CN115261330B - Flutriafol artificial antigen, monoclonal antibody, hybridoma cell strain and application thereof - Google Patents
Flutriafol artificial antigen, monoclonal antibody, hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention provides an flutriafol artificial antigen, a monoclonal antibody, a hybridoma cell strain and application thereof, and belongs to the technical field of immunochemistry. The hybridoma cell lines have the preservation numbers of: cgmccno.45107. The invention synthesizes the flutriafol hapten, synthesizes the complete antigen by coupling the hapten and carrier protein, then mixes and emulsifies the complete antigen with equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multi-point injection at the neck and back. Fusing spleen cells of a high-titer and high-sensitivity mouse with myeloma cells of the mouse, and screening out hybrid cells after fusing the two cells by adopting a selection medium; and then screening cells by an indirect competitive ELISA method, and performing subcloning for a plurality of times to finally obtain a hybridoma cell. The monoclonal antibody secreted by the cell strain has better detection sensitivity (IC) to flutriafol 50 A value of 1.02 ng/mL) and specificity, can be used for detecting the residual of flutriafol.
Description
Technical Field
The invention relates to the technical field of pesticide residue immunodetection, in particular to a flutriafol artificial antigen, a monoclonal antibody, a hybridoma cell strain and application thereof.
Background
The English generic name of flutriafol is: flutriasol, CAS:76674-21-0, chemical name: (RS) -2,4' -difluoro-a- (1H-1, 2, 4-triazole-1-methyl) diphenylmethanol. The flutriafol is a triazole fungicide, has broad-spectrum bactericidal activity and strong systemic property, is conducted to the top in a plant body, and has the effects of protecting and treating diseases. Can be used for preventing and treating diseases of stem and leaf, ear and seed of cereal crops (mainly including wheat, barley, rye, corn, etc.), such as powdery mildew, rust disease, leaf spot, net blotch, smut, etc. Has special effect on powdery mildew of cereal, and especially has the function of removing spore piles of powdery mildew of wheat. The main action mechanism is that the ergosterol can be effectively inhibited from biosynthesis, the cell wall of fungi can be broken, and the ergosterol has good protection and treatment effects on a plurality of diseases caused by basidiomycetes and ascomycetes and has a certain fumigating effect. In order to improve the yield and quality of crops, pesticides are often required to be used, and the non-standard use of the pesticides leads pesticide residues in the crops to exceed standards, so that the health of the crops is endangered.
At present, the flutriafol detection method mainly comprises instrument detection, and commonly used methods include gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Although these chromatographic-based methods have high sensitivity and specificity, there are drawbacks such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Thus, there is a need for a rapid, simple method of detecting flutriafol residues.
The ELISA is a very efficient, sensitive and rapid detection method, and has the advantages of simple pretreatment of samples during detection, few purification steps, large analysis capacity, low detection cost and simple and convenient operation, and is suitable for the on-site rapid detection of a large number of samples, thus being widely applied to the analysis of drug residues. On the premise of detecting the flutriafol by using an enzyme-linked immunosorbent assay, a monoclonal antibody with high specificity and high sensitivity to the flutriafol is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the flutriafol is very critical. The inventor tries to prepare the flutriafol monoclonal antibody through hybridoma cells, but in the process of preparing a hybridoma cell strain capable of secreting the flutriafol monoclonal antibody, how to prepare flutriafol hapten and complete antigen and how to enable a mouse to generate a strong immune effect needs further research; how to enable the prepared hybridoma cell strain to successfully secrete the flutriafol monoclonal antibody is further researched; how to make the secreted flutriafol monoclonal antibody have strong specificity and high sensitivity, and further research is also needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides an flutriafol artificial antigen, a monoclonal antibody, a hybridoma cell strain and application thereof. The flutriafol monoclonal antibody secreted by the hybridoma cell strain has good detection sensitivity (IC) to flutriafol 50 The value is 1.02 ng/mL), can be used for establishing an immunological detection method of the flutriafol, and can be used for detecting the residual flutriafol in food.
The first object of the present invention is to provide a hybridoma cell strain secreting the flutriafol monoclonal antibody, wherein the hybridoma cell strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.45107 and the preservation address of North West road No. 1,3 in the Korean region of Beijing city, and the preservation date of 2022, 03 and 03 days.
The second object of the present invention is to provide a method for preparing the hybridoma cell strain secreting the flutriafol monoclonal antibody, comprising the following steps:
step 1: preparing flutriafol complete antigen by using flutriafol hapten, and emulsifying the obtained flutriafol complete antigen with Freund's adjuvant or incomplete Freund's adjuvant to prepare immunogen;
step 2: injecting the obtained immunogen into an animal body through subcutaneous multiple points of the neck and back for multiple times, wherein complete Freund's adjuvant is selected for the first immunization, and incomplete Freund's adjuvant is selected for the booster immunization;
step 3: the animals subjected to the immunization process are subjected to blood sampling, the immune titer and the immune suppression capacity of the serum of the animals are detected through indirect ELISA, and the animals with high sensitivity to flutriafol in the serum are screened out;
step 4: the screened animals are subjected to sprint immunity through intraperitoneal injection, wherein the sprint immunity adopts flutriafol complete antigen without Freund's adjuvant;
step 5: and (3) fusing spleen cells and myeloma cells of the animals subjected to the sprint immunization in the step (3) to obtain the hybridoma cell strain.
In one embodiment of the invention, the flutriafol hapten has the following molecular structure:
in one embodiment of the invention, the molecular structure of the flutriafol complete antigen is as follows:
in one embodiment of the invention, the method for preparing the flutriafol complete antigen by using the flutriafol hapten is as follows: dissolving flutriafol hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in an organic solvent, and mixing for reaction to obtain an activated flutriafol hapten solution, namely solution A; diluting the coupling protein with carbonate buffer solution to obtain solution B; and adding the solution A into the solution B for reaction to obtain the flutriafol complete antigen.
In one embodiment of the invention, the coupling protein is selected from bovine serum albumin, chicken serum albumin, human serum albumin, keyhole limpet hemocyanin or synthetic polylysine.
In one embodiment of the invention, the mole ratio of flutriafol hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1:1-5:1-5.
In one embodiment of the invention, the animal is selected from BALB/c mice.
In one embodiment of the present invention, the first immunization and the boost in the steps 2 and 4 are separated by one month, the boost is separated by 21 days, and the boost is separated by 18-21 days.
In one embodiment of the invention, the first immunization dose in steps 2 and 4 is 100 μg/dose, the booster immunization dose is 50 μg/dose, and the sprint immunization dose is 25 μg/dose.
In one embodiment of the invention, the immunization process in steps 2,4 comprises 1 first immunization, at least 4 booster immunizations, 1 sprint immunization;
in one embodiment of the present invention, the blood collection in the step 3 is performed on the 7 th day after the 3 rd immunization course is completed.
In one embodiment of the invention, the cell fusion in step 5 is performed 3 to 5 days after the termination of the sprint immunization.
In one embodiment of the present invention, the cell fusion in step 5 is performed by a polyethylene glycol (PEG 4000) method.
In one embodiment of the present invention, the medium in the step 5 is RPMI-1640 medium.
In one embodiment of the present invention, the number of subcloning in the step 5 is 4.
The third object of the invention is to provide an application of the hybridoma cell strain in preparing the flutriafol monoclonal antibody.
The fourth object of the present invention is to provide a flutriafol monoclonal antibody, which is secreted by the hybridoma cell strain with the preservation number of CGMCC No.45107.
The fifth object of the present invention is to provide a preparation method of flutriafol monoclonal antibody, comprising the following steps: taking BALB/c mice, injecting paraffin oil into the abdominal cavity, injecting hybridoma cell strain with the preservation number of CGMCC No.45107 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained monoclonal antibody at low temperature.
In one embodiment of the present invention, the preparation method of the flutriafol monoclonal antibody comprises the following specific steps: the method comprises taking 8-10 week-old BALB/c mice, injecting paraffin oil into abdominal cavity of each mouse 1mL, and injecting paraffin oil into abdominal cavity of each mouse 1×10 after 7 days 6 Collecting ascites from 7 th day of hybridoma cell strain with preservation number of CGMCC No.45107, purifying the ascites by octanoic acid-ammonium sulfate method, and preserving the obtained monoclonal antibody at-20deg.C.
The sixth object of the present invention is to provide a composition comprising said flutriafol monoclonal antibody.
The seventh object of the present invention is to provide a kit comprising the flutriafol monoclonal antibody or the composition.
The eighth object of the invention is to provide the application of the flutriafol monoclonal antibody, the composition or the kit in the identification of flutriafol.
The hybridoma cell strains are classified and named as monoclonal cell strains.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) The flutriafol monoclonal antibody obtained by the invention is p-flutriafolHas better detection sensitivity (IC) 50 A value of 1.02 ng/mL);
(2) The flutriafol monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIG. 1 is a standard curve of inhibition of flutriafol by the flutriafol monoclonal antibodies of the invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The following examples relate to the following media:
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, D-calcium 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, sodium bicarbonate 2000.
The reagents involved in the following examples were as follows:
carbonate buffer: weighing Na 2 CO 3 1.59 g，NaHCO 3 2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, mixing uniformly, adjusting the pH value to 9.6, adding the double distilled water to 1000mL, and storing at 4 ℃ for later use.
Phosphate buffer: 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9gNa 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
phosphate buffer T: phosphate buffer containing 0.05% tween 20;
antibody dilution: phosphate buffer containing 0.1% gelatin;
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4. 12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The solution B is prepared from the following components in percentage by weight: 1 are mixed to obtain TMB color development liquid, and are mixed when in use.
The detection method involved in the following examples is as follows:
the method for detecting the inhibition rate of flutriafol comprises the following steps: the most appropriate 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 μg/mL with carbonate buffer and the antibody diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the optimal working point, the flutriafol standard is diluted to 8 concentrations (0,0.037,0.111,0.333,1,3,9 and 27 ng/mL), and the flutriafol standard inhibition curve is obtained by plotting OrigingPro 8.5 (the result is shown in FIG. 1) according to the IC-ELISA procedure, and IC is calculated 50 。
The method for measuring the flutriafol cross reaction comprises the following steps: antigen was diluted to 0.01,0.03,0.1 and 0.3 μg/mL with carbonate buffer and antibody diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution, measured by ic-ELISA, standard solutions of different structural analogs were added and the crossover rate of the prepared flutriafol monoclonal antibodies to the structural analogs was measured (results are shown in Table 1).
Example 1: synthesis of flutriafol hapten
Because the flutriafol small molecules have no immunogenicity, the mice cannot be stimulated to generate immune responses, and then antibodies are generated, the flutriafol is coupled to proteins through a protein connection technology, so that the flutriafol is immunogenic; the commonly used active groups in the protein coupling technology are amino, carboxyl, hydroxyl, sulfhydryl and the like, and the flutriafol is derived in view of the fact that the flutriafol molecular structural formula does not contain the active groups.
200mg of flutriafol is dissolved in 5mL of anhydrous pyridine, 80mg of succinic anhydride is added, the solution is stirred, and 10mg of 4-dimethylaminopyridine is added. After sealing, stirring for 72h at 100 ℃, and blowing the reactant by nitrogen. 5mL of water was added and the pH was adjusted to 3 with 6mol/L HCl solution. Additional 5mL of dichloromethane were added to extract 3 times and the organic phase was collected. Drying the extracting solution by anhydrous sodium sulfate, concentrating under reduced pressure to obtain a small amount of yellow viscous waxy solid, and drying to obtain the flutriafol hapten.
Example 2: synthesis of flutriafol immunogen
9.2mg of flutriafol hapten and 4.4mg of N-hydroxysuccinimide are weighed and dissolved in 200 mu L N and N-dimethylformamide, and the mixture is stirred and reacted for 10min at room temperature; then 8.4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and added into the flutriafol hapten solution, and the mixture is stirred at room temperature for reaction for 6-8 hours for activation. 6mg of keyhole limpet hemocyanin was taken and added to 3mL of 0.01M carbonate buffer, and the mixture was dissolved well, and the activated hapten was slowly added to the KLH-dissolved diluent and stirred overnight at room temperature. Then, the solution is dialyzed by 0.01M phosphate buffer solution to remove unreacted micromolecular substances, thus obtaining purer complete antigen and being identified by an ultraviolet absorption scanning method.
Example 3: synthesis of flutriafol coating
Dissolving 4.5mg of flutriafol hapten and 3.1mg of N-hydroxysuccinimide in 200 mu L N, N-dimethylformamide, and stirring at room temperature for reaction for 10min; dissolving 4.3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the solution, stirring at room temperature for reaction for 6-8 hours to obtain hapten activating solution; dissolving 6mg bovine serum albumin in carbonate buffer; the hapten-activated solution was slowly added to the protein dilution and stirred overnight at room temperature. Then, the reaction solution was dialyzed against 0.01M phosphate buffer solution to remove unreacted small molecular substances, thereby obtaining a coating antigen.
Example 4: preparation of hybridoma cell strain secreting flutriafol monoclonal antibody
1. Acquisition of animal immunity
Mixing and emulsifying flutriafol complete antigen and equivalent Freund's adjuvant, and performing subcutaneous multipoint injection immunization (except sprint immunization) on the back of the neck of a BALB/c mouse; the first immunization is carried out by using complete Freund's adjuvant, and the dosage is 100 mug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 μg/dose; the sprint immunity does not need adjuvant, and the dosage is halved to 25 mug/patient after the sprint immunity is directly diluted by normal saline for intraperitoneal injection; one month is separated from the first immunization and the second immunization, 21 days is separated from the multiple times of the immunization, and 18-21 days is separated from the sprint immunization and the last immunization; observing the immune effect of the mice by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), namely detecting the titer and inhibition of the serum of the mice;
2. cell fusion
Three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
a. immediately placing the mice after killing the mice by a cervical dislocation method into 75% alcohol for disinfection, soaking for about 5min, taking out spleens of the mice by aseptic operation, moderately grinding the spleens by a rubber head of a syringe, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells for three times by using RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifuging, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in 10% fetal bovine serum RPMI-1640 medium in 5% CO 2 Amplifying in incubator to reach SP2/0 tumor cell number of 1-4×10 before fusion 7 Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion, collecting tumor cells during fusion, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. fusion process 7min: 1min, 1mL of PEG4000 was added dropwise to the cells from slow to fast; standing for 2 min; dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 was added dropwise every 10s for cultureA base. The solution was continuously shaken for other times than 2 min. Then carrying out warm bath at 37 ℃ for 5min; centrifuging (800 rpm,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200 μl/well to 96-well cell plate, and standing at 37deg.C and 5% CO 2 Culturing in an incubator.
3. Cell screening and cell strain establishment
The cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 after cell fusion, full-replacement with a 100 XHT RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection.
Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ic-ELISA method, and the second step is to select flutriafol as a standard substance, and to measure the inhibition effect of positive cells by using the ic-ELISA method.
Selecting cell holes with better inhibition on the flutriafol standard, subcloning by adopting a limiting dilution method, and detecting by using the same method after seven days.
And (3) performing subcloning at least three times according to the method to finally obtain the flutriafol monoclonal antibody cell strain.
Example 5: preparation and identification of flutriafol monoclonal antibody
Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 The flutriafol hybridoma cells collect ascites from the seventh day, and antibody purification is carried out on the ascites by an octanoic acid-saturated ammonium sulfate method.
Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating monoclonal antibody of IgG type with ammonium sulfate solution of equal saturation, centrifuging, discarding supernatant, dissolving with 0.01M phosphate buffer solution (pH 7.4), dialyzing for desalting, and storing at-20deg.C.
Determination of IC of flutriafol monoclonal antibodies using an indirect competition ELISA 50 The value is 1.02ng/mL, which shows that the flutriafol-containing high sensitivity can be used for flutriafolAnd (5) performing immunoassay detection.
Example 6: application of flutriafol monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain through in vivo ascites is applied to ELISA (enzyme-linked immunosorbent assay) additive recovery test of flutriafol, and the specific steps are as follows:
(1) Coating 96-well ELISA plates with coating raw materials diluted by carbonate buffer solution and having the concentration of 0.1 mug/mL, wherein each well is 100 mug, baking for 2 hours at 37 ℃, washing the plates with phosphate buffer solution T for three times, each well is 200 mug, each time is 3min, and drying;
(2) Blocking with carbonate buffer solution containing 0.2% gelatin, baking at 37deg.C for 2 hr, washing the plate with phosphate buffer solution T three times, 200 μl each time, 3min each time, and drying;
(3) Preparing 0,0.037,0.111,0.333,1,3,9 and 27ng/mL of flutriafol standard solution by using phosphate buffer (phosphate buffer), respectively adding the standard solution and the sample extracting solution to be detected into a sealed ELISA plate, repeating 3 holes for each sample by 50 mu L per hole, adding 50 mu L of flutriafol monoclonal antibody diluted to 0.1 mu g/mL per hole, reacting for 30min at 37 ℃, washing the plate, and beating to dryness;
(4) 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with phosphate buffer containing 0.1% gelatin was added to each well, reacted at 37deg.C for 30min, and then washed with plates and patted dry;
(5) 100. Mu.L of TMB developing solution was added to each well, and after developing at 37℃for 15min, 50. Mu.L of 2M H was added to each well 2 SO 4 Stop solution, measuring light absorption value at 450 nm;
(6) And (3) adding and recycling and sample pretreatment:
wheat was selected as the test sample.
Pulverizing sample to be tested, sieving with 60 mesh standard sieve, weighing three samples, 1g each, adding 5ppb, 10ppb, 50ppb flutriafol standard (according to antibody linear range and IC) 50 Set the addition concentration), adding 2.5mL of water, mixing uniformly by vortex, standing for 30min, and filtering. Adding 2.5mL of acetone into the sample, shaking on an electric shaker for 30min, filtering with quick qualitative filter paper in a beaker, and re-using 2.5m of residueL acetone was extracted once as above, the residue was washed twice with 2.5mL of acetone, the wash solution was incorporated into a beaker, concentrated to near dryness on a 50℃water bath, and reconstituted with 5mL of 10% acetone phosphate buffer solution (i.e., five times diluted to reduce the effect of sample matrix).
The recovery of the additives was 97.4%,92.7% and 105.7% respectively by indirect competition ELISA.
Example 7 method for measuring flutriafol cross-reaction
TABLE 1 Cross-reaction of flutriafol monoclonal antibodies to structural analogues
As can be seen from Table 1, the monoclonal antibody obtained in the present invention inhibits flutriafol alone, IC 50 The value is 1.02ng/mL, the crossing rate of the monoclonal antibody to the analogue is less than 5%, and the monoclonal antibody has high sensitivity and specificity.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (7)
1. The hybridoma cell strain secreting the flutriafol monoclonal antibody is characterized by being preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45107 and the preservation address of North Xiyi Hirscho No. 1 in the Korean region of Beijing city of 3, and the preservation date of 2022, 03 and 03 days.
2. The use of the hybridoma cell line of claim 1 for preparing flutriafol monoclonal antibodies.
3. The flutriafol monoclonal antibody is secreted by the hybridoma cell strain with the preservation number of CGMCC No.45107 as claimed in claim 1.
4. The preparation method of the flutriafol monoclonal antibody is characterized by comprising the following steps of: injecting paraffin oil into the abdominal cavity of an animal, injecting the hybridoma cell strain in claim 1 into the abdominal cavity, collecting ascites after injection, and purifying the ascites to obtain the monoclonal antibody.
5. A composition comprising the flutter monoclonal antibody of claim 3.
6. A kit comprising the flutter monoclonal antibody of claim 3 or the composition of claim 5.
7. Use of the tebuconazole monoclonal antibody of claim 3, the composition of claim 5 or the kit of claim 6 for identifying tebuconazole.
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| CN105572348A (en) * | 2015-12-23 | 2016-05-11 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit |
| CN108998422A (en) * | 2018-08-14 | 2018-12-14 | 江南大学 | It is a kind of secrete Diacloden monoclonal antibody hybridoma cell strain and its application |
| CN113717950A (en) * | 2021-09-29 | 2021-11-30 | 江南大学 | Hybridoma cell strain secreting penconazole monoclonal antibody and application thereof |
| CN114317451A (en) * | 2022-01-18 | 2022-04-12 | 江南大学 | Hybridoma cell strain secreting diuron monoclonal antibody and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105572348A (en) * | 2015-12-23 | 2016-05-11 | 中国烟草总公司郑州烟草研究院 | Enzyme linked immunosorbent assay kit for detecting triadimenol and application of enzyme linked immunosorbent assay kit |
| CN108998422A (en) * | 2018-08-14 | 2018-12-14 | 江南大学 | It is a kind of secrete Diacloden monoclonal antibody hybridoma cell strain and its application |
| CN113717950A (en) * | 2021-09-29 | 2021-11-30 | 江南大学 | Hybridoma cell strain secreting penconazole monoclonal antibody and application thereof |
| CN114317451A (en) * | 2022-01-18 | 2022-04-12 | 江南大学 | Hybridoma cell strain secreting diuron monoclonal antibody and preparation method and application thereof |
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