active group to react with aminocaproic acid to synthesize a hapten containing a 1-naphthyl-N-carbamate structure, the specific method is:

①Synthesis of the hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea:

Take 1-naphthyl isocyanic acid and 6-aminocaproic acid mixed in tetrahydrofuran (THF), the molar ratio of the three is 1:120-130:120-130, stir at 22-25°C for 12-16 hours , and then filtered to obtain the hapten, and finally the solid was recrystallized in acetonitrile, and the solvent was evaporated to obtain the pure compound hapten;

②Synthesis of hapten N-(2-naphthoyl)6-aminocaproic acid (c):

Weigh 2-naphthoyl chloride and dissolve it in 1,4-dioxane, in an ice bath, add this material dropwise to the aqueous solution containing 6-aminocaproic acid and potassium hydroxide and mix to make 2- The molar ratio of naphthoyl chloride, 1,4-dioxane and 6-aminocaproic acid is 1:12-13:1, the mixture is stirred at 22-25°C for 4 hours, and then acidified with hydrochloric acid , to obtain a white solid hapten by filtration, and finally extract the filtrate with ether, from which a white crystal hapten is crystallized;

(2) Synthesis of artificial antigen

The hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea was used to connect to ovalbumin (OA) or hemocyanin, respectively, by the active ester method with the amide group CONH as a bridge (keyhole limpethemocyanin, KLH), synthesize the artificial antigen; the specific method is: take the hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea, dissolve it in N,N-dimethylformamide In DMF, add N-hydroxysuccinimide (NHS) dissolved in DMF solution and N, N-dicyclohexylcarbodiimide dissolved in DMF solution, so that the hapten 1-(5-carboxypentyl)-3- The molar ratio of (1-naphthyl)urea, N-hydroxysuccinimide (NHS) and N, N-dicyclohexylcarbodiimide is 1:4-5:3-4, at 22-25°C Stir and react for 1 hour, then put it in a refrigerator at 4°C for 18 hours, centrifuge to remove the precipitate, then add the upper activated ester solution to the protein PBS solution with pH=7.0, and the molar ratio of hapten to protein is 10-50:1 , reacted at 4°C for 5 hours after stirring, and finally put the reaction solution into a dialysis bag, dialyzed in PBS with pH=7.4 at 4°C, accurately measured the volume of the protein conjugate solution, measured the concentration and binding ratio, and analyzed Pack and store at -20°C.

(3) Preparation of enzyme-labeled antigen

Link the hapten N-(2-naphthoyl)-6-aminocaproic acid with horseradish peroxidase to make an enzyme-labeled antigen for color reaction, the specific method is the same as the synthesis of artificial antigen (2);

(4) Immunization and specific antibody preparation

Immunization: Female white rabbits are selected as the immunized animals. Subcutaneous and intramuscular injections are used for immunization. Four booster immunizations are carried out after the primary immunization. The specific methods are as follows:

Initial immunization: Take 1 mg of the above-mentioned artificial antigen and dissolve it in a solution prepared by equal volumes of 0.9% NaCl solution and Freund's complete adjuvant for animal immunization;

Booster immunization: use 0.5 mg of the above-mentioned artificial antigen dissolved in 0.9% NaCl solution and an equal volume of Freund's incomplete adjuvant to immunize animals; booster immunization is performed 2 weeks, 4 weeks and 6 weeks after the initial immunization Immunizations were given three times, with one-month intervals thereafter. Nine days after the fifth immunization, blood was collected from the rabbit's ear vein for titer detection;

Antibody purification: regularly monitor the animal antibody titer, when the antibody reaches a suitable titer for a certain coated antigen, collect blood and centrifuge to obtain antiserum, use G-Sepharose protein affinity chromatography column to purify the antiserum, and prepare IgG antibodies.

The pesticide carbaryl antibody prepared above can be used for immunodetection of carbaryl.

Establishment of immunoassay method and optimization of assay conditions:

The square array experiment was used to determine the antigen-antibody binding titer, affinity, and the optimal titer of the antibody and enzyme marker of the ELISA method. The present invention establishes the rapid immunoassay method and laboratory standard detection method of carbaryl, and analyzes the factors affecting the measurement such as pH value and ionic strength, determines the best working conditions, and establishes a standard curve: that is, the target analyte concentration vs. Correlation curve of antibody inhibition rate (or binding rate B/B ₀ ). Inhibition rate of target analyte concentration on antibody I=(A _max -A _min )-(A _i -A _min )/(A _max -A _min )*100

The binding rate of antibody to antigen B/B ₀ =(A _i -A _min )/(A _max -A _min )*100

In the formula: _Amax is the average absorbance value of the blank well; _Amin is the average absorbance value of the serum-free control well before immunization. A _i is the average absorbance value of the sample well. Draw the standard curve with the inhibition rate or binding rate B/B ₀ as the vertical axis and the analyte concentration C as the horizontal axis.

Antibody specificity: the degree of cross-reactivity between the antibody and structurally similar compounds, expressed as a percentage of the ratio of the concentration I _50i of the target analyte required to inhibit 50% of the maximum binding rate of the antibody to the concentration I _50M of various structurally similar compounds required , that is, the cross-reactivity rate CR (%).

CR(%)=I _50i /I _50M *100

The smaller the cross-reactivity, the higher the specificity of the antibody.

The pesticide carbaryl antibody prepared above can be used in the immunoassay method of carbaryl, and its method is as follows:

Coating: Dissolve the prepared antibody in 50mmol, pH9-10 carbonic acid buffer to prepare a 10mg/mL coating solution, add 100μl coating solution to each well of the enzyme-labeled strip for 12-16 hours, and Each well of the coated enzyme-labeled strip was washed three times with PBST, 0.05% (v/v) of phosphate buffer saline, and Tween20 washing solution;

Blocking: add 150-200 μl, 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour;

Adding samples: Dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the standard sample of carbaryl in PBS containing 1% BSA. When adding samples, add 50 μl of the above sample or standard sample to be tested;

Competitive reaction: Dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the test sample or standard sample, add 50 μl enzyme-labeled antigen solution to each well, and incubate for 10 minutes or 60 minutes Wash three times with plate washing solution;

Color development: Tetramethylbenzidine (TMB) is used as the chromogenic substrate, and 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution (5 mg tetramethylbenzidine solution is dissolved in 1 ml substrate buffer) is added to each well. After 5 minutes or 30 minutes of color development, 50 μl of 5 mol/L sulfuric acid was added to each well to stop; the reaction solution was read on an automatic microplate reader.

It should be noted:

The above-mentioned sample to be tested is the pesticide carbaryl extract of the item to be tested, and the rapid extraction method of the extract adopts the methanol extraction method.

The specific method of methanol extraction method is: use 5 times the volume of methanol to soak the unground sample for 12-16 hours; or soak 10g of the crushed sample in 50ml of methanol and shake for 2 minutes; or use 10g of the crushed sample Soak in 50ml of methanol, stir for 2 minutes, let stand, take the solution, and then get the sample to be tested.

Description of drawings:

Figure 1: Relationship curve between different concentrations of carbaryl and inhibition rate

Beneficial effect:

Compared with other similar methods, the present invention has the characteristics of specificity, sensitivity, accuracy, rapidity, convenience and cheapness. The designed and synthesized hapten has a high degree of similarity to the target analyte, and the characteristic structure of the analyte remains intact, laying a foundation for the preparation of an antibody with good specificity. The antibody has good specificity and sensitivity, the detection limit reaches 1/1x10 ¹⁰ ng/ml, and the dilution of the antibody can reach 1/1x10 ⁶ . The obtained antibody and enzyme-labeled antigen are stable and have the advantage of long storage time at room temperature.

It has been verified by experiments that the synthesis method of the above-mentioned haptens is simple, and the main raw materials used such as 1-naphthoyl chloride, 1,4-dioxane, and 6-aminocaproic acid are relatively cheap and easy to obtain. Reagent companies are available for purchase. Due to the high synthesis efficiency and few reaction steps, the hapten can be synthesized in only three steps, thereby improving the controllability of the reaction, and the reaction yields reach 96% and 86% respectively. In addition, the extraction and purification methods of the synthetic product are simple and convenient, and the high-purity target product can be obtained only by crystallizing the synthetic product. Therefore, compared with other methods, the method for synthesizing haptens of the present invention is easier to popularize.

The rapid detection method provided by the invention is easy and fast to operate, and it only takes 40-60 minutes to complete the entire detection operation process, and the detection accuracy can reach more than 90%, which is very suitable for the needs of on-site detection. Antibodies and enzyme-labeled antigens can be stored at room temperature for 1 week, and enzyme-labeled antigen-packed enzyme-labeled plates can be stored at 4°C for more than 6 months, which provides great opportunities for centralized detection of large-scale samples. convenient. Therefore, the present invention not only performs well in laboratory testing, but also lays a foundation for developing an ELISA rapid detection tool with low cost, high detection efficiency and easy operation, and has good application prospects; it has both economic benefits and social benefits.

Detailed ways

Example 1:

1. Hapten Synthesis

In the present invention, the structure of 1-naphthyl-N-carbamate (b) is selected as the basic structure of the hapten, and its molecular weight MW is 300. The design synthesized:

1) Hapten 41-(5-carboxypentyl)-3-(1-naphthyl)urea

Mix 1.7g (0.01mol) of 1-naphthyl isocyanic acid and 0.1g (1.30mol) of 6-aminocaproic acid in 100mL of tetrahydrofuran (THF), stir for 12 hours at 22°C, and filter to obtain 2.0 g of the hapten as a solid; recrystallized in acetonitrile and evaporation of the solvent gave 0.9 g of pure compound in an overall yield of 96%.

2) Hapten 3 (N-(2-naphthoyl)-6-aminocaproic acid)

3.8g (0.02mol) of 2-naphthoyl chloride was dissolved in 20ml of 1,4-dioxane, and in an ice bath, this material was added dropwise to 100ml containing 2.6g (0.02mol) of 6- Aminocaproic acid and 8.0 g (0.30 mol) of potassium hydroxide were mixed in an aqueous solution. The mixture was stirred at 25°C for 4 hours. After acidification with hydrochloric acid, white solid hapten 3 was obtained by filtration, from which a white crystal hapten was crystallized from the ether extraction filtrate, and the total yield was 86%.

2. Synthesis of artificial antigen

The hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea was used to connect to hemocyanin (keyhole limpet hemocyanin, KLH) respectively by the active ester method (AE method for short), Synthetic artificial antigens; specifically:

Accurately weigh 10 mg of the hapten 1-(5-carboxypentyl)-3-(1-naphthyl) urea and dissolve it in 300 μl N, N-dimethylformamide DMF, add 13.6 mg (0.14 mmol) N-hydroxy Succinimide (NHS) (dissolved in 300 μl DMF solution) and 24 mg N, N-dicyclohexylcarbodiimide (dissolved in 300 μl DMF solution), at 22 ° C, stirred for 1 hour, and then placed at 4 ° C In the refrigerator, wait for 18 hours, there is turbidity, centrifuge to remove the precipitate, add the upper activated ester solution to 5ml protein PBS solution (pH=7.0, 0.01mol/ml) with a concentration of 4mg/ml, stir at 4°C React for 5 hours, then put the reaction solution into a dialysis bag, dialyze in 0.01mol/ml PBS with pH=7.4 at 4°C, then accurately measure the volume of the protein conjugate solution, measure the concentration and binding ratio, and pack , stored at -20°C.

3. Preparation of enzyme-labeled antigen

The hapten N-(2-naphthoyl)-6-aminocaproic acid is linked with horseradish peroxidase to make enzyme-labeled antigen for color reaction. The specific method is the same as the synthesis of artificial antigen (2);

4. Immunization and specific antibody preparation

·immunity:

As animals, male white rabbits, 3 months old and 1.5 kg in weight, were raised in a standard experimental animal room, observed continuously for 3 days, and immunized after confirming that their physical condition was normal.

Initial immunization: Accurately weigh 1 mg of artificial antigen dissolved in 0.5 ml of 0.9% NaCl solution and 0.5 ml of complete Freund's adjuvant for immunization.

For booster immunization, a solution prepared by dissolving 0.5 mg artificial antigen in 0.5 ml 0.9% NaCl solution and 0.5 ml Freund's incomplete adjuvant was used. Booster immunizations were given three times at 2 weeks, 4 weeks and 6 weeks after the initial immunization, with an interval of one month thereafter. Nine days after the fifth immunization, blood was collected from the rabbit's ear vein for titer detection.

Preparation of antibodies purified from antiserum

The animal antibody titer was monitored regularly, and when the endpoint titer reached more than 200,000, whole blood was collected from the carotid artery. The collected whole blood was rested at 22 °C for 2 hours and then at 4°C for 8 hours, then centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected and stored at -20°C. The serum obtained after centrifugation was filtered with G-Sepharose CL-4B immunoaffinity chromatography column, and the antiserum was purified to prepare IgG antibody.

5. Establishment of immunoassay method

The items to be tested are three kinds of grains: sorghum, barley and wheat. Using the rapid immunoassay method, the steps of detecting their carbaryl content are as follows:

1) Extract the sample to be tested—methanol extraction method①

10 g of unground samples of barley, wheat, and sorghum were soaked in 50 ml of methanol, respectively, for 12 hours. The soaking solution is the sample to be tested.

2) Detection method and square matrix test evaluation method

Adopt rapid immunoassay method (the test uses 8-well enzyme labeling strip):

Coating: Dissolve the prepared antibody in 50 mmol, pH 9.6 carbonic acid buffer to prepare a coating solution of 10 μg/mL. Add 100 μl of coating solution to each well of the strip for 12 hours. Each well of the coated enzyme-labeled strip was washed three times with PBST (phosphate buffer saline 0.05% (v/v), Tween 20) washing solution.

Blocking: add 150 μl of 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour.

·Adding samples: dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the carbaryl standard sample in PBS containing 1% BSA. When adding samples, add 50 μl of the above-mentioned sample or standard sample to each well.

Competitive reaction: dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the sample or standard sample to be tested, add 50 μl enzyme-labeled antigen solution to each well, incubate for 10 minutes and wash the plate Washed three times.

Color development: Tetramethylbenzidine (TMB) was used as a color development substrate. Add 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution (5 mg tetramethylbenzidine solution dissolved in 1 ml substrate buffer) to each well, add 50 μl 5 mol/L sulfuric acid to each well after 5 minutes of color development to stop. The reaction solution was read on an automatic microplate reader.

)分别为1％的BSA-PBS标样、大麦提取液、高梁提取液和小麦提取液的西维因抑制率标准曲线。从试验结果可以看出，西维因抗体的特异性很好。The square array test results of the above detection method are shown in the standard curve of inhibition rate of carbaryl as follows. In Figure 1 (■), (●), (▲), (

) are the standard curves of carbaryl inhibition rate of 1% BSA-PBS standard sample, barley extract, sorghum extract and wheat extract respectively. It can be seen from the test results that the specificity of the carbaryl antibody is very good.

·Sensitivity and specificity analysis:

(1) Sensitivity

See the table below for the sensitivity analysis results of the carbaryl detection method:

Carbaryl sensitivity analysis results Table 1

The analysis results in Table 1 show that the sensitivity of the rapid detection method is lower than that of the standard detection method, but it can meet the sensitivity requirements of rapid detection.

(2) specificity

Antibodies prepared by the present invention and established immunoassay methods have no cross-reaction to commonly used pesticides such as chlorpyrifos, dichlorvos, fenitrothion, and structurally similar pesticides bimauron, 1-naphthol, naphthalene, propoxur, etc., which shows that the present invention The prepared antibody is highly specific to the target detection object carbaryl.

6. Antibody Stability Test

The rapid detection method has high requirements on the stability of antibodies and enzyme-labeled antigens at room temperature, so the present invention has carried out verification experiments on this.

Stability test results of freeze-dried antibodies Table 2

^* The results shown in Table 2 are the relative value of the preservation board and the current cladding board (the current cladding board is 100%)

Stability test results of lyophilized and solution state enzyme-labeled antigen Table 3

*The results shown in Table 3 are the relative values of treatment and 4°C storage liquid HRP (4°C storage liquid HRP=100%)

The test results show that various storage conditions have no obvious effect on the activity of the antibody and the enzyme-labeled antigen, and the activities of the antibody and the enzyme-labeled antigen are very stable under various storage conditions, which can meet the needs of rapid detection methods.

Example 2:

During the immunoassay, extract the sample using the methanol extraction method ② that is: use 10g of crushed barley, wheat and sorghum to be tested, respectively, soak in 50ml of methanol and shake for 2 minutes, let it stand, and take the solution to obtain the sample to be tested . The resulting soaking solution is the sample to be tested. Others are with embodiment 1.

Example 3:

During the immunoassay, extract the samples using the methanol extraction method ③ that is: use 10g of crushed barley, wheat and sorghum to be tested, soak them in 50ml of methanol and stir for 2 minutes, let stand, and take the solution to obtain the samples to be tested . The resulting soaking solution is the sample to be tested. Others are with embodiment 1.

Example 4:

Artificial antigen synthesis, using ovalbumin (OA);

The detection method adopts the laboratory detection method (the test uses 8-well enzyme labeling strip):

Coating: Dissolve the prepared antibody in 50mmol, pH 9.6 carbonic acid buffer to prepare a 10mg/mL coating solution. Add 100 μl of coating solution to each well of the enzyme-labeled strip overnight. Each well of the coated enzyme-labeled strip was washed three times with PBST (phosphate buffer saline 0.05% (v/v), Tween 20) washing solution.

Blocking: add 150 μl of 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour.

·Adding samples: dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the carbaryl standard sample in PBS containing 1% BSA. When adding samples, add 50 μl of the above-mentioned sample or standard sample to each well.

Competitive reaction: dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the sample or standard sample to be tested, add 50 μl enzyme-labeled antigen solution to each well, incubate for 60 minutes and wash the plate Washed three times.

Color development: Tetramethylbenzidine (TMB) was used as a color development substrate. Add 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution (5 mg tetramethylbenzidine solution dissolved in 1 ml substrate buffer) to each well, add 50 μl 5 mol/L sulfuric acid to each well after 30 minutes of color development to stop. The reaction solution was read on an automatic microplate reader. Others are with embodiment 1.

Example 5:

The detection method adopts the laboratory detection method (the test uses 8-well enzyme labeling strip):

Coating: Dissolve the prepared antibody in 50 mmol, pH 9.6 carbonic acid buffer to prepare a 10 mg/mL coating solution. Add 100 μl of coating solution to each well of the enzyme-labeled strip overnight. Each well of the coated enzyme-labeled strip was washed three times with PBST (phosphate buffer saline 0.05% (v/v), Tween 20) washing solution.

Blocking: add 150 μl of 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour.

·Adding samples: dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the carbaryl standard sample in PBS containing 1% BSA. When adding samples, add 50 μl of the above-mentioned sample or standard sample to each well.

Competitive reaction: dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the sample or standard sample to be tested, add 50 μl enzyme-labeled antigen solution to each well, incubate for 60 minutes and wash the plate Washed three times.

Color development: Tetramethylbenzidine (TMB) was used as a color development substrate. Add 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution (5 mg tetramethylbenzidine solution dissolved in 1 ml substrate buffer) to each well, add 50 μl 5 mol/L sulfuric acid to each well after 30 minutes of color development to stop. The reaction solution was read on an automatic microplate reader. Others are with embodiment 2.

Embodiment 6:

The detection method adopts the laboratory detection method (the test uses 8-well enzyme labeling strip):

Coating: Dissolve the prepared antibody in 50mmol, pH 9.6 carbonic acid buffer to prepare a 10mg/mL coating solution. Add 100 μl of coating solution to each well of the enzyme-labeled strip overnight. Each well of the coated enzyme-labeled strip was washed three times with PBST (phosphate buffer saline 0.05% (v/v), Tween 20) washing solution.

Blocking: add 150 μl of 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour.

·Adding samples: dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the carbaryl standard sample in PBS containing 1% BSA. When adding samples, add 50 μl of the above-mentioned sample or standard sample to each well.

Competitive reaction: dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the sample or standard sample to be tested, add 50 μl enzyme-labeled antigen solution to each well, incubate for 60 minutes and wash the plate Washed three times.

Color development: Tetramethylbenzidine (TMB) was used as a color development substrate. Add 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution (5 mg tetramethylbenzidine solution dissolved in 1 ml substrate buffer) to each well, add 50 μl 5 mol/L sulfuric acid to each well after 30 minutes of color development to stop. The reaction solution was read on an automatic microplate reader. Others are the same as embodiment 3.

Claims (6)

1. Pesticide carbaryl artificial antigen is characterized in that with 1-(5-carboxypentyl)-3-(1-naphthyl)urea as hapten, it is connected with carrier protein and the synthetic molecular formula is

compounds are artificial antigens. 2. Pesticide carbaryl antibody, characterized in that 1-(5-carboxypentyl)-3-(1-naphthyl)urea is used as a hapten and is connected to a carrier protein to synthesize an artificial antigen

After animal immunization, blood was collected, anti-whole serum was separated, and antibodies were purified. 3. the preparation method of pesticide carbaryl artificial antigen described in claim 1 is characterized in that it is to make with following steps: (1) Hapten design and synthesis Using a naphthyl-containing compound with a formyl active group to react with aminocaproic acid to synthesize a hapten containing a 1-naphthyl-N-carbamate structure, the specific method is: Synthesis of Hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea Take 1-naphthyl isocyanic acid and 6-aminocaproic acid mixed in tetrahydrofuran (THF), the molar ratio of the three is 1:120-130:120-130, stir at 22-25°C for 12-16 hours , and then filtered to obtain the hapten, and finally the solid was recrystallized in acetonitrile, and the solvent was evaporated to obtain the pure compound hapten; (2) Synthesis of artificial antigen The hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea was used to connect to ovalbumin (OA) or hemocyanin, respectively, by the active ester method with the amide group CONH as a bridge (keyhole limpethemocyanin, KLH), synthetic artificial antigen: the specific method is: Take the hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea and dissolve it in N,N-dimethylformamide DMF, add N-hydroxysuccinimide (NHS) and dissolve it in DMF solution and N, N-dicyclohexylcarbodiimide were dissolved in DMF solution, so that the hapten 1-(5-carboxypentyl)-3-(1-naphthyl)urea, N-hydroxysuccinimide (NHS ) and N, N-dicyclohexylcarbodiimide in a molar ratio of 1:4-5:3-4, stirred and reacted for 1 hour at 22-25°C, and then placed in a refrigerator at 4°C for 18 hours, Centrifuge to remove the precipitate, then add the upper activated ester solution into the protein PBS solution with pH=7.0, the molar ratio of hapten to protein is 10-50:1, react at 4°C for 5 hours after stirring, and finally put the reaction solution into Put it into a dialysis bag, dialyze in PBS with pH=7.4 at 4°C, accurately measure the volume of the protein conjugate solution, measure the concentration and binding ratio, aliquot, and store at -20°C. 4. the preparation method of pesticide carbaryl antibody described in claim 2 is characterized in that immunity and specific antibody preparation method are as follows: Immunization: Female white rabbits were selected as immunized animals. Subcutaneous and intramuscular injections were used for immunization. Four booster immunizations were carried out after the primary immunization. The booster immunizations were immunized three times after 2 weeks, 4 weeks, and 6 weeks after the initial immunization, and thereafter an interval of one month month, 9 days after the fifth immunization, blood was taken from the rabbit's ear vein for titer detection; the specific method is: Initial immunization: Dissolve 1 mg of the above-mentioned artificial antigen in a solution prepared by 0.9% NaCl solution and Freund's complete adjuvant in equal volumes to immunize animals; Booster immunization: immunize animals with a solution prepared by dissolving 0.5 mg of the above-mentioned artificial antigen in 0.9% NaCl solution and an equal volume of Freund's incomplete adjuvant; Antibody purification: regularly monitor the animal antibody titer, when the antibody reaches a suitable titer for a certain coated antigen, collect blood and centrifuge to obtain antiserum, use G-Sepharose protein affinity chromatography column to purify the antiserum, and prepare IgG antibodies. 5. the application of the pesticide carbaryl artificial antigen or antibody described in claim 1 or 2 in the immunoassay method of carbaryl is characterized in that the method is as follows: Coating: Dissolve the above antibody in 50mmol, pH9-10 carbonic acid buffer to prepare a 10mg/mL coating solution, add 100μl coating solution to each well of the enzyme labeling strip for 12-16 hours, and coat the For each well of the enzyme-labeled strip, wash three times with PBST, phosphate buffered saline 0.05% (v/v), Tween20 washing solution; Blocking: add 150-200 μl, 1% bovine serum albumin (BSA)/PBS blocking solution to each well, and block for one hour; Adding samples: Dissolve the sample to be tested in PBS containing 1% BSA, and dissolve the standard sample of carbaryl in PBS containing 1% BSA. When adding samples, add 50 μl of the above sample or standard sample to be tested; Competitive reaction: Dissolve the enzyme-labeled antigen in 0.1% fish skin glue (FG)-PBS buffer, add the test sample or standard sample, add 50 μl enzyme-labeled antigen solution to each well, and incubate for 10 minutes or 60 minutes Wash three times with plate washer: Color development: use tetramethylbenzidine (TMB) as the substrate for color development, add 150 μl tetramethylbenzidine (TMB)-hydrogen peroxide solution to each well, add 50 μl 5mol/L per well after color development for 5 minutes or 30 minutes Sulfuric acid terminated, wherein the tetramethylbenzidine (TMB)-hydrogen peroxide solution used was prepared by dissolving 5 mg of tetramethylbenzidine solution in 1 ml of substrate buffer; the reaction solution was read on an automatic microplate reader. 6. according to the application of pesticide carbaryl artificial antigen or antibody in carbaryl immunoassay method according to claim 5, it is characterized in that the sample to be tested is the pesticide carbaryl extract of the article to be tested, the extract The rapid extraction method adopts the methanol extraction method. The specific method is: use 5 times the volume of methanol to soak the unground sample for 12-16 hours; or soak 10g of the ground sample in 50ml methanol and shake for 2 minutes; or use Soak 10 g of crushed samples in 50 ml of methanol, stir for 2 minutes, let stand, and take the supernatant.