CN116082258A - Indirect competition ELISA kit for detecting prometryn, and detection method and application thereof - Google Patents
Indirect competition ELISA kit for detecting prometryn, and detection method and application thereof Download PDFInfo
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- CN116082258A CN116082258A CN202111309927.XA CN202111309927A CN116082258A CN 116082258 A CN116082258 A CN 116082258A CN 202111309927 A CN202111309927 A CN 202111309927A CN 116082258 A CN116082258 A CN 116082258A
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- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 title claims abstract description 165
- 238000008157 ELISA kit Methods 0.000 title claims abstract description 11
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- DBAKFASWICGISY-BTJKTKAUSA-N Chlorpheniramine maleate Chemical compound OC(=O)\C=C/C(O)=O.C=1C=CC=NC=1C(CCN(C)C)C1=CC=C(Cl)C=C1 DBAKFASWICGISY-BTJKTKAUSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
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- 241000196324 Embryophyta Species 0.000 description 2
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- 101500021084 Locusta migratoria 5 kDa peptide Proteins 0.000 description 1
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
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- 125000002816 methylsulfanyl group Chemical group [H]C([H])([H])S[*] 0.000 description 1
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
- C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
- C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
- C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
- C07D251/40—Nitrogen atoms
- C07D251/48—Two nitrogen atoms
- C07D251/52—Two nitrogen atoms with an oxygen or sulfur atom attached to the third ring carbon atom
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
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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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- 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
- C07K14/77—Ovalbumin
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- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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)
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Abstract
The invention provides an indirect competition ELISA kit for detecting prometryn, a detection method and application thereof. Firstly, synthesizing a hapten of prometryn by taking prometryn as a raw material, and coupling the prometryn with OVA or BSA by an activated ester method to obtain a complete antigen of the prometryn. And then, obtaining a prometryn specific antibody by using prometryn-BSA immunization, establishing a competition ELISA detection method for detecting prometryn based on the prometryn complete antigen and the specific antibody, and providing a corresponding competition ELISA detection kit. The detection kit disclosed by the invention can accurately, sensitively and conveniently quantitatively detect the prometryn, the detection range is 0.9-241 mug/mL, and an important tool is provided for the detection work of the prometryn.
Description
Technical Field
The invention belongs to the field of environmental detection, and particularly relates to an indirect competition ELISA kit for detecting prometryn, a detection method and application thereof.
Background
Pesticides play an important role in agricultural production, and in recent years, pesticide pollution has become a hot spot of interest to students. The prometryn as one kind of triazine compound has the features of high weed selectivity, wide weed killing spectrum, etc. and is one kind of herbicide and water quality improver. Because the prometryn has stable chemical property, the prometryn can exist in the environment for a long time and can be collected through surface and underground runoffs, so that the prometryn in the water environment is frequently detected. For example, the concentration of prometryn in a water collecting area of a Hartbesespot dam in south Africa is 367-527 ng/L; the concentration of the surface layer of the Laizhou bay is 21.2 ng/L. Studies show that the prometryn with environmental concentration can cause abnormal physiological and biochemical indexes and tissue damage of carps. In addition, prometryn has high bioaccumulation, and higher levels of prometryn are detected in infant umbilical cord blood, and it is confirmed that prometryn is associated with slow hearing development in infants. In view of the high concentration and toxic effects of prometryn in the environment, it is highly desirable to establish a method for detecting prometryn.
The current detection method of prometryn mainly comprises a gas chromatography and a liquid chromatography. These methods generally require pretreatment of the sample, wherein the most cost-effective solid phase extraction column is available from Watertian technologies, inc., which is too expensive and the pretreatment process is complex. Expensive equipment is required in the detection process, and the detection method belongs to a labor and resource intensive detection method. ELISA is a detection method established by utilizing the principle of antigen-antibody interaction, has low cost and high detection speed, and does not need complex instruments and equipment and pretreatment methods, and is widely applied to the detection field. Whereas prometryn belongs to a small molecular substance, and does not have a coupling group, the prometryn needs to be modified to be coupled with a macromolecular protein to obtain a hapten, so that an antibody is prepared. However, the existing immunological detection methods are hapten obtained by directly modifying the prometryn group, which may cause the loss of the prometryn specific group and influence the affinity and the specificity. Therefore, in order to make up the defects of the existing detection method, the invention uses the prometryn as a raw material, uses 3-mercaptopropionic acid to reform, and adds a section of C chain to the methylthio group of prometryn for coupling with macromolecular protein. Through the structure, on the premise of retaining the specificity group of the prometryn, the recognition capability of the antibody to the prometryn hapten is effectively improved. Then, the invention establishes the competition ELISA kit of prometryn based on the antigen antibody, compared with the chromatographic detection method, for this purpose, the invention firstly coats prometryn-OVA on the solid phase carrier, then adds prometryn specific antibody and prometryn standard/sample mixture to be tested, the prometryn-OVA on the solid phase carrier can compete with prometryn antibody in the mixture for combination, and can quantify the prometryn content after adding HRP marked goat anti-mouse antibody. Compared with the chromatographic detection method, the kit developed by the invention is simpler, more convenient and faster, and simultaneously compared with the prior direct competition ELISA, the preparation steps and the cost of the ELISA kit are reduced, and a reliable, simple and convenient detection method is provided for the detection work of prometryn.
Disclosure of Invention
The invention aims to provide a prometryn hapten, a complete antigen prepared according to the hapten and an anti-prometryn antibody prepared according to the prometryn complete antigen, and further provides a kit for performing competition ELISA detection for detecting prometryn by using the prometryn complete antigen and the antibody, so as to make up for the defects of the existing prometryn detection method.
The invention synthesizes a prometryn hapten, which is characterized by having a structural formula shown in figure 1.
The invention also prepares the prometryn complete antigen according to the synthesized prometryn hapten, which is characterized in that the prometryn hapten carboxyl is coupled with the amino of carrier protein. The invention also provides carrier proteins, including bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen, or gelatin, that can be coupled to the prometryn hapten of the invention.
The invention also provides a prometryn specific antibody prepared by using the prometryn complete antigen, which can be a monoclonal antibody, a polyclonal antibody or a genetic engineering antibody.
The invention also provides application of the prometryn complete antigen in preparation of prometryn specific antibodies.
The invention also provides application of the prometryn complete antigen or the prometryn specific antibody in prometryn immunodetection. The immunodetection is carried out by an enzyme-linked immunosorbent assay method, a colloidal gold test strip method, an immunohistochemical method, a radioimmunoassay method, a chemiluminescent enzyme-linked immunosorbent assay method or an immunosensor method.
In addition, the invention also provides a method for detecting the prometryn by indirect competitive enzyme-linked immunosorbent assay, which is characterized by comprising the following steps of,
and step 2, sealing the ELISA plate by using sealing liquid,
when the prometryn specific antibody in the step 4 is non-enzyme-labeled, the method also comprises a step 5, and an enzyme-labeled secondary antibody is added, wherein the secondary antibody is IgG of the antibody source species added in the step 4,
step 7, adding a color development liquid to develop color,
step 9, determining the wavelength according to the color reagent added in the step 7 and measuring the absorbance value at the wavelength,
and step 10, judging the content of prometryn in the sample to be tested according to a standard curve.
Further, in step 1 of the detection method of the prometryn indirect competitive enzyme-linked immunosorbent assay, the prometryn complete antigen coated ELISA plate with the concentration of 0.5-10 mug/mL, preferably 1 mug/mL, is used, and in step 3, the dilution factor is 1: 1000-1: 1024000, preferably 1: the prometryn specific antibody of 256000 was mixed with the test sample.
In addition, the invention provides an indirect competitive enzyme-linked immunosorbent assay kit for detecting prometryn, which comprises an ELISA plate, a coating liquid, an optional sealing liquid, a washing liquid, a color development liquid and a termination liquid, and is characterized by further comprising the prometryn complete antigen and the prometryn specific antibody.
Drawings
FIG. 1 shows a prometryn hapten according to the present invention.
FIG. 2 is a diagram showing the synthesis of the prometryn hapten and the complete antigen of the present invention.
FIG. 3 is a graph showing the results of ultraviolet identification and polyacrylamide gel electrophoresis verification of the prometryn complete antigen of the present invention.
FIG. 4 is a graph showing the optimal results of dilution of the coating antigen and the specific antibody in the method for detecting prometryn by indirect competitive ELISA according to the present invention.
FIG. 5 is a standard curve of an indirect competitive ELISA assay kit of the invention.
Detailed Description
The invention is further illustrated by the following examples and figures.
Compared with the prior art, the invention adopts the chlorphenamine maleate as a synthesis raw material, and utilizes the 3-mercaptopropionic acid to reform to obtain the hapten of the prometryn, and the structural formula of the hapten is shown in figure 1.
The hapten is added with a connecting arm on the basis of retaining a prometryn structure, and the recognition capability of an antibody on prometryn specific groups is ensured on the premise that the prometryn hapten can be effectively connected with macromolecular proteins.
The synthetic prometryn hapten can be used to prepare a prometryn complete antigen, for example, by coupling the prometryn hapten carboxyl group to the amino group of a carrier protein to obtain the prometryn complete antigen. Carrier proteins that can be used for the purposes of the present invention are very numerous, such as, but not limited to, bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen, gelatin, and the like.
It is well known in the art that the prometryn complete antigen of the present invention can be used to prepare prometryn specific antibodies, wherein the antibodies can be monoclonal antibodies, such as mouse monoclonal antibodies, rabbit monoclonal antibodies, and the like, and the polyclonal antibodies can be mouse polyclonal antibodies, rabbit polyclonal antibodies, sheep polyclonal antibodies, donkey polyclonal antibodies, camel polyclonal antibodies, and the like.
It is well known in the art that the detection of prometryn in a sample can be performed using the prometryn complete antigen and specific antibody. The detection method may be any immunological detection method using immunological binding of antigen and antibody, for example, immunological detection methods which may be used for the purpose of the present invention include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), colloidal gold test strip method, immunohistochemical method, radioimmunoassay, chemiluminescent enzyme-linked immunosensor method, or the like.
The invention also provides a method for detecting the prometryn by indirect competitive enzyme-linked immunosorbent assay, which is characterized by comprising the following steps,
and step 2, sealing the ELISA plate by using sealing liquid,
when the prometryn specific antibody in the step 4 is non-enzyme-labeled, the method also comprises a step 5, and an enzyme-labeled secondary antibody is added, wherein the secondary antibody is IgG of the antibody source species added in the step 4,
step 7, adding a color development liquid to develop color,
step 9, determining the wavelength according to the color reagent added in the step 7 and measuring the absorbance value at the wavelength,
and step 10, judging the content of prometryn in the sample to be tested according to a standard curve.
As is well known in the art, in indirect competitive ELISA assays, ELISA plates that can be used for the purposes of the present invention include, but are not limited to, 96-well plates, 48-well plates, 24-well plates, 6-well plates, and the like. Coating liquids include, but are not limited to, naHCO 3 、Na 2 CO 3 Etc., it is well known in the art how to formulate coating solutions. Blocking may or may not be performed after antigen coating, and blocking fluids include, but are not limited to, BSA, OVA, gelatin, and the like. The sealing operation is not carried out, and the test result is not affected. In this method, an enzyme-labeled prometryn antibody may be used, followed by measurement by a color reaction, or a non-enzyme-labeled prometryn specific antibody may be used, followed by addition of an enzyme-labeled secondary antibody against the non-enzyme-labeled antibody, followed by color development and measurement. When a non-enzyme-labeled prometryn antibody is used, the enzyme-labeled secondary antibody used is an anti-non-enzyme-labeled prometryn specific antibody source species IgG, and it is common knowledge in the art to select an enzyme-labeled secondary antibody for a non-enzyme-labeled prometryn antibody. Enzymes for labelling prometryn-specific antibodies or secondary antibodies are also well known in the artIncluding but not limited to horseradish peroxidase or alkaline phosphatase, and the like. It is also known in the art how to prepare a color-developing solution and a wavelength for measurement according to the labeling enzyme used in the previous step is well known in the art. In addition, the stop solution may be H 2 SO 4 HCl or HNO 3 Etc., it is well known in the art how to formulate a stop solution. As is well known in the art for determining the content of an analyte in a sample, generally, a series of diluted analyte standards are used to perform experimental operations and determine the corresponding absorbance values, then a standard curve is made according to Log values and absorbance values of the concentration of the sample, the absorbance of the analyte in the sample is determined, and the content of prometryn in the sample is determined by querying the standard curve.
Further, in the step 1 of the detection method of the prometryn indirect competitive enzyme-linked immunosorbent assay, the prometryn complete antigen coated ELISA plate with the concentration of 0.5-10 mu g/mL, preferably 1 mu g/mL is used, and in the step 3, the dilution multiple is 1: 1000-1: 1024000, preferably 1: the prometryn specific antibody of 256000 was mixed with the test sample.
In addition, the invention provides a prometryn detection kit based on the indirect competitive ELISA method, which comprises an ELISA plate, a coating liquid, an optional blocking liquid, a washing liquid, a color development liquid and a termination liquid, and further comprises a prometryn complete antigen and a prometryn specific antibody.
Compared with the chromatograph, the prometryn indirect competition ELISA detection method adopted by the invention has lower cost and simple required equipment, so the invention provides a reliable tool for prometryn detection work in the environment and biological tissues.
The present invention is further described below with reference to the examples and the accompanying drawings, but the following examples do not limit the scope of the invention.
Example 1: preparation of prometryn antigen and antibody
(1) Synthesis of prometryn hapten
0.233 g of chlorphenamine is weighed and dissolved in 22.5 mL of absolute ethanol, stirred, then added with 0.224 g of 3-mercaptopropionic acid and 0.24 g of potassium hydroxide, and heated to reflux overnight. Stopping the reaction the next day, cooling to room temperature, filtering, spin-drying the filtrate with a spin dryer, and separating with 10:1 dichloromethane/methanol (v/v) column to obtain prometryn hapten with structural formula shown in figure 1.
(2) Synthesis of prometryn complete antigen
The complete prometryn antigen was synthesized according to the synthetic diagram of FIG. 2. Weighing hapten of 10 mg prometryn, using 500 mu L N, dissolving N-Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with equal mass, reacting at normal temperature for 2 h, adding 15 mg chicken Ovalbumin (OVA) or 10 mg Bovine Serum Albumin (BSA), reacting at 4 ℃ overnight, centrifuging at 4000 r/min for 10 min, taking supernatant, dialyzing in 0.01M PBS for 48 h, and centrifuging the solution again to obtain supernatant, wherein prometryn-BSA is an immune antigen, and prometryn-OVA is a coating antigen. The ultraviolet spectrum scanning is carried out on the prepared complete antigen, the result is shown in figure 3, the ultraviolet absorption peak of the complete antigen after being coupled with BSA is obviously shifted, in addition, the result of polyacrylamide gel electrophoresis also shows that the molecular weight of the complete antigen is obviously larger than that of BSA or OVA, and the results prove that the complete antigen of prometryn is prepared.
(3) Preparation of prometryn specific antibodies
4 healthy female Kunming mice were selected as experimental organisms, and 0.1 mL containing 150 μg of immune antigen and an equal volume of Freund's complete adjuvant were emulsified and injected into the abdominal cavity of the mice. The Kunming mice were then immunized 8 times at 7 days intervals with the same dose of immunizing antigen fully emulsified with Freund's incomplete adjuvant. Three days after the last immunization is finished, the mice are boosted, the mice are killed by an eyeball blood taking method, the blood of the mice is collected, the mice are kept stand at room temperature for 2 h, and the supernatant is taken after 4000 r/min centrifugation for 10 min, so that the prometryn specific antibody is obtained.
Example 2: establishment of competitive ELISA for detecting prometryn
(1) Determination of optimal coating antigen and antibody dilution
The optimal dilution factors of the coating antigen and the antibody are determined by using a chessboard method, 10 mu g/mL of the coating antigen is diluted to 0.5 mu g/mL by using a coating liquid (0.05M carbonate buffer solution pH9.6) according to a double-ratio dilution method, and after the coating antigen is added into a 96-well plate, the coating antigen is incubated overnight at 4 ℃. The next day, the coating solution was discarded, after washing 3 times with washing solution (PBS containing 0.05% Tween-20), 200. Mu.L of blocking solution (PBS containing 2% BSA and 1% Tween-20) was added to each well to block 1 h, and after discarding the blocking solution, washing 3 times. The prometryn specific antibodies were then diluted according to the fold ratio from 1:1000 to 1:256000 times, after incubation at 37 ℃ for 1 h, washing for 5 times; after adding HRP-labeled goat anti-mouse IgG antibody diluted 1:2000 and incubating for 1:1 h and washing for 5 times, adding TMB color development liquid for 10 min, adding 100 mu L of termination liquid into each hole to terminate the color development reaction, and measuring the ultraviolet absorption value at the position of 450: 450 nm by using an enzyme label instrument. As a result, as shown in FIG. 4, it was found that when the dilution concentration of the coating antigen was 1. Mu.g/mL and the dilution factor of the prometryn-specific antibody was 1:256000, the OD value of the competition ELISA was closest to 1.0, and it was confirmed that the conditions were the optimal concentration of the coating antigen and the dilution factor of the antibody.
(2) Determination of a competitive ELISA Standard Curve
Example 3: use of prometryn competition ELISA kit
The prometryn competition ELISA kit comprises the following components:
(1) Blank ELISA plate
(2) The prometryn carrier protein conjugate is diluted to 1 mug/mL by coating liquid before use;
(3) The prometryn pure product is diluted to the required concentration by a diluent before use;
(4) Prometryn specific antibody, diluted 256000-fold before use;
(5) Horseradish peroxidase-labeled goat anti-mouse IgG antibody, diluted 2000-fold before use;
(6) The kit further comprises a coating liquid, a sealing liquid, a washing liquid, a sample diluting liquid, a color development liquid and a stopping liquid, wherein the coating liquid is 50mM carbonate buffer solution with the pH value of 9.6: 1.59 g Na 2 CO 3 ，2 .93g NaHCO 3 Distilled water was added to 1000mL and the wash was 150mM pH7.4 phosphate buffer containing 0.05% Tween-20: 8.0g NaCl,0.2g KCl,2.9g Na 2 HPO 4 ·12H 2 O， 0.2g KH 2 PO 4 0.5 mL Tween-20, distilled water to 1000 mL; the blocking solution was pH7.4 phosphate buffer containing 2% BSA: 0.2g BSA was dissolved in 10 mL phosphate buffer pH7.4; the diluent is 150mM phosphate buffer solution containing 0.05% Tween-20, 1% BSA and 10% dimethyl sulfoxide, and pH7.4; the color developing solution is TMB single-component color developing solution produced by Beijing Nobolide technology Co., ltd; h of termination liquid 2M 2 SO 4 An aqueous solution.
The competition ELISA kit for detecting the prometryn is used for detecting the prometryn in a sample, and the detection steps comprise:
(1) According to the results of FIG. 4, the complete antigen of prometryn was diluted to 1. Mu.g/ml using the coating solution in the kit, 100. Mu.L was added to each well, and after coating overnight at 4 ℃, the coating solution was discarded and washed 3 times;
(2) 200 mu L of blocking solution is added to each well, after incubation at 37 ℃ for 1 h, the blocking solution is discarded and washed 3 times;
(3) Diluting the prometryn pure product to 0.94.1.89, 3.77, 7.54, 15.08, 30.16, 60.33, 120.65 and 241.3 mug/mL by using a diluent; diluting the prometryn antibody 256000 times, mixing with a standard substance or a sample to be detected, adding into a 96-well ELISA plate, wherein each well contains 100 mu L of the standard substance or the sample to be detected and the prometryn specific antibody, incubating for 1 hour at 37 ℃, discarding the solution in the well, and washing for 5 times;
(4) Adding 100 mu L of 2000-fold diluted goat anti-mouse IgG antibody marked by horseradish peroxidase into each well of a 96-well ELISA plate, incubating for 1 hour at 37 ℃, discarding the solution in the well, and washing for 5 times;
(5) Adding 100 mu L of color development liquid into a 96-well ELISA plate, and reacting for 10 min at 37 ℃ in a dark place at room temperature;
(6) Adding a stop solution after the yellow color appears obviously, wherein 100 mu L/hole;
(7) Measuring the absorbance value of each hole under the wavelength of 450 nm by using an enzyme-labeled instrument, wherein the measurement is carried out within 10 minutes after the stop solution is added;
(8) And (3) calculating: and (3) taking the logarithmic value of the concentration of the standard substance as an abscissa and the OD value as an ordinate as a standard curve, calculating a linear regression equation of the standard curve by using the concentration of the standard substance and the OD value, substituting the OD value of the sample into the equation, calculating the concentration of the sample, and multiplying the calculated concentration by the dilution multiple to obtain the actual concentration of the prometryn in the sample. ELISA detection results of the kit provided by the invention on the prometryn standard substance are shown in FIG. 5.
As can be seen from FIG. 5, the standard curve shows a good linear relationship when the concentration of the prometryn is 0.9-241 mu g/mL, so that the detection range is 0.9-241 mu g/mL, and meanwhile, the detection kit can accurately, sensitively and conveniently quantitatively detect the prometryn, thereby providing an important tool for the detection work of the prometryn.
Claims (11)
1. A prometryn hapten characterized by the following structural formula:
。
2. a prometryn complete antigen, characterized in that a prometryn hapten according to claim 1 is coupled to the amino group of the carrier protein via a carboxyl group.
3. The prometryn complete antigen according to claim 2, characterized in that the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroglobulin, fibrinogen or gelatin.
4. A prometryn specific antibody prepared using a prometryn complete antigen according to claim 2 or 3.
5. The prometryn specific antibody according to claim 4 wherein the prometryn specific antibody is a monoclonal, polyclonal or genetically engineered antibody.
6. Use of a prometryn complete antigen according to claim 2 or 3 for the preparation of a prometryn specific antibody.
7. Use of a prometryn complete antigen according to claim 2 or 3 or a prometryn specific antibody according to claim 4 or 5 for prometryn immunodetection.
8. The use according to claim 7, wherein said immunodetection is performed by an enzyme-linked immunosorbent assay method, a colloidal gold test strip method, an immunohistochemical method, a radioimmunoassay method, a chemiluminescent enzyme linked immunosorbent assay method or an immunosensor method.
9. The method for detecting the prometryn by indirect competitive enzyme-linked immunosorbent assay is characterized by comprising the following steps of,
step 1, coating the prometryn complete antigen according to claim 2 or 3 on an ELISA plate with a coating solution,
and step 2, sealing the ELISA plate by using sealing liquid,
step 3, washing the ELISA plate with a washing solution,
step 4, mixing the prometryn specific antibody according to claim 4 or 5, which is enzyme-labeled or non-enzyme-labeled, with the sample to be tested, adding the prometryn complete antigen coated in step 1 and prometryn in the sample to be tested to competitively bind the prometryn specific antibody,
when the prometryn specific antibody in the step 4 is non-enzyme-labeled, the method also comprises a step 5, and an enzyme-labeled secondary antibody is added, wherein the secondary antibody is IgG of the antibody source species added in the step 4,
step 6, washing the ELISA plate with a washing solution,
step 7, adding a color development liquid to develop color,
step 8, adding a stop solution to stop developing,
step 9, determining the wavelength according to the color reagent added in the step 7 and measuring the absorbance value at the wavelength,
and step 10, judging the content of prometryn in the sample to be tested according to a standard curve.
10. The method for detecting prometryn by indirect competitive enzyme-linked immunosorbent assay according to claim 9, wherein in step 1, the prometryn complete antigen with a concentration of 0.5-10 μg/mL, preferably 1 μg/mL is used for coating the elisa plate, and in step 3, the dilution factor is 1: 1000-1: 1024000, preferably 1: the prometryn specific antibody of 256000 was mixed with the test sample.
11. An indirect competitive enzyme-linked immunosorbent assay kit for detecting prometryn, comprising an enzyme-labeled plate, a coating liquid, an optional blocking liquid, a washing liquid, a chromogenic liquid, and a stop liquid, characterized by further comprising a prometryn complete antigen according to claim 2 or 3 and a prometryn specific antibody according to claim 4 or 5.
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