CN112595850A - Application of clothianidin artificial antigen in enzyme linked immunosorbent assay kit - Google Patents
Application of clothianidin artificial antigen in enzyme linked immunosorbent assay kit Download PDFInfo
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- CN112595850A CN112595850A CN202011291511.5A CN202011291511A CN112595850A CN 112595850 A CN112595850 A CN 112595850A CN 202011291511 A CN202011291511 A CN 202011291511A CN 112595850 A CN112595850 A CN 112595850A
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- clothianidin
- solution
- enzyme
- linked immunosorbent
- immunosorbent assay
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- 239000005888 Clothianidin Substances 0.000 title claims abstract description 81
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
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- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting clothianidin, which comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a clothianidin standard solution, a clothianidin antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is a clothianidin coupled antigen, the enzyme conjugate is an enzyme-labeled clothianidin antibody, and the clothianidin antibody is obtained by immunizing animals with immunogen. The invention also discloses a method for detecting clothianidin by applying the enzyme linked immunosorbent assay kit, which comprises the following steps: firstly, preprocessing a sample, then detecting by using a kit, and finally analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of clothianidin in crop samples, is simple and convenient to operate, low in cost, high in sensitivity, capable of being monitored on site and suitable for screening a large number of samples.
Description
Technical Field
The invention relates to an application technology of clothianidin artificial antigen in an enzyme linked immunosorbent assay kit, in particular to a clothianidin artificial antigen molecular structure and an enzyme linked immunosorbent assay kit for detecting clothianidin, which can qualitatively and quantitatively detect the residual quantity of clothianidin drugs in crops.
Background
Clothianidin is an insecticide in neonicotinoids, is a novel efficient, safe and high-selectivity insecticide, has similar action to nicotinic acetylcholine receptors, and has contact poisoning, stomach toxicity and systemic activity. The pesticide is mainly used for preventing and controlling hemiptera, coleopteran, dipteran and certain lepidopteran pests such as aphids, leafhoppers, thrips, plant hoppers and the like on rice, vegetables, fruit trees and other crops, has the advantages of high efficiency, broad spectrum, low dosage, low toxicity, long efficacy duration, no phytotoxicity to the crops, safe use, no cross resistance with conventional pesticides and the like, has excellent systemic and osmotic effects, and is another variety for replacing high-toxicity organophosphorus pesticides. The pesticide has novel and special structure and more excellent performance compared with the traditional nicotine pesticide, and is likely to become a worldwide large-scale pesticide variety.
The method for detecting clothianidin comprises high performance liquid chromatography-tandem mass spectrometry, gas chromatography and the like, and compared with the method, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample processing, automatic operation realization and the like. The invention discloses a method for preparing clothianidin artificial antigen, which is applied to an enzyme linked immunosorbent assay kit, has the advantages of short time, simple operation and lower cost, and is suitable for detecting samples in bulk by a basic unit.
Disclosure of Invention
The invention aims to provide an enzyme linked immunosorbent assay kit capable of detecting clothianidin drug residue in crops, and provides a qualitative and quantitative detection method which is efficient, accurate, simple and convenient and is suitable for screening large-batch samples.
The kit of the invention comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a clothianidin standard solution, a clothianidin antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution, wherein the coating antigen is a clothianidin coupled antigen, and the enzyme conjugate is an enzyme-labeled clothianidin antibody.
The clothianidin specific antibody is prepared by taking a clothianidin artificial antigen as an immunogen, and can be a clothianidin monoclonal antibody or a clothianidin polyclonal antibody, wherein the clothianidin monoclonal antibody is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and clothianidin antibody.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a clothianidin standard solution, a substrate developing solution, a stop solution and a washing solution.
The concentration of the clothianidin standard solution in 6 bottles is 0 mug/L, 1 mug/L, 2 mug/L, 4 mug/L, 8 mug/L and 16 mug/L respectively.
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 A is hydrogen peroxide or carbamide peroxide, the B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution or hydrochloric acid buffer 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 liquid preferably has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
The coating buffer solution used in the preparation process of the ELISA plate is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is carbonate buffer solution with the pH value of 7.1-7.5, contains 1-3% of casein and 0.1-0.3 mol/L of phosphate buffer solution, and the percentage is weight volume percentage.
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 into each hole, incubating for 2h in a dark place at 25 ℃ or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 h in a dark place at 25 ℃, pouring off liquid in the holes, patting to dry, drying, and performing vacuum sealing and storage by using an aluminum film.
The detection principle of the invention is as follows:
the kit adopts an ELISA method, conjugate antigens are pre-coated on an enzyme label plate microporous strip, residual clothianidin in a sample and the conjugate antigens pre-coated on the enzyme label plate microporous strip compete for an enzyme conjugate resisting clothianidin, TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual clothianidin contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple to obtain the residual quantity of the clothianidin in the sample.
The invention also provides a method for detecting clothianidin by applying the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) detecting by using the kit;
(2) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting clothianidin mainly adopts an ELISA method to qualitatively or quantitatively detect the content of clothianidin 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; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, 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 screening of large-batch samples.
Drawings
FIG. 1: molecular structural formula of clothianidin hapten
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. Preparation of clothianidin hapten
Taking 2.49g of clothianidin, adding 200ml of methanol for dissolving, taking 1.2ml of 37% formaldehyde, adding 20ml of pure water for diluting, adding 3ml of 1mol/L hydrochloric acid, fully stirring for 1h, adding into the clothianidin solution, fully and uniformly mixing, and continuously stirring for 2h to obtain solution A; dissolving 1.03g of aminobutyric acid in 10ml of water, dropwise adding the solution into the solution A, heating in an oil bath, carrying out reflux reaction for 3 hours, stopping the reaction, adding 200ml of water, adding 200ml of ethyl acetate, extracting, washing the organic phase with 100ml of water multiplied by 3 for three times, evaporating the organic phase to dryness, applying to a silica gel column, and eluting and separating dichloromethane/methanol (v/v,10/1) to obtain a butyric acid-clothianidin hapten product of 0.63g with the yield of 17.3%.
2. Preparation of antigens
Immunogen preparation-clothianidin hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Dissolving 14mg of butyric acid-clothianidin hapten in 1ml of DMF (dimethyl formamide), adding 20 microliters of triethylamine and 95 microliters of isobutyl chloroformate, cooling to 0-5 ℃, and reacting for 3 hours to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M PB buffer solution for dissolving to obtain solution B, dripping the solution A into the solution B, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02M PBS, and changing the solution three times every day to obtain the clothianidin-BSA conjugate which is the immunogen, and storing at-20 ℃ for later use.
Preparation of coating antigen-coupling clothianidin hapten and Ovalbumin (OVA) to obtain immunogen.
Taking 8mg of butyric acid-clothianidin hapten, adding 1ml of DMSO for dissolving, adding 7.1mg of NHS and 16mg of DCC, and reacting at room temperature for 3 hours to obtain a hapten solution A; dissolving egg serum albumin (OVA)50mg in 0.05M PB buffer solution to obtain solution B, dripping A solution into solution B, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02M PBS for 3 days, and changing solution three times per day to obtain clothianidin-OVA conjugate, which is coating antigen, and storing at-20 deg.C for use.
Clothianidin and amino acid (straight-chain amino acid including aminobutyric acid, aminopropionic acid, aminocaproic acid and the like) are subjected to acid catalysis in the presence of formaldehyde to carry out Mannich reaction, so that a spacer arm with a carboxyl group is connected to an imine outlet, and then the carrier protein is coupled to obtain the immunogen and the coating antigen.
3. Preparation of clothianidin monoclonal antibody
Animal immunization: injecting the immunogen obtained in the above steps into Balb/c mice, wherein the immunization dose is 150 mug/mouse, so that antiserum is generated.
Cell fusion and cloning: after the serum determination result of the mouse is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), indirect competitive ELISA is adopted to determine cell supernatant, and positive holes are screened. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the clothianidin monoclonal antibody.
Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
Production and purification of monoclonal antibodies: injecting sterilized paraffin oil 0.5 mL/mouse into the abdominal cavity of Balb/c mouse, and injecting stable monoclonal hybridoma cell strain 5X 10 into the abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
4. Preparation of enzyme conjugates
Taking a goat as an immune animal and taking a clothianidin monoclonal antibody as an immunogen to immunize a goat without a pathogen to obtain the clothianidin antibody. The clothianidin antibody is coupled with Horse Radish Peroxidase (HRP) to obtain an enzyme conjugate.
5. Preparation of ELISA plates
Diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 200 mu l of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
Example 2 construction of enzyme-linked immunosorbent assay kit for detecting clothianidin
An enzyme linked immunosorbent assay kit for detecting clothianidin is constructed, and comprises the following components:
(1) an ELISA plate coated with clothianidin coupled antigen;
(2) 6 bottles of clothianidin standard solution, wherein the concentrations are 0 mug/L, 1 mug/L, 2 mug/L, 4 mug/L, 8 mug/L and 16 mug/L respectively;
(3) a clothianidin antibody labeled by horseradish peroxidase;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
example 3 detection of clothianidin in crops
1. Detection with a kit
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. Adding 50 mul of standard substance/sample into corresponding micropores, then adding 50 mul/pore of the working solution of the enzyme conjugate, and reacting for 30min at 25 ℃ in a dark environment. And (5) spin-drying the liquid in the holes, washing for 4-5 times, and patting dry. Adding 50 mul/hole of the substrate solution A, adding 50 mul/hole of the substrate solution B, mixing uniformly, and reacting for 15min at 25 ℃ in a dark environment. Adding 50 mul/well of stop solution, mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each well.
2. Analysis of detection results
The percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance value of the standard or sample. And drawing a standard curve graph by taking the percent absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mu g/L) of the clothianidin standard substance as an abscissa. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution times to obtain the actual concentration of the clothianidin in the sample.
Example 4 determination of clothianidin technical parameters
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, the range of a standard curve is 1-16 mu g/L, and IC50The floating range of (50% inhibition concentration) is 2.3-3.6 mu g/L; and (3) detecting 20 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 sample concentration to represent the detection limit, wherein the result shows that the detection limit of the method for clothianidin in crops is 1 mu g/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 and used as a precision evaluation index. The calculation formula is as follows: recovery (%) — actual measurement/theoretical value × 100%, where 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 Chinese cabbage sample is subjected to addition recovery measurement according to clothianidin with concentration of 1 mu g/kg and 2 mu g/kg, 4 samples are subjected to parallel measurement by using three different reagents, and the average recovery rate and precision result of the calculated sample are shown in the following table.
TABLE 1 crop sample precision and accuracy test
The Chinese cabbage sample is subjected to addition recovery measurement according to clothianidin with two concentrations of 1 mu g/kg and 2 mu g/kg, and the average recovery rate is 83.6-90.2%; 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 clothianidin addition of the kit are 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.
4. Kit specificity test
The cross-reaction rate with imidacloprid and triazophos was determined with clothianidin test paper and the results are shown in Table 2.
TABLE 2 specificity test
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting clothianidin, which is characterized by comprising: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, a clothianidin standard solution, a clothianidin antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a washing solution, wherein the coating antigen is a clothianidin coupled antigen, the enzyme conjugate is an enzyme-labeled clothianidin antibody, the clothianidin antibody is obtained by immunizing an animal with immunogen, the clothianidin coupled antigen is obtained by coupling clothianidin hapten and carrier protein, and the clothianidin hapten is obtained by carrying out a series of chemical reactions on clothianidin, aminobutyric acid and other substances.
2. The kit of claim 1, wherein the clothianidin hapten has the molecular formula:
3. the kit of claim 1, wherein said hapten is prepared by the following method:
taking 2.49g of clothianidin, adding 200ml of methanol for dissolving, taking 1.2ml of 37% formaldehyde, adding 20ml of pure water for diluting, adding 3ml of 1mol/L hydrochloric acid, fully stirring for 1h, adding into the clothianidin solution, fully and uniformly mixing, and continuously stirring for 2h to obtain solution A; dissolving 1.03g of aminobutyric acid in 10ml of water, dropwise adding the solution into the solution A, heating in an oil bath, carrying out reflux reaction for 3 hours, stopping the reaction, adding 200ml of water, adding 200ml of ethyl acetate, extracting, washing the organic phase with 100ml of water multiplied by 3 for three times, evaporating the organic phase to dryness, applying to a silica gel column, and eluting and separating dichloromethane/methanol (v/v,10/1) to obtain a butyric acid-clothianidin hapten product of 0.63g with the yield of 17.3%.
4. A method for detecting the content of clothianidin in a sample, comprising the following steps:
(1) detecting with the kit according to any one of claims 1 to 3;
(2) and analyzing the detection result.
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