Hapten for detecting content of isofenphos-methyl and application thereof

Abstract

The invention discloses a hapten for detecting the content of isofenphos-methyl and application of the hapten, particularly relates to the hapten for detecting the content of isofenphos-methyl and a preparation method of the hapten, and further discloses application of the hapten in the aspects of antibodies and kits. The enzyme linked immunosorbent assay kit established by aiming at the hapten for detecting the content of isofenphos-methyl can realize rapid detection of a large batch of samples to be detected, is convenient to use and low in detection cost, the detection method is efficient, accurate and rapid, can simultaneously detect a large batch of samples, and is suitable for on-site monitoring of isofenphos-methyl in fruits, vegetables and traditional Chinese medicinal materials and screening of a large number of samples.

Description

Technical Field

[0001] The present application relates to the technical field of pesticide residue detection, and in particular to a hapten for detecting the content of isofosfos-methyl and its application. Background Art

[0002] Methyl isofosfos is a highly toxic organic phosphorus soil insecticide with strong contact and stomach poisoning effects on pests. It is mainly used to control ground pests such as aphids, wireworms, and nematodes. A large part of it will remain in the soil during application. It poses a great threat to human health during application. It can strongly inhibit the activity of cholinesterase in the human body and cause serious toxic mycotic, nicotinic, and central nervous system symptoms. Therefore, it is necessary to establish an efficient, sensitive, and accurate detection and analysis method for methyl isofosfosfos in vegetables, fruits, and Chinese medicinal materials.

[0003] At present, the main detection methods for isofosfos-methyl residues at home and abroad are gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc. This type of instrumental analysis method has the advantages of high detection sensitivity and strong specificity, but the pre-treatment of the detection sample is cumbersome and time-consuming, and the sample needs to be extracted and purified. At the same time, the instrumental detection method requires expensive large-scale instruments and equipment, equipped with professional detection technicians for operation and management, and cannot be carried out on-site large-scale detection. It has poor timeliness and is difficult to promote.

[0004] Immunological detection and analysis technology has been widely used in the field of drug residue detection due to its advantages of high sensitivity, high specificity, rapidity and ease of operation, and has many advantages over instrumental detection methods. However, the existing technology lacks detection reagents that have a specific reaction to methyl isofenphos, which hinders the application of immunoassay detection in the detection of methyl isofenphos.

[0005] When establishing an immunological detection method and applying it to detect methyl isofenphos, the key technology is to be able to obtain antibodies with strong specificity and high sensitivity. To achieve this goal, the prerequisite is to synthesize and prepare a suitable methyl isofenphos hapten. Summary of the invention

[0006] The present application provides a hapten for detecting the content of isofenphos-methyl and its application, which is used to solve the technical problems in the prior art of isofenphos-methyl detection methods, such as cumbersome sample pretreatment, low detection efficiency, inability to achieve rapid and large-scale detection, and lack of an enzyme-linked immunosorbent assay hapten specific for isofenphos-methyl.

[0007] The present application provides a hapten for detecting the content of isofosfomethyl, the chemical structure of which is:

[0008]

[0009] The method for preparing the hapten for detecting the content of isofosfomethyl comprises the following steps:

[0010] 2-Hydroxy-5-nitrobenzoic acid isopropyl ester is used as a raw material, and N-isopropylphosphoramidochlorosulfate O-methyl ester is subjected to a phosphorylation reaction under alkaline conditions, followed by hydrogenation reduction, and finally reacted with succinic anhydride to obtain the hapten for detecting the methyl isoflavone content.

[0011] The hapten with the structural formula can be treated by a common method in the prior art to obtain an antibody, and the antibody can produce a specific reaction to methyl isofenphos contained in various vegetables, fruits and Chinese medicinal materials that have been pretreated. According to this characteristic, the methyl isofenphos standard can be gradiently diluted to obtain methyl isofenphos standard solutions of different concentrations, and the OD value of each standard solution is measured at 450nm using an enzyme marker to obtain a standard curve, and then the sample to be tested is pretreated, the OD value at the same wavelength is measured, and the corresponding concentration is obtained through the standard curve, thereby achieving quantitative, effective and rapid detection of the methyl isofenphos content contained in the sample.

[0012] The reaction can be used to obtain a hapten with a high purity. The reagents and reaction conditions used in the preparation method can be selected according to the reagents and conditions required for the existing phosphorylation reaction, reduction reaction, and addition reaction.

[0013] The present application provides that when the hapten is made into a kit and used to detect methyl isofenphos in vegetables, fruits and Chinese medicinal materials, the sensitivity is 0.05 μg / L, indicating that after the hapten is made into a kit, the detection sensitivity of methyl isofenphos is high; after the kit is made into a kit according to the existing method, the detection limit of methyl isofenphos in vegetables, fruits and Chinese medicinal materials is 5 μg / kg, and it can be used to effectively detect the content of methyl isofenphos in vegetables, fruits and Chinese medicinal materials; after the hapten is made into a kit, the recovery rate of methyl isofenphos is 100%±20%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%, the accuracy is good, and can meet the detection needs.

[0014] After the obtained hapten is made into a test kit, the cross-reactivity to other organophosphorus pesticides is less than 1%, while the cross-reactivity to methyl isofenphos can reach 100%, indicating that the hapten can specifically detect methyl isofenphos and avoid the interference of other structural analogs on the test results, which can well fill the lack of a hapten with obvious specificity for methyl isofenphos in the prior art.

[0015] Preferably, the method for preparing the hapten for detecting the content of isofosfos-methyl comprises the following steps:

[0016] 1) adding chloroform to dissolve isopropyl 2-hydroxy-5-nitrobenzoate, adding N-isopropylphosphoramidochlorosulfate O-methyl ester and triethylamine, reacting at 55° C. for 2 h, stopping the reaction, evaporating to dryness, applying to a silica gel column, eluting and separating with a methanol-dichloromethane mixed solution with a volume ratio of 10:1, to obtain an intermediate 1, wherein the intermediate 1 has the structural formula

[0017] 2) Add ethanol to dissolve the intermediate 1, add zinc powder and 1 mol / L hydrochloric acid, stir well, react at 80°C for 2h, stop the reaction, neutralize with 1 mol / L sodium hydroxide to neutrality, remove ethanol by rotary evaporation, add chloroform and water, shake, stand, separate the water phase, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness, and obtain the intermediate 2, which has the structural formula

[0018] 3) adding pyridine to dissolve the intermediate 2, adding succinic anhydride, reacting at 90° C. for 4 h, stopping the reaction, rotary evaporation to remove pyridine, applying to a silica gel column, eluting and separating with a dichloromethane-methanol mixed solution with a volume ratio of 6:1 to obtain a hapten for detecting the content of methyl isofenphos, wherein the hapten for detecting the content of methyl isofenphos has a structural formula

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

[0020]

[0021] From the above, it can be seen that the reaction can occur normally and a hapten having the above structure can be produced.

[0022] To achieve effective application of haptens, the following applications can be performed:

[0023] On the other hand, the present application also provides an antigen for detecting the content of isofosfomethyl, which is a conjugate obtained by coupling the above-mentioned hapten with a carrier protein.

[0024] Preferably, the carrier protein is bovine serum albumin or ovalbumin. The use of the above types of carrier proteins can effectively play the role of hapten detection.

[0025] The coupling method of the hapten and the carrier protein used in the present invention can be the existing commonly used method for preparing the antigen from the hapten. For example, the coupling method using bovine serum albumin as the carrier protein can be:

[0026] Take 33.3 mg of the above hapten, add 2 mL of N,N-dimethylformamide to dissolve and clarify, add 18 mg of N-hydroxysuccinimide and 31 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation solution A; take 100 mg of bovine serum albumin (BSA), add 4 mL of 0.1 mol / L, pH 8.0 PB buffer to dissolve, and obtain solution B; add solution A drop by drop into solution B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS buffer for 3 days, change the solution 3 times a day, centrifuge and pack, and obtain methyl isoflavone antigen coupled with bovine serum albumin, which is stored at -20°C.

[0027] The coupling method using ovalbumin as carrier protein comprises the following steps:

[0028] Take 19.84 mg of the above hapten, add 2 mL of N,N-dimethylformamide to dissolve and clarify, add 18 mg of N-hydroxysuccinimide and 31 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation solution A; take 100 mg of ovalbumin (OVA), add 4 mL of 0.1 mol / L, pH 8.0 PB buffer to dissolve, and obtain solution B; add solution A drop by drop into solution B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS buffer for 3 days, change the solution 3 times a day, centrifuge and pack, and obtain methyl isoflavone antigen coupled with ovalbumin, which is stored at -20°C.

[0029] Another aspect of the present invention provides an antibody for detecting the content of isofenphos-methyl, which is an antibody obtained by immunizing an animal with the above antigen; the antibody has a specific immune reaction with isofenphos-methyl.

[0030] Preferably, the antibody is isofosfosinate-methyl monoclonal antibody.

[0031] The method for preparing antibodies from the above antigens used in the present invention can be a commonly used animal immunization method in the art, for example comprising the following steps:

[0032] (1) Animal immunization: The methyl isoflavone antigen coupled to bovine serum albumin was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce polyclonal antibody serum.

[0033] (2) Cell fusion and cloning: After the mouse serum test results are high, the spleen cells are taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1. The cell supernatant is measured by indirect competitive ELISA to screen positive wells. The positive wells are cloned by limiting dilution method until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0034] (3) Cell freezing and thawing: Monoclonal hybridoma cell lines of isofosfos-methyl were prepared with freezing solution at 1×10 9 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryopreserved tubes were 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 to culture bottles for culture.

[0035] (4) Production and purification of monoclonal antibodies: Balb / c mice were intraperitoneally injected with 0.5 mL of sterile paraffin oil. Seven days later, 6 × 10 monoclonal hybridoma cells of isofosfate-methyl were intraperitoneally injected. 5 The ascites was collected 7 days later and purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C.

[0036] (5) Determination of monoclonal antibody titer: The antibody titer was determined by indirect competitive ELISA method and was 1:512000.

[0037] The antibody is put into use in the form of a monoclonal antibody and has a high titer.

[0038] Another aspect of the present invention provides an enzyme-linked immunosorbent assay kit for detecting the content of isofenphos-methyl, comprising: an enzyme-labeled plate and an enzyme-labeled antibody, wherein the enzyme-labeled plate is coated with the above-mentioned antigen for detecting the content of isofenphos-methyl; and the enzyme-labeled antibody is the above-mentioned antibody for detecting the content of isofenphos-methyl labeled with horseradish peroxidase.

[0039] The kit is prepared according to an existing method, and the specific method can be prepared by those skilled in the art by searching the existing enzyme-linked immunosorbent assay kit production method.

[0040] Preferably, the enzyme-linked immunosorbent assay kit for detecting the content of isofosfo-methyl further comprises: isofosfo-methyl standard solution with gradient concentrations, substrate solution A, substrate solution B, stop solution, washing solution, and reconstitution solution.

[0041] Preferably, the gradient concentrations of the isofosfos-methyl standard solution are 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, and 4.05 μg / L, respectively;

[0042] Substrate liquid A is carbamide peroxide, and substrate liquid B is tetramethylbenzidine;

[0043] The stop solution is 2 mol / L sulfuric acid solution;

[0044] The washing solution has a pH of 7.2 and contains 0.8% to 1.2% Tween-20, 0.01‰ to 0.03‰ thimerosal preservative, and 0.1 to 0.3 mol / L carbonate buffer;

[0045] The reconstitution solution has a pH of 7.6 and contains 8% to 12% casein and 0.1 to 0.3 mol / L phosphate buffer.

[0046] The test kit made of the above components has high detection efficiency and good accuracy, and is suitable for testing large quantities of samples on site.

[0047] The room temperature in this application ranges from 20 to 25°C, based on the thermometer reading at the time of operation.

[0048] The beneficial effects that the present invention can produce include:

[0049] 1) The hapten for detecting the content of isofenphos-methyl provided by the present invention, when the hapten is made into a kit and used to detect isofenphos-methyl contained in vegetables, fruits and Chinese medicinal materials, the sensitivity is 0.05 μg / L, indicating that after the hapten is made into a kit, the detection sensitivity of isofenphos-methyl is high; after the kit is made into a kit according to the existing method, the detection limit of isofenphos-methyl in vegetables, fruits and Chinese medicinal materials is 5 μg / kg, which can be used for the effective detection of the content of isofenphos-methyl contained in vegetables, fruits and Chinese medicinal materials; after the hapten is made into a kit, the recovery rate of isofenphos-methyl is 100%±20%, the intra-batch variation coefficient is less than 10%, and the inter-batch variation coefficient is less than 15%, the accuracy is good, and it can meet the detection needs. The hapten can retain the characteristic structure of isofenphos-methyl to the greatest extent. After the artificial antibody is prepared with the hapten, the antibody has the characteristics of high sensitivity and strong specificity for isofenphos-methyl detection.

[0050] 2) The hapten for detecting the content of isofenphos-methyl provided by the present invention, and the antibody prepared by using the hapten as a raw material, show strong immunogenicity for the detection of isofenphos-methyl residues due to the use of the hapten as a raw material; the hapten is used as a raw material to prepare an artificial antigen system suitable for animal immunization to immunize animals, and the titer, specificity and affinity of the obtained antibody are relatively good, and the detection sensitivity of isofenphos-methyl can reach 0.05 μg / L.

[0051] 3) The antibody and kit for detecting the content of isofosfos-methyl provided by the present invention are simple to operate, and there is no need to purchase large-scale instrumental analysis equipment. The detection of samples can be completed only through the kit, which is suitable for on-site mass detection.

[0052] 4) The enzyme-linked immunosorbent assay kit provided by the present invention is made of the obtained antibodies, is easy to use, has low detection cost, and has an efficient, accurate, and rapid detection method. It can detect a large number of samples at the same time and is suitable for on-site monitoring of isofenphos-methyl in vegetables, fruits, and Chinese medicinal materials and screening of a large number of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The figure is the hydrogen nuclear magnetic resonance spectrum of the hapten obtained in the example of the present invention.

[0054] Figure 2 The standard curve is obtained by using the antibody provided by the present invention to detect gradient concentrations of isofosfo-methyl standard solutions in the application of the enzyme-linked immunosorbent assay kit obtained in the embodiment of the present invention in the detection of vegetables, fruits and Chinese medicinal materials. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and its accompanying drawings. The contents not described in detail in the hapten preparation antigen, antigen preparation antibody, and antibody preparation kit of the present invention are all carried out according to the methods disclosed in the prior art, and are not repeated here.

[0056] Unless otherwise specified, the materials and instruments used in the following examples were purchased from commercial channels.

[0057] Example 1 Preparation of hapten for detection of isofosfomethyl content

[0058] Prepare the hapten for detecting the content of isofosfomethyl as follows:

[0059] 1) 2.25 g of isopropyl 2-hydroxy-5-nitrobenzoate (CAS No.: 508211-75-4) was dissolved in 200 mL of chloroform, and then 2.05 g of N-isopropylphosphoramidochlorosulfate O-methyl ester (CAS No.: 35236-17-0) and 1.3 mL of triethylamine were added. The mixture was reacted at 55° C. for 2 h, and the reaction was stopped. The mixture was evaporated to dryness by rotary evaporation, and applied to a silica gel column. The mixture was eluted and separated with a methanol-dichloromethane mixed solution with a volume ratio of 10:1 to obtain 2.16 g of intermediate 1;

[0060] 2) Dissolve 2.16 g of intermediate 1 in 150 mL of ethanol, add 0.65 g of zinc powder and 1 mL of 1 mol / L hydrochloric acid, stir well, react at 80 °C for 2 h, stop the reaction, neutralize with 1 mol / L sodium hydroxide to neutrality, remove ethanol by rotary evaporation, add 200 mL of chloroform and 100 mL of water, shake, let stand, separate the water phase, dry the organic phase with anhydrous sodium sulfate, and evaporate to dryness to obtain intermediate 2;

[0061] 3) Add 50 mL of pyridine to dissolve all the intermediate 2, add 1.14 g of succinic anhydride, react at 90° C. for 4 h, stop the reaction, remove pyridine by rotary evaporation, apply to a silica gel column, and elute and separate with a mixed solution of dichloromethane-methanol in a volume ratio of 6:1 to obtain the above-mentioned hapten for detecting the content of methyl isofosfate.

[0062] H NMR ( 1 H-NMR) determination, the results are as follows Figure 1 As shown: 1H NMR (500MHz, Chloroform-d): δ (ppm) 9.29 (s, 1H), 8.11 (d, J = 2.2Hz, 1H), 7.52 (dd, J = 8.4, 2.2Hz, 1H), 7.25 (d, J = 8.5Hz, 1H), 5.05 (dq, J = 11. 5,5.9Hz,1H), 5.00(d,J=7.7Hz,1H), 3.87(d,J=11.0Hz,2H), 3.64(dp,J=7.7,6.6Hz,1H), 2.63–2.50(m,4H), 1.29(dd,J=12.5,6.1Hz,12H). The presence of chemical shift δ2.63–2.50 (m, 4H) and 9.29 (s, 1H) absorption peaks in the H NMR spectrum proved that the spacer arm was successfully coupled and the hapten structure was correct.

[0063] Example 2 Preparation of antigen for detecting the content of methyl isofosfate coupled to bovine serum albumin

[0064] The following steps are involved:

[0065] Take 33.3 mg of the hapten for detecting the content of methyl isofosfate prepared in Example 1, add 2 mL of N, N-dimethylformamide to dissolve and clarify, add 18 mg of N-hydroxysuccinimide and 31 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation solution A; take 100 mg of bovine serum albumin (BSA), add 4 mL of 0.1 mol / L, pH 8.0 PB buffer to dissolve to obtain solution B; add solution A drop by drop into solution B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS buffer for 3 days, change the solution 3 times a day, centrifuge and pack to obtain methyl isofosfate antigen coupled to bovine serum albumin, and store at -20°C.

[0066] Example 3 Preparation of antigen for detecting the content of isofosfomethyl coupled to ovalbumin

[0067] The following steps are involved:

[0068] Take 19.84 mg of the hapten for detecting the content of methyl isoflavone prepared in Example 1, add 2 mL of N, N-dimethylformamide to dissolve and clarify, add 18 mg of N-hydroxysuccinimide and 31 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation liquid A liquid; take 100 mg of ovalbumin (OVA), add 4 mL of 0.1 mol / L, pH 8.0 PB buffer to dissolve, and obtain liquid B; add liquid A dropwise into liquid B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 mol / L PBS buffer for 3 days, change the liquid 3 times a day, centrifuge and pack, and obtain methyl isoflavone antigen coupled to ovalbumin, which is stored at -20°C.

[0069] Example 4 Preparation of monoclonal antibody for detection of isofosfos-methyl content

[0070] The following steps are involved:

[0071] (1) Animal immunization: The isofosfosinate-methyl antigen coupled to bovine serum albumin prepared in Example 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce polyclonal antibody serum.

[0072] (2) Cell fusion and cloning: After the mouse serum test results are high, the spleen cells are taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1. The cell supernatant is measured by indirect competitive ELISA to screen positive wells. The positive wells are cloned by limiting dilution method until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0073] (3) Cell freezing and thawing: Monoclonal hybridoma cell lines of isofosfos-methyl were prepared with freezing solution at 1×10 9 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryopreserved tubes were 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 to culture bottles for culture.

[0074] (4) Production and purification of monoclonal antibodies: Balb / c mice were intraperitoneally injected with 0.5 mL of sterile paraffin oil. Seven days later, 6 × 10 monoclonal hybridoma cells of isofosfate-methyl were intraperitoneally injected. 5 The ascites was collected 7 days later and purified using the caprylic acid-saturated ammonium sulfate method and stored at -20°C.

[0075] (5) Determination of monoclonal antibody titer: The antibody titer was determined by indirect competitive ELISA method and was 1:512000.

[0076] Indirect competitive ELISA method: the ELISA plate was coated with the ovalbumin-coupled methyl isofenphos antigen prepared in Example 3, and the methyl isofenphos standard solution and the monoclonal antibody working solution were added, and the reaction was carried out at 4°C for 30 minutes. The liquid in the wells was poured out, and the wells were washed with PBST washing solution for 3 to 5 times, and patted dry with absorbent paper; goat anti-mouse anti-antibody labeled with horseradish peroxidase was added, and the reaction was carried out at 25°C for 30 minutes, and the plate washing step was repeated; the substrate solution was added, and the reaction was carried out at 25°C for 15 minutes, and the stop solution was added to stop the reaction; the ELISA reader was set to measure the absorbance value of each well at a wavelength of 450nm.

[0077] Example 5 Preparation of ELISA kit for detecting isofosfomethyl content

[0078] The kit includes:

[0079] (1) an ELISA plate coated with isofosfate-methyl antigen coupled to ovalbumin;

[0080] (2) Horseradish peroxidase-labeled monoclonal antibody against methyl isofosfate;

[0081] (3) 6 bottles of isofosfos-methyl standard solution, with concentrations of 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, and 4.05 μg / L respectively;

[0082] (4) The substrate solution consists of solution A and solution B, wherein solution A is urea peroxide and solution B is tetramethylbenzidine;

[0083] (5) The stop solution is 2 mol / L sulfuric acid solution;

[0084] (6) The washing solution is pH 7.2, containing 1.0% Tween-20, 0.02‰ thimerosal preservative, and 0.2 mol / L carbonate buffer;

[0085] (7) The reconstitution solution has a pH of 7.6 and contains 10% casein and 0.2 mol / L phosphate buffer.

[0086] The main reagents of this kit are provided in the form of working solution. The test method is convenient and easy to use, and has the characteristics of high specificity, high sensitivity, high precision and high accuracy. It is convenient for rapid batch testing of large quantities of vegetable, fruit and Chinese medicinal materials samples.

[0087] Example 6 Application of the obtained enzyme-linked immunosorbent assay kit for detecting the content of isofosfomethyl in the detection of vegetables, fruits and Chinese medicinal materials and its performance detection

[0088] 1. Testing method of the kit obtained in Example 5

[0089] 1. Sample pretreatment

[0090] Homogenize the sample; weigh (1.0±0.05) g of the homogenate, add 4 mL of acetonitrile, vortex for 1 min, and centrifuge at 3000 r / min at room temperature for 5 min; take 2 mL of the supernatant into a centrifuge tube and blow dry in a 50°C water bath under a nitrogen flow; add 500 μL of 0.1 mol / L phosphate buffer (pH 7.2), vortex for 30 s, and mix well for analysis.

[0091] 2. Detection operation steps of the kit obtained in Example 5

[0092] Add 50 μL / well of the test solution to the microwells of the ELISA plate (coated with the methyl isoflavone antigen coupled to ovalbumin obtained in Example 3), then add 50 μL / well of the methyl isoflavone monoclonal antibody working solution labeled with horseradish peroxidase (prepared in Example 4), cover the plate with a cover film, and react at 25° C. in the dark for 30 minutes; 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 seconds, repeat the washing operation 5 times, and pat dry with absorbent paper;

[0093] Add 50 μL of substrate solution A (urea peroxide) and 50 μL of substrate solution B (tetramethylbenzidine (TMB)) to each well, shake gently to mix, cover the plate with a cover film and place at 25°C in the dark for 15 min, add 50 μL of stop solution 2 mol / L sulfuric acid solution to each well, shake gently to mix, set the wavelength of the microplate reader at 450 nm, and measure the absorbance (OD value) of each well.

[0094] 3. Detection and analysis of results

[0095] According to the above operation steps, the methyl isofosfosinate standard solution and the pre-treated sample with gradient concentrations are used as the test solution for detection;

[0096] The absorbance value in percentage was obtained by dividing the average absorbance value (B) of the standard solution of each concentration obtained by the average absorbance value (B0) of the first standard solution (0 standard) and then multiplying by 100%.

[0097] The standard curve is drawn with the logarithmic value of the concentration of methyl isofosphate standard (μg / L) as the X-axis and the percentage absorbance value as the Y-axis, as shown in the figure below: Figure 2 shown.

[0098] The same method is used to calculate the percent absorbance value of the pretreated sample solution obtained by the test, and then the concentration corresponding to the pretreated sample is read from the standard curve of the methyl isofenphos standard. The actual concentration of methyl isofenphos in the pretreated sample is obtained by multiplying it by the corresponding dilution factor.

[0099] 2. Sensitivity determination of the kit obtained in Example 5

[0100] In this application, sensitivity refers to the minimum content of isofenphos-methyl in the analyte that can be detected by the kit. The sensitivity of the quantitative kit itself can usually be understood as the concentration value of the standard with the lowest concentration in the kit standard except the "0" standard or the minimum standard concentration corresponding to the establishment of the standard curve. According to the existing method, the sensitivity of the kit provided in this application to isofenphos-methyl is 0.05 μg / L.

[0101] III. Determination of the detection limit of the kit obtained in Example 5

[0102] Detection limit: usually refers to the minimum detection amount of the actual sample determined by the test kit product. Its theoretical definition is: according to the reasonable pre-treatment method, 20 negative (blank) samples are measured, and the average value X and 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 this kit for isofosfos-methyl in vegetables, fruits and Chinese medicinal materials is 5μg / kg.

[0103] IV. Determination of Accuracy and Precision of the Kit Obtained in Example 5

[0104] Accuracy and precision: The accuracy of ELISA is expressed as recovery rate, and the precision is expressed as coefficient of variation. Blank leek, cowpea, apple, pear, honeysuckle, and wolfberry samples were taken, and methyl isoflavone was added to them at three concentrations of 5, 10, and 20 μg / kg according to the existing method for recovery test. The experimental results showed that the recovery rate of the sample prepared by the kit using the hapten provided by the application was 100% ± 20%, the intra-batch coefficient of variation was <10%, and the inter-batch coefficient of variation was <15%.

[0105] It is shown that the kit prepared by using hapten provided in the present application has good accuracy and precision and can accurately detect methyl isoflavone contained in the sample.

[0106] V. Determination of cross-reactivity of antibodies in the kit obtained in Example 5

[0107] The existing indirect competitive ELISA method was used to determine the following substances with similar structures to isothiophos-methyl: isocarbophos, phoxim, malathion, phorate, methamidophos, ethophos, acephate, monocrotophos, methyl parathion, demeton, parathion and mefenphos. The results showed that except for isothiophos-methyl which was 100%, the others were all <1%.

[0108] It is shown that the antibody prepared by using the hapten provided in the present application has good specificity for isofenphos-methyl, can accurately detect isofenphos-methyl contained in the sample, and is not affected by other structural analogs.

[0109] VI. Test on the shelf life of the kit obtained in Example 5

[0110] After the kit was stored at 2-8°C for 12 months, its maximum absorbance value (zero standard), 50% inhibition concentration, and actual measured value of isofosfos-methyl were tested, and all values ​​were within the normal range.

[0111] It has been determined that the kit provided in this application can be stored for at least 12 months.

[0112] From the above test results, it can be seen that the antibody prepared from the hapten provided by the present invention and made into a kit has a relatively accurate detection effect in the detection of methyl isoflurane residues in vegetables, fruits and traditional Chinese medicines. The kit provided by the present invention can be used for effective detection in various pre-treated plant products.

[0113] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hapten for detecting the content of isofosfosinate-methyl, characterized in that: The chemical structure is:

2. A method for preparing a hapten for detecting the content of methyl isofosphate, which is to use 2-hydroxy-5-nitrobenzoic acid isopropyl ester as a raw material, and N-isopropylphosphoramido chlorosulfate O-methyl ester is subjected to a phosphorylation reaction under alkaline conditions, followed by hydrogenation reduction, and finally reacting with succinic anhydride, wherein: The specific steps include: 1) adding chloroform to dissolve isopropyl 2-hydroxy-5-nitrobenzoate, adding N-isopropylphosphoramidochlorosulfate O-methyl ester and triethylamine, reacting at 55° C. for 2 h, stopping the reaction, evaporating to dryness, applying to a silica gel column, eluting and separating with a methanol-dichloromethane mixed solution with a volume ratio of 10:1, to obtain an intermediate 1, wherein the intermediate 1 has the structural formula 2) Add ethanol to dissolve the intermediate 1, add zinc powder and 1 mol / L hydrochloric acid, stir well, react at 80°C for 2h, stop the reaction, neutralize with 1 mol / L sodium hydroxide to neutrality, remove ethanol by rotary evaporation, add chloroform and water, shake, stand, separate the water phase, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness, and obtain the intermediate 2, which has the structural formula 3) adding pyridine to dissolve the intermediate 2, adding succinic anhydride, reacting at 90° C. for 4 h, stopping the reaction, rotary evaporation to remove pyridine, applying to a silica gel column, eluting and separating with a dichloromethane-methanol mixed solution with a volume ratio of 6:1 to obtain a hapten for detecting the content of methyl isofenphos, wherein the hapten for detecting the content of methyl isofenphos has a structural formula 3. An antigen for detecting the content of isofosfosinate-methyl, characterized in that: The conjugate is obtained by coupling the hapten as claimed in claim 1 with a carrier protein; the carrier protein is bovine serum albumin or ovalbumin.

4. An antibody for detecting the content of isofosfosinate-methyl, characterized in that: The invention relates to an antibody obtained by immunizing an animal with the antigen as claimed in claim 3; the antibody undergoes a specific immune reaction with methyl isoflavone.

5. The antibody for detecting the content of isofosfos-methyl according to claim 4, characterized in that: The antibody is a monoclonal antibody against isofosfosinate-methyl.

6. An enzyme-linked immunosorbent assay kit for detecting the content of isofosfos-methyl, comprising: ELISA plate, enzyme-labeled antibody, gradient concentration of isofosfos-methyl standard solution, substrate solution A, substrate solution B, stop solution, washing solution, and reconstitution solution, characterized in that: The ELISA plate is coated with the antigen for detecting the content of isofosfos-methyl as claimed in claim 3; The enzyme-labeled antibody is the antibody for detecting the content of isofosfomethyl as claimed in claim 4 or 5 labeled with horseradish peroxidase; The gradient concentrations of the isofosfos-methyl standard solution are 0 μg / L, 0.05 μg / L, 0.15 μg / L, 0.45 μg / L, 1.35 μg / L, and 4.05 μg / L respectively; The substrate liquid A is carbamide peroxide, and the substrate liquid B is tetramethylbenzidine; The stop solution is 2 mol / L sulfuric acid solution; The washing solution has a pH of 7.2 and contains 0.8% to 1.2% Tween-20, 0.01‰ to 0.03‰ thimerosal preservative, and 0.1 to 0.3 mol / L carbonate buffer; The reconstituted solution has a pH of 7.6 and contains 8% to 12% casein and 0.1 to 0.3 mol / L phosphate buffer.

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