CN114277000B - Hybridoma cell strain secreting isoprothiolane monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting isoprothiolane monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain secreting a isoprothiolane monoclonal antibody and application thereof, and belongs to the technical field of immunochemistry. The invention mixes and emulsifies the complete antigen of the isoprothiolane and equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multi-point injection on the neck and back. The first immunization (100. Mu.g/dose) was emulsified with complete Freund's adjuvant, multiple booster immunization with incomplete Freund's adjuvant, and the last immunization with blasticidin complete antigen sprint (intraperitoneal injection). 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 screening cells by an indirect competitive ELISA method, and performing subcloning for multiple times to finally obtain a monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity to the isoprothiolane, and can be used for detecting the residue of the isoprothiolane.
Description
Technical Field
The invention belongs to the technical field of food safety immunodetection, and particularly relates to a hybridoma cell strain secreting a isoprothiolane monoclonal antibody and application thereof.
Background
The English generic name of isoprothiolane is: isoprothiolane, chemical name: diisopropyl 1, 3-dithiolan-2-ylidene malonate; mechanism of action: after the rice plants absorb, the phospholipid N-methyltransferase is inhibited, so that the formation of fatty acid, glycerol and phospholipids is blocked. The main application is as follows: the product is a systemic bactericide, has special effect on neck blast of rice, is effective on rice seedling blast and micrococcus sclerotinia, and can be used for treating rice planthoppers and sogatella furcifera in a large area. The composition is mainly used for preventing and treating rice blast, is effective for both panicle rice blast and She Dao blast, and has better effect on panicle rice blast.
At present, the isoprothiolane 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. Therefore, a rapid and simple method for detecting isoprothiolane residues is needed.
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 isoprothiolane by using an enzyme-linked immunosorbent assay, the monoclonal antibody with high specificity and high sensitivity to the isoprothiolane is obtained, so that the method for preparing the monoclonal antibody with high specificity and high sensitivity to the isoprothiolane is very critical. The inventor tries to prepare the isoprothiolane monoclonal antibody through the hybridoma cells, but in the process of preparing the hybridoma cell strain capable of secreting the isoprothiolane monoclonal antibody, how to prepare the isoprothiolane hapten and the complete antigen and how to make mice generate strong immune effect needs further research; how to successfully secrete the isoprothiolane monoclonal antibody from the prepared hybridoma cell strain is further required to be studied; how to make the secreted isoprothiolane 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 a hybridoma cell strain secreting a isoprothiolane monoclonal antibody and application thereof.
A hybridoma cell strain secreting a isoprothiolane monoclonal antibody, wherein the hybridoma cell strain is preserved in a China general microbiological culture Collection center (CGMCC) No.22339 monoclonal cell strain, the preservation date is 2021, 05 months and 13 days, and the preservation address is North Chen Xi Lu 1,3 in the Chaoyang region of Beijing city.
The preparation method of the hybridoma cell strain secreting the isoprothiolane monoclonal antibody comprises the following steps:
(1) Preparing a isoprothiolane hapten and a isoprothiolane complete antigen, emulsifying the obtained isoprothiolane complete antigen and a complete Freund adjuvant to obtain an immunogen 1, and emulsifying the isoprothiolane complete antigen and an incomplete Freund adjuvant to obtain an immunogen 2;
(2) Performing first subcutaneous immunization on the mice by using the immunogen 1 obtained in the step (1), performing booster immunization by using the immunogen 2, collecting blood of the mice subjected to the immunization process, detecting serum immune titer and immunosuppressive ability of the mice by indirect ELISA, screening the mice with high sensitivity of the isoprothiolane antibodies in the serum, and performing sprint immunization by using the isoprothiolane complete antigen;
(3) Fusing spleen cells and myeloma cells of the mice subjected to sprint immunization in the step (2), screening and culturing the fused cells by using a HAT culture medium, detecting positive cell holes by using an indirect ELISA (enzyme-linked immunosorbent assay), further measuring the inhibition effect of the positive cell holes by using an indirect competition ELISA method, subcloning the positive cell holes with the best inhibition by using a limiting dilution method, and finally screening out hybridoma cell strains capable of secreting high-sensitivity isoprothiolane monoclonal antibodies.
In one embodiment of the present invention, in step (1), the isoprothiolane hapten formula:
in one embodiment of the present invention, in step (1), the isoprothiolane complete antigen has the formula:
in one embodiment of the invention, the isoprothiolane complete antigen is prepared by the following method: dissolving the isoprothiolane hapten, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an organic solvent, stirring and reacting to obtain an activated isoprothiolane hapten solution, adding the isoprothiolane hapten solution into a keyhole limpet hemocyanin solution, and mixing and reacting to obtain the isoprothiolane complete antigen.
In one embodiment of the invention, the organic solvent is MDF.
In one embodiment of the present invention, in step (2), the first immunization is separated from the boost by one month, the boost is separated from the boost by 21 days, and the boost is separated from the sprint by 18-21 days.
In one embodiment of the invention, in step (2), the first immunization dose 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, step (2) comprises 1 first immunization, 4 booster immunizations, and 1 sprint immunization.
In one embodiment of the invention, in step (3), cell fusion is performed 3 days after the termination of sprint immunization.
In one embodiment of the invention, the fusion in step (3) is performed by the polyethylene glycol (PEG 4000) method.
In one embodiment of the invention, in step (3), the number of subclones is 4.
The hybridoma cell strain is applied to preparation of the isoprothiolane monoclonal antibody.
A monoclonal antibody of isoprothiolane is secreted by a hybridoma cell strain with a preservation number of CGMCC No. 22339.
The invention provides application of the hybridoma cell strain secreting the isoprothiolane monoclonal antibody or the preparation method of the hybridoma cell strain secreting the isoprothiolane monoclonal antibody in preparation of the isoprothiolane monoclonal antibody.
The invention provides a preparation method of a isoprothiolane monoclonal antibody, which comprises the steps of taking a BALB/c mouse, injecting paraffin oil into the abdominal cavity, injecting hybridoma cell strain with the preservation number of CGMCC No22339 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 method is to take 8-10 week old BALB/c mice, each mouse was intraperitoneally injected with 1mL of paraffin oil, and each mouse was intraperitoneally injected with 1X 10 after 7 days 6 Collecting ascites from 7 th day of hybridoma cell strain with preservation number of CGMCC No.22339, purifying the ascites by octanoic acid-ammonium sulfate method, and preserving the obtained monoclonal antibody at-20deg.C.
The monoclonal antibody of the isoprothiolane is applied to the detection of the isoprothiolane.
A composition comprising said monoclonal antibodies to isoprothiolane.
The composition is applied to the detection of isoprothiolane.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the monoclonal antibody of the isoprothiolane obtained by the invention has better detection sensitivity (IC) 50 A value of 0.88 ng/mL); the isoprothiolane monoclonal antibody cell strain obtained by the invention can be used for immunoassay detection; the hybridoma cell strain belongs to a monoclonal cell strain.
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 the inhibition of isoprothiolane by the 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 (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use.
Phosphate Buffer (PBS): 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;
PBST: PBS containing 0.05% Tween 20;
antibody dilution: PBS 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 the isoprothiolane 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 (CBS) 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 isoprothiolane standard is diluted to 8 concentrations (0,0.037,0.111,0.333,1,3,9 and 27 ng/mL), and the isoprothiolane standard inhibition curve is obtained by finally mapping OrigingPro 8.5 (the result is shown in FIG. 1) according to the IC-ELISA operation steps, and IC is calculated 50 。
Example 1: synthesis of isoprothiolane hapten
Because the small molecule of the isoprothiolane has no immunogenicity, the small molecule of the isoprothiolane can not stimulate a mouse to generate immune response so as to generate antibodies, the isoprothiolane is coupled to protein through a protein connection technology, so that the isoprothiolane can obtain immunogenicity; the commonly used active groups in the protein coupling technology are amino, carboxyl, hydroxyl, sulfhydryl and the like, and the isoprothiolane is derived in view of the fact that the isoprothiolane molecular structural formula does not contain the active groups.
Pyricularia oryzae (2 g,6.89mmol,1.00 eq) was dissolved in absolute ethanol (15 mL) and cooled to-15 ℃. The freshly prepared filtered KOH (405 mg,1.05 eq) was slowly added at 25 ℃. The resulting mixture was stirred at 25 ℃ for 17h and then concentrated to give some white sticky material. Treated with water and extracted with ethyl acetate, then Na 2 SO 4 Drying, concentrating on a rotary evaporator, and purifying on a silica gel column to give a white solid (500mg g,2.01mmol,1.0eq) dissolved in anhydrous N, N-dimethylformamide (20 mL) at 0deg.C, 6-bromohexanoic acid (350 mg,0.9 eq) and K were slowly added 2 CO 3 (418 mg,1.5 eq) was stirred for 10min and allowed to warm to room temperature overnight. The mixture was filtered through a pad of celite and the solid was rinsed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL) and washed with water (3×100 mL) and brine (100 mL) in this order. The organic layer was separated, dried over sodium sulfate, filtered and concentrated in vacuo to give the isoprothiolane derivative (60 mg, 8%).
Example 2: synthesis of Pyricularia oryzae complete antigen
Weighing 6mg of isoprothiolane hapten, 4.4mg of N-hydroxysuccinimide (NHS), dissolving in 200 mu LN and N-Dimethylformamide (DMF), and stirring and reacting for 10min at room temperature; then 7.5mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is weighed and added into the isoprothiolane hapten solution, and the mixture is stirred and reacted for 6 to 8 hours at room temperature for activation. 6mg of Keyhole Limpet Hemocyanin (KLH) was added to 3mL of 0.01M Carbonate Buffer (CBS) and dissolved well, and the activated hapten was slowly added to the KLH-dissolved dilution and stirred overnight at room temperature. Then, the solution is dialyzed by 0.01M PBS to remove unreacted micromolecular substances, thus obtaining purer complete antigen, and the complete antigen is identified by an ultraviolet absorption scanning method.
Example 3: synthesis of Pyricularia oryzae coating antigen
3.2mg of isoprothiolane hapten and 2.4mg of N-hydroxysuccinimide (NHS) are dissolved in 200 mu L of anhydrous N, N-Dimethylformamide (DMF) and stirred at room temperature for reaction for 10min; dissolving 4.2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in the solution, and stirring at room temperature for reaction for 6-8 hours to obtain hapten activating solution; 6mg chicken Ovalbumin (OVA) was dissolved in Carbonate Buffer (CBS); 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 PBS to remove unreacted small molecular substances, thereby obtaining a coating antigen.
Example 4: preparation of Magnaporthe grisea monoclonal antibody-secreting hybridoma cell line
1. Acquisition of animal immunity
After mixing and emulsifying the finishing antigen of the isoprothiolane and equivalent Freund's adjuvant, performing subcutaneous multipoint injection immunization (except sprint immunization) on the back of the neck of the 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. taking blood from the tail, killing a mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out the spleen of the mouse by aseptic operation, moderately grinding the spleen by using the 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 an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume after the last centrifuging, and counting for later use;
b. collecting SP2/0 cells: SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium at 5% CO 7-10 days prior to fusion 2 Amplifying in incubator to reach SP2/0 tumor cell number of 1-4×10 before fusion 7 Ensure melting ofBefore confluence, SP2/0 tumor cells are in the logarithmic growth phase, and when confluence, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
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 medium was added dropwise every 10 s. 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 isoprothiolane as a standard substance and to measure the inhibition effect of positive cells by using the ic-ELISA method.
Selecting a cell hole with better inhibition on a isoprothiolane standard substance, 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 isoprothiolane monoclonal antibody cell strain.
Example 5: preparation and identification of Magnaporthe grisea 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 Pyricularia oryzae hybridoma cells, collecting ascites from the seventh day, and purifying the ascites by 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 PBS solution (pH 7.4), dialyzing for desalting, and storing at-20deg.C.
Determination of IC for Magnaporthe grisea monoclonal antibody Using Indirect competition ELISA 50 The value is 0.88ng/mL, which shows that the high sensitivity to the isoprothiolane is good, and the high sensitivity can be used for the immunoassay detection of the isoprothiolane.
Example 6: application of isoprothiolane monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strains through in-vivo ascites is applied to ELISA (enzyme-linked immunosorbent assay) additive recovery test of isoprothiolane, and the specific steps are as follows:
(1) Coating 96-well ELISA plates with coating raw materials diluted by Carbonate Buffer Solution (CBS) and having the concentration of 0.1 mug/mL, wherein 100 mug of each well is baked for 2 hours at 37 ℃, and then washing the plates with PBST washing liquid three times, 200 mug of each well is performed for 3min, and the plates are dried by beating;
(2) Blocking with CBS containing 0.2% gelatin, baking at 37deg.C for 2 hr, washing the plate with PBST lotion three times, 200 μl each time, 3min each time, and drying;
(3) Phosphate Buffer (PBS) is used for preparing 0ng/mL,0.037ng/mL,0.111ng/mL,0.333ng/mL,1ng/mL,3ng/mL,9ng/mL and 27ng/mL of isoprothiolane standard solutions, the standard solutions and the sample extracting solutions to be detected are respectively added into the sealed ELISA plates, 50 mu L of each hole is formed, 3 holes are repeated for each sample, 50 mu L of isoprothiolane monoclonal antibody diluted to 0.1 mu g/mL is added into each hole, and after reaction is carried out for 30min at 37 ℃, the plates are washed and patted dry;
(4) 100 μl of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin was added to each well, reacted at 37deg.C for 30min, and then washed with a plate and dried;
(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:
rice was selected as the test sample.
Pulverizing sample to be tested, sieving with 20 mesh standard sieve, weighing three samples, each 20g, adding 5ppb, 10ppb, 50ppb of isoprothiolane standard (according to antibody linear range and IC 50 Setting the addition concentration), adding 10mL of water, mixing uniformly by vortex, standing for 30min, and carrying out suction filtration. The sample was then added with 50mL of acetone, shaken on an electric shaker for 30min, filtered through a rapid qualitative filter paper in a beaker, the residue was extracted once with 30mL of acetone as described above, the residue was washed twice with 30mL of acetone, the wash was incorporated into the beaker, concentrated to near dryness on a 50℃water bath, and reconstituted with 5mL of 10% acetone in PBS (i.e., five times diluted to reduce the effect of the sample matrix).
The recovery rate of the addition was 104%,96% and 106% respectively by indirect competition ELISA.
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 (6)
1. The hybridoma cell strain secreting the isoprothiolane monoclonal antibody is characterized by being preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.22339 and the preservation address of North Chen West Lu No. 1 in the Korean area of Beijing city.
2. The use of the hybridoma cell line of claim 1 for the preparation of a monoclonal antibody to isoprothiolane.
3. The monoclonal antibody of isoprothiolane is characterized by being secreted by a hybridoma cell strain with a preservation number of CGMCC No. 22339.
4. Use of the monoclonal antibody of isoprothiolane of claim 3 for detecting isoprothiolane.
5. A composition comprising a isoprothiolane monoclonal antibody as claimed in claim 3.
6. Use of the composition according to claim 5 for the detection of isoprothiolane.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000086654A (en) * | 1998-09-11 | 2000-03-28 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of isoprothiolane, antibody and assay |
| CN108866009A (en) * | 2018-08-16 | 2018-11-23 | 江南大学 | One plant of metalaxyl monoclonal antibody hybridoma cell strain and its application |
| 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 |
| CN113788814A (en) * | 2021-10-15 | 2021-12-14 | 云南省烟草质量监督检测站 | Hapten for detecting content of isoprothiolane, preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000086654A (en) * | 1998-09-11 | 2000-03-28 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of isoprothiolane, antibody and assay |
| 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 |
| CN108866009A (en) * | 2018-08-16 | 2018-11-23 | 江南大学 | One plant of metalaxyl monoclonal antibody hybridoma cell strain and its application |
| CN113788814A (en) * | 2021-10-15 | 2021-12-14 | 云南省烟草质量监督检测站 | Hapten for detecting content of isoprothiolane, preparation method and application thereof |
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