amisulbrom hapten, artificial antigen and antibody, and preparation method and application thereof

Abstract

The application provides a flumetover half antigen, artificial antigen and antibody and a preparation method of the flumetover half antigen, artificial antigen and antibody, and application of the flumetover half antigen, artificial antigen and antibody to an enzyme-linked immunoassay kit, the kit comprising: an enzyme-labeled plate coated with a coating agent, a flumetover standard solution, a flumetover antibody, an enzyme conjugate concentrate, an enzyme conjugate diluent, a substrate developing solution, a termination solution and a washing solution, wherein the coating agent is a flumetover conjugate antigen, the enzyme conjugate is an enzyme-labeled flumetover antibody, and the flumetover antibody is obtained by immunizing animals with an immunogen. The application further discloses a method for detecting flumetover by using the enzyme-linked immunoassay kit, which comprises the following steps: firstly, sample pretreatment, then, detection by using the kit, and finally, analysis of the detection result. The enzyme-linked immunoassay kit provided by the application can be used for detecting the content of flumetover in vegetable and fruit samples, and has the advantages of simple operation, low cost, high sensitivity, on-site monitoring and suitability for screening of a large number of samples.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of fludioxonil haptens, artificial antigen and antibody and its preparation method and application technology, specifically to a kind of fludioxonil artificial antigen molecular structure and for detecting fludioxonil enzyme-linked immunosorbent assay kit, qualitative, quantitative detection leek, cucumber, grape, mango and other vegetables and fruits fludioxonil drug residue. BACKGROUND

[0002] Fludioxonil is a new type of nicotinamide fungicide developed by BASF Company in Germany, chemical name is 2-chloro-N-(4'-chlorobenzene-2-yl) nicotinamide, mainly used for preventing and treating powdery mildew, gray mold, various rot diseases, brown rot and root rot, etc., fungicidal spectrum is wide, almost all types of fungal diseases are active, and other agents resistant bacteria are also effective, mainly used for the prevention and treatment of diseases including rape, grape, fruit tree, vegetable and field crops, etc.The drug has been registered in the United Kingdom, Germany and Switzerland in 2004.

[0003] Fludioxonil can cause serious harm to human health, therefore, in order to ensure the interests of consumers and the safety of animal-derived food, some countries and regions have stipulated the maximum residue limit (MRLs) of fludioxonil, GB 2763-2021 food safety national standard "maximum residue limits of pesticides in food" stipulates that the maximum residue limit of fludioxonil in raw milk is 0.1 mg / kg, and the maximum residue limit of fludioxonil in vegetables and fruits is 0.1 mg / kg-50 mg / kg.China has also formulated corresponding national standards for detecting the residue of fludioxonil in food.China GB 23200.68-2016 food safety national standard "determination of fludioxonil residue in food by gas chromatography-mass spectrometry" stipulates that the quantitative limit of fludioxonil in spinach, carrot, strawberry, peanut, chestnut, tea, onion, chicken, cod and honey is 0.01 mg / kg.GB / T 22979-2008 "determination of fludioxonil residue in milk and milk powder by gas chromatography-mass spectrometry" stipulates that the detection limit of fludioxonil in milk is 0.01 mg / kg, and the detection limit of fludioxonil in milk powder is 0.08 mg / kg.

[0004] The methods for detecting fludioxonil include gas chromatography-mass spectrometry, high performance liquid chromatography, ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry, etc.Compared with the above methods, the immunochemical detection method has the advantages of rapidity, specificity, sensitivity, accuracy, batch monitoring, simple sample processing and automation, etc.The prerequisite for detecting fludioxonil by immunochemical method is to have antibody against fludioxonil, the present application invents a method for preparing fludioxonil artificial antigen, and applies it to enzyme-linked immunosorbent assay kit, which is short in time, simple in operation, low in cost and suitable for large-scale detection of samples in basic units. SUMMARY

[0005] The present application aims to provide an enzyme-linked immunoassay kit capable of detecting the amount of fludioxonil drug residues in vegetables and fruits (leeks, cucumbers, grapes, mangos), and to provide a qualitative and quantitative detection method which is efficient, accurate, simple and suitable for screening a large number of samples.

[0006] The kit of the present application comprises: an enzyme-labeled plate coated with a coating agent, a fludioxonil standard solution, a fludioxonil antibody, an enzyme conjugate concentrate, an enzyme conjugate diluent, a substrate developing solution, a stop solution, and a washing solution, wherein the coating agent is a fludioxonil conjugate antigen, and the enzyme conjugate is an enzyme-labeled fludioxonil antibody.

[0007] The fludioxonil specific antibody is prepared by using a fludioxonil artificial antigen as an immunogen, and the fludioxonil specific antibody is a fludioxonil monoclonal antibody or a fludioxonil polyclonal antibody.

[0008] The labeled enzyme of the enzyme conjugate is horseradish peroxidase or a bacterial extract alkaline phosphatase, and the labeled enzyme is preferably horseradish peroxidase; the enzyme conjugate is obtained by coupling an enzyme and a fludioxonil antibody.

[0009] In order to facilitate on-site monitoring and screening of a large number of samples, the kit further comprises a fludioxonil standard solution, a substrate developing solution, a stop solution, and a washing solution.

[0010] The fludioxonil standard solution comprises six bottles, and the concentrations are 0 μg / L, 0.2 μg / L, 0.6 μg / L, 1.8 μg / L, 5.4 μg / L, and 16.2 μg / L, respectively.

[0011] When the labeled enzyme is horseradish peroxidase, the substrate developing solution is composed of a substrate solution A and a substrate solution B, the A is hydrogen peroxide or urea peroxide, the B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is a 1-2 mol / L sulfuric acid solution or a hydrochloric acid buffer solution; when the labeled enzyme is a bacterial extract alkaline phosphatase, the substrate developing solution is a p-nitrophenyl phosphate buffer solution, and the stop solution is a 1-2 mol / L sodium hydroxide solution.

[0012] The washing solution preferably has a pH value of 7.4, contains 0.5-1.0% Tween-20, 0.01‰-0.03‰ sodium azide preservative, and 0.1-0.3 mol / L phosphate buffer, and the percentages are weight / volume percentages.

[0013] In the preparation of the enzyme-labeled plate, the coating buffer solution is a 0.05 mol / L carbonate buffer solution with a pH value of 9.6, and the blocking solution is a 1-3% casein phosphate buffer solution with a pH value of 7.1-7.5, and the percentages are weight / volume percentages.

[0014] The preparation process of the enzyme label plate in the application is as follows: the coating agent is diluted to 30 μg / mL with coating buffer, 100 μl is added to each well, incubated at 25℃ for 2 hours or overnight at 4℃ in the dark, the liquid in the well is poured out, washed with washing liquid for 2 times, 30 seconds each time, patted dry, then 150-200 μl of blocking liquid is added to each well, incubated at 25℃ for 1-2 hours in the dark, the liquid in the well is poured out and patted dry, and then the enzyme label plate is stored in a vacuum sealed aluminum film after drying.

[0015] The detection principle of the application is as follows:

[0016] The kit adopts ELISA method, the antigen is pre-coated on the enzyme label plate micro-well strip, the residual fludioxonil in the sample and the pre-coated antigen on the enzyme label plate micro-well strip compete for the enzyme conjugate of anti-fludioxonil, the color is developed by TMB substrate, the absorbance value of the sample is negatively correlated with the content of the residual fludioxonil, compared with the standard curve, and then multiplied by the corresponding dilution factor, so as to obtain the residual amount of fludioxonil in the sample.

[0017] The application also provides a method for detecting fludioxonil by using the above-mentioned enzyme-linked immunoassay kit, which comprises the following steps:

[0018] (1) detecting by using the kit;

[0019] (2) analyzing the detection result.

[0020] The enzyme-linked immunoassay kit for detecting fludioxonil in the application mainly adopts ELISA method to qualitatively or quantitatively detect the content of fludioxonil in the sample; the pretreatment requirement of the sample is low, the sample pretreatment process is simple, and a large number of samples can be quickly detected at the same time; the main reagent is provided in the form of working solution, the detection method is convenient and easy to operate, and has the characteristics of high specificity, high sensitivity, high accuracy, high accuracy and the like. The enzyme-linked immunoassay kit in the application has the characteristics of simple structure, convenient use, low price, convenient carrying, efficient detection method, accuracy, simplicity and suitability for large-scale sample screening of qualitative and quantitative. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : the molecular structural formula of fludioxonil hapten DETAILED DESCRIPTION

[0022] The application will be further described in combination with specific examples. It should be understood that these examples are only used to illustrate the application, and are not used to limit the scope of the application.

[0023] Example 1: Preparation of kit components

[0024] 1. Preparation of fludioxonil hapten

[0025] Take 4'-chloro-2-amino biphenyl 2.0 g, add pyridine 50 mL to dissolve, drop 2-chloro-5-aldehyde-nicotinoyl chloride 2.4 g in pyridine 50 mL, heat in oil bath, 90℃ for 4 h, stop the reaction, rotary evaporation, remove pyridine, get red oil, elute on silica gel column, volume ratio of dichloromethane to methanol is 5:1, get aldehyde-ambam product 2.7 g, yield 72.9%, which is hapten product.

[0026] 2. Preparation of antigen

[0027] Preparation of immunogen: coupling ambam hapten with bovine serum albumin (BSA) to get immunogen.

[0028] Take 13.8 mg of aldehyde-ambam hapten product, add methanol 2 mL to dissolve, get solution A; take bovine serum albumin BSA 50 mg, add 0.1 mol / L PB 5.8 buffer 5 mL to dissolve, drop all solution A, react at room temperature for 12 h, add 5.8 mg sodium borohydride aqueous solution, continue to stir for 2 h, purify by dialysis with 0.02 M PB buffer, change liquid 3 times a day, dialysis for 3 days, centrifuge, and aliquot to get ambam-BSA conjugate, which is immunogen.

[0029] Preparation of coating antigen: coupling ambam hapten with ovalbumin OVA to get coating antigen.

[0030] The same as the preparation method of immunogen, condense 16.44 mg ambam hapten with 100 mg ovalbumin OVA, reduce by sodium borohydride to get ambam-OVA conjugate, which is coating antigen.

[0031] 4'-chloro-2-amino biphenyl and 2-chloro-5-aldehyde-nicotinoyl chloride in pyridine, acylation reaction to get aldehyde-ambam hapten, condense with carrier protein, then reduce by sodium borohydride to get ambam antigen.

[0032] 3. Preparation of ambam monoclonal antibody

[0033] Animal immunization: inject the immunogen obtained in the above step into Balb / c mice, the immunization dose is 150 μg per mouse, so that the mice produce antisera.

[0034] 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 (quantity ratio), determine the cell supernatant by indirect competitive ELISA, screen positive wells, and use limited dilution method to clone the positive wells until the hybridoma cell strain secreting ambam monoclonal antibody is obtained.

[0035] Cell freezing and recovery: make the monoclonal hybridoma cell strain into 1×106 Cell suspensions of cells / mL were stored long-term in liquid nitrogen. Upon thawing, the cryovials were removed 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 flasks for incubation.

[0036] Production and purification of monoclonal antibodies: Balb / c mice were intraperitoneally injected with 0.5 mL of sterile paraffin oil per mouse. Seven days later, stable monoclonal hybridoma cell lines (5 × 10⁶ cells / mL) were injected intraperitoneally. 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] 4. Preparation of enzyme conjugates

[0038] Goats were used as immunized animals, and boscalid monoclonal antibody was used as an immunogen to immunize pathogen-free goats to obtain boscalid antibody. The boscalid antibody was then conjugated with horseradish peroxidase (HRP) to obtain an enzyme conjugate.

[0039] 5. Preparation of ELISA plates

[0040] Dilute the coating agent to 30 μg / mL with coating buffer, add 100 μl to each well, incubate at 25°C in the dark for 2 h, discard the liquid in the well, wash twice with washing buffer for 30 s each time, blot dry, then add 200 μl of blocking buffer to each well, incubate at 25°C in the dark for 2 h, discard the liquid in the well, blot dry, and then vacuum seal with aluminum foil for storage.

[0041] Example 2: Construction of an enzyme-linked immunosorbent assay kit for detecting boscalid.

[0042] Construct an enzyme-linked immunosorbent assay (ELISA) kit for the detection of boscalid, comprising the following components:

[0043] (1) ELISA plate coated with boscalidamine-conjugated antigen;

[0044] (2) Six bottles of cyazofamid standard solution with concentrations of 0 μg / L, 0.2 μg / L, 0.6 μg / L, 1.8 μg / L, 5.4 μg / L, and 16.2 μg / L, respectively;

[0045] (3) Horseradish peroxidase-labeled cyazofamid antibody;

[0046] (4) The substrate color development solution consists of solution A and solution B. Solution A is urea peroxide and solution B is tetramethylbenzidine;

[0047] (5) The stop solution is 2 mol / L sulfuric acid;

[0048] (6) The washing liquid is a phosphate buffer with pH value of 7.4, containing 0.5%-1.0% Tween-20, 0.01‰-0.03‰ sodium azide preservative, 0.1-0.3 mol / L, and the percentage is weight volume percentage;

[0049] Example 3 Detection of boscalid in vegetables and fruits (leek, cucumber, grape, mango)

[0050] 1. Detection by using the kit

[0051] The micro-wells corresponding to the samples and standard samples are numbered in order, and each sample and standard sample is made in two holes in parallel, and the positions of the standard holes and sample holes are recorded. Add 50 μl of standard sample / sample to the corresponding micro-well, then add 50 μl of enzyme conjugate working solution per hole, and react for 30 min in a 25℃ dark environment. Shake off the liquid in the hole, wash 4-5 times, and pat dry. Add 50 μl of substrate solution A per hole, then add 50 μl of substrate solution B per hole, mix well, and react for 15 min in a 25℃ dark environment. Add 50 μl of stop solution per hole, mix well, set the enzyme marker at 450 nm, and measure the OD value of each hole.

[0052] 2. Analysis of detection results

[0053] The percentage absorbance of the standard sample or sample is equal to the average value of the absorbance value of the standard sample or sample (two holes) divided by the average value of the absorbance value of the first standard sample (0 standard), multiplied by 100%, to obtain the percentage absorbance value of the standard sample or sample. The standard curve is drawn with the standard sample percentage absorbance as the vertical coordinate and the logarithm of the boscalid standard sample concentration (μg / L) as the horizontal coordinate. The percentage absorbance of the sample is substituted into the standard curve, and the corresponding concentration of the sample is read from the standard curve, multiplied by the corresponding dilution factor, which is the actual concentration of boscalid in the sample.

[0054] Example 4 Determination test of boscalid technical parameters

[0055] 1. Sensitivity and detection limit of the kit

[0056] The sensitivity of the kit is determined according to the conventional method, the range of the standard curve is 0.2-16.2 μg / L, the IC 50 (50% inhibition concentration) floating range is 0.89-1.62 μg / L; 20 samples are detected, the concentration corresponding to each percentage absorbance value is found from the standard curve, and the detection limit is represented by the average value of the concentration of the 20 samples plus 3 times the standard deviation. The results show that the detection limit of this method for boscalid in vegetables and fruits (leek, cucumber, grape, mango) is 1 μg / kg.

[0057] 2. Sample precision and accuracy test

[0058] The relative standard deviation (RSD%) of the detection results of a certain concentration sample in repeated determination was used as the precision evaluation index. The calculation formula was: recovery (%) = actual measured value / theoretical value x 100%, wherein the theoretical value was the added concentration of the sample; relative standard deviation RSD% = SD / X x 100%, wherein SD was the standard deviation, and X was the average value of the measured data.

[0059] The fludioxonil was added to the vegetable and fruit (leek, cucumber, grape and mango) samples at two concentrations of 1.0 μg / kg and 2.0 μg / kg, four parallel samples were prepared for each sample, and three batches of different reagents were used for determination. The average recovery rate and precision results of the samples are shown in the following table.

[0060] Table 1 Precision and accuracy test of vegetable and fruit samples

[0061]

[0062]

[0063] The fludioxonil was added to the vegetable and fruit (leek, cucumber, grape and mango) samples at two concentrations of 1.0 μg / kg and 2.0 μg / kg, and the recovery rate ranged from 72.6% to 105.7%; the intra-batch and inter-batch relative standard deviations were less than 10%.

[0064] 3. Kit stability test

[0065] The storage condition of the kit was 2-8°C. After 12 months of determination, the maximum absorbance value (zero standard), 50% inhibition concentration and actual measured value of fludioxonil addition of the kit were all within the normal range. Considering that abnormal storage conditions may occur during transportation and use, the kit was placed at 37°C for 7 days for accelerated aging test, and the results showed that all the indexes of the kit met the requirements. Considering the freezing condition of the kit, the kit was placed in a refrigerator at-20°C for 7 days, and the determination results also showed that all the indexes of the kit were completely normal. From the above results, it can be concluded that the kit can be stored at 2-8°C for at least 12 months or more.

[0066] 4. Kit specificity test

[0067] The cross-reactivity rate with the drugs (as the possible combined drugs in vegetables and fruits) of boscalid kit, chlorothalonil, kresoxim-methyl, difenoconazole, imidacloprid, penthiopyrad, zoxamide, pyraclostrobin, pyrametostrobin, glyphosate, anilozine, chlorpyrifos, parathion, carbendazim, triazophos, procymidone, fenpropimorph, myclobutanil, etc. was determined by using the kit, and the results are shown in Table 2. The test results show that the kit does not react with the possible combined drugs in vegetables and fruits, and thus, in practical application, the kit has good specificity, avoiding unnecessary cross-reaction.

[0068]

[0069] Table 2 Specificity test

[0070] Drug name Cross-reactivity rate boscalid 100％ chlorothalonil ＜1％ chlorpyrifos ＜1％ chlorpyrifos-methyl ＜1％ clofentezine ＜1％ cyflumetofen ＜1％ cyproconazole ＜1％ cyprodinil ＜1％ dimethomorph ＜1％ dimoxystrobin ＜1％ flamprop-m ＜1％ flamprop-m-isopropyl ＜1％ flamprop-m-methyl ＜1％ flamprop-m-richter ＜1％ flamprop-m-richter-isopropyl ＜1％ flamprop-m-richter-methyl ＜1％ flamprop-m-isopropyl ＜1％ flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop-m-isopropyl flamprop ＜1％

Claims

1. A hapten for the detection of boscalid in vegetables and fruits, characterized in that The structural formula is as follows:

2. An enzyme-linked immunoassay kit for detecting the amino acid in the vegetable and fruit as claimed in claim 1.

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