CN114395534A - Hybridoma cell strain secreting prometryn monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting prometryn monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting prometryn monoclonal antibody and application thereof in the field of food safety immunodetection. The hybridoma cell strain CPZ secreting the prometryn monoclonal antibody has the preservation number of CGMCC NO.45022, and the prometryn monoclonal antibody produced by secretion has good affinity, high specificity and high sensitivity (the IC50 value is 0.65ng/mL), can be used for preparing an immunoassay kit of abscisic acid and a colloidal gold test strip, establishes an immunological detection method of prometryn, and provides a powerful detection method for detecting prometryn residues in food.
Description
Technical Field
The invention belongs to the field of food safety immunodetection, and particularly relates to a hybridoma cell strain secreting prometryn monoclonal antibody and application thereof.
Background
Prometryn is a selective s-triazine herbicide for both paddy fields and dry lands, and has a systemic conduction effect. Can be absorbed from roots, or permeated into plants from stems and leaves, and transported to green leaves to inhibit photosynthesis, so that weeds are green and dry and die. The duration is as long as 20-70 days, and the herbicide is suitable for crops such as rice, wheat, soybean, cotton, sugarcane, fruit trees, vegetables and the like, and is used for preventing and killing annual broadleaf weeds, grass, nutgrass flatsedge and certain perennial weeds such as crabgrass, green bristlegrass, barnyard grass, monochoria vaginalis, Equisetum ramosissimum and the like, and perennial eyedrops, hairyvein agropyron and the like.
At present, the prometryn detection method mainly adopts instrument detection, and high performance liquid chromatography, gas chromatography-mass spectrometry and the like are commonly used. Although these chromatographic-based methods have high sensitivity and specificity, they suffer from drawbacks such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, there is a need to develop a rapid and simple method for analyzing prometryn residue.
The enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, has the advantages of simple pretreatment of a sample during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for field rapid detection of a large number of samples, thereby being widely applied. The precondition for detecting prometryn by using an enzyme-linked immunosorbent assay is that a monoclonal antibody with high specificity and high sensitivity to prometryn is obtained, so that the key is to find a method for preparing the monoclonal antibody with high specificity and high sensitivity to prometryn. The inventor tries to prepare prometryn monoclonal antibody through hybridoma cells, but in the process of preparing a hybridoma cell strain capable of secreting the prometryn monoclonal antibody, how to prepare prometryn hapten and prometryn complete antigen and how to enable a mouse to generate strong immunity need further research; further research is needed on how to successfully secrete prometryn monoclonal antibody from the prepared hybridoma cell strain; how to make the secreted prometryn monoclonal antibody have strong specificity and high sensitivity also needs further research.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a hybridoma cell strain secreting prometryn monoclonal antibody and application thereof.
The invention provides a hybridoma cell strain CPZ for secreting prometryn monoclonal antibody in a first aspect, and the preservation number of the hybridoma cell strain CPZ is CGMCC NO. 45022.
The invention provides a prometryn monoclonal antibody secreted and produced by the hybridoma cell strain CPZ in the second aspect.
The third aspect of the invention provides an application of the hybridoma cell strain CPZ or the prometryn monoclonal antibody in prometryn detection, an application in preparation of a prometryn immunoassay kit or an application in preparation of a prometryn detection colloidal gold test strip.
The fourth aspect of the invention provides a prometryn immunodetection kit, which comprises the hybridoma cell strain CPZ or the prometryn monoclonal antibody.
Further, the prometryn immunoassay kit further comprises an enzyme label plate, a prometryn coating antigen, a prometryn standard solution, an enzyme-labeled secondary antibody and a substrate reaction solution.
Further, the prometryn coating antigen has the following structure:
the invention provides a prometryn detection colloidal gold test strip in a fifth aspect, which comprises a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold combined pad is coated with a prometryn monoclonal antibody.
Further, the detection line is printed by prometryn coating antigen.
Further, the prometryn coating antigen has the following structure:
the sixth aspect of the invention provides an application of the prometryn immunoassay kit or the prometryn detection colloidal gold test strip in prometryn detection.
The preparation method of the hybridoma cell strain secreting prometryn monoclonal antibody comprises the following steps:
(1) designing and preparing prometryn hapten;
(2) preparing a prometryn complete antigen, and preparing a Freund adjuvant and an incomplete Freund adjuvant from the obtained prometryn complete antigen;
(3) injecting the Freund's adjuvant into BALB/c mouse via back subcutaneous injection for multiple times of immunization, wherein complete Freund's adjuvant is adopted for the first immunization, and incomplete Freund's adjuvant is adopted for boosting immunization;
(4) collecting blood from the mice subjected to the immunization process, detecting the serum immune titer and the immune suppression capacity of the mice through indirect ELISA, and screening the mice with high prometryn antibody content in the serum to obtain immunized mice;
(5) performing last boosting immunization on the screened mice by using incomplete Freund adjuvant, and performing sprint immunization by intraperitoneal injection, wherein the sprint immunization is performed by using a prometryn complete antigen without Freund adjuvant;
(6) fusing splenocytes of BALB/c mice subjected to sprint immunization with myeloma cells, culturing the fused cells through a culture medium, detecting positive cell holes by using indirect ELISA, further determining the inhibition effect of the positive cell holes by using an indirect competitive ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain capable of secreting prometryn monoclonal antibodies.
In the preparation method of the present invention, the interval between the primary immunization and the booster immunization in the steps (2) and (4) is one month, the interval between the booster immunization is 21 days, and the interval between the booster immunization and the sprint immunization is 18-21 days.
In the above preparation method of the present invention, the primary immunization dose in the steps (3) and (5) is 100. mu.g/mouse, the booster immunization dose is 50. mu.g/mouse, and the sprint immunization dose is 25. mu.g/mouse.
In the above preparation method of the present invention, the immunization process in the steps (3) and (5) comprises 1 primary immunization, 4 booster immunizations and 1 sprint immunization;
in the above preparation method of the present invention, the blood collection in the step (4) is performed on the 7 th day after the 3 rd immunization course is completed.
In the above preparation method of the present invention, the cell fusion in the step (6) is performed 3 days after the completion of the boosting immunization.
In the above preparation method of the present invention, the cell fusion in the step (6) is performed by a polyethylene glycol (PEG1450) method.
In the above production method of the present invention, the medium in the step (6) is RPMI-1640 medium.
In the above production method of the present invention, the number of subclonings in the step (6) is 3.
The invention has the beneficial effects that:
the prometryn monoclonal antibody secreted by the hybridoma cell strain CPZ has good affinity, high specificity and high sensitivity (the IC50 value is 0.65ng/mL), can be used for preparing an immunoassay kit and a colloidal gold test strip for abscisic acid, establishes an immunological detection method for prometryn, and provides a powerful detection method for detecting prometryn residues in food.
Drawings
FIG. 1 is a standard inhibition curve of prometryn monoclonal antibody;
FIG. 2 shows the LC-MS identification result of prometryn hapten.
Deposit description
The prometryn monoclonal antibody secreting hybridoma cell strain CPZ is deposited in the China general microbiological culture Collection center (CGMCC), China institute of microbiology, national institute of sciences, China institute of sciences, No. 3, West Lu 1, North Cheng, south China, Beijing, and has a collection number of CGMCC No.45022 and a collection date of 2021, 12 and 16 days.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
The culture media involved in the examples of the present invention are as follows:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-pantothenate 0.25, Folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, and sodium bicarbonate 2000.
The reagents involved in the examples of the invention are as follows:
carbonate Buffer (CBS): weighing Na2CO3 1.59g，NaHCO32.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
PBST: PBS containing 0.05% tween 20;
antibody dilution: PBS containing 0.1% gelatin;
TMB color development liquid: solution A: na (Na)2HPO4·12H218.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. And the volume ratio of the solution B to the solution B is 5:1 to obtain the TMB color developing solution which is mixed at present.
The detection method involved in the embodiment of the invention is as follows:
the prometryn inhibition rate detection method comprises the following steps: the most suitable antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3. mu.g/mL with Carbonate Buffer (CBS) and the antibody was diluted to 0.01, 0.03, 0.1 and 0.3. mu.g/mL with antibody diluent. After selecting the optimal working point, the prometryn standard was diluted to 8 concentrations (0, 0.03, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3ng/mL), according to the IC-ELISA procedure, and finally the originPro 8.5 was used as a graph to obtain the prometryn standard inhibition curve, and IC was calculated50。
Example 1: synthesis of prometryn hapten
Cyanuric chloride (10g, 54.2mmoL) was dissolved in tetrahydrofuran (350mL) as a solvent, N-diisopropylethylamine (24.5g, 189.8mmoL) and isopropylamine (8.05g, 135.6mmoL) were added, the reaction was stirred at room temperature for 72 hours, water was added to the reaction mixture to precipitate the desired product, and 8.5g of an off-white solid (Compound 1) was collected by suction filtration. Uniformly mixing the compound 1(2.3g, 10.0mmoL) and ethanol (60mL) in a 150mL flask, slowly adding KOH (2.8g, 50.1mmoL) and 3-mercaptopropionic acid (1.5g, 15.0mmoL), and heating and refluxing for reacting for 3 hours; after the reaction is finished, the solvent is removed by reduced pressure distillation; dissolving the residue with 5% NaOH solution, and extracting with chloroform (10mL) for 3 times; taking the water phase, acidifying with 1M HCl aqueous solution to pH 4-5, extracting with ethyl acetate (3 × 15mL) for three times, combining the organic layers, and adding Na2SO4Drying, filtration and concentration of the filtrate crystallized from hexane to give 1.1g of compound 2 as a white solid, i.e. prometryn hapten. LC-MS identification is carried out on prometryn hapten by adopting a negative ion mode. The retention time of the target was 2.80min, at which the peak at mass to charge ratio (m/z)298.1 in the corresponding mass spectrum (fig. 2) corresponds to the actual relative molecular mass of prometryn hapten (relative molecular mass 299.1) reduced hydrogen, confirming the success of prometryn hapten derivatization.
Example 2: synthesis of prometryn complete antigen
Weighing 2.7mg prometryn hapten and 3.1mg N-hydroxysuccinimide NHS, dissolving in 300 mu L N N-dimethylformamide DMF, and stirring at room temperature for reaction for 5 min; then 5.2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC is added, and the mixture is stirred at room temperature and reacts for 6 to 8 hours to obtain solution A; taking 6mg of hemocyanin KLH, and diluting the hemocyanin KLH to 3mg/mL by using 0.01M carbonate buffer CBS to obtain solution B; slowly adding the solution A into the solution B drop by drop, and reacting at room temperature for 12 h; then dialyzed with 0.01M PBS solution to remove unreacted small molecule hapten, thus obtaining prometryn complete antigen-immunogen (as shown in the following formula).
Example 3: synthesis of prometryn coating antigen
Weighing 1.3mg prometryn hapten and 1.5mg N-hydroxysuccinimide NHS, dissolving in 300 mu L N N-dimethylformamide DMF, and stirring at room temperature for 5 min; then adding 2.6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC, stirring and reacting at room temperature for 6-8h to obtain solution A; taking 10mg BSA, diluting to 3mg/mL with 0.01M carbonate buffer CBS, and obtaining solution B; slowly adding the solution A into the solution B drop by drop, and reacting at room temperature for 12 h; then dialyzed with 0.01M PBS solution to remove unreacted small molecule hapten, thus obtaining prometryn-coated antigen (shown as the following formula).
Example 4: preparation of hybridoma cell strain secreting prometryn monoclonal antibody
(1) Obtaining animal immunity: mixing and emulsifying a prometryn complete antigen and an equivalent amount of Freund adjuvant, and performing neck-back subcutaneous multipoint injection immunization (except for sprint immunization) on a BALB/c mouse; complete Freund adjuvant is used for the first immunization, and the dosage is 100 mug/mouse; incomplete Freund's adjuvant is used for multiple times of boosting immunization, and the dosage is reduced by half to be 50 mu g/mouse; the thorny immunity does not use an adjuvant, the thorny immunity is directly diluted by normal saline and then injected into the abdominal cavity, and the dosage is reduced by half to obtain 25 mu g/mouse; one month is separated between the first immunization and the second boosting immunization, 21 days are separated between the multiple boosting immunizations, and 18-21 days are separated between the sprint immunization and the last boosting immunization; collecting blood 7 days after 3 rd boosting immunization, observing the immune effect of the mice by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), namely detecting the titer and inhibition of the serum of the mice, and screening the mice with high prometryn antibody content in the serum to obtain the immunity; the selected mice are subjected to the last boosting immunization, and then the thorny immunization is carried out through intraperitoneal injection. The whole immunization process comprises 1 first immunization, 4 booster immunizations and 1 sprint immunization.
(2) Cell fusion: after three days of the spurting immunity, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight 1450) method, and the specific steps are as follows:
a. cutting the tail, taking blood, immediately putting the mouse into 75% alcohol for disinfection after the mouse is killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mouse through aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2Culturing in an incubator, wherein the number of SP2/0 tumor cells is required to reach (1-4) x 10 before fusion7Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion, collecting the tumor cells during fusion, suspending the tumor cells in RPMI-1640 basic culture solution, and counting the cells;
c. the fusion process is 7 min: mixing SP2/0 cells and splenocytes according to a counting ratio of 2-5: 10, centrifuging, and fusing with PEG. 1min, 1mL of PEG1450 was added to the cells dropwise from slow to fast; standing for 2 min; dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1 was added dropwise every 10s640 a culture medium; then carrying out warm bath at 37 ℃ for 5 min; centrifuging (800rpm, 8min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum and 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, placing at 37 ℃ and 5% CO2Culturing in an incubator.
(3) Cell screening and cell line establishment
On day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected and screened.
The screening is divided into two steps: firstly, screening out positive cell holes by an ic-ELISA method, secondly, selecting prometryn as a standard substance, and measuring the inhibition effect of the positive cells by the ic-ELISA method.
And selecting a cell hole with better inhibition on the prometryn standard substance, performing subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days.
And carrying out subcloning for three times according to the method to finally obtain the prometryn monoclonal antibody cell strain CPZ.
Example 5: preparation and identification of prometryn monoclonal antibody
Taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106Prometryn hybridoma cells, ascites fluid was collected from the seventh day, and antibody purification was performed on the ascites fluid by the octanoic acid-saturated ammonium sulfate method.
Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, and the supernatant was dissolved in a 0.01M PBS solution (pH7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
Determination of IC of prometryn monoclonal antibody Using Indirect competitive ELISA50The value was 0.65ng/mL and its IC was verified for prometryn analogue50And the results are shown in Table 1, which shows the effects on prometrynHas good sensitivity and specificity, and can be used for prometryn immunoassay detection.
Table 1: identification of antibody specificity
Example 6: application of prometryn monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain CPZ through in vivo ascites is applied to an ELISA detection test of prometryn, and the specific steps are as follows:
(1) coating the 96-well enzyme label plate with the coating antigen with the concentration of 0.01 mu g/mL diluted by Carbonate Buffer Solution (CBS), coating 100 mu L of the coated antigen in each well at 37 ℃ for 2h, washing the plate with PBST washing liquor for three times, wherein 200 mu L of the coated antigen in each well is used for 3min each time, and drying by beating;
(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;
(3) respectively preparing 0, 0.03, 0.1, 0.3, 0.9, 2.7, 8.1 and 24.3ng/mL prometryn standard solution by using Phosphate Buffered Saline (PBS), respectively adding the standard solution and a sample extracting solution to be detected into an enzyme label plate which is sealed, wherein each hole is 50 mu L, each sample is repeatedly provided with 3 holes, 50 mu L of an anti-prometryn monoclonal antibody diluted to 0.01 mu g/mL is added into each hole, reacting at 37 ℃ for 0.5h, washing and drying the plate;
(4) adding 100 μ L of HRP-labeled goat anti-mouse IgG secondary antibody diluted with PBS containing 0.1% gelatin at a ratio of 1:3000 into each well, reacting at 37 ℃ for 0.5h, washing and drying;
(5) adding 100 μ L of TMB color developing solution into each well, developing at 37 deg.C for 15min, and collecting the solutionAdd 50. mu.L of 2M H2SO4Stop solution, absorbance at 450 nm.
Using originPro 8.5 as a graph, a prometryn standard inhibition curve (the result is shown in FIG. 1) was obtained, and IC of the prometryn monoclonal antibody against prometryn50The concentration is 0.65ng/mL, which indicates that the prometryn derivative has good sensitivity and can be used for immune analysis and detection of prometryn. And determining the prometryn content in the sample to be detected according to the prometryn standard inhibition curve and the OD450nm value of the sample to be detected.
Example 7: prometryn immunodetection kit
The embodiment provides a prometryn immunodetection kit, which comprises the prometryn monoclonal antibody prepared in the embodiment 5, an ELISA plate, a prometryn coating antigen, a prometryn standard solution, an HRP-labeled goat anti-mouse IgG secondary antibody and a TMB color developing solution.
The prometryn immunodetection kit for detecting prometryn has the following principle: and detecting the content of prometryn in the sample to be detected by adopting an indirect competitive ELISA method. The prometryn envelope antigen is pre-enveloped in the micropore of the ELISA plate, prometryn standard solution or a sample to be detected, a prometryn monoclonal antibody, a goat anti-mouse IgG secondary antibody marked by HRP and TMB developing solution are added to prepare a prometryn standard inhibition curve, and the prometryn content in the sample to be detected is determined according to the prometryn standard inhibition curve and the absorbance value of the sample to be detected. The prometryn can be detected by adopting a method commonly used in the field for operation.
Example 8: prometryn detection colloidal gold test strip
The embodiment provides a colloidal gold test strip, and it includes sample pad, colloidal gold conjugate pad, nitrocellulose membrane and water absorption pad, be equipped with detection line and quality control line on the nitrocellulose membrane in proper order, the last parcel of colloidal gold conjugate pad has the prometryn monoclonal antibody of embodiment 5 preparation. The detection line is obtained by printing prometryn coating antigen. The quality control line is obtained by printing a goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled in a way commonly used in the field.
The principle of the prometryn detection colloidal gold test strip for detecting prometryn is as follows: and detecting whether the sample to be detected contains prometryn by using the principle of an indirect competition method. If the sample to be detected contains prometryn, the detection line is not colored, and the quality control line is colored. If the sample to be detected does not contain prometryn, the detection line and the quality control line are both colored. The prometryn can be detected by adopting a method commonly used in the field for operation.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A hybridoma cell strain CPZ secreting prometryn monoclonal antibody is characterized in that the preservation number is CGMCC NO. 45022.
2. A prometryn monoclonal antibody secreted and produced by the hybridoma cell line CPZ of claim 1.
3. The hybridoma cell strain CPZ of claim 1 or the prometryn monoclonal antibody of claim 2, and the application thereof in prometryn detection, the preparation of prometryn immunoassay kit or the preparation of prometryn detection colloidal gold test strip.
4. A prometryn immunoassay kit, which is characterized by comprising the hybridoma cell strain CPZ of claim 1 or the prometryn monoclonal antibody of claim 2.
5. The prometryn immunoassay kit of claim 4, wherein the prometryn immunoassay kit further comprises an ELISA plate, a prometryn coating antigen, a prometryn standard solution, an enzyme-labeled secondary antibody and a substrate reaction solution.
6. The prometryn immunoassay kit of claim 5, wherein the prometryn coating antigen has the structure shown as follows:
7. a prometryn detection colloidal gold test strip is characterized by comprising a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the prometryn monoclonal antibody of claim 2 is coated on the colloidal gold combined pad.
8. The prometryn detection colloidal gold test strip of claim 7, wherein the detection line is printed with prometryn coating antigen.
9. A prometryn detection colloidal gold test strip according to claim 8, wherein the prometryn coating antigen has the structure shown as follows:
10. use of the prometryn immunoassay kit of claim 4 or the prometryn detection colloidal gold test strip of claim 7 in prometryn detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114853687A (en) * | 2022-06-07 | 2022-08-05 | 中国农业科学院农业质量标准与检测技术研究所 | Prometryn hapten, complete antigen, antibody, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030064389A1 (en) * | 2001-05-18 | 2003-04-03 | Yasuhiro Goda | Method of selecting an antibody, a hybridoma, a monoclonal antibody and use thereof |
| CN105572341A (en) * | 2014-10-08 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Prometryn detection enzyme linked immunoassay kit |
| CN109868261A (en) * | 2017-12-04 | 2019-06-11 | 中国医学科学院药用植物研究所 | The preparation of mass selection monoclonal antibody and its application of anti-eight kinds of triazines |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030064389A1 (en) * | 2001-05-18 | 2003-04-03 | Yasuhiro Goda | Method of selecting an antibody, a hybridoma, a monoclonal antibody and use thereof |
| CN105572341A (en) * | 2014-10-08 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Prometryn detection enzyme linked immunoassay kit |
| CN109868261A (en) * | 2017-12-04 | 2019-06-11 | 中国医学科学院药用植物研究所 | The preparation of mass selection monoclonal antibody and its application of anti-eight kinds of triazines |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114853687A (en) * | 2022-06-07 | 2022-08-05 | 中国农业科学院农业质量标准与检测技术研究所 | Prometryn hapten, complete antigen, antibody, preparation method and application |
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