CN113176403A - Double-antibody sandwich ELISA kit for detecting acetamiprid and application thereof - Google Patents
Double-antibody sandwich ELISA kit for detecting acetamiprid and application thereof Download PDFInfo
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- CN113176403A CN113176403A CN202110432287.5A CN202110432287A CN113176403A CN 113176403 A CN113176403 A CN 113176403A CN 202110432287 A CN202110432287 A CN 202110432287A CN 113176403 A CN113176403 A CN 113176403A
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- acetamiprid
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
- G01N33/18—Water
- G01N33/1826—Water organic contamination in water
- G01N2033/184—Water organic contamination in water herbicides, pesticides, fungicides, insecticides, or the like
Abstract
The invention discloses a double-antibody sandwich ELISA kit for detecting acetamiprid and application thereof, wherein the kit comprises: a capture agent consisting of a specific monoclonal antibody and/or polyclonal antibody against acetamiprid, an enzyme-labeled secondary antibody, a sample diluent and a substrate. The kit prepared by the scheme of the invention can quickly detect the concentration of the acetamiprid, has simple and convenient detection procedure, can detect a plurality of samples simultaneously, and has short detection time and high sensitivity.
Description
Technical Field
The invention relates to an enzyme-linked immunosorbent assay technology, in particular to a double-antibody sandwich ELISA kit for detecting acetamiprid and application thereof.
Background
The nicotine pesticide is an important pesticide following organophosphorus, carbamate and pyrethroid insecticides, and mainly blocks normal conduction of the central nervous system of insects by selectively controlling nicotinic acetylcholinesterase receptors (nAChRs) of the central nervous system of the insects, so that the insects are paralyzed systemically and die. Acetamiprid is a representative agent of a neonicotinoid insecticide, is widely used for preventing and controlling various pests in seeds, leaf surfaces and soil in agricultural production, has ideal effect on preventing and controlling pests such as aphids, leafhoppers, bemisia tabaci, leaf miners and the like, and is mainly used for preventing and controlling myzus persicae in tobacco. Pesticide residue control is an important content of product quality safety control and is a key point of common attention of government agencies, manufacturing enterprises and consumers. Acetamiprid is widely used in recent years, has high detection rate and is frequently over-limited.
Although the traditional detection method can accurately determine the content of the acetamiprid, the traditional detection method needs special instruments and equipment, has complex steps, high detection cost and long time consumption, and samples need to be subjected to complex pretreatment and completed by special technicians, so that the requirements on rapid field detection of environment and market products are difficult to meet. The immunoassay is a novel detection method, and compared with the traditional detection method, the immunoassay has the advantages of time saving, labor saving, low cost, convenience in carrying, easiness in operation and the like.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a double-antibody sandwich ELISA kit for detecting acetamiprid, which can be used for quickly detecting the residue of the acetamiprid in crops.
The invention also provides application of the double-antibody sandwich ELISA kit for detecting acetamiprid.
According to the first aspect of the invention, the double-antibody sandwich ELISA kit for acetamiprid comprises a capture agent consisting of a monoclonal antibody and/or polyclonal antibody specific to acetamiprid, an enzyme-labeled secondary antibody, a sample diluent and a substrate.
In some embodiments of the invention, the capture agent is a mouse anti-acetamiprid antibody IgG.
In some embodiments of the invention, the mouse anti-acetamiprid antibody IgG is diluted with a sample diluent, and the diluted solution is a capture reagent.
In some embodiments of the invention, the secondary antibody is a labeled rabbit anti-acetamiprid antibody.
In some embodiments of the invention, the labeled enzyme-labeled secondary antibody is HRP.
In some embodiments of the invention, the HRP-labeled secondary enzyme-labeled antibody is diluted with a sample diluent and used as a secondary enzyme-labeled antibody.
In some embodiments of the invention, the acetamiprid double antibody sandwich ELISA kit comprises a capture agent attached to a solid support.
In some embodiments of the invention, the acetamiprid double-antibody sandwich ELISA kit comprises a solid support and a reagent kit, wherein the solid support is an ELISA plate or a biochip.
In some embodiments of the invention, the component of the diluent is a phosphate buffer containing 8% to 12% by volume FBS (calf serum).
In some embodiments of the invention, the components of the diluent are phosphate buffered saline at ph7.4 containing 10% FBS (calf serum) by volume fraction.
In some embodiments of the invention, the acetamiprid double-antibody sandwich ELISA kit comprises the following components in the dosage ratio of the capture agent to the enzyme-labeled secondary antibody of 1: (1-3).
In some embodiments of the invention, the acetamiprid double-antibody sandwich ELISA kit comprises the following components in the dosage ratio of the capture agent to the enzyme-labeled secondary antibody of 1: 1.
in some embodiments of the invention, the antibody is prepared by: the first immunization adopts antigen immunization and acetamiprid injection; and (3) performing boosting immunization after the first immunization, performing boosting immunization for 3-6 times in total, and taking blood after the last boosting immunization.
The application of the double-antibody sandwich ELISA kit for detecting acetamiprid according to the second aspect embodiment of the invention is the application in detecting the content of acetamiprid.
In some embodiments of the invention, the application is the application of the kit in detecting acetamiprid residues in food.
A method for detecting the content of acetamiprid comprises the following steps:
s1, taking out the enzyme label plate coated by the rat anti-acetamiprid antibody, adding a sample solution, removing liquid in the hole after incubation, washing with a washing solution, and then drying by beating;
s2, adding a rabbit anti-acetamiprid antibody marked by HRP, discarding liquid in the hole after incubation is finished, and drying by beating after washing liquid is cleaned;
s3, adding a color developing liquid for color development;
s4, adding a stop solution;
s5, determining the light absorption value of the reaction liquid, and obtaining the content of the acetamiprid in the sample to be detected according to the standard curve.
In some embodiments of the invention, the color developing solution is 3,3',5,5' -Tetramethylbenzidine (TMB).
In some embodiments of the invention, the stop solution is sulfuric acid.
In some embodiments of the invention, the concentration of the acetamiprid antigen standard is 0 ng/mL-80 ng/mL.
In some embodiments of the invention, the concentration of the HRP-labeled rabbit anti-acetamiprid antibody is 0.5-1.5. mu.g/mL.
The invention has the beneficial effects that: the invention provides a double-antibody sandwich ELISA kit for detecting acetamiprid, and a method and application thereof. Through the high monoclonal antibody of coating specificity, detect the polyclonal antibody of HRP mark for the antibody, can short-term test acetamiprid's concentration, detection procedure is simple and convenient, and detection time is short, and one-time operation time is no longer than 2h, can detect a plurality of samples simultaneously, and detectivity is high.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
EXAMPLE 1 preparation of acetamiprid immunogen
Synthesis of acetamiprid hapten
Dissolving 0.50g of N-noracetamiprid in 50mL of methanol, adding 0.30g of aminobutyric acid, stirring, adding 0.37mL of 37% formaldehyde water solution, stirring, uniformly mixing, and reacting for 4 hours at 80 ℃; stopping reaction, removing methanol by rotary evaporation, adding 50mL of water, extracting by adding 100mL of ethyl acetate, extracting for three times, combining organic phases, drying by rotary evaporation, loading on a silica gel column, and eluting and separating by dichloromethane/methanol (10/1) to obtain a hapten product.
The acetamiprid hapten and Bovine Serum Albumin (BSA) are coupled to obtain the immunogen.
Dissolving 11mg of hapten carboxyl acetamiprid in 1mL of dimethyl sulfoxide, adding 0.18mL of isobutyl chloroformate and 0.1mL of triethylamine, and reacting at 0-4 ℃ for 1h to obtain a hapten activating solution A; taking 50mg of BSA, and adding 3mL of 0.8% saline to dissolve the BSA to obtain solution B; dripping the A solution into the B solution, continuously stirring for reaction for 5h, dialyzing and purifying 0.02mol/L PB for 3 days, changing the solution 3 times per day to obtain immunogen, subpackaging, and storing at-20 ℃.
Example 2 preparation of mouse anti-acetamiprid antibody serum and Rabbit anti-acetamiprid antibody serum
The specific immunization method is as follows:
(1) antigen emulsification: adding the adjuvant and the antigen into a centrifugal tube according to the volume ratio of 1:1, quickly stirring in an ice bath by using a miniature stirrer for emulsification, and not diffusing when the emulsion is dropped into water.
(2) First immunization: antigen emulsification is carried out by adopting Freund complete adjuvant, subcutaneous multipoint immunization is carried out on the back of guinea pigs or rabbits, 0.2mg of acetamiprid immunogen is injected into the former, and 0.5mg of acetamiprid immunogen is injected into the latter.
(3) And (3) boosting immunity: the first immunization is carried out for 14 days, then the antigen emulsification is carried out by Freund incomplete adjuvant, other steps are the same as the first immunization, the interval time of each boosting immunization is 10 days, and 3 boosting immunizations are carried out totally.
(4) On the 5 th day after the third booster immunization, a heart blood was collected, guinea pigs or rabbits were fixed, and each abdominal cavity was anesthetized with a 20% gulose anesthetic. After complete anesthesia, the thoracic cavity is cut off by scissors, the heart position is exposed, a disposable syringe is used for inserting a needle from the apex of the heart, the needle point enters the ventricle, the syringe is slowly drawn back, blood is pumped out, the needle head is pulled out, the syringe is slowly pushed against the wall of the centrifugal tube, and the blood in the syringe flows into the centrifugal tube along the wall of the centrifugal tube.
(5) The collected blood was kept at 4 ℃ overnight, the next day at 3000rpm, centrifuged for 10min, and the supernatant was stored at-70 ℃.
Example 3 measurement of antibody serum titer by Indirect ELISA method
The specific operation is as follows:
(1) the murine anti-acetamiprid antibody was coated in a 96-well plate at 20. mu.g/mL using a coating solution, 100. mu.L was added to each well, left overnight at 4 ℃, washed 3 times with a wash solution, and blotted dry.
(2) Add 200. mu.L of blocking solution to each well, incubate for 2 hours at 37 ℃, wash 3 times with wash solution, and pat dry.
(3) Rabbit anti-acetamiprid antibody serum and mouse anti-acetamiprid antibody serum were mixed according to a ratio of 1: 10000. 1: 50000. 1: 100000 and 1: 200000 dilution (negative control serum was diluted in equal fold ratio), 100. mu.L of each well was added, incubated at 37 ℃ for 1 hour, washed 3 times with wash solution, and patted dry.
(4) Adding an HRP-labeled goat anti-rabbit 1: 10000, adding an HRP-labeled goat anti-mouse 1 into a mouse anti-acetamiprid antibody serogroup: 10000, 100 mu L of the extract is added into each hole, the mixture is incubated for 1h at 37 ℃, washed 6 times by washing liquid and patted dry.
(5) 100 μ L of TMB color developing solution was added to each well, and color development reaction was carried out at room temperature for 10 min.
(6) The reaction was stopped by adding 50. mu.L of stop solution to each well.
(7) OD at 450nm was measured on a microplate reader. When the sample absorbance/negative absorbance >2.1, it is considered positive, and the titer is calculated.
Antibody titer: the ELISA antiserum titer reaches 1: 50000, which shows that the immunization is qualified, and the antiserum titer of the mice is 1: over 20000; the antiserum titer of the rabbits is 1: 100000 or more.
Antibody purification an antibody purification kit from sermera femtology was used.
EXAMPLE 4 plotting of Standard Curve
In this embodiment, a double antibody sandwich method for detecting acetamiprid is used to detect OD450 values of different samples, and the specific operation method is as follows:
(1) adding a coating antibody on the ELISA plate, wherein the adding amount of the coating antibody is 100 mu L/hole, allowing the coating antibody to coat the ELISA plate after overnight standing at 4 ℃, washing the plate, and washing off the coating antibody which is not combined with the ELISA plate, wherein the coating antibody is an acetamiprid monoclonal antibody;
(2) adding a sealing solution, sealing for 1-2 hours at 37 ℃, sealing redundant binding sites on the ELISA plate by the sealing solution, washing the plate and then washing the redundant sealing solution on the ELISA plate, wherein the addition amount of the sealing solution is 200 mu L/hole;
(3) adding a sample on the ELISA plate, and washing the plate after incubation; in this example, a total of 9 samples were used for detection, wherein the 9 samples were 1 detection sample and 8 acetamiprid standard dilutions with concentrations of 5ng/mL, 10ng/mL, 25ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 250ng/mL and 300ng/mL, respectively. The test sample was a PBS blank sample.
(4) Adding a rat anti-acetamiprid antibody serving as a primary antibody into an enzyme label plate, wherein the adding amount of the primary antibody is 100 mu L/hole, reacting at 37 ℃ for 1h, and washing the plate; then, rabbit anti-acetamiprid antibody marked by horseradish peroxidase is used as an enzyme-labeled secondary antibody, the addition amount of the enzyme-labeled secondary antibody is 100 mu L/hole, and the plate is washed after the reaction is carried out at 37 ℃ for 30 min;
(5) adding color development liquid to develop the enzyme label plate, wherein the addition amount is 100 mu L/hole, and developing in the dark at 37 ℃ for 10 min; then adding sulfuric acid with the concentration of 2mol/L to carry out termination reaction, wherein the adding amount of the sulfuric acid is 100 mu L/hole; detection of OD Using microplate reader450。
The specific operation process of washing the plate in the above steps is as follows: pouring out liquid in the holes of the enzyme label plate, patting the enzyme label plate dry on absorbent paper, filling the holes with PBST containing 0.05% Tween-20, standing for 3min, and repeatedly washing for 3-5 times.
According to the detected OD450 values of 8 standard solutions and the acetamiprid concentration in 9 samples, a standard curve of the acetamiprid can be correspondingly drawn, wherein Y is 3.66X +0.136, and R is20.994. The unit of acetamiprid concentration is mug/mL.
And secondly, by adopting the same steps, the OD450 value of the article to be detected can be detected only by replacing the sample with the article to be detected, and the acetamiprid content of the article to be detected is calculated corresponding to the standard curve.
Test example
1. Sensitivity detection
Determining the sensitivity of the kit according to a conventional method, wherein the lowest point of a standard curve of the kit is 5ng/mL, and the range of the standard curve is 5-300 ng/mL; and (3) detecting 20 parts of the blank water sample, finding out the concentration corresponding to each percent absorbance value from the standard curve, and using the average value of the concentrations of the 20 parts of samples plus 3 times of standard deviation to represent the detection limit, wherein the detection limit of the method to the blank water sample is 2.8 ng/mL.
2. Experiment of specificity
The kit prepared by the scheme of the invention is used for measuring 3 kinds of nicotine pesticides (imidacloprid, thiacloprid and nitenpyram). The sample is diluted according to a certain concentration gradient, and the detection method is shown in example 4, and the result shows that neither imidacloprid, thiacloprid nor nitenpyram can react with the antibody prepared by the scheme, so that the antibody prepared by the scheme has high specificity.
3. Sample detection
Preparing acetamiprid artificially-contaminated food: the acetamiprid solution is directly added with normal saline, fruit juice and milk respectively to be mixed uniformly to be used as polluted water, fruit juice and milk. And (3) diluting the water sample, the fruit juice and the milk which are added with the acetamiprid solution by 10 times, and eliminating the influence of the matrix. OD measurement by the Rapid ELISA method established herein450And (3) detecting the content of the acetamiprid in the sample and calculating the recovery rate and the relative standard deviation of the sample. Recovery rate is measured concentration/actual concentration x 100%. Three replicates of each sample were run, and three different kits were used to determine the mean, the results are shown in table 1.
TABLE 1
The detection results are shown in table 1, and it can be seen from the table that the water sample, the fruit juice and the milk have better recovery rates, the recovery rate is earlier within the range of 92.6-102.3%, and the relative standard deviation in batch and between batches is less than 10%. The result shows that the double-antibody sandwich ELISA method established by the scheme of the invention has high accuracy and precision, and is suitable for rapid detection of acetamiprid residue in food samples.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A double-antibody sandwich ELISA kit for detecting acetamiprid is characterized by comprising: a capture agent consisting of a specific monoclonal antibody and/or polyclonal antibody against acetamiprid, an enzyme-labeled secondary antibody, a sample diluent and a substrate.
2. The kit of claim 1, wherein the capture agent is a mouse anti-acetamiprid antibody IgG.
3. The kit of claim 1, wherein the capture agent is attached to a solid support.
4. The kit of claim 3, wherein the solid support is an elisa plate or a biochip.
5. The kit of claim 1, wherein the enzyme-labeled secondary antibody is a rabbit anti-acetamiprid antibody.
6. The kit of claim 1, wherein the dosage ratio of the capture agent to the enzyme-labeled secondary antibody is 1: (1-3).
7. The kit according to claim 1, wherein the components of the diluent are phosphate buffer containing 8 to 12% by volume of FBS.
8. The kit according to claim 1, wherein the antibody is prepared by a method comprising the steps of: the first immunization adopts antigen immunization and acetamiprid injection; and (3) performing boosting immunization after the first immunization, performing boosting immunization for 3-6 times in total, and taking blood after the last boosting immunization.
9. Use of a kit according to any one of claims 1 to 8 for detecting acetamiprid content.
10. Use of a kit according to any one of claims 1 to 8 for detecting acetamiprid residue in food.
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Citations (4)
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| US20050170446A1 (en) * | 2002-04-29 | 2005-08-04 | Niklas Ahlborg | Sandwich assay and kit |
| US20080305559A1 (en) * | 2005-12-15 | 2008-12-11 | Gualberto Gonzalez-Sapienza | Non-Competitive Immunoassays to Detect Small Molecules |
| US20170362305A1 (en) * | 2014-12-17 | 2017-12-21 | Siemens Healthcare Diagnostics Inc. | Sandwich assay design for small molecules |
| US20180306828A1 (en) * | 2015-10-29 | 2018-10-25 | Siemens Healthcare Diagnostics Inc. | Sandwich assay for small molecules |
-
2021
- 2021-04-21 CN CN202110432287.5A patent/CN113176403A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170446A1 (en) * | 2002-04-29 | 2005-08-04 | Niklas Ahlborg | Sandwich assay and kit |
| US20080305559A1 (en) * | 2005-12-15 | 2008-12-11 | Gualberto Gonzalez-Sapienza | Non-Competitive Immunoassays to Detect Small Molecules |
| US20170362305A1 (en) * | 2014-12-17 | 2017-12-21 | Siemens Healthcare Diagnostics Inc. | Sandwich assay design for small molecules |
| US20180306828A1 (en) * | 2015-10-29 | 2018-10-25 | Siemens Healthcare Diagnostics Inc. | Sandwich assay for small molecules |
Non-Patent Citations (2)
| Title |
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| 杨唐斌等: "小分子抗原酶免疫分析方法研究进展", 《中国生物工程杂志》 * |
| 陈志宝等: "《药物残留检测》", 31 August 2011 * |
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Application publication date: 20210727 |