CN111304174A - Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof - Google Patents
Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof Download PDFInfo
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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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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- 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
Abstract
A triazolone monoclonal antibody hybridoma cell strain B11S and application thereof belong to the technical field of food safety immunodetection. The triazolone monoclonal antibody hybridoma cell strain B11S of the invention has been preserved in China general microbiological culture Collection center (CGMCC), is named as a monoclonal cell strain in classification, and is preserved for 14 months and 10 months in 2019The serial number is CGMCC No. 18518. BALB/c mice were immunized with the triazolone complete antigen. High potency, low IC50And fusing the mouse spleen cells with myeloma cells by a PEG method, and screening and carrying out third subcloning by an indirect competitive enzyme-linked immunosorbent assay to obtain a hybridoma cell strain B11S. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) on triazolone50The value is 5.25 ng/mL), provides raw materials for immunodetection of triazolone residues in food, and has practical application value.
Description
Technical Field
The invention relates to a triazolone monoclonal antibody hybridoma cell strain B11S and application thereof, and belongs to the technical field of food safety immunodetection.
Background
Triazolone (TDF) is an efficient broad-spectrum azole bactericide, has effects of preventing, treating and eradicating, and has strong systemic effect (can be conducted up and down). Has special effects on powdery mildew, rust disease and smut, and has the bactericidal effect of inhibiting the biosynthesis of ergosterol. Is mainly used for preventing and treating diseases of crops such as wheat, fruit trees, vegetables, melons, flowers and the like.
The maximum residual limit standards (MRL) of triazolone in various foods are specified in GB 2763-2016 (maximum residual limit of pesticide in food), wherein the maximum residual limit standards (MRL) in wheat (except for wheat) and dry grain (except for corn) are respectively 0.2mg/kg, the maximum residual limit standards (MRL) in solanaceous vegetables are respectively 1mg/kg, and the maximum residual limit standards (MRL) in citrus, apple and banana are respectively 1 mg/kg.
In order to effectively monitor the use condition of triadimefon in food, a determination method with good specificity and high sensitivity is needed, but the prior detection methods such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry and the like have complex sample pretreatment process and high cost, and more interferents exist in food, so the instrumental method is not suitable for field detection. Therefore, the establishment of a rapid and portable triazolone detection method has important significance. The enzyme-linked immunosorbent assay (ELISA) is a low-cost, rapid and portable immunological detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for rapidly detecting results on the spot of a large number of samples. Establishing an efficient immunological detection method, and screening a monoclonal antibody with high specificity is an important prerequisite.
Disclosure of Invention
The invention aims to provide a triazolone monoclonal antibody hybridoma cell strain B11S and application thereof, and the antibody prepared by the cell strain has higher detection sensitivity on triazolone and can be used for establishing an immunological detection method of triazolone.
The technical scheme of the invention is that a triazolone monoclonal antibody hybridoma cell strain B11S is deposited in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms, and the institute of microbiology, China academy of sciences, No. 3, Xilu No.1, Beijing, the morning area, is classified and named as a monoclonal cell strain, the preservation date is 2019, 10 months and 14 days, and the preservation number is CGMCC No. 18518.
The triazolone monoclonal antibody is secreted and generated by the triazolone monoclonal antibody hybridoma cell strain B11S with the preservation number of CGMCC No. 18518.
The preparation of the triazolone monoclonal antibody hybridoma cell strain B11S provided by the invention comprises the following basic steps:
(1) synthesis of hapten:
the synthetic scheme for the triazolone hapten is shown in the formula above. The derivation procedure is briefly as follows: dissolving sodium chloroacetate and anhydrous sodium carbonate in 2mL of pure water to obtain a solution A, and dissolving triadimenol which is a structural analogue of triadimefon in 95% ethanol with mass concentration to obtain a solution B. And slowly adding the solution A into the solution B, and stirring at room temperature for 10 hours to obtain solution C. The reaction was filtered and the filtrate evaporated to dryness in a water bath at 80 ℃. The precipitate TDF was then stored at 4 ℃ for use.
(2) Preparation of complete antigen:
preparation of immunogen TDF-BSA: 6.3mg of TDF, 10.3mg of 1-ethylcarbodiimide hydrochloride and 6.2mg of N-hydroxysuccinimide were weighed, dissolved in 400. mu.L of anhydrous N, N-dimethylformamide (referred to as solution A), and the reaction was stirred at room temperature for 6 hours. Taking 10mg of bovine serum albumin BSA, adding 3mL of boric acid buffer solution (called B solution), slowly adding the A solution into the B solution at room temperature, stirring at room temperature for overnight reaction to obtain a conjugate TDF-BSA mixed solution, and separating a complete antigen and unconjugated micromolecules through dialysis;
preparation of original TDF-OVA: 2.4mg of TDF, 3.9mg of 1-ethylcarbodiimide hydrochloride and 2.4mg of N-hydroxysuccinimide were weighed, dissolved in 300. mu.L of anhydrous N, N-dimethylformamide (referred to as "solution C"), and the reaction was stirred at room temperature for 6 hours. Weighing 10mg of chicken ovalbumin OVA, dissolving the OVA in 2mL of boric acid buffer solution (called D solution), dropwise adding the C solution into the D solution at room temperature, stirring at room temperature for reacting overnight to obtain a conjugate TDF-OVA mixed solution, and separating complete antigen and unconjugated micromolecules through dialysis.
(3) Immunization of mice: the TDF-BSA complete antigen was mixed with an equal volume of Freund's adjuvant and emulsified, and then injected subcutaneously into BALB/c mice through the back of the neck. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for multiple booster immunizations. The interval between the first immunization and the second boosting immunization is one month, and the interval between the boosting immunization is 21 days. The final immunization with TDF-BSA complete antigen (without adjuvant) prick; the serum titer and inhibition rate were measured by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
(4) Cell fusion and cell line establishment: fusing mouse spleen cells and myeloma cells by a PEG method, selectively culturing by HAT culture medium, detecting the positive and inhibition rates of cell holes by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), carrying out three times of subcloning on the positive cell holes with higher positive and inhibition rates by a limiting dilution method, and finally screening to obtain the triazolone monoclonal antibody hybridoma cell strain B11S.
(5) And (3) identification of the properties of hybridoma cell strains: the sensitivity was determined by ic-ELISA.
Get high-efficient low IC50The spleen cells of the mice are fused with myeloma cells of the mice by a PEG method, and a hybridoma cell strain is obtained by screening and three times of subcloning by an indirect competitive enzyme-linked immunosorbent assay.
The invention has the beneficial effects that: the monoclonal antibody secreted by the cell strain B11S has better specificity and detection sensitivity (IC) on triazolone50The value is 5.25 ng/mL), can realize the detection of the residual quantity of the triazolone in fruits, vegetables and grains, provides raw materials for the immunodetection of the residual quantity of the triazolone in food, and has practical application value.
Biological material sample preservation: a triazolone monoclonal antibody hybridoma cell strain B11S, which is preserved in China general microbiological culture Collection center, and has the following address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, is classified and named as monoclonal cell strain, the preservation date is 2019, 10 months and 14 days, and the preservation number is CGMCC No. 18518.
Drawings
FIG. 1B 11S standard curve for inhibition of triazolone by monoclonal antibody.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
According to the invention, a mice is immunized by the triazolone complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by ic-ELISA, so that the monoclonal antibody hybridoma cell strain with high sensitivity to triazolone is finally obtained.
Example 1 preparation of a triazolone monoclonal antibody hybridoma cell strain B11S
(1) Synthesis of hapten:
the synthetic scheme for the triazolone hapten is shown above. The derivation procedure is briefly as follows: sodium chloroacetate and anhydrous sodium carbonate were dissolved in 2mL of pure water to give a solution A, and triadimenol, a structural analogue of triadimefon, was dissolved in 95% ethanol to give a solution B. And slowly adding the solution A into the solution B, and stirring at room temperature for 10 hours to obtain solution C. The reaction was filtered and the filtrate evaporated to dryness in a water bath at 80 ℃. The precipitate TDF was then stored at 4 ℃ for use.
(2) Preparation of complete antigen:
preparation of immunogen TDF-BSA: 6.3mg of TDF, 10.3mg of 1-ethylcarbodiimide hydrochloride and 6.2mg of N-hydroxysuccinimide were weighed, dissolved in 400. mu.L of anhydrous N, N-dimethylformamide (referred to as solution A), and the reaction was stirred at room temperature for 6 hours. Taking 10mg of bovine serum albumin BSA, adding 3mL of boric acid buffer solution (called B solution), slowly adding the A solution into the B solution at room temperature, stirring at room temperature for overnight reaction to obtain a conjugate TDF-BSA mixed solution, and separating a complete antigen and unconjugated micromolecules through dialysis;
preparation of original TDF-OVA: 2.4mg of TDF, 3.9mg of 1-ethylcarbodiimide hydrochloride and 2.4mg of N-hydroxysuccinimide were weighed, dissolved in 300. mu.L of anhydrous N, N-dimethylformamide (referred to as "solution C"), and the reaction was stirred at room temperature for 6 hours. Weighing 10mg of chicken ovalbumin OVA, dissolving the OVA in 2mL of boric acid buffer solution (called D solution), dropwise adding the C solution into the D solution at room temperature, stirring at room temperature for reacting overnight to obtain a conjugate TDF-OVA mixed solution, and separating complete antigen and unconjugated micromolecules through dialysis.
(3) Immunization of mice: the TDF-BSA complete antigen was mixed with an equal volume of Freund's adjuvant and emulsified, and then injected subcutaneously into BALB/c mice through the back of the neck. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for multiple booster immunizations. The interval between the first immunization and the second boosting immunization is one month, and the interval between the boosting immunization is 21 days. The final immunization with TDF-BSA complete antigen (without adjuvant) prick; the serum titer and inhibition rate were measured by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA).
(4) Cell fusion: after three days of spurting immunization, cell fusion is carried out according to a conventional PEG method, and the specific steps are as follows:
a. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleens of the mice through aseptic operation, properly grinding the spleens by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a splenocytes suspension, collecting, centrifuging (1200 rpm, 8 min), washing the splenocytes for three times by using an RPMI-1640 culture medium, diluting the splenocytes 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% CO2An incubator. Before fusion, the number of SP2/0 tumor cells is required to reach 1-4 multiplied by 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7 min. 1min, 1mL of PEG 1500 is added to the cells from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; dripping 2mLRPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifuging (800 rpm, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C and 5% CO2Culturing in an incubator.
(5) Cell screening and cell strain 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 for screening. The screening is divided into two steps: firstly, screening out positive cell holes by using ic-ELISA, secondly, selecting triazolone as a standard substance, and measuring the inhibition effect of the positive cells by using ic-ELISA. And selecting cell wells with good inhibition to the triazolone standard, subcloning by a limiting dilution method, and detecting by the same method. This was repeated three times to obtain cell line B11S.
(6) Preparation and identification of 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 106Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-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 (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
a. Coating: diluting the coated original TDF-OVA by using 0.05M carbonate buffer solution with pH9.6 from 1 mug/mL to 100 mug/hole in a multiple ratio, and reacting for 2h at 37 ℃;
b. washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
c. and (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. Drying for later use after washing;
d. sample adding: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
e. color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
f. termination and measurement: the reaction was stopped by adding 50. mu.L of a stop solution to each well, and the OD of each well was measured by a microplate reader450The value is obtained.
Determination of IC of monoclonal antibody triazolone by IC-ELISA50The concentration is 5.25 ng/mL, which shows that the kit has good sensitivity to triazolone and can be used for immunoassay detection of triazolone.
Solution preparation: carbonate Buffer (CBS): weighing Na2CO31.59 g，NaHCO32.93 g 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 Na2HPO4·12 H2Dissolving 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;
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. Mixing the liquid B according to the volume ratio of 1:5 to obtain TMB, and mixing the liquid B at the present time.
Claims (5)
1. A triazolone monoclonal antibody hybridoma cell strain B11S is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences (China institute of microbiology, institute of sciences, No. 3, west Lu 1, north Cheng, south China, Beijing City, south China, and has a preservation date of 2019, 10 months and 14 days and a preservation number of CGMCC No. 18518.
2. A triazolone monoclonal antibody characterized by: it is secreted and produced by triazolone monoclonal antibody hybridoma cell strain B11S with the preservation number of CGMCC No.18518 as claimed in claim 1.
3. The synthesis of triazolone hapten is characterized by comprising the following steps: dissolving sodium chloroacetate and anhydrous sodium carbonate in 2mL of pure water to obtain a solution A, and dissolving triadimefon analogue triadimenol in 95% ethanol to obtain a solution B; slowly adding the solution A into the solution B, and stirring at room temperature for 10h to obtain solution C; the reaction was filtered, the filtrate evaporated to dryness in a water bath at 80 ℃ and the precipitate TDF was stored for use at 4 ℃.
4. The synthesis of triazolone complete antigen is characterized by comprising the following steps:
(1) preparation of immunogen TDF-BSA: weighing 6.3mg of TDF, 10.3mg of 1-ethyl carbodiimide hydrochloride and 6.2mg of N-hydroxysuccinimide, dissolving the TDF, 1-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide in 400 mu L of anhydrous N, N-dimethylformamide, and stirring the mixture at room temperature for reacting for 6 hours to obtain solution A; taking 10mg of bovine serum albumin BSA, adding 3mL of boric acid buffer solution, namely B solution, slowly adding the A solution into the B solution at room temperature, stirring at room temperature for overnight reaction to obtain a conjugate TDF-BSA mixed solution, and separating a complete antigen and unconjugated micromolecules through dialysis;
(2) preparation of original TDF-OVA: weighing 2.4mg of TDF, 3.9mg of 1-ethyl carbodiimide hydrochloride and 2.4mg of N-hydroxysuccinimide, dissolving the TDF, 1-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide in 300 mu L of anhydrous N, N-dimethylformamide, and stirring the mixture at room temperature for reacting for 6 hours to obtain solution C; weighing 10mg of chicken ovalbumin OVA, and dissolving in 2mL of boric acid buffer solution to obtain solution D; and (3) dropwise adding the solution C into the solution D at room temperature, stirring at room temperature for overnight reaction to obtain a conjugate TDF-OVA mixed solution, and separating a complete antigen and unconjugated micromolecules by dialysis.
5. Use of the triazolone monoclonal antibody according to claim 2, characterized in that: the method is used for analyzing and detecting the residual triazolone in food safety detection.
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| CN112266901A (en) * | 2020-10-26 | 2021-01-26 | 江南大学 | Azoxystrobin monoclonal antibody hybridoma cell strain and application thereof |
| CN112280745A (en) * | 2020-10-26 | 2021-01-29 | 江南大学 | Hybridoma cell strain capable of secreting pyridaben monoclonal antibody and application thereof |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
| CN112574957A (en) * | 2020-12-29 | 2021-03-30 | 江南大学 | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof |
| CN112778420A (en) * | 2020-11-26 | 2021-05-11 | 苏州诚检生物科技有限公司 | Pyridaben monoclonal antibody and application thereof |
| CN113717949A (en) * | 2021-09-22 | 2021-11-30 | 江南大学 | Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof |
| CN114480296A (en) * | 2022-01-28 | 2022-05-13 | 中国农业科学院农业质量标准与检测技术研究所 | Hybridoma cell strain, monoclonal antibody, detection kit and detection method |
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| CN112280745A (en) * | 2020-10-26 | 2021-01-29 | 江南大学 | Hybridoma cell strain capable of secreting pyridaben monoclonal antibody and application thereof |
| CN112266901A (en) * | 2020-10-26 | 2021-01-26 | 江南大学 | Azoxystrobin monoclonal antibody hybridoma cell strain and application thereof |
| CN112778420A (en) * | 2020-11-26 | 2021-05-11 | 苏州诚检生物科技有限公司 | Pyridaben monoclonal antibody and application thereof |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
| CN112574957A (en) * | 2020-12-29 | 2021-03-30 | 江南大学 | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof |
| CN112574957B (en) * | 2020-12-29 | 2022-09-27 | 江南大学 | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof |
| CN113717949A (en) * | 2021-09-22 | 2021-11-30 | 江南大学 | Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof |
| CN113717949B (en) * | 2021-09-22 | 2023-06-30 | 江南大学 | Hybridoma cell strain secreting ketoconazole monoclonal antibody and application thereof |
| CN114480296A (en) * | 2022-01-28 | 2022-05-13 | 中国农业科学院农业质量标准与检测技术研究所 | Hybridoma cell strain, monoclonal antibody, detection kit and detection method |
| CN114480296B (en) * | 2022-01-28 | 2023-09-15 | 北京壹拾智检生物科技有限公司 | Hybridoma cell strain, monoclonal antibody, detection kit and detection method |
| CN114957141A (en) * | 2022-06-20 | 2022-08-30 | 云南省烟草质量监督检测站 | Hapten for detecting content of triadimenol and application thereof |
| CN114957141B (en) * | 2022-06-20 | 2023-08-29 | 云南省烟草质量监督检测站 | Hapten for detecting triadimenol content and application thereof |
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