Hapten for detecting phorate content and application thereof

Abstract

The application discloses a semi-antigen for detecting phorate content and application thereof, and particularly relates to a semi-antigen for detecting phorate content and a preparation method thereof, and further discloses application of the semi-antigen in antibodies and kits. The enzyme-linked immunoassay kit established for the semi-antigen for detecting phorate content can realize rapid detection of a large number of samples to be detected, is convenient to use, low in detection cost, efficient, accurate, rapid, capable of simultaneously detecting a large number of samples, and suitable for on-site monitoring of phorate in vegetables and fruits and screening of a large number of samples.

Description

TECHNICAL FIELD

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

[0002] Phorate is a highly toxic and broad-spectrum organophosphorus insecticide, which is mainly used for preventing and treating underground pests and aphids. It is widely used in the process of planting agricultural products due to its high insecticidal efficiency and low price. Direct consumption of agricultural products containing phorate and its metabolites can inhibit cholinesterase activity and cause physiological function disorders. Phorate that is not absorbed by plants is transferred to surface water, rivers and lakes, forming secondary pollution. Phorate can be metabolized to produce phorate sulfone and phorate sulfoxide, which are more toxic and more difficult to degrade, causing greater harm to the ecological environment and human health. GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Foods" stipulates that the maximum residue content of phorate, phorate sulfoxide and phorate sulfone in vegetables and fruits shall not exceed 0.01 mg / kg. Therefore, it is necessary to establish an efficient, sensitive and accurate method for detecting phorate and its metabolites in fruits and vegetables.

[0003] At present, the detection methods for phorate residues at home and abroad mainly include fluorescence spectrophotometry, gas chromatography, gas chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography, liquid chromatography-tandem mass spectrometry and the like. Such instrumental analysis methods have the advantages of high detection sensitivity and strong specificity, but the pretreatment of the detection sample is complicated and time-consuming, and the sample needs to be extracted and purified. At the same time, the instrumental detection method needs expensive large instruments and equipment, and is operated and managed by professional detection technicians. It is difficult to carry out large-scale detection on site, and the timeliness is poor, which is difficult to promote.

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

[0005] In the establishment of immunological detection method and the application of the detection method for detecting phorate, 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 phorate hapten. SUMMARY

[0006] The application provides a hapten for detecting phorate content and an application thereof, and aims at solving the technical problems of complicated sample pretreatment, low detection efficiency, and inability to realize rapid and large-batch detection in the prior art.

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

[0008]

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

[0010] O-ethyl S-[(ethylthio) methyl] ester is used as a raw material, and is subjected to condensation reaction with N-(2-aminopropyl) maleimide under the catalysis of pyridine, so that the hapten for detecting phorate content is obtained.

[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 phorate contained in various vegetables and fruits after pretreatment. According to the characteristics, a phorate standard product is gradiently diluted to obtain a phorate standard product solution with different concentrations, an enzyme label instrument is used to measure the OD values of the standard product solutions at 450 nm to obtain a standard curve, a sample to be detected is pretreated, the OD value at the same wavelength is measured, and the corresponding concentration is obtained through the standard curve, so that the content of phorate contained in the sample is quantitatively and effectively and rapidly detected.

[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 phorate contained in vegetables and fruits after being made into a kit, the sensitivity is 0.1 μg / L, which indicates that the sensitivity of the hapten for detecting phorate is high after being made into a kit. According to the existing method, the detection limit of phorate in vegetables and fruits is 5 μg / kg, and the hapten can be used for effectively detecting the content of phorate contained in fruits and vegetables. After the hapten is made into a kit, the recovery rate of phorate is 100% ± 20%, the within-batch coefficient of variation is less than 10%, the between-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 to other organophosphorus pesticides is less than 1%, and the cross-reactivity to phorate is 100%, and the cross-reactivity to metabolites phorate-sulfone and phorate-sulfoxide is 80%, which indicates that the hapten can specifically detect phorate and its metabolites and avoid the interference of other structural analogues on the detection results, and can well fill the lack of a hapten with obvious specificity for phorate in the prior art.

[0015] Preferably, the preparation method of the phorate content detection hapten comprises the following steps:

[0016] 1) Take O-ethyl S-[(ethylthio) methyl] ester and add pyridine to dissolve;

[0017] 2) Add N-(2-aminopropyl) maleimide, stir well, install a reflux condenser, and react at 80°C for 4h, and stop the reaction to obtain a reaction solution;

[0018] 3) The obtained reaction solution is separated and purified to obtain the phorate content detection hapten.

[0019] The reaction equation of the hapten preparation method of 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: rotary evaporation of the obtained reaction solution to remove the organic solvent, and elution separation on a silica gel column with a mixed solution of dichloromethane-methanol with a volume ratio of 10:1 to obtain the phorate content detection hapten.

[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 a phorate content detection antigen, 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 effect of the hapten.

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

[0027] Take 100 mg of bovine serum albumin (BSA), dissolve in 10 mL of PB buffer with pH 8.0, drop 1 mL of an aqueous solution containing 7.2 mg of tris(2-carboxyethyl)phosphine hydrochloride, and react at room temperature for 2 hours to obtain solution A; take 9.34 mg of the above-mentioned hapten, dissolve in 0.5 mL of N,N-dimethylformamide, and then drop into solution A, react at 4°C for 12 hours, and then purify by dialysis with 0.02 mol / L PB buffer for 3 days, with liquid change for 3 times per day, to obtain the methamidophos antigen coupled with bovine serum albumin, which is divided and stored at -20°C.

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

[0029] Take 100 mg of ovalbumin (OVA), dissolve in 10 mL of PB buffer with pH 8.0, drop 1 mL of an aqueous solution containing 3.17 mg of tris(2-carboxyethyl)phosphine hydrochloride, and react at room temperature for 2 hours to obtain solution A; take 4.08 mg of the above-mentioned hapten, dissolve in 0.5 mL of N,N-dimethylformamide, and then drop into solution A, react at 4°C for 12 hours, and then purify by dialysis with 0.02 mol / L PB buffer for 3 days, with liquid change for 3 times per day, to obtain the methamidophos antigen coupled with ovalbumin, which is divided and stored at -20°C.

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

[0031] Preferably, the antibody is a methamidophos monoclonal antibody.

[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 comprises the following steps, for example:

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

[0034] (2) Cell fusion and cloning: after the mouse serum determination result is high, take the spleen cells, 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 the hybridoma cell strain secreting monoclonal antibody is obtained.

[0035] (3) Cell cryopreservation and recovery: the methamidophos monoclonal hybridoma cell strain is prepared into a cell suspension of 1×10 9 / mL with a cryopreservation solution, and is stored in liquid nitrogen for a long time. When recovered, the cryopreservation tube is taken out immediately and is placed into a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cell suspension is transferred into a culture bottle for culture.

[0036] (4) Production and purification of monoclonal antibodies: Balb / c mice were injected intraperitoneally with 0.5 mL of sterile paraffin oil per mouse, and 7 days later, they were injected intraperitoneally with 6 × 10⁶ monoclonal hybridoma cells containing phorate. 5 Ascites fluid was collected from each animal after 7 days. The ascites fluid was purified using the octanoic acid-saturated ammonium sulfate method and stored at -20°C.

[0037] (5) Determination of monoclonal antibody titer: The antibody titer was determined by indirect competitive ELISA at a ratio of 1:256000.

[0038] In another aspect, the present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for detecting phorate content, comprising: an ELISA plate and an antibody, wherein the ELISA plate is coated with the antigen for detecting phorate content as described above; and the antibody is the antibody for detecting phorate content as described above.

[0039] This kit is prepared using existing methods, the specific methods of which can be obtained by those skilled in the art through searching existing enzyme-linked immunosorbent assay (ELISA) kit manufacturing methods.

[0040] Preferably, the enzyme-linked immunosorbent assay kit for detecting phorate content further includes: phorate standard solutions of varying concentrations, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, stop solution, washing solution, and reconstitution solution.

[0041] Preferably, the gradient concentrations of the phorate standard solution are 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L, respectively.

[0042] The enzyme-labeled secondary antibody was a horseradish peroxidase-labeled goat anti-mouse anti-antibody.

[0043] Substrate solution A is urea peroxide, and substrate solution B is tetramethylbenzidine;

[0044] The stop solution is a 2 mol / L sulfuric acid solution;

[0045] The washing solution has a pH of 7.2 and contains 0.8%–1.2% Tween-20, 0.01‰–0.03‰ thimerosal preservative, and 0.1–0.3 mol / L carbonate buffer.

[0046] The reconstitution solution is pH 7.6 and contains 8%–12% casein and 0.1–0.3 mol / L phosphate buffer.

[0047] The reagent kit made from the above-mentioned components has high detection efficiency and accuracy, and is suitable for testing large batches of samples on-site.

[0048] In this application, the room temperature range is 20 to 25°C, based on the thermometer reading at the time of operation.

[0049] The beneficial effects that can be produced by the present application include:

[0050] 1) The hapten for detecting phorate content provided by the present application, when the hapten is made into a kit, the sensitivity for detecting phorate contained in vegetables and fruits is 0.1 μg / L, which indicates that the sensitivity for detecting phorate is high after the hapten is made into a kit; the detection limit for phorate in vegetables and fruits is 5 μg / kg after the hapten is made into a kit according to the existing method, which can be used for effectively detecting the content of phorate contained in fruits and vegetables; the recovery rate of phorate after the hapten is made into a kit is 100% ± 20%, the within-batch coefficient of variation is < 10%, the between-batch coefficient of variation is < 15%, and the accuracy is good, which can meet the detection needs. The hapten can maximize the retention of the characteristic structure of phorate, and the antibody prepared from the hapten has the characteristics of high detection sensitivity and strong specificity for phorate.

[0051] 2) The hapten for detecting phorate content provided by the present application, the antibody prepared from the hapten as raw material has strong immunogenicity for phorate residue detection; the antibody prepared from the hapten as raw material has good titer, specificity and affinity, and the detection sensitivity for phorate can reach 0.1 μg / L.

[0052] 3) The antibody and kit for detecting phorate content provided by the present application, the detection operation is simple, and large-scale instrument analysis equipment does not need to be purchased, and only the detection of the sample can be completed through the kit, which is suitable for on-site mass detection.

[0053] 4) The enzyme-linked immunoassay kit provided by the present application is made of the obtained antibody, which is convenient to use, low in detection cost, efficient, accurate, fast, and can detect a large number of samples at the same time, and is suitable for on-site monitoring and screening of a large number of samples of phorate in fruits and vegetables. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the hapten obtained in the embodiments of the present application.

[0055] Figure 2 The standard curve obtained by detecting gradient concentration phorate standard solution using the antibody provided by the present application in the use of the enzyme-linked immunoassay kit obtained in the embodiments of the present application in fruit and vegetable detection. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Any aspects not detailed in the preparation of haptens, antibodies, and antibody preparation kits of the present invention are performed according to methods disclosed in the prior art, and will not be repeated here.

[0057] Unless otherwise specified, all materials and instruments used in the following examples were purchased from commercial sources.

[0058] Example 1: Preparation of hapten for methamidophos content detection

[0059] Prepare the hapten for phorate content detection according to the following steps:

[0060] 1) Dissolve 2.5g of O-ethyl S-[(ethylthio)methyl] ester (CAS No.: 181257-76-1) in 150mL of pyridine;

[0061] 2) Add 2.31g of N-(2-aminopropyl)maleimide (CAS No.: 110008-25-8), stir thoroughly, attach a reflux condenser, react at 80℃ for 4h, stop the reaction, and obtain the reaction solution;

[0062] 3) The organic solvent in the obtained reaction solution is removed by rotary evaporation, and the solution is loaded onto a silica gel column and eluted with a 10:1 volume ratio of dichloromethane-methanol mixed solution to obtain the hapten for the detection of phorate content.

[0063] Hydrogen nuclear magnetic resonance (HNMR) of the hapten 1 H-NMR) determination, results are as follows Figure 1 As shown: 1 ¹H NMR (500MHz, Chloroform-d): δ (ppm) 6.71 (s, 2H), 4.42 (t, J = 4.6 Hz, 1H), 4.26 (dq, J = 8.4, 7.0 Hz, 2H), 3.92 (qt, J = 16.9, 5.1 Hz, 2H), 3.80 (s, 2H), 3.20 (q, J = 4.3 Hz, 2H), 2.57 (q, J = 5.9 Hz, 2H), 2.00–1.93 (m, 2H), 1.32 (td, J = 7.0, 0.7 Hz, 3H), 1.22 (t, J = 5.8 Hz, 3H). The chemical shift δ = 6.71 (s, 2H) is the absorption peak of the double-bonded hydrogen on the spacer arm. The presence of this peak proves successful coupling and the correct hapten structure.

[0064] Example 2: Preparation of antigen for phorate content detection coupled with bovine serum albumin

[0065] Includes the following steps:

[0066] Take 100 mg of bovine serum albumin (BSA), dissolve in 10 mL of PB buffer at pH 8.0, drop 1 mL of an aqueous solution containing 7.2 mg of tris (2-carboxyethyl) phosphine hydrochloride, react at room temperature for 2 h to obtain A solution; take 9.34 mg of the phosphamidon content detection hapten prepared in Example 1, dissolve in 0.5 mL of N,N-dimethylformamide, then drop into A solution, react at 4°C for 12 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change the solution 3 times a day, to obtain phosphamidon antigen coupled with bovine serum albumin, divide and store at -20°C.

[0067] Preparation of phosphamidon content detection antigen coupled with ovalbumin in Example 3

[0068] The following steps are included:

[0069] Take 100 mg of ovalbumin (OVA), dissolve in 10 mL of PB buffer at pH 8.0, drop 1 mL of an aqueous solution containing 3.17 mg of tris (2-carboxyethyl) phosphine hydrochloride, react at room temperature for 2 h to obtain A solution; take 4.08 mg of the phosphamidon content detection hapten prepared in Example 1, dissolve in 0.5 mL of N,N-dimethylformamide, then drop into A solution, react at 4°C for 12 h, purify by dialysis with 0.02 mol / L PB buffer for 3 days, change the solution 3 times a day, to obtain phosphamidon antigen coupled with ovalbumin, divide and store at -20°C.

[0070] Preparation of phosphamidon content detection monoclonal antibody in Example 4

[0071] The following steps are included:

[0072] (1) Animal immunization: inject the phosphamidon antigen coupled with bovine serum albumin prepared in Example 2 into Balb / c mice, the immunization dose is 150 μg per mouse, so that the mice produce polyclonal antibody serum.

[0073] (2) Cell fusion and cloning: after the mouse serum determination result is high, take the spleen cells, 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 the hybridoma cell strain secreting monoclonal antibody is obtained.

[0074] (3) Cell cryopreservation and recovery: the monoclonal hybridoma cell strain of phosphamidon is made into a cell suspension of 1×10 9 / mL with cryopreservation solution, and stored in liquid nitrogen for a long time. When recovering, take out the cryopreservation tube and immediately put it into a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, transfer into a culture bottle for culture.

[0075] (4) Monoclonal antibody production and purification: 0.5 mL sterile paraffin oil was injected into the abdominal cavity of Balb / c mice, 7 days later, 6 x 10 5 monoclonal hybridoma cell lines of phorate were injected into the abdominal cavity, 7 days later, the ascites were collected. The ascites were purified by caprylic acid-saturated ammonium sulfate method, and stored at -20 °C.

[0076] (5) Determination of monoclonal antibody titer: the titer of the antibody was determined by indirect competitive ELISA method to be 1:256000.

[0077] Indirect competitive ELISA method: the enzyme-labeled plate coated with phorate antigen coupled with ovalbumin prepared in Example 3 was added with phorate standard solution and monoclonal antibody working solution, and reacted at 4 °C for 30 min, the liquid in the wells was poured out, and the plate was washed 3-5 times with PBST washing solution and dried with absorbent paper; horseradish peroxidase-labeled goat anti-mouse antibody was added, and reacted at 25 °C for 30 min, and the plate was taken out and the washing step was repeated; substrate solution was added, and reacted at 25 °C for 15 min, then stop solution was added to terminate the reaction; the enzyme-labeled instrument was set at wavelength 450 nm to determine the absorbance value of each well.

[0078] Example 5 Preparation of enzyme-linked immunosorbent kit for detection of phorate content

[0079] The obtained kit includes:

[0080] (1) enzyme-labeled plate coated with phorate antigen coupled with ovalbumin;

[0081] (2) phorate monoclonal antibody working solution;

[0082] (3) horseradish peroxidase-labeled goat anti-mouse antibody;

[0083] (4) 6 bottles of phorate standard solution, with concentrations of 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L, respectively;

[0084] (5) substrate solution composed of A and B, A is urea peroxide, and B is tetramethyl benzidine;

[0085] (6) stop solution is 2 mol / L sulfuric acid solution;

[0086] (7) washing solution is pH 7.2, containing 1.0% Tween-20, 0.02‰ thiomersal preservative, and 0.2 mol / L carbonate buffer;

[0087] (8) reconstitution solution is pH 7.6, containing 10% casein, and 0.2 mol / L phosphate buffer.

[0088] The main reagent of the kit is provided in the form of a working solution, the test method is convenient and easy to operate, has the characteristics of high specificity, high sensitivity, high accuracy, high accuracy, and is convenient for rapid batch detection of a large number of fruit and vegetable samples.

[0089] Application of the enzyme-linked immunoassay kit for detecting phorate content obtained in Example 6 in fruit and vegetable detection and performance detection thereof

[0090] I. Test method of the kit obtained in Example 5

[0091] 1. Sample pretreatment

[0092] Homogenize the fruit and vegetable samples; weigh (2.0 ± 0.05) g of the homogenate, add 6 mL of 0.02 mol / L phosphate buffer, and shake for 5 min using a shaker at 3000 r / min or above at room temperature; centrifuge for 5 min; remove 100 μL of the supernatant and add 900 μL of 0.02 mol / L phosphate buffer; vortex for 2 min using a vortex mixer; mix well and use for analysis.

[0093] 2. Detection operation steps of the kit obtained in Example 5

[0094] Add 50 μL of the sample solution to be detected to each well of the enzyme-labeled plate (coated with the phorate antigen coupled with ovalbumin obtained in Example 3), then add 50 μL of the phorate monoclonal antibody working solution prepared in Example 4 to each well, cover the plate with a cover film, and react at 25°C in the dark for 30 min; pour out the liquid in the wells, add 250 μL of washing solution to each well, pour out the liquid in the wells after 30 s, and repeat the operation to wash the plate 5 times; pat dry with a water-absorbing paper; add 100 μL of horseradish peroxidase-labeled goat anti-mouse antibody to each well, mix gently, cover the plate with a cover film, and react at 25°C in the dark for 30 min; repeat the plate washing steps.

[0095] Add 50 μL of substrate solution A (urea peroxide) and 50 μL of substrate solution B (tetramethylbenzidine, TMB) to each well, mix gently, cover the plate with a cover film, and develop color at 25°C in the dark for 15 min; add 50 μL of stop solution (2 mol / L sulfuric acid solution) to each well, mix gently, and set the wavelength of the enzyme-labeled instrument at 450 nm to measure the absorbance value (OD value) of each well.

[0096] 3. Detection and result analysis

[0097] According to the above operation steps, gradient concentrations of phorate standard solution and pretreated samples were detected as the sample solution to be detected.

[0098] Divide the average absorbance value (B) of each concentration of the standard solution obtained by the average absorbance value (B0) of the first standard solution (0 standard) and multiply by 100% to obtain the percentage absorbance value.

[0099] The standard curve is plotted with the logarithm of the concentration of the standard phorate sample (μg / L) as the X axis and the percentage absorbance value as the Y axis, as shown in Figure 1. Figure 2

[0100] The percentage absorbance value of the pretreated sample solution is calculated in the same way, and the corresponding concentration of the pretreated sample is read from the standard curve of the phorate standard sample. The actual concentration of phorate in the pretreated sample is obtained by multiplying the corresponding dilution factor.

[0101] II. Sensitivity determination of the kit obtained in Example 5

[0102] Sensitivity in the present application: refers to the minimum content of phorate in the sample that can be detected by the kit. The sensitivity of the quantitative kit itself can be understood as the concentration of the smallest standard sample or the lowest standard sample concentration corresponding to the standard curve when the standard curve is established. According to the existing method, the sensitivity of the kit provided in the present application for phorate is 0.1 μg / L.

[0103] III. Detection limit determination of the kit obtained in Example 5

[0104] 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 existing method, the detection limit of the kit for phorate in vegetables and fruits is 5 μg / kg.

[0105] IV. Accuracy and precision determination of the kit obtained in Example 5

[0106] Accuracy and precision: the accuracy of ELISA is represented by the recovery rate, and the precision is represented by the coefficient of variation. Blank cowpea, chives, celery, orange, and apple samples are added with phorate at concentrations of 5, 10, and 20 μg / kg according to the existing method. The experimental results: the recovery rate of the kit prepared by the hapten provided in the present application for the samples is 100%±20%, the within-batch coefficient of variation is <10%, and the between-batch coefficient of variation is <15%.

[0107] It is shown that the kit prepared by the hapten provided in the present application has good accuracy and precision, and can accurately detect the phorate contained in the sample.

[0108] V. Antibody cross-reactivity determination of the kit obtained in Example 5

[0109] ​The indirect competitive ELISA method was used to determine the results of phorate, phorate sulfone, phorate sulfoxide, glyphosate, chlorpyrifos, acephate, sulfotep, methyl isofenphos, parathion, methyl parathion and fenthion with similar structures to phorate. The results showed that phorate was 100%, phorate sulfone and phorate sulfoxide were 80%, and the rest were <1%.

[0110] It is shown that the antibody prepared from the hapten has good specificity for phorate and its metabolites, and can accurately detect phorate and its metabolites in samples without being affected by other structural analogues.

[0111] Six, the storage period test of the kit obtained in Example 5

[0112] After storing the kit at 2-8℃ for 12 months, the maximum absorbance value (zero standard), 50% inhibition concentration and the actual determination value of phorate were detected, and each value was within the normal range.

[0113] It is determined that the kit provided by the present application can be stored for at least 12 months.

[0114] From the above detection results, it can be seen that the antibody prepared from the hapten provided by the present application has a more accurate detection effect in the detection of phorate residues in vegetables and fruits, and the kit provided by the present application can be used for effective detection in various pretreated fruits and vegetables.

[0115] 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 phorate content, characterized in that, The chemical structural formula is: Equation (1); The preparation method of the hapten for the detection of phorate content includes the following steps: O-ethyl S-[(ethylthio)methyl] ester was used as a raw material and condensed with N-(3-aminopropyl)maleimide under pyridine catalysis to obtain the hapten for detecting phorate content; the chemical structure of the O-ethyl S-[(ethylthio)methyl] ester was... The formula is .

2. The hapten for detecting phorate content according to claim 1, characterized in that, The preparation method of the hapten for the detection of phorate content includes the following steps: 1) Dissolve O-ethyl S-[(ethylthio)methyl] ester in pyridine; 2) Add N-(3-aminopropyl)maleimide, stir thoroughly, attach a reflux condenser, react at 80°C 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 phorate content.

3. The hapten for detecting phorate content according to claim 2, characterized in that, The separation and purification operation in step 3) includes the following steps: the organic solvent is removed by rotary evaporation of the obtained reaction solution, the solution is loaded onto a silica gel column, and eluted with a dichloromethane-methanol mixed solution with a volume ratio of 10:1 to obtain the hapten for the detection of phorate content.

4. An antigen for detecting phorate content, characterized in that, It is a conjugate obtained by conjugating the hapten with a carrier protein as described in any one of claims 1 to 3.

5. The antigen for detecting phorate content according to claim 4, characterized in that, The carrier protein is bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.

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