CN109324187B

CN109324187B - Enzyme linked immunosorbent assay kit for detecting metalaxyl and application thereof

Info

Publication number: CN109324187B
Application number: CN201811104473.0A
Authority: CN
Inventors: 范子彦; 陈黎; 李旭; 朱亮; 刘惠民; 唐纲岭; 潘立宁; 颜权平; 冯才伟
Original assignee: Beijing Kwinbon Biotechnology Co Ltd; Zhengzhou Tobacco Research Institute of CNTC; National Tobacco Quality Supervision and Inspection Center
Current assignee: Beijing Kwinbon Biotechnology Co Ltd; Zhengzhou Tobacco Research Institute of CNTC; National Tobacco Quality Supervision and Inspection Center
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2021-08-24
Anticipated expiration: 2038-09-21
Also published as: CN109324187A

Abstract

An enzyme linked immunosorbent assay kit for detecting metalaxyl and application thereof are characterized in that: the kit comprises: an ELISA plate coated with a metalaxyl coupling antigen, a metalaxyl monoclonal antibody, an enzyme-labeled anti-antibody, a metalaxyl standard solution, a substrate developing solution, a stop solution, a washing solution and a re-solution. The metalaxyl monoclonal antibody is prepared by taking a metalaxyl coupling antigen as an immunogen, the metalaxyl coupling antigen is obtained by coupling a metalaxyl hapten and a carrier protein, and the metalaxyl hapten is obtained by reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenylmetalaxyl and then reducing the nitrophenyl metalaxyl by zinc powder. The invention also discloses a method for detecting metalaxyl residual quantity by using the enzyme linked immunosorbent assay kit. The enzyme linked immunosorbent assay kit can be used for detecting the residual quantity of metalaxyl in tobacco leaves, has the advantages of simple and convenient operation, low cost and high sensitivity, can be monitored on site and is suitable for screening a large number of samples.

Description

Enzyme linked immunosorbent assay kit for detecting metalaxyl and application thereof

Technical Field

The invention relates to an enzyme-linked immunoassay technology, in particular to an enzyme-linked immunoassay kit for detecting metalaxyl and application thereof, which are particularly suitable for detecting the residual quantity of the metalaxyl in tobacco.

Background

Metalaxyl (Metalaxyl) is a strong systemic substituted benzamide fungicide, has a chemical name of N- (2-methoxyacetyl) -N- (dimethylphenyl) -rac-aminobenzoic acid, belongs to amide fungicides, has high efficiency and low toxicity, and is mainly used for various downy mildew, late blight, early blight, damping-off, blight, fruit rot and the like caused by oomycetes, phycomycetes and fungi. Mainly inhibits the synthesis of protein in the hypha of the pathogenic bacteria, so that the hypha of the pathogenic bacteria is lack of nutrition and can not grow normally to die. The plant pesticide has strong systemic and osmotic force, can be conducted in the plant body in two directions up and down 30 min after the pesticide is applied, has the protection and treatment effects on plant diseases, and has better curative effects on controlling frost virus diseases and epidemic diseases of melons, fruits, vegetables and tobacco.

However, metalaxyl is one of the important pollutants in the environment, and poses potential health threats to human beings and pollutes the ecological environment, so that the residual problem in the production of fruits, vegetables and tobaccos is receiving more and more attention. China sets the maximum residue limit standard of metalaxyl for different crops, wherein the maximum residue limit of cucumber, pepper and tomato is 0.5 mg/kg, the maximum residue limit of brown rice is 0.1 mg/kg, and the maximum residue limit of other grains is 0.05 mg/kg. The international cooperation center for tobacco science research (CORESTA) stipulates that the directive residual limit of metalaxyl in tobacco is 2 mg/kg, and in actual production, 2 mg/kg is taken as the maximum residual quantity judgment standard of tobacco.

At present, the detection methods for metalaxyl residue at home and abroad mainly comprise a gas chromatography-mass spectrometry combined method, a high performance liquid chromatography-mass spectrometry combined method, a gas chromatography and a high performance liquid chromatography. The instrument and the method have the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of a detection sample is complicated and time-consuming, the sample also needs to be extracted and purified, and meanwhile, the instrument and the detection method need expensive large-scale instruments and equipment and are equipped with professional detection technicians for operation and management, so that the field large-scale detection cannot be carried out, the timeliness is poor, and the popularization is difficult. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples.

The invention patent (201510529378.5) discloses an enzyme linked immunosorbent assay kit for detecting metalaxyl residues and a using method thereof, and the invention is the most different from the enzyme linked immunosorbent assay kit in the structure of hapten. It is known that the key to establishing an immunoassay method for small molecular compounds is the ability to prepare antibodies with high affinity and high selectivity to small molecular compounds, and the synthesis of haptens with end groups as functional groups and connecting arms of a certain length is an important process for preparing antibodies against small molecular compounds and establishing a corresponding immunoassay method. Any structural differences between the hapten and the target analyte, such as topological characteristics of molecular size, shape, composition, configuration, conformation, polarity, electron cloud density, etc., can greatly affect the properties of the corresponding antibody. Whether hapten with better performance and effect can be designed and synthesized or not is the focus of the invention, so that the antibody with high sensitivity and good specificity can be obtained.

Disclosure of Invention

The invention aims to provide an enzyme linked immunosorbent assay kit for metalaxyl residual quantity detection, which has the advantages of simple structure, convenient use, low price and convenient carrying, and provides a qualitative and quantitative detection method which is efficient, accurate, simple and convenient and is suitable for screening large-batch samples based on the prior art.

The enzyme linked immunosorbent assay kit for detecting metalaxyl comprises: an ELISA plate coated with a metalaxyl coupling antigen, a metalaxyl monoclonal antibody, an enzyme-labeled anti-antibody, a metalaxyl standard solution, a substrate color development solution, a stop solution, a washing solution and a redissolution; the metalaxyl monoclonal antibody is prepared by taking a metalaxyl coupling antigen as an immunogen, the metalaxyl coupling antigen is obtained by coupling a metalaxyl hapten and a carrier protein, the carrier protein is thyroid protein, bovine serum albumin, rabbit serum protein, human serum protein, ovalbumin or hemocyanin, the metalaxyl hapten is obtained by reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenyl metalaxyl, and reducing by zinc powder, and the molecular structural formula is as follows:

。

the specific preparation method of the metalaxyl hapten comprises the following steps:

1) taking 2.00 g of N- (2, 6-dimethylphenyl) alanine methyl ester, adding pyridine to fully dissolve the N- (2, 6-dimethylphenyl) alanine methyl ester, adding 2.30 g of (4-nitrophenoxy) acetyl chloride, stirring, heating in an oil bath, reacting at 60 ℃ for 2 hours, detecting by TLC (thin layer chromatography), stopping the reaction when the raw materials basically react completely, recovering to room temperature, carrying out rotary evaporation, removing pyridine, adding 60 mL of water, adjusting the pH value to 5 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for extraction, drying an organic phase anhydrous sodium sulfate, loading the organic phase on a silica gel column, and eluting and separating by using ethyl acetate/N-hexane with the volume ratio of 1:10 to obtain 3.68 g of an intermediate product, namely nitrophenylmetalaxyl;

2) taking 3.60 g of the intermediate product nitrophenyl metalaxyl, and adding ethanol to dissolve the intermediate product nitrophenyl metalaxyl to obtain solution A; taking 1.80 g of zinc powder, adding 10 mL of distilled water, adding 0.5 mL of diluted hydrochloric acid, activating for 20 min at 60 ℃, adding the solution A, continuing stirring for 3h, detecting by TLC, stopping the reaction after all the raw materials are reacted, performing suction filtration to remove the zinc powder, evaporating the filtrate to dryness, adding 50 mL of water, adjusting the pH value to 7 by using sodium carbonate, adding 50 mL of ethyl acetate for extraction, washing an organic phase with water, drying and evaporating anhydrous sodium sulfate, and recrystallizing by using dichloromethane/n-hexane with the volume ratio of 1:2 to obtain 3.22 g of a metalaxyl hapten product.

The anti-antibody in the enzyme-labeled anti-antibody is a goat anti-mouse anti-antibody.

The labeled enzyme in the enzyme-labeled anti-antibody is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme-labeled anti-antibody is obtained by coupling a labeled enzyme and the anti-antibody by a glutaraldehyde method or a sodium periodate method.

In order to facilitate field monitoring and large-scale sample screening, the kit further comprises metalaxyl standard solution, substrate developing solution, stopping solution, washing solution and compound solution.

The concentration of the metalaxyl standard substance solution is 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively in 6 bottles.

When the labeled enzyme is horseradish peroxidase, the substrate color development solution consists of a substrate solution A and a substrate solution B, wherein the substrate solution A is hydrogen peroxide or carbamide peroxide, the substrate solution B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid or hydrochloric acid solution; when the marker enzyme is bacteria extracted alkaline phosphatase, the substrate color developing solution is a para-nitrophosphate buffer solution, and the stop solution is 1-2 mol/L sodium hydroxide solution.

The washing solution is preferably 0.02 mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent of Tween-20 and 0.01 per thousand of thimerosal preservative, wherein the percentage is mass volume percentage and unit g/mL.

The re-solution is preferably phosphate buffer solution with pH value of 7.0 and 0.1 mol/L.

The preparation process of the ELISA plate comprises the following steps: diluting the coating source into 20 mu g/mL by using a coating buffer solution, adding 100 mu L of the coating source into each hole, incubating for 2 hours at 37 ℃ in the dark or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution, patting the liquid for 30 s each time, then adding 150-200 mu L of a sealing solution into each hole, incubating for 1-2 hours at 37 ℃ in the dark, pouring off the liquid in the holes, patting the liquid for dryness, drying and then sealing and storing in vacuum by using an aluminum film.

Wherein the coating buffer solution used in the preparation process of the ELISA plate is 0.05 mol/L carbonate buffer solution with the pH value of 9.6, and the confining solution is 7.4 and contains 1-3% (g/mL) casein and 0.1-0.3 mol/L phosphate buffer solution.

The detection principle of the invention is as follows:

pre-coating metalaxyl coupling antigen on the microporous strip, adding sample solution or standard solution, then adding metalaxyl monoclonal antibody solution, adding enzyme-labeled anti-antibody for amplification, developing with developing solution, wherein the absorbance value of the sample is negatively correlated with the residual amount of metalaxyl, and comparing with a standard curve to obtain the residual amount of metalaxyl in the sample; meanwhile, the concentration range of the residual quantity of metalaxyl in the sample can be roughly judged by comparing the color of the elisa plate with the colors of the standard solutions with series concentrations.

The invention also provides a method for detecting metalaxyl residual quantity by applying the enzyme linked immunosorbent assay kit, which comprises the following steps:

1) sample pretreatment;

2) detecting by using the kit;

3) and analyzing the detection result.

The hapten structure designed by the invention is obviously different from the modification sites designed by 201510529378.5, and the spatial structures of hapten modifications formed by different modification sites are different, namely, antigenic determinants exposed on the surface of the artificial antigen generated after the hapten is combined with a carrier are different, so that the generated antibody has differences in titer, affinity and specificity.

The enzyme linked immunosorbent assay kit for detecting metalaxyl mainly adopts an indirect competitive ELISA method to qualitatively or quantitatively detect the residual quantity of metalaxyl in a sample; the pretreatment requirement on the sample is low, the pretreatment process of the sample is simple, and a large amount of samples can be detected quickly at the same time; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement; the hapten has proper terminal active groups, and the length of a modification site and a spacer arm is selected properly, and can simulate the molecular structure of metalaxyl to the maximum extent, and a kit developed on the basis of the hapten has the characteristics of high specificity, high sensitivity, high accuracy and the like. The enzyme linked immunosorbent assay kit has the advantages of simple structure, convenient use, low price, convenient carrying, high efficiency, accuracy, simplicity and convenience of the detection method, and is suitable for qualitative and quantitative detection of large-scale sample screening.

Drawings

FIG. 1: a route chart for synthesizing metalaxyl hapten,

FIG. 2: standard graph of kit (the figure is as abstract figure).

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLE 1 preparation of kit Components

1. Synthesis and identification of metalaxyl hapten (synthetic route is shown in figure 1)

Taking 2.00 g of N- (2, 6-dimethylphenyl) alanine methyl ester, adding pyridine to fully dissolve the N- (2, 6-dimethylphenyl) alanine methyl ester, adding 2.30 g of (4-nitrophenoxy) acetyl chloride, stirring, heating in an oil bath, reacting at 60 ℃ for 2 hours, detecting by TLC (thin layer chromatography), stopping the reaction when the raw materials basically react completely, recovering to room temperature, carrying out rotary evaporation, removing pyridine, adding 60 mL of water, adjusting the pH value to 5 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate to extract, drying an organic phase anhydrous sodium sulfate, loading the organic phase on a silica gel column, eluting and separating by using ethyl acetate/N-hexane with the volume ratio of 1:10 to obtain 3.68 g of an intermediate product, namely nitrophenylmetalaxyl, and;

taking 3.60 g of the intermediate product nitrophenyl metalaxyl, and adding ethanol to dissolve the intermediate product nitrophenyl metalaxyl to obtain solution A; taking 1.80 g of zinc powder, adding 10 mL of distilled water, adding 0.5 mL of diluted hydrochloric acid, activating at 60 ℃ for 20 min, adding the solution A, continuously stirring for 3h, detecting by TLC, stopping the reaction after all the raw materials are reacted, performing suction filtration to remove the zinc powder, evaporating the filtrate to dryness, adding 50 mL of water, adjusting the pH value to 7 by using sodium carbonate, adding 50 mL of ethyl acetate for extraction, washing an organic phase with water, drying and evaporating anhydrous sodium sulfate, recrystallizing by using dichloromethane/n-hexane with the volume ratio of 1:2 to obtain 3.22 g of a metalaxyl hapten product, wherein the yield is 96.99%.

Nuclear magnetic identification¹H NMR（CDCl₃300 MHz) δ: 4.778 (2H, q, J = 7.047), 3.646 (3H), 1.104 (3H, d, J = 7.047), 4.52 (t, 1H), 7.313 (1H, dd, J = 7.888), 2.260 (t, 6H), 7.41 (1H, dd, J = 7.888), 6.865 (1H, t, J = 7.888), 6.858 (1H, ddd, J =8.804, J =0.545, J = 0.000), 6.272 (t, 2H). In the diagram, the chemical shift delta =6.272 is the resonance absorption peak of the phenyl ring amino hydrogen on the spacer arm,the resonance absorption peaks of benzene ring hydrogen on the spacer arm are delta =6.858 and 6.856, and the existence of the peaks proves that the spacer arm coupling is successful and the metalaxyl hapten is correct in structure.

2. Synthesis and identification of metalaxyl coupling antigen

Immunogen preparation-metalaxyl hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.

Adding 0.1 mL of 1 mol/L diluted hydrochloric acid into 8 mg of metalaxyl hapten, adding 0.8 mL of distilled water, stirring at the low temperature of 0-5 ℃ for 20 min, adding 0.1 mL of aqueous solution containing 1.7 mg of sodium nitrite, and continuously stirring for 1h to obtain solution A; and (3) dissolving 50 mg of BSA in 6 mL of 0.1 mol/L sodium carbonate solution, stirring at 0-5 ℃ and balancing the temperature for 20 min to obtain solution B, dripping the solution A into the solution B, and continuing to react for 2 h. Stopping reaction, dialyzing with 0.02 mol/L PBS for 3 d, changing liquid three times per day, and packaging to obtain immunogen, and storing at-20 deg.C.

Preparation of coating antigen-coupling metalaxyl hapten and Ovalbumin (OVA) to obtain the coating antigen.

Taking 6 mg metalaxyl hapten, adding 0.7 mL of 1 mol/L diluted hydrochloric acid, adding 0.8 mL of distilled water, stirring at the low temperature of 0-5 ℃ for 20 min, adding 0.1 mL of aqueous solution containing 1.3 mg of sodium nitrite, and continuously stirring for 1h to obtain solution A; and (3) taking 60 mg of OVA, adding 6 mL of 0.1 mol/L sodium carbonate solution for dissolving, stirring at 0-5 ℃ for balancing the temperature for 20 min to obtain a solution B, dripping the solution A into the solution B, and continuing to react for 2 h. Stopping reaction, dialyzing with 0.02 mol/L PBS for 3 d, changing liquid three times per day, and packaging to obtain coating antigen, and storing at-20 deg.C for use.

And (3) carrying out ultraviolet (200-400 nm) scanning measurement according to the proportion of the hapten, the carrier protein and the coupling product used in the synthetic metalaxyl coupling antigen reaction, and calculating the binding ratio of the hapten, the carrier protein and the coupling product by comparing the absorbance values of the hapten, the carrier protein and the coupling product at 260 nm and 280 nm respectively. The maximum absorption peak of the conjugate metalaxyl hapten-carrier protein is obviously changed compared with the maximum absorption peaks of the metalaxyl hapten and the carrier protein, which indicates that the synthesis of the metalaxyl hapten-carrier protein is successful. The binding ratio of hapten to BSA was calculated to be 13:1 and binding ratio to OVA was calculated to be 10: 1.

3. Preparation of metalaxyl monoclonal antibody

1) Obtaining hybridoma cells

First immunization: the metalaxyl hapten-BSA conjugate (immunogen) was emulsified well with an equal amount of Freund's complete adjuvant and injected subcutaneously into 6-week-old Balb/c mice, 0.2 mL each;

two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;

after one week of last boosting immunization, measuring the titer and inhibition in fundus venous blood sampling, and performing the following last immunization when the titer reaches more than 1: 10000: injecting 0.1 mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;

and (3) measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the metalaxyl monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging in a freezing tube, and storing in liquid nitrogen for a long time.

2) Preparation of monoclonal antibodies

Cell recovery: taking out the frozen tube of the monoclonal antibody hybridoma cell strain of metalaxyl, immediately putting the frozen tube into a water bath at 37 ℃ for medium-speed melting, centrifuging to remove frozen stock solution, and transferring the frozen stock solution into a culture bottle for culture;

preparing ascites and purifying antibodies: injecting sterilized paraffin oil 0.5 mL/mouse in Balb/c mouse (8 weeks old) into abdominal cavity by in vivo induction method, injecting hybridoma cells 5 × 10 into abdominal cavity 7 days later⁵Ascites were collected 7 days later. Purifying by octanoic acid-saturated ammonium sulfate method to obtain metalaxyl monoclonal antibody solution (preservation at-20 deg.C).

3) Determination of the potency of monoclonal antibodies

The titer of the antibody is 1 (100000-300000) by using an indirect competition ELISA method.

Indirect competitive ELISA method: coating an enzyme label plate with a metalaxyl hapten-OVA conjugate, adding a metalaxyl standard substance solution, a metalaxyl monoclonal antibody solution and a horseradish peroxidase-labeled goat anti-mouse anti-antibody solution, reacting for 30 min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.

4) Determination of monoclonal antibody specificity

Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.

In the experiment, metalaxyl and compounds (propyzamide, alachlor, acetochlor, metolachlor, pretilachlor and butachlor) with similar structures are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained by analysis₅₀Then, the cross-reactivity was calculated as follows:

the results show that the cross-reactivity of metalaxyl and its structural analogues is: 100 percent of metalaxyl, less than 1 percent of propyzamide, less than 1 percent of alachlor, less than 1 percent of acetochlor, less than 1 percent of metolachlor, less than 1 percent of pretilachlor and less than 1 percent of butachlor. The antibody of the invention has no cross reaction to compounds with similar structures to metalaxyl, such as propyzamide, alachlor, acetochlor, metolachlor, pretilachlor, butachlor and the like, and only has specific binding to the metalaxyl.

4. Preparation of goat anti-mouse anti-antibody

The sheep is used as an immune animal, and the pathogen-free sheep is immunized by using the murine antibody as an immunogen to obtain the goat anti-mouse antibody.

5. Preparation of enzyme-labeled anti-antibody

The goat anti-mouse antibody and horseradish peroxidase (HRP) are coupled by a modified sodium periodate method. The traditional sodium periodate method requires that the molar concentration ratio of enzyme to antibody in a reaction system is 4:1, and horseradish peroxidase generates a plurality of sites for binding with the antibody under the strong oxidation effect, so that the activated horseradish peroxidase molecules serve as bridges for connecting the molecules, the enzyme activity of an enzyme marker is reduced, and a plurality of polymers are mixed in the prepared conjugate. To solve this problem, we modified the conventional method, namely:

1) the blocking process of the amino group is omitted, because the amino group capable of generating self amino group connection is practically few;

2) the molar concentration ratio of the horseradish peroxidase to the antibody is reduced to 2:1, the improved method is simpler than the traditional method, and the loss of enzyme activity is reduced.

6. Preparation of ELISA plates

Diluting the coating antigen (metalaxyl hapten-OVA conjugate) to 20 mu g/mL by using a coating buffer solution, adding 100 mu L into each hole, incubating for 2h in the dark at 37 ℃, pouring off liquid in the hole, washing for 2 times by using a washing solution for 30 s each time, patting dry, then adding 200 mu L of a confining liquid into each hole, incubating for 2h in the dark at 37 ℃, pouring off liquid in the hole, patting dry, drying, and performing vacuum sealing and storage by using an aluminum film.

Example 2 construction of enzyme-linked immunosorbent assay kit for detecting metalaxyl

An enzyme linked immunosorbent assay kit for detecting metalaxyl is constructed, and comprises the following components:

1) an ELISA plate coated with metalaxyl coupling antigen;

2) metalaxyl standard solution with the concentration of 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively;

3) metalaxyl monoclonal antibody working solution;

4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;

5) the substrate color developing solution consists of a solution A and a solution B, wherein the solution A is carbamide peroxide, and the solution B is tetramethylbenzidine;

6) the stop solution is a 2 mol/L sulfuric acid buffer solution;

7) the washing liquid is 0.02 mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent of Tween-20 and 0.01 per thousand of thimerosal preservative, and the percentage is weight volume percentage;

8) the complex solution is phosphate buffer solution with pH value of 7.0 and 0.1 mol/L.

Example 3 detection of residual amount of metalaxyl in tobacco leaf sample

1. Sample pretreatment

Homogenizing the tobacco leaf sample with a homogenizer; weighing 1.0 +/-0.05 g of homogenized sample into a 15 mL polystyrene centrifuge tube, adding 5 mL of methanol, whirling for 3 min by using a vortex instrument, and uniformly mixing; centrifuging at 3000 g room temperature (20-25 deg.C) for 5 min, collecting 10 μ L supernatant, adding 1990 μ L complex solution, vortexing with vortex instrument for 30 s, and mixing; 50 μ L was taken for analysis.

2. Detection with a kit

Adding 50 mu L/hole of metalaxyl standard solution or pretreated sample solution into micropores of an ELISA plate coated with metalaxyl coupling antigen, then adding 50 mu L/hole of metalaxyl monoclonal antibody working solution, lightly oscillating and uniformly mixing, and placing the mixture in a dark environment at 25 ℃ for reaction for 30 min after covering a plate with a cover plate film; pouring out liquid in the holes, adding 250 mu L of washing liquid into each hole, fully washing for 4-5 times at intervals of 10 s each time, and patting dry by using absorbent paper; adding 100 mu L/hole of goat anti-mouse anti-antibody marked by horseradish peroxidase, lightly oscillating and uniformly mixing, covering a plate with a cover plate film, reacting for 30 min in a dark environment at 25 ℃, taking out and repeating the plate washing step; adding 50 mu L of substrate color development liquid A liquid carbamide peroxide and 50 mu L of substrate color development liquid B liquid tetramethylbenzidine into each hole, lightly oscillating and uniformly mixing, covering a plate by a cover plate film, placing the plate in a dark environment at 25 ℃ for color development for 15 min, adding 50 mu L of stop solution 2 mol/L sulfuric acid into each hole, lightly oscillating and uniformly mixing, setting the wavelength at 450 nm by using an enzyme-linked immunosorbent assay, setting the reference wavelength at 620 nm, and measuring the absorbance value (OD value) of each hole.

3. Analysis of detection results

Dividing the average absorbance (B) obtained for each concentration of the standard solution by the absorbance value (B) of the first standard solution (0 standard)₀) And then multiplied by 100 percent to obtain the percent absorbance value. A standard curve is drawn by taking the concentration (mu g/L) of the metalaxyl standard substance as an X axis and the percent absorbance value as a Y axis, and is shown in figure 2. By the same methodCalculating the percent absorbance value of the sample solution, and reading the residual metalaxyl corresponding to each sample from the standard curve.

Example 4 determination of technical parameters of metalaxyl ELISA kit

1. Sensitivity and detection limit of kit

The sensitivity of the kit is determined according to a conventional method, the lowest point of a standard curve of the kit is 0.5 mug/L, the range of the standard curve is 0.5-40.5 mug/L, and the IC is₅₀(50% inhibitory concentration) the floating range is 1.5-3.0 mug/L; and (3) detecting 20 parts of blank tobacco leaf samples, finding out the concentration corresponding to each percent absorbance value from the standard curve, and adding 3 times of standard deviation to the average value of the 20 parts of sample concentration to represent the detection limit, wherein the detection limit of the method on the tobacco leaf samples is 0.5 mg/kg.

2. Sample precision and accuracy testing

The recovery rate is used as an accuracy evaluation index, and the relative standard deviation (RSD%) of the detection result of a sample with a certain concentration is repeatedly measured as a precision evaluation index. The calculation formula is as follows: recovery (%) — actual measurement/theoretical value × 100%, where the theoretical value is the added concentration of the sample; relative standard deviation RSD% ═ SD/X × 100%, where SD is the standard deviation and X is the average of the measured data.

The blank tobacco leaf samples are subjected to addition recovery measurement according to metalaxyl with three concentrations of 0.5 mg/kg, 1 mg/kg and 2 mg/kg, 4 samples are parallelly measured by three different kits, the average recovery rate and the precision of the samples are calculated, and the results are shown in the following table.

TABLE 1 precision and accuracy tests

Adding a blank tobacco leaf sample with metalaxyl with three concentrations of 0.5, 1 and 2 mg/kg, wherein the average recovery rate is 86.9-105.2%; the relative standard deviation in each batch and between batches is less than 10 percent.

3. Stability test of kit

The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), the 50% inhibition concentration and the actual measurement value of metalaxyl addition of the kit are all within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.

Claims

1. An enzyme linked immunosorbent assay kit for detecting metalaxyl, which is characterized in that: comprises an ELISA plate coated with a metalaxyl coupling antigen, a metalaxyl monoclonal antibody, an enzyme-labeled anti-antibody, a metalaxyl standard solution, a substrate developing solution, a stop solution, a washing solution and a redissolution; the metalaxyl monoclonal antibody is prepared by taking a metalaxyl coupling antigen as an immunogen, the metalaxyl coupling antigen is obtained by coupling a metalaxyl hapten and a carrier protein, the carrier protein is thyroid protein, bovine serum albumin, rabbit serum protein, human serum protein, ovalbumin or hemocyanin, the metalaxyl hapten is obtained by reacting N- (2, 6-dimethylphenyl) alanine methyl ester with (4-nitrophenoxy) acetyl chloride to generate nitrophenyl metalaxyl, and reducing by zinc powder, and the molecular structural formula is as follows:

the preparation reaction process of the metalaxyl hapten is as follows:

。

2. the enzyme linked immunosorbent kit of claim 1, wherein: the anti-antibody in the enzyme-labeled anti-antibody is a goat anti-mouse anti-antibody.

3. The enzyme linked immunosorbent kit of claim 1, wherein: the enzyme-labeled anti-antibody is characterized in that a labeling enzyme in the enzyme-labeled anti-antibody is horseradish peroxidase or bacteria extracted alkaline phosphatase, when the labeling enzyme is horseradish peroxidase, a substrate color development solution consists of a substrate solution A and a substrate solution B, the substrate solution A is hydrogen peroxide or carbamide peroxide, the substrate solution B is o-phenylenediamine or tetramethylbenzidine, and a stop solution is 1-2 mol/L sulfuric acid or hydrochloric acid solution; when the marker enzyme is bacteria to extract alkaline phosphatase, the substrate color development solution is a para-nitrophosphate buffer solution, and the stop solution is 1-2 mol/L sodium hydroxide solution.

4. The enzyme linked immunosorbent kit of claim 1, wherein: the washing solution is 0.02 mol/L phosphate buffer solution with the pH value of 7.4 and containing 0.05 percent of Tween-20 and 0.01 per thousand of thimerosal preservative; the compound solution is phosphate buffer solution with the pH value of 7.0 and 0.1 mol/L, and the percentage in the washing solution is weight volume percentage.

5. The enzyme linked immunosorbent kit of claim 1, wherein: the concentration of the metalaxyl standard solution is 0 mug/L, 0.5 mug/L, 1.5 mug/L, 4.5 mug/L, 13.5 mug/L and 40.5 mug/L respectively.

6. A method for detecting residual quantity of metalaxyl in tobacco leaves by using the enzyme linked immunosorbent assay kit of claim 1, which is characterized in that: the method comprises the following steps:

1) pretreating a sample;

2) detecting by using the kit;

3) and analyzing the detection result.