Hapten for detecting the content of trifloxystrobin and application thereof

Abstract

The application discloses an oxycarbamazepine content detection hapten and application thereof, and particularly relates to an oxycarbamazepine content detection hapten and a preparation method thereof, and further discloses application of the hapten in antibodies and kits. The enzyme-linked immunoassay kit established for the oxycarbamazepine content detection hapten can realize rapid detection of a large number of samples to be detected, is convenient to use, low in detection cost, efficient, accurate, rapid, can simultaneously detect a large number of samples, and is suitable for on-site monitoring and screening of a large number of samples of oxycarbamazepine in agricultural products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide residue detection, and particularly relates to a hapten for detecting the content of trifloxystrobin and application thereof. BACKGROUND

[0002] Trifloxystrobin belongs to the methoxy acrylate fungicide, and plays an inhibitory role on bacteria by preventing the synthesis of cell adenosine triphosphatase and inhibiting the respiration of mitochondria. Trifloxystrobin has a strong inhibitory effect on most fungi. Due to the characteristics of high efficiency, broad spectrum and long persistence, and safety to crops and environment, trifloxystrobin is often used for preventing and treating rice blast, rice smut and sheath blight. However, the residues caused by the unreasonable use of trifloxystrobin will destroy the soil and aquatic ecosystems. Although trifloxystrobin has low toxicity, due to the development of food safety science and the increasing attention of people to food safety, many countries have given relevant maximum residue limits (MRL) for the residues of trifloxystrobin in food and crops. The MRL of trifloxystrobin in vegetables and fruits is 0.05-15 mg / kg in the national standard GB 2763 of China.

[0003] At present, the detection methods for trifloxystrobin residues at home and abroad mainly include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. Such instrument analysis methods have the advantages of high detection sensitivity and strong specificity. However, the sample pretreatment is complicated and time-consuming, and the sample needs to be extracted and purified. At the same time, expensive large instruments and equipment are needed for instrument detection methods, and professional detection technicians are needed for operation and management. The instrument detection method cannot be used for large-scale detection on site, has poor timeliness, and is difficult to popularize.

[0004] The immunological detection and analysis technology has been widely used in the field of drug residue detection due to the advantages of high sensitivity, high specificity, rapidness and simple operation. Compared with the instrument detection method, the immunological detection and analysis technology has many advantages. However, there is a lack of detection reagents with specific reaction to trifloxystrobin in the prior art, which hinders the application of the immunological detection and analysis method in the detection of trifloxystrobin.

[0005] In the establishment of the immunological detection method and the application of the detection method for detecting trifloxystrobin, the key technology is to obtain an antibody with high specificity and high sensitivity. To achieve this goal, the prerequisite is to synthesize and prepare a suitable hapten of trifloxystrobin. SUMMARY

[0006] The present application provides a hapten for detecting the content of trifloxystrobin and application thereof, and solves the technical problems in the prior art that the sample pretreatment of the trifloxystrobin detection method is complicated, the detection efficiency is low, and rapid large-scale detection cannot be achieved.

[0007] The application provides a hapten for detecting content of trifloxystrobin, and a chemical structural formula is as follows:

[0008]

[0009] The preparation method of the hapten for detecting content of trifloxystrobin comprises the following steps:

[0010] The hapten for detecting content of trifloxystrobin is obtained by condensation reaction of methyl 2-[2-[(4-acetylphenoxy)methyl]phenyl]-2-methoxyiminoacetate as a raw material and 2-(aminoxy)acetic acid under catalysis of pyridine.

[0011] The hapten with the structural formula can be processed by a method commonly used in the prior art to obtain an antibody, and the antibody can specifically react with trifloxystrobin contained in various vegetables and fruits after pretreatment. According to the characteristics, a standard sample solution of different concentrations of trifloxystrobin can be obtained by gradient dilution of a standard sample of trifloxystrobin, and a standard curve can be obtained by measuring OD values of the standard sample solutions at 450 nm by using an enzyme label instrument. After pretreatment of a sample to be measured, OD values at the same wavelength are measured, and corresponding concentrations are obtained through the standard curve, so that quantitative and effective and rapid detection of the content of trifloxystrobin contained in the sample can be realized.

[0012] The reaction can obtain a hapten with high purity. The reagents and reaction conditions used in the preparation method can be selected according to the reagents and conditions required by the existing condensation reaction.

[0013] When the hapten is used to detect trifloxystrobin contained in vegetables and fruits after being made into a kit, the sensitivity is 0.02 μg / L, which indicates that the sensitivity of the hapten for detecting trifloxystrobin is high after being made into a kit. The detection limit of trifloxystrobin in vegetables and fruits is 0.5 μg / kg after being made into a kit according to the existing method, and the kit can be used for effective detection of the content of trifloxystrobin contained in fruits and vegetables. The recovery rate of trifloxystrobin is 100%±20% after the hapten is made into a kit, the intra-batch coefficient of variation is less than 10%, the inter-batch coefficient of variation is less than 15%, the accuracy is good, and the detection needs can be met.

[0014] The cross-reactivity of the obtained hapten made into a kit to other methoxy acrylate fungicides is less than 1%, and the cross-reactivity to trifloxystrobin can reach 100%, which indicates that the hapten can specifically detect trifloxystrobin and avoid the interference of other structural analogues on the detection results, and can well fill the lack of haptens with obvious specificity for trifloxystrobin in the prior art.

[0015] Preferably, the preparation method of the hapten for detecting content of trifloxystrobin comprises the following steps:

[0016] 1) adding ethanol to dissolve 2-[2-[(4-acetylphenoxy)methyl]phenyl]-2-methoxyiminoacetic acid methyl ester;

[0017] 2) adding water to dissolve 2-(aminooxy)acetic acid, adding pyridine, stirring well, and then adding dropwise to the solution obtained in 1), and reacting at room temperature for 4 hours to obtain a reaction solution;

[0018] 3) separating and purifying the reaction solution to obtain the hapten for detecting the content of trifloxystrobin.

[0019] The reaction equation of the preparation method of the hapten in the present application is shown in formula (2):

[0020]

[0021] As can be seen from the above, the reaction can occur normally, and the hapten with the above structure is prepared.

[0022] Preferably, the separation and purification operation in step 3) of the preparation method comprises the following steps: removing ethanol and pyridine from the reaction solution by rotary evaporation, adding 200 mL of water and 100 mL of ethyl acetate for extraction, separating the water phase, evaporating the organic phase to dryness, eluting and separating on a silica gel column with a petroleum ether-ethyl acetate mixed solution with a volume ratio of 3:1 to obtain the hapten for detecting the content of trifloxystrobin.

[0023] To realize the effective application of the hapten, the following applications can be carried out:

[0024] Another aspect of the present application also provides an antigen for detecting the content of trifloxystrobin, which is a conjugate obtained by coupling the above-mentioned hapten with a carrier protein.

[0025] Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin. The use of the above types of carrier proteins can effectively exert the detection function of the hapten.

[0026] The coupling method of the hapten with the carrier protein in the present application can be the conventional method for preparing an antigen from a hapten, for example, the coupling method using bovine serum albumin as the carrier protein can be as follows:

[0027] Take the above hapten 15.44 mg, add 1 mL of N, N-dimethylformamide, add N-hydroxysuccinimide 8.58 mg, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride 14.29 mg, mix well with a pipette, react at room temperature for 2 h to obtain hapten activation solution A solution; Take bovine serum albumin (BSA) 50 mg, add 0.1 mol / L pH 8.0 PB buffer 4 mL to dissolve; Add all of A solution dropwise, react at room temperature for 6 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change liquid 3 times a day, centrifuge to obtain oxycarbamide antigen coupled with bovine serum albumin, aliquot, store at -20℃.

[0028] The coupling method of the ovalbumin as the carrier protein includes the following steps:

[0029] Take the above hapten 15.44 mg, add 1 mL of N, N-dimethylformamide, add N-hydroxysuccinimide 8.58 mg, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride 14.29 mg, mix well with a pipette, react at room temperature for 2 h to obtain hapten activation solution A solution; Take bovine serum albumin (BSA) 50 mg, add 0.1 mol / L pH 8.0 PB buffer 4 mL to dissolve; Add all of A solution dropwise, react at room temperature for 6 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change liquid 3 times a day, centrifuge to obtain oxycarbamide antigen coupled with bovine serum albumin, aliquot, store at -20℃.

[0030] Another aspect of the present application also provides an antibody for detecting the content of oxycarbamide, which is an antibody obtained by immunizing an animal with the above-mentioned antigen; the antibody specifically reacts with oxycarbamide.

[0031] Preferably, the antibody is a monoclonal antibody of oxycarbamide.

[0032] The method for preparing the antibody from the above-mentioned antigen in the present aspect can be a commonly used animal immunization method in the art, which includes the following steps, for example:

[0033] (1) Animal immunization: inject the oxycarbamide antigen coupled with bovine serum albumin into a Balb / c mouse, and the immunization dose is 150 μg per mouse, so that the mouse produces polyclonal antibody serum.

[0034] (2) Cell fusion and cloning: after the mouse serum determination result is high, take the spleen cells thereof, fuse with SP2 / 0 myeloma cells at a ratio of 8:1, determine the cell supernatant by indirect competitive ELISA, and screen positive wells. The positive wells are cloned by limited dilution method until a hybridoma cell strain secreting a monoclonal antibody is obtained.

[0035] (3) Cell freezing and recovery: the monoclone hybridoma cell strain of trifloxystrobin is made into 1×10 9 individuals, and is stored in liquid nitrogen. When recovered, the frozen tube is taken out and immediately put into a 37℃ water bath for quick thawing. After centrifugation to remove the freezing solution, it is moved into a culture bottle for culture.

[0036] (4) Production and purification of the monoclone antibody: the Balb / c mice are injected with 0.5 mL of sterilized paraffin oil per individual, and 6×10 5 individuals of the monoclone hybridoma cell strain of trifloxystrobin are injected into the abdominal cavity after 7 days. The ascites is collected after 7 days. The ascites is purified by the caprylic acid-saturated ammonium sulfate method, and is stored at -20℃.

[0037] (5) Determination of the titer of the monoclone antibody: the titer of the antibody is determined to be 1:512000 by indirect competitive ELISA.

[0038] The antibody is used in the form of a monoclone antibody, and has a high titer.

[0039] Another aspect of the present application further provides an enzyme-linked immunoassay kit for detecting the content of trifloxystrobin, comprising: an enzyme-labeled plate coated with an antigen for detecting the content of trifloxystrobin as described above; and an antibody as described above.

[0040] The kit is prepared according to the existing method, and the specific method can be prepared by searching the existing production method of the enzyme-linked immunoassay kit by those skilled in the art.

[0041] Preferably, the enzyme-linked immunoassay kit for detecting the content of trifloxystrobin further comprises: a standard solution of trifloxystrobin with gradient concentrations, an enzyme-labeled secondary antibody, a substrate solution A, a substrate solution B, a termination solution, a washing solution, and a reconstitution solution.

[0042] Preferably, the gradient concentrations of the standard solution of trifloxystrobin with gradient concentrations are 0 μg / L, 0.02 μg / L, 0.06 μg / L, 0.18 μg / L, 0.54 μg / L, and 1.62 μg / L, respectively.

[0043] The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody;

[0044] The substrate solution A is urea peroxide, and the substrate solution B is tetramethyl benzidine;

[0045] The termination solution is a 2 mol / L sulfuric acid solution;

[0046] The washing solution is a pH 7.2 solution containing 0.8% to 1.2% Tween-20, 0.01‰ to 0.03‰ thiomersal preservative, and 0.1 to 0.3 mol / L carbonate buffer;

[0047] The reconstituted solution is a phosphate buffer solution with a pH of 7.6, containing 8%-12% casein and 0.1-0.3 mol / L.

[0048] The kit prepared from the components has high detection efficiency and good accuracy, and is suitable for detecting a large number of samples on site.

[0049] In the present application, the room temperature ranges from 20 to 25 DEG C, and the temperature reading of the thermometer at the time of operation is used as the reference.

[0050] The beneficial effects of the present application include:

[0051] 1) The hapten for detecting the content of trifloxystrobin provided by the present application has a sensitivity of 0.02 ug / L when used in the detection of trifloxystrobin contained in vegetables and fruits, which indicates that the hapten has high detection sensitivity for trifloxystrobin. The detection limit of trifloxystrobin in vegetables and fruits is 0.5 ug / kg when the hapten is prepared into a kit according to the prior art, and the kit can be used for effective detection of the content of trifloxystrobin contained in fruits and vegetables. The recovery rate of trifloxystrobin is 100%+20% when the hapten is prepared into a kit, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%, which indicates that the kit has good accuracy and can meet the detection requirements. The hapten can retain the characteristic structure of trifloxystrobin to the greatest extent, and the artificial antibody prepared from the hapten has the characteristics of high detection sensitivity and strong specificity for trifloxystrobin.

[0052] 2) The hapten for detecting the content of trifloxystrobin provided by the present application has high immunogenicity for the detection of trifloxystrobin residues, and the antibody prepared from the hapten has good titer, specificity and affinity, and the detection sensitivity for trifloxystrobin can reach 0.02 ug / L.

[0053] 3) The antibody and kit for detecting the content of trifloxystrobin provided by the present application have simple detection operation, do not need to purchase large-scale instrument analysis equipment, and can complete the detection of samples through the kit, which is suitable for on-site large-scale detection.

[0054] 4) The enzyme-linked immunoassay kit provided by the present application is prepared from the obtained antibody, and has the advantages of convenient use, low detection cost, efficient, accurate, rapid detection method, and can simultaneously detect a large number of samples, which is suitable for on-site monitoring and screening of a large number of samples of trifloxystrobin in fruits and vegetables. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the hapten obtained in the embodiments of the present application is shown.

[0056] Figure 2The antibody provided in the application is used to detect the standard curve of the gradient concentration of the standard solution of the trifloxystrobin after detection. DETAILED DESCRIPTION

[0057] To make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be described clearly and completely below with reference to the embodiments of the application and the drawings. The contents not described in detail in the preparation of the antigen from the hapten, the preparation of the antibody from the antigen and the preparation of the kit from the antibody are performed according to the methods disclosed in the prior art, which will not be repeated here.

[0058] EMBODIMENT

[0059] The materials and instruments used in the following embodiments are commercially available unless otherwise specified.

[0060] Embodiment 1 Preparation of the hapten for the detection of the content of trifloxystrobin

[0061] The hapten for the detection of the content of trifloxystrobin is prepared according to the following steps:

[0062] 1) 3.41 g of 2-[2-[(4-acetylphenoxy) methyl] phenyl]-2-methoxyiminoacetic acid methyl ester (CAS No. 132738-52-4) is dissolved in 200 mL of ethanol;

[0063] 2) 1.27 g of 2-(aminooxy) acetic acid (CAS No. 645-88-5) is dissolved in 20 mL of water, 50 mL of pyridine is added, and the mixture is stirred thoroughly, then the solution obtained in 1) is added dropwise, and the reaction is carried out at room temperature for 4 h, and the reaction is stopped, and the reaction solution is obtained;

[0064] 3) The ethanol and pyridine in the obtained reaction solution are removed by rotary evaporation, 200 mL of water and 100 mL of ethyl acetate are added for extraction, the water phase is separated, the organic phase is evaporated to dryness, and the obtained product is separated by silica gel column and eluted with a mixture of petroleum ether and ethyl acetate (volume ratio 3:1) to obtain the hapten for the detection of the content of trifloxystrobin.

[0065] The yield of the hapten for the detection of the content of trifloxystrobin is 63.77%, and the hydrogen nuclear magnetic resonance (H-NMR) determination result of the hapten is shown as follows: 1 H-NMR Figure 1 1 ​H NMR (500 MHz, Chloroform-d): δ (ppm) 7.72-7.66 (m, 1H), 7.69-7.61 (m, 2H), 7.51 (ddt, J = 7.4, 2.1, 1.0 Hz, 1H), 7.45-7.36 (m, 2H), 6.99-6.93 (m, 2H), 5.30 (d, J = 0.9 Hz, 2H), 4.66 (s, 2H), 3.95 (s, 2H), 3.85 (s, 2H), indicating that the hapten structure is correct.

[0066] Preparation of the antigen for the detection of the content of trifloxystrobin coupled with bovine serum albumin

[0067] The following steps are included:

[0068] Take 15.44 mg of the hapten for the detection of the content of trifloxystrobin prepared in Example 1, dissolve in 1 mL of N,N-dimethylformamide, add 8.58 mg of N-hydroxysuccinimide and 14.29 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, mix well by blowing with a pipette, and react at room temperature for 2 h to obtain hapten activation solution A. Take 50 mg of bovine serum albumin (BSA), dissolve in 4 mL of 0.1 mol / L PB buffer at pH 8.0; add all of the A solution dropwise, react at room temperature for 6 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change the solution 3 times a day, centrifuge, and obtain the trifloxystrobin antigen coupled with bovine serum albumin, which is divided into portions and stored at -20°C.

[0069] Preparation of the antigen for the detection of the content of trifloxystrobin coupled with ovalbumin

[0070] The following steps are included:

[0071] Take 20.24 mg of the hapten for the detection of the content of trifloxystrobin prepared in Example 1, dissolve in 1 mL of N,N-dimethylformamide, add 8.58 mg of N-hydroxysuccinimide and 14.29 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, mix well by blowing with a pipette, and react at room temperature for 2 h to obtain hapten activation solution A. Take 100 mg of ovalbumin (OVA), dissolve in 4 mL of 0.1 mol / L PB buffer at pH 8.0; add all of the A solution dropwise, react at room temperature for 6 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change the solution 3 times a day, centrifuge, and obtain the trifloxystrobin antigen coupled with ovalbumin, which is divided into portions and stored at -20°C.

[0072] Preparation of the monoclonal antibody for the detection of the content of trifloxystrobin

[0073] The following steps are included:

[0074] (1) Animal immunization: The oxycarbamide antigen coupled with bovine serum albumin prepared in Example 2 was injected into Balb / c mice to produce polyclonal antibody serum, with an immunization dose of 150 μg per mouse.

[0075] (2) Cell fusion and cloning: After the mouse serum was determined to have a high result, the spleen cells were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1. The supernatant of the cells was determined by indirect competitive ELISA to screen positive wells. The positive wells were cloned by limited dilution until a hybridoma cell strain secreting monoclonal antibody was obtained.

[0076] (3) Cell cryopreservation and recovery: The monoclonal hybridoma cell strain of oxycarbamide was prepared into a cell suspension of 1 x 10 9 cells / mL with a cryopreservation solution and stored in liquid nitrogen for a long time. When recovered, the cryopreservation tube was taken out and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred into a culture bottle for culture.

[0077] (4) Production and purification of monoclonal antibody: The Balb / c mice were injected with 0.5 mL of sterilized paraffin oil per mouse in the abdominal cavity. After 7 days, the mice were injected with 6 x 10 5 cells of the monoclonal hybridoma cell strain of oxycarbamide per mouse in the abdominal cavity. After 7 days, the ascites were collected. The ascites were purified by the caprylic acid-saturated ammonium sulfate method and stored at -20°C.

[0078] (5) Determination of the titer of monoclonal antibody: The titer of the antibody was determined by indirect competitive ELISA to be 1:512,000.

[0079] Indirect competitive ELISA method: The oxycarbamide antigen coupled with ovalbumin prepared in Example 3 was used to coat the enzyme-labeled plate. Oxycarbamide standard solution and monoclonal antibody working solution were added and reacted at 4°C for 30 min. The liquid in the wells was poured out and washed 3-5 times with PBST washing solution and then patted dry with absorbent paper. Horseradish peroxidase-labeled goat anti-mouse antibody was added and reacted at 25°C for 30 min. The plate was taken out and the plate washing step was repeated. Substrate solution was added and reacted at 25°C for 15 min. The reaction was terminated by adding a termination solution. The absorbance value of each well was determined by setting the enzyme-labeled instrument at a wavelength of 450 nm.

[0080] Example 5 Preparation of an enzyme-linked immunoassay kit for detecting the content of oxycarbamide

[0081] The obtained kit includes:

[0082] (1) an enzyme-labeled plate coated with oxycarbamide antigen coupled with ovalbumin;

[0083] (2) oxycarbamide monoclonal antibody working solution;

[0084] (3) horseradish peroxidase-labeled goat anti-mouse antibody;

[0085] (4) 6 bottles of trifloxystrobin standard solution with concentrations of 0 μg / L, 0.02 μg / L, 0.06 μg / L, 0.18 μg / L, 0.54 μg / L and 1.62 μg / L respectively;

[0086] (5) The substrate solution is composed of A solution and B solution, wherein the A solution is urea peroxide, and the B solution is tetramethyl benzidine;

[0087] (6) The termination solution is 2 mol / L sulfuric acid solution;

[0088] (7) The washing solution is pH 7.2, containing 1.0% Tween-20, 0.02‰ thiomersal preservative and 0.2 mol / L carbonate buffer;

[0089] (8) The reconstitution solution is pH 7.6, containing 10% casein and 0.2 mol / L phosphate buffer.

[0090] The main reagents of the kit are provided in the form of working solution, the test method is convenient and easy to operate, and has the characteristics of high specificity, high sensitivity, high accuracy, high accuracy and the like, and is convenient for rapid batch detection of a large number of fruit and vegetable samples.

[0091] Application of the enzyme-linked immunoassay kit for detecting the content of trifloxystrobin obtained in Example 6 in fruit and vegetable detection and performance detection thereof

[0092] I. Test method of the kit obtained in Example 5

[0093] 1. Sample pretreatment

[0094] Homogenize the fruit and vegetable sample; weigh (1.0 ± 0.05) g of the homogenate, and then add 5 mL of 0.02 mol / L phosphate buffer, oscillate for 5 min with a shaker at 3000 r / min or above, and centrifuge at room temperature for 5 min; remove 100 μL of supernatant, add 900 μL of 0.02 mol / L phosphate buffer, and vortex for 2 min with a vortex instrument, mix well, and then use for analysis.

[0095] 2. Detection operation steps of the kit obtained in Example 5

[0096] Add 50 μL of sample solution to each well of the enzyme-labeled plate (coated with the trifloxystrobin antigen coupled with ovalbumin obtained in Example 3), and then add 50 μL of the trifloxystrobin monoclonal antibody working solution prepared in Example 4 to each well, cover the plate with a cover film, and then react at 25°C in the dark for 30 min; pour out the liquid in the well, add 250 μL of washing solution to each well, pour out the liquid in the well after 30 s, and repeat the operation for 5 times to wash the plate; pat dry with a water-absorbing paper; add 100 μL of horseradish peroxidase-labeled goat anti-mouse antibody to each well, and mix gently; cover the plate with a cover film, and then react at 25°C in the dark for 30 min; repeat the plate washing step;

[0097] Add 50 μL of substrate solution A, 50 μL of substrate solution B (tetramethyl benzidine, TMB) to each well, mix gently, cover with cover film, develop color for 15 min at 25°C in dark, add 50 μL of stop solution (2 mol / L sulfuric acid) to each well, mix gently, and measure the absorbance (OD value) at 450 nm using a microplate reader.

[0098] 3. Detection and analysis of results

[0099] According to the above operation steps, gradient concentrations of the standard oxycarbamide solution and the pretreated sample were detected.

[0100] The average absorbance (B) of each concentration of the standard solution was divided by the average absorbance (B0) of the first standard solution (0 standard), and then multiplied by 100% to obtain the percentage absorbance value.

[0101] The logarithmic value of the oxycarbamide standard concentration (μg / L) was taken as the X axis, and the percentage absorbance value was taken as the Y axis to draw a standard curve, as shown in Figure 2 .

[0102] The percentage absorbance value of the pretreated sample solution was calculated in the same way, and the corresponding concentration of the pretreated sample was read from the standard curve of the oxycarbamide standard, and then multiplied by the corresponding dilution factor to obtain the actual concentration of oxycarbamide in the pretreated sample.

[0103] II. Sensitivity determination of the kit obtained in Example 5

[0104] Sensitivity in this application: refers to the minimum content of oxycarbamide in the detected substance that can be detected by the kit. The sensitivity of the quantitative kit itself can be understood as the concentration of the smallest standard in the kit except for the "0" standard or the lowest standard concentration corresponding to the standard curve. According to the existing method, the sensitivity of the kit provided in this application to oxycarbamide is 0.02 μg / L.

[0105] III. Detection limit determination of the kit obtained in Example 5

[0106] Detection limit: usually refers to the minimum detection amount of the kit product for determining the actual sample. Its theoretical definition is: according to a reasonable pretreatment method, 20 negative (blank) samples are determined, and the average value X and the standard deviation (SD) are calculated. The result obtained by the formula X+3SD is the detection (lower) limit of the sample. Therefore, according to the above existing method, the detection limit of the kit for oxycarbamide in vegetables and fruits is 0.5 μg / kg.

[0107] IV. Accuracy and precision determination of the kit obtained in Example 5

[0108] Accuracy and precision: the accuracy of ELISA was expressed by recovery rate, and the precision was expressed by coefficient of variation. The leeks, onions, asparagus, citrus, apple samples were taken, and oxycarboxin was added to them at the concentrations of 0.5, 1.0, 2.0 μg / kg to carry out recovery test according to the existing method. The experimental results: the recovery rate of the sample detected by the kit prepared by the hapten provided in the application was 100% ± 20%, the within-batch coefficient of variation was <10%, and the between-batch coefficient of variation was <15%.

[0109] It is illustrated that the kit prepared by the hapten provided in the application has good accuracy and precision, and can accurately detect oxycarboxin contained in the sample.

[0110] V. Determination of antibody cross-reactivity of the kit obtained in Example 5

[0111] The existing indirect competitive ELISA method was used to determine the cross-reactivity of the kit obtained in Example 5, and the results showed that the cross-reactivity of the kit obtained in Example 5 was <1% except for oxycarboxin which was 100%.

[0112] It is illustrated that the antibody prepared by the hapten provided in the application has good specificity for oxycarboxin, can accurately detect oxycarboxin contained in the sample, and is not affected by other structural analogues.

[0113] VI. Comparison of determination results of the kit obtained in Example 5 with the existing instrument determination method

[0114] Twenty leek, onion, asparagus, citrus, and apple samples were taken, and the same sample was divided into 20 parts and numbered as No. 1-20. The same sample was used as a detection sample, and the kit obtained in Example 5 and the instrument detection method in Comparative Example 1 were used for detection.

[0115] Comparative Example 1

[0116] The content of oxycarboxin in the sample was detected by the instrument detection method.

[0117] The instrument method refers to: "GB / T 20769-2008 Determination of 450 Pesticides and Related Chemicals Residues in Fruits and Vegetables by Liquid Chromatography-Tandem Mass Spectrometry".

[0118] The detection limit of the kit was used as the positive judgment line. If it was lower than the positive judgment line, it was considered as not detected, and "-" was used to represent it. If it was higher than the positive judgment line, the actual result was used to represent it.

[0119] The results obtained by the experimental group and the control group are shown in the following table.

[0120] Table 1 Comparison of detection results of the kit and the instrument method (μg / kg)

[0121]

[0122]

[0123] From the above table, it can be seen that the detection results of the kit provided by the present application are basically consistent with the instrument detection results, the error is within the acceptable range, which shows that the kit provided by the present application can replace the existing cumbersome instrument detection method, improve the detection efficiency, and obtain accurate detection results.

[0124] Seven, the shelf life condition test of the kit obtained in Example 5

[0125] After storing the kit at 2-8℃ for 12 months, the maximum absorbance value (zero standard), 50% inhibition concentration and the actual measured value of the trifloxystrobin were detected, and each value was within the normal range.

[0126] It is determined that the kit provided by the present application can be stored for at least 12 months.

[0127] From the above detection results, it can be seen that the antibody prepared from the hapten provided by the present application, and the kit prepared therefrom, has a relatively accurate detection effect in the detection of the residual amount of trifloxystrobin in vegetables and fruits, and the kit provided by the present application can be used for effective detection in various pretreated fruits and vegetables.

[0128] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A hapten for detecting oxime ester content, characterized in that, The chemical structural formula is: Equation (1).

2. A method for preparing a hapten for detecting oxime ester content, characterized in that, The hapten for detecting oxime ester content is obtained by condensing methyl 2-[2-[(4-acetylphenoxy)methyl]phenyl]-2-methoxyiminoacetic acid with 2-(aminooxy)acetic acid under the catalysis of pyridine.

3. The method for preparing the hapten for oxime ester content detection according to claim 2, characterized in that, The preparation method specifically includes the following steps: 1) Dissolve methyl 2-[2-[(4-acetylphenoxy)methyl]phenyl]-2-methoxyiminoacetate in ethanol; 2) Dissolve 2-(aminooxy)acetic acid in water, add pyridine and stir thoroughly, then add dropwise to the solution obtained in 1) above, react at room temperature for 4 hours, stop the reaction, and obtain the reaction solution; 3) The obtained reaction solution is separated and purified to obtain the hapten for the detection of oxime ester content.

4. The method for preparing the hapten for oxime ester content detection according to claim 3, characterized in that, The separation and purification operation in step 3) includes the following steps: evaporating the obtained reaction solution by rotary evaporation to remove ethanol and pyridine, extracting with 200 mL of water and 100 mL of ethyl acetate, separating the aqueous phase, evaporating the organic phase to dryness, loading onto a silica gel column, and eluting with a petroleum ether-ethyl acetate mixed solution with a volume ratio of 3:1 to obtain the hapten for detecting the oxime ester content.

5. An antigen for detecting oxime ester content, characterized in that, It is a conjugate obtained by conjugating the hapten with a carrier protein as described in claim 1; wherein the carrier protein is bovine serum albumin or ovalbumin.

6. An antibody for detecting oxime ester content, characterized in that, The antibody is obtained by immunizing an animal with the antigen as described in claim 5; the antibody undergoes a specific immune response with oxime ester.

7. The antibody for detecting oxime ester content according to claim 6, characterized in that, The antibody is an oxime ester monoclonal antibody.

8. An enzyme-linked immunosorbent assay (ELISA) kit for detecting oxime ester content, comprising: The enzyme-labeled plate, antibody, graded concentrations of oxime ester standard solutions, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, stop solution, washing solution, and reconstitution solution are characterized in that... The enzyme-labeled plate is coated with the antigen for detecting oxime ester content as described in claim 5; The antibody is the oxime ester content detection antibody as described in claim 6 or 7; The gradient concentrations of the azoxystrobin standard solutions were 0 μg / L, 0.02 μg / L, 0.06 μg / L, 0.18 μg / L, 0.54 μg / L, and 1.62 μg / L, respectively. The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-mouse anti-antibody; The substrate solution A is urea peroxide, and the substrate solution B is tetramethylbenzidine; The terminating solution is a 2 mol / L sulfuric acid solution; The washing solution has a pH of 7.2 and contains 0.8%~1.2% Tween-20, 0.01‰~0.03‰ thimerosal preservative, and 0.1~0.3mol / L carbonate buffer solution; The reconstitution solution is pH 7.6 and contains 8%–12% casein and 0.1–0.3 mol / L phosphate buffer.

Images