CN111505297A - Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit - Google Patents
Application of endosulfan artificial antigen in enzyme linked immunosorbent assay kit Download PDFInfo
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- CN111505297A CN111505297A CN202010400219.6A CN202010400219A CN111505297A CN 111505297 A CN111505297 A CN 111505297A CN 202010400219 A CN202010400219 A CN 202010400219A CN 111505297 A CN111505297 A CN 111505297A
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- endosulfan
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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
- G01N33/53—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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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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
- G01N33/53—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
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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)
Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting endosulfan, which comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a endosulfan standard solution, endosulfan antibodies, 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 endosulfan coupled antigen, the enzyme conjugate is an enzyme-labeled endosulfan antibody, and the endosulfan antibody is obtained by immunizing an animal with immunogen. The invention also discloses a method for detecting endosulfan by using 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 endosulfan in vegetable and fruit 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 endosulfan artificial antigen in an enzyme linked immunosorbent assay kit, in particular to an endosulfan artificial antigen molecular structure and an enzyme linked immunosorbent assay kit for detecting endosulfan, which can qualitatively and quantitatively detect the residual amount of endosulfan medicines in vegetables and fruits.
Background
Endosulfan is an artificially synthesized organochlorine compound and is widely used as a pesticide. The medicine has the advantages of wide insecticidal range, low price, low toxicity to bees and the like, and is widely applied to various crops. China began to produce endosulfan in the last 90 th century, and by 2011, 47 endosulfan products which are registered and used relate to 43 families of medicine enterprises, which are called the second major producing country of the medicine. The medicine has long shelf life, biological enrichment effect, great harm to fish and shellfish, and cumulative toxicity to mammal. There have been a number of reports in the literature on the detection of endosulfan residues in crops such as vegetables and fruits.
Currently, there are 70 countries worldwide that prohibit the production and use of endosulfan. According to the notice No. 1586 issued by five committees of Ministry of agriculture in China, the registration of endosulfan on apple trees and tea trees is cancelled. This means that endosulfan must not continue to be used on the withdrawn crop and will phase out the production and use of endosulfan.
Solid phase extraction and gas chromatography detection are mostly adopted in the research methods of endosulfan at home and abroad. Compared with the two methods, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like for the detection of the medicine. The invention discloses a method for preparing artificial antigen of endosulfan, 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 the residual quantity of endosulfan in vegetables and fruits, 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 endosulfan standard solution, endosulfan antibodies, 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 endosulfan coupled antigen, and the enzyme conjugate is enzyme-labeled endosulfan antibodies.
The endosulfan specific antibody is prepared by taking an endosulfan artificial antigen as an immunogen, and can be an endosulfan monoclonal antibody or an endosulfan polyclonal antibody, wherein the endosulfan 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 endosulfan antibody.
In order to facilitate field monitoring and large-scale sample screening, the kit further comprises a endosulfan standard solution, a substrate developing solution, a stop solution and a washing solution.
The concentration of the standard endosulfan solution in 6 bottles is respectively 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L.
When the marker enzyme is horseradish peroxidase, the substrate color developing 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, the stop solution is a sulfuric acid solution or a hydrochloric acid buffer solution with the concentration of 1-2 mol/L, 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 a sodium hydroxide solution with the concentration of 1-2 mol/L.
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 enzyme label plate is carbonate buffer solution with the pH value of 9.6 and 0.05 mol/L, and the confining solution is phosphate buffer solution with the pH value of 7.1-7.5 and containing 1-3% of casein and 0.1-0.3 mol/L, wherein the percentages are weight volume percentages.
The preparation process of the ELISA plate comprises the steps of diluting a coating source into 20 mu g/m L by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2 hours at 25 ℃ in the dark or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting the liquid dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 hours at 25 ℃ in the dark, pouring off liquid in the holes, patting the liquid dry, drying and then sealing and storing in vacuum by using an aluminum film.
The detection principle of the invention is as follows:
the kit adopts a direct competition E L ISA method, pre-coats a coupling antigen on an enzyme label plate micropore strip, the residual endosulfan in a sample and the coupling antigen pre-coated on the enzyme label plate micropore strip compete for an enzyme conjugate resisting endosulfan, a TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the residual endosulfan 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 endosulfan in the sample.
The invention also provides a method for detecting endosulfan by using the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using the kit;
(3) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the endosulfan mainly adopts an E L ISA method to qualitatively or quantitatively detect the content of the endosulfan in a sample, has low requirement on the pretreatment of the sample, is simple in the pretreatment process of the sample, can simultaneously and quickly detect a large number of samples, is provided in the form of working solution as a main reagent, is convenient and easy to carry, and has the characteristics of high specificity, high sensitivity, high accuracy and the like.
Drawings
FIG. 1: molecular structural formula of endosulfan 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 endosulfan hapten
Taking 3.6g of thiodan alcohol, adding 100ml of 1, 4-dioxane for dissolving, fully stirring, adding 3.0g of molecular sieve, adding 1.02g of succinic semialdehyde, fully stirring, introducing hydrochloric acid gas, stirring at room temperature in a dark place for 5 hours, stopping the reaction, adding 0.5 mol/L NaOH solution, fully shaking 200ml of solution, adding 200ml of ethyl acetate, fully shaking, standing, separating an aqueous phase, washing with 80ml of organic phase water, standing after shaking, separating the aqueous phase, drying and evaporating the organic phase anhydrous sodium sulfate to dryness, loading to a silica gel column, eluting and purifying dichloromethane/methanol (v/v, 10/1) to obtain 2.1g of the product of the succinyl acetal thiodan hapten, wherein the yield is 45.75%.
2. Preparation of antigens
Immunogen preparation-endosulfan hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Taking 16mg of succinyl acetal endosulfan, adding 1ml of DMF for dissolving, adding 18.7mg of NHS and 33mg of DDC, and reacting for 3 hours at room temperature 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 A solution 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 a endosulfan-BSA conjugate which is an immunogen and is stored at-20 ℃ for later use.
Preparation of coating antigen-coupling of endosulfan hapten and Ovalbumin (OVA) to obtain immunogen.
Dissolving Succinal acetal endosulfan 10mg in DMSO 1ml, adding NHS6.7mg and EDC16mg, and reacting at room temperature for 3 hr to obtain 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 endosulfan-OVA conjugate, which is coating antigen, and storing at-20 deg.C.
3. Preparation of endosulfan 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, namely, after the determination result of mouse serum is higher, taking splenocytes, fusing the splenocytes with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), adopting indirect competition E L ISA to determine cell supernatant, screening positive holes, and cloning the positive holes by using a limiting dilution method until obtaining a hybridoma cell strain secreting endosulfan monoclonal antibodies.
Freezing and thawing the cell, namely preparing the frozen solution of the monoclonal hybridoma cell strain into 1 × 106The cell suspension of L per m is preserved in liquid nitrogen for a long time, the frozen tube is taken out during recovery, the frozen tube is immediately put into a water bath at 37 ℃ for fast melting, and after the frozen solution is removed by centrifugation, the frozen tube is transferred into a culture bottle for culture.
Production and purification of monoclonal antibody, injecting sterilized paraffin oil 0.5m L/mouse into Balb/c mouse abdominal cavity, injecting stable monoclonal hybridoma cell strain 5 × 10 into abdominal cavity 7 days later5Ascites 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 endosulfan monoclonal antibody as an immunogen to immunize the goat without pathogens to obtain the endosulfan antibody. And (3) coupling the endosulfan antibody with horseradish peroxidase (HRP) to obtain an enzyme conjugate.
5. Preparation of ELISA plates
Diluting the coating source to 20 μ g/m L with a coating buffer solution, adding 100 μ l into each well, incubating at 25 deg.C in the dark for 2h, decanting off the liquid in the well, washing with a washing solution for 2 times, each time for 30s, patting to dryness, adding 200 μ l of blocking solution into each well, incubating at 25 deg.C in the dark for 2h, decanting off the liquid in the well, patting to dryness, drying, and vacuum sealing with aluminum film for storage.
EXAMPLE 2 construction of enzyme-linked immunoassay kit for detecting endosulfan
An enzyme linked immunosorbent assay kit for detecting endosulfan is constructed, and comprises the following components:
(1) an ELISA plate coated with endosulfan coupling antigen;
(2) 6 bottles of endosulfan standard solution with the concentrations of 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L respectively;
(3) a endosulfan antibody labeled with 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 2 mol/L sulfuric acid;
(6) the washing liquid is phosphate buffer solution with the pH value of 7.4, containing 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L, and the percentage is weight volume percentage;
example 3 detection of endosulfan in vegetables and fruits
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 value (double holes) of the absorbance values of the standard or sample divided by the average value of the absorbance values of the first standard (0 standard), and then multiplied by 100 percent to obtain the percent absorbance value of the standard or sample, the percent absorbance of the standard is taken as the ordinate, the logarithm of the concentration (mu g/L) of the endosulfan standard is taken as the abscissa, a standard curve graph is drawn, the percent absorbance of the sample is substituted into the standard curve, the concentration corresponding to the sample is read from the standard curve, and the dilution multiple corresponding to the percent absorbance is multiplied to obtain the actual concentration of endosulfan in the sample.
EXAMPLE 4 determination of endosulfan technical parameters
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, and the range of a standard curve is 1-81 mu g/L50The (50% inhibition concentration) floating range is 4.5-7.5 mug/L, 20 samples are detected, the concentration corresponding to each percent absorbance value is found out from a standard curve, the detection limit is represented by adding 3 times of standard deviation to the average value of the 20 sample concentration, and the result shows that the detection limit of the method for endosulfan in vegetables and fruits is 50 mug/kg and 100 mug/kg respectively.
2. Sample precision and accuracy testing
The calculation formula is that the recovery ratio (%) is × 100% of actual measured value/theoretical value, wherein the theoretical value is the added concentration of the sample, and the relative standard deviation RSD% is SD/X × 100%, wherein SD is the standard deviation, and X is the average value of the measured data.
The vegetable sample is subjected to addition recovery measurement according to endosulfan with two concentrations of 50 mug/kg and 100 mug/kg, the fruit sample is subjected to addition recovery measurement according to endosulfan with two concentrations of 100 mug/kg and 200 mug/kg, 4 samples are parallelly measured by three different reagents, and the average recovery rate and precision result of the samples are calculated and shown in the following table.
TABLE 1 vegetable and fruit sample precision and accuracy tests
Adding and recovering the vegetable sample according to the endosulfan with the concentration of 50 mug/kg and 100 mug/kg, and adding and recovering the fruit sample according to the endosulfan with the concentration of 100 mug/kg and 200 mug/kg, wherein the average recovery rates are 81.2% -92.3% and 74.8% -87.9% respectively; the relative standard deviation in each batch and between batches is less than 15 percent.
3. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and the actual measurement value of the added endosulfan 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.
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting endosulfan is characterized by comprising: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, a endosulfan standard solution, endosulfan antibodies, 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 an endosulfan conjugate antigen, the enzyme conjugate is an enzyme-labeled endosulfan antibody, the endosulfan antibody is obtained by immunizing an animal with an immunogen, the endosulfan conjugate antigen is obtained by coupling endosulfan hapten and carrier protein, and the endosulfan hapten is obtained by carrying out a series of chemical reactions on endosulfan and 1, 4-dioxane and other substances.
2. The kit of claim 1, wherein the endosulfan hapten has the molecular formula:
3. the kit according to claim 1, characterized in that the immunogen is prepared as follows:
dissolving Succinal acetal endosulfan 16mg in DMF 1ml, adding NHS18.7mg and DDC33mg, and reacting at room temperature for 3 hr to obtain hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.05M PB buffer solution for dissolving to obtain solution B, dripping A solution 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 a endosulfan-BSA conjugate which is an immunogen and is stored at-20 ℃ for later use.
4. A method for detecting the content of endosulfan in a sample comprises the following steps:
(1) pretreating a sample;
(2) detecting with the kit according to any one of claims 1 to 3;
(3) and analyzing the detection result.
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