CN113637081B - Hybridoma cell strain secreting anti-pendimethalin monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting anti-pendimethalin monoclonal antibody 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
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/52—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
- C07C229/54—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
- C07C229/60—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring with amino and carboxyl groups bound in meta- or para- positions
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/18—Water
- G01N33/1826—Water organic contamination in water
- G01N2033/184—Water organic contamination in water herbicides, pesticides, fungicides, insecticides, or the like
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A hybridoma cell strain secreting anti-pendimethalin monoclonal antibody and application thereof belong to the field of food safety immunodetection. The invention provides a hybridoma cell strain secreting anti-pendimethalin monoclonal antibodies, which is classified and named as a monoclonal cell strain, and the preservation date is 9/27/2020, and the preservation number is CGMCC No.20791. The invention mixes and emulsifies the pendimethalin complete antigen and equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multipoint injection at the neck and back. Will have high potency and low IC 50 The spleen cells of the mice are fused with myeloma cells of the mice by a PEG method, and a selection medium is adopted to screen out hybrid cells after the two cells are fused; and screening cells by an indirect competitive ELISA method and subcloning the cells for three times to finally obtain a monoclonal antibody hybridoma cell strain ACE. The monoclonal antibody secreted by the hybridoma cell strain ACE has better affinity and higher sensitivity to pendimethalin, and provides a powerful detection method and means for detecting pendimethalin in animal-derived foods.
Description
Technical Field
The invention relates to a hybridoma cell strain secreting anti-pendimethalin monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.
Background
Dimevalonate dinitroaniline herbicide is named as N- (1-ethyl propyl) -2, 6-binitro-3, 4-dimethylaniline, and is also named as pendimethalin, shi Tian patch, degerming, etc. Pendimethalin is a high-efficiency and low-toxicity herbicide, and the action mechanism is that pendimethalin enters a plant body to be combined with tubulin, so that division and elongation of plant cells are inhibited. In recent years, the herbicide is widely applied to weeding of various crops such as corn, potato, rice, soybean, tobacco, vegetables and the like. Its widespread use has led to detection in soil, groundwater, surface water and air, creating significant hazards to the ecosystem and human health.
The detection method of pendimethalin residue in fruits and vegetables has been reported in small amounts at home and abroad, mainly by high performance liquid chromatography-fluorescence detector detection method and liquid chromatography-tandem mass spectrometry method. The extraction methods are also different, and include liquid-liquid extraction, liquid-solid extraction, etc. The instrument detection method can perform quantitative analysis and has lower detection limit, but expensive instruments and complex operation are usually required, and the pretreatment and detection time are long, so that the popularization of the detection methods is severely restricted. The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirements on technicians and the like, so that the method is suitable for rapid screening of a large number of samples.
CN10961147a discloses a test strip for detecting pendimethalin, its preparation method and application, which also uses immunoassay method to detect, the adopted pendimethalin hapten is that N- (1-ethyl) -3, 4-dimethylaniline reacts with ethyl 4-bromobutyrate to generate hydrocarbylated N- (1-ethyl propyl) -3, 4-dimethylaniline, then reacts with concentrated nitric acid to generate nitroalkylated N- (1-ethyl propyl) -3, 4-dimethylaniline, then reacts with potassium hydroxide to obtain the product; the hapten is carboxyl derived from the imino site in the middle part of pendimethalin, and the invention directly uses the structural analogue of pendimethalin, namely 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid as the hapten, wherein the analogue is very similar to the pendimethalin in structure, and a carboxyl is added on the side of the pendimethalin for binding protein so as to prepare the immunogen on the basis of completely retaining the chemical structure of the pendimethalin. Compared with two hapten structures, the hapten disclosed by the invention has the advantages that the original structure of pendimethalin is reserved to a greater extent, and after the carboxyl on the side is coupled with the protein, the specific recognition site of pendimethalin is easier to be exposed, so that the antibody aiming at pendimethalin with higher sensitivity is generated. The hapten can be directly obtained from the existing compound without complicated chemical synthesis steps, so that the cost is saved.
CN109324182 a discloses a fluorescent microsphere immunochromatographic test strip for detecting pendimethalin, a preparation method and application thereof, the adopted pendimethalin hapten is obtained by reacting 3- (1-ethyl alanyl) -6-methyl-2, 4-dinitrobenzaldehyde with 3-hydrazinopropionic acid, and compared with the invention, the structure is that carboxyl for binding protein is derived on the basis of pendimethalin analogue. However, the final hapten structure is increased with unnecessary structures such as double bond, imino and the like compared with pendimethalin, and the design principle of the hapten is that the original structure is kept to the greatest extent as possible, so that the antibody with high sensitivity can be obtained. Therefore, the hapten of the invention has better consistency with pendimethalin compared with the hapten. The hapten of the invention is thus more advantageous for the production of highly sensitive antibodies.
CN109232286 a discloses a preparation method and application of pendimethalin hapten and antigen, wherein N- (1-ethylpropyl) -3, 4-dimethylaniline reacts with ethyl 4-bromobutyrate to generate hydrocarbylated N- (1-ethylpropyl) -3, 4-dimethylaniline, then reacts with concentrated nitric acid to generate nitroalkylated N- (1-ethylpropyl) -3, 4-dimethylaniline, then reacts with potassium hydroxide to obtain hapten, and carboxyl is derived from the imino site in the middle part of pendimethalin; the invention directly uses the structural analogue of pendimethalin, namely 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid as hapten, the analogue is extremely similar to the pendimethalin in structure, and a carboxyl group is added on the side of the pendimethalin for binding protein on the basis of completely retaining the chemical structure of the pendimethalin so as to prepare the immunogen. Compared with two hapten structures, the hapten disclosed by the invention has the advantages that the original structure of pendimethalin is reserved to a greater extent, and after the carboxyl on the side is coupled with the protein, the specific recognition site of pendimethalin is easier to be exposed, so that the antibody aiming at pendimethalin with higher sensitivity is generated. The hapten can be directly obtained from the existing compound without complicated chemical synthesis steps, so that the cost is saved.
Disclosure of Invention
The invention aims to overcome the defects, and provides a hybridoma cell strain secreting an anti-pendimethalin monoclonal antibody and application thereof, and the monoclonal antibody prepared by the cell strain has better affinity and sensitivity to pendimethalin, and can be used for establishing a pendimethalin ELISA detection method or a colloidal gold immunochromatography test strip rapid detection method.
According to the technical scheme, a hybridoma cell strain ACE secreting anti-pendimethalin monoclonal antibodies is preserved in China General Microbiological Culture Center (CGMCC) of China Committee for culture Collection of microorganisms, and is classified and named as a monoclonal cell strain by the national institute of microbiology, national academy of sciences of China, no. 3, north Chen West Lu No. 1, the area of Beijing, and the preservation date of the hybridoma cell strain is 2020, 9 months and 27 days, and the preservation number of the hybridoma cell strain is CGMCC No.20791.
The pendimethalin monoclonal antibody is secreted by the hybridoma cell strain ACE with the preservation number of CGMCC No.20791.
The pendimethalin hapten is 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid; the structural formula is as follows:
the pendimethalin complete antigen has the following structural formula:
the preparation method of the pendimethalin complete antigen comprises the steps of taking 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid as a raw material, connecting amino groups of an activated ester method protein carrier, and separating the complete antigen and unconjugated small molecule hapten through dialysis after the reaction is finished to obtain the pendimethalin complete antigen.
The preparation method of the pendimethalin complete antigen comprises the following steps: 4-6mg of 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid is taken, 5-10mg of EDC 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 3-5mg of NHS N-hydroxysuccinimide are added, DMF N, N-dimethylformamide is used for dissolution, stirring is carried out at room temperature, and activation is carried out for 6-8h; dissolving 10-15mg BSA bovine serum albumin in 3-5mL CB carbonate buffer solution with the concentration of 0.05M and the pH of 9.6; the activating solution is added into BSA solution drop by drop, stirred at room temperature for reaction overnight, and then the immunogen PBS is taken out for dialysis for 2-3 days, and split charging and storage are carried out at-20 ℃.
The preparation method of the hybridoma cell strain ACE secreting the anti-pendimethalin monoclonal antibody comprises the following steps:
(1) Immunization of mice: selecting BALB/c mice with 6-8 weeks of age for immunization; taking pendimethalin complete antigen as an immunogen, emulsifying the immunogen and Freund's adjuvant completely, and immunizing a mouse by subcutaneous multipoint injection; the first immunization adopts Freund's complete adjuvant, the boost immunization adopts Freund's incomplete adjuvant, the immunization dose is half of the previous immunization dose during the sprint immunization, and the mixture is uniformly mixed with normal saline and then directly injected into the abdominal cavity; the immunization interval is three weeks; after the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition;
(2) Cell fusion and cell strain establishment: fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol method, culturing by 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 for three times, and finally screening to obtain hybridoma cell strains ACE;
(3) Identification of hybridoma cell line properties: adopting enzyme-labeled secondary antibody suit determination for mouse monoclonal antibody Ig class/subclass identification; IC (integrated circuit) 50 The values, cross-reactivity and affinity were determined by ELISA.
The hybridoma cell strain ACE secreting the anti-pendimethalin monoclonal antibody or the preparation method of the hybridoma cell strain ACE secreting the anti-pendimethalin monoclonal antibody is applied to the aspect of preparing the pendimethalin monoclonal antibody.
The application of the anti-pendimethalin monoclonal antibody is used for detecting pendimethalin residues in foods.
The invention has the beneficial effects that: the anti-pendimethalin monoclonal antibody prepared by the method has better detection sensitivity and affinity to pendimethalin and 50% inhibition concentration IC of pendimethalin 50 2.05ng/mL, can be used for preparing an immunity detection kit of pendimethalin and a colloidal gold test strip, and provides a powerful detection method and means for the detection of pendimethalin in animal-derived foods;
the invention provides a novel method for synthesizing pendimethalin immunogen, which has more simplified and effective synthesis steps and provides ideas and methods for synthesizing immunogen for the research of people in future.
Preservation of biological material samples: the hybridoma cell strain ACE secreting anti-pendimethalin monoclonal antibody is preserved in China general microbiological culture Collection center (CGMCC), the institute of microbiological research, national academy of sciences, no. 3, north Chenxi, korea, beijing, and the preservation date 2020, 9 months and 27 days, and the preservation number CGMCC No.20791.
Drawings
FIG. 1 is a standard inhibition curve for pendimethalin monoclonal antibodies.
Detailed Description
The following examples of the present invention are merely further illustrative of the present invention and are not intended to limit the scope or spirit of the present invention. The invention is further illustrated by the following examples.
According to the invention, mice are immunized by the pendimethalin complete antigen, through cell fusion and HAT selective medium culture, cell supernatants are screened through indirect ELISA and indirect competition ELISA, and finally the monoclonal antibody hybridoma cell strain ACE with better affinity and sensitivity to pendimethalin is obtained.
Example 1: preparation of anti-pendimethalin monoclonal antibody hybridoma cell strain ACE
1. Complete antigen synthesis: 4.5mg of 4- [ (1-ethylpropyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid was taken, 5.0mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7mg of NHS (N-hydroxysuccinimide) were added thereto, dissolved in DMF (N, N-dimethylformamide), stirred at room temperature and activated for 6 hours; another 15mg BSA (bovine serum albumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer) solution at pH 9.6; the activating solution is added into BSA solution drop by drop, stirred at room temperature for reaction overnight, and then the immunogen PBS is taken out for dialysis for 3 days, and split charging and storage are carried out at-20 ℃.
2. Animal immunization: healthy 608 week old BALB/c mice were selected for immunization. Full antigen of pendimethalin (1 mg/mL) was emulsified with equal amount of Freund's adjuvant and immunized by subcutaneous multi-point injection into BALB/c mice, 100. Mu.L each. The first immunization adopts Freund's complete adjuvant, the boost immunization adopts Freund's incomplete adjuvant, the immunization dose is half of the previous immunization dose during the sprint immunization, and the mixture is uniformly mixed with normal saline and then directly injected into the abdominal cavity; each immunization interval was three weeks. After the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition; mice with the best inhibition were selected and were challenged 18 days after five days to prepare for fusion.
3. Cell fusion: after three days of sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 2000-4000) method, specifically as follows:
(1) Taking a mouse spleen in a sterile mode, grinding and passing through a 200-mesh cell screen to obtain spleen cell suspension, and performing cell counting;
(2) Collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
(3) Mixing spleen cells and SP2/0 cells at a ratio of 2-0:1, centrifuging, fusing with PEG for 1min, adding RPMI-1640 basal culture solution from slow to fast, centrifuging, suspending in RPMI-1640 screening culture solution containing 20% fetal bovine serum and 2% 50×HAT, adding into 96-well cell culture plate, and placing at 37deg.C and 5% CO 2 Is cultured in an incubator of (a).
4. Cell screening and cell strain establishment: the cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full-replacement with a 100 XHT-containing 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 indirect ELISA, and the second step is to use pendimethalin as a standard substance, and to measure the inhibition effect of positive cells by indirect competition ELISA. Selecting a cell hole with better inhibition on pendimethalin, subcloning by adopting a limiting dilution method and detecting by adopting the same method. The cell line ACE was obtained by repeating three times.
5. Preparation and identification of monoclonal antibodies: taking 8-10 week-old BALB/c mice, and injecting paraffin oil into the abdominal cavity of each mouse by 1mL; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, collecting ascites from day 7, purifying the ascites by octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20deg.C.
Immunoglobulin subtype identification was performed on the monoclonal antibodies obtained by ascites purification using a mouse monoclonal antibody subtype identification kit, the subtype of which was of the IgG2a type, as shown in table 1.
TABLE 1 subtype identification of pendimethalin monoclonal antibodies
| Antibody subclasses | OD value |
| IgA | 0.204 |
| IgG1 | 0.203 |
| IgG2a | 0.533 |
| IgG2b | 2.221 |
| IgG3 | 0.212 |
| IgM | 0.053 |
Determination of monoclonal antibody to pendimethalin IC using indirect competition ELISA method 50 2.05ng/mL, and validated its IC for diclazuril et al 50 And the cross-reactivity is shown in Table 2.
TABLE 2 IC of pendimethalin monoclonal antibody to pendimethalin, trifluralin, butralin and flumetralin 50 Cross-reactivity ratio
6. Antibody application: the monoclonal antibody prepared from hybridoma cell strain ACE through in vivo ascites is applied to a pendimethalin ELISA (enzyme-linked immunosorbent assay) additive recovery test, and the specific steps are as follows:
(1) Coating a 96-well ELISA plate with 0.1 mu g/mL pendimethalin diluted by Carbonate Buffer (CBS) as a coating raw material, coating 100 mu L of each well at 37 ℃ for 2 hours, washing the plate with PBST washing liquid three times, 200 mu L of each well for 3min, and drying;
(2) Blocking with CBS containing 0.2% gelatin, blocking at 37deg.C for 2 hr, washing the plate with PBST wash solution three times, 200 μl each time, 3min each time, and drying;
(3) 0,0.03,0.1,0.3,1,3, 10, 30,90ng/mL of pendimethalin standard solution were prepared with Phosphate Buffered Saline (PBS), respectively. Respectively adding the standard solution and the sample extracting solution to be detected into the sealed ELISA plate, wherein each hole is 50 mu L, each sample is repeated for 3 holes, and then 50 mu L of 1 is added into each hole: after 16000 diluted anti-pendimethalin monoclonal antibody reacts for half an hour at 37 ℃, washing the plate and beating;
(4) mu.L of PBS 1 with 0.1% gelatin was added to each well: after a reaction of 3000 dilution of HRP-marked goat anti-mouse IgG secondary antibody at 37 ℃ for half an hour, washing the plate and beating;
(5) Adding 100 mu LTMB color development liquid into each well, developing at 37 ℃ for 15min, adding 50 mu L2M H into each well 2 SO 4 Stop solution, measuring light absorption value at 450 nm;
(6) And (3) adding and recycling and sample pretreatment: fresh cucumber samples of 5g were taken and three different doses of oxadixyl standard were added, 5ng, 10ng, 20ng respectively. The mixture was placed in a 50mL centrifuge tube, 1mL of 50% potassium hydroxide solution was slowly dropped, the mixture was sufficiently shaken on a vortex mixer, 20mL of ethyl acetate was slowly dropped, the mixture was shaken on the vortex mixer for 10 minutes, and the mixture was then placed in a centrifuge and centrifuged at 3000r/min for 5 minutes. 4mL of supernatant was removed from the tube and dried with nitrogen, 1mL of PBS containing 10% methanol was added for reconstitution, and 50. Mu.L was taken for detection. The recovery of the additives was 92.2%, 101.1% and 95.7% respectively by indirect competition ELISA.
Solution preparation:
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): 8.00gNaCl,0.2g KCl,0.24g KH 2 PO 4 ，3.62gNa 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;
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 volume ratio of the solution B is 1:5, mixing to obtain TMB color development liquid, and mixing immediately.
The foregoing description is merely illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Equivalent changes and modifications are within the scope of the present invention.
Claims (4)
1. The hybridoma cell strain ACE secreting anti-pendimethalin monoclonal antibody is preserved in China general microbiological culture Collection center (CGMCC), the institute of microbiological research, national academy of sciences, no. 3, north Chenxi, korea, beijing, and the preservation date 2020, 9 months and 27 days, and the preservation number CGMCC No.20791.
2. An anti-pendimethalin monoclonal antibody, characterized in that: it is secreted by hybridoma cell strain ACE with preservation number of CGMCC No.20791 as claimed in claim 1.
3. Use of the hybridoma cell strain ACE secreting the anti-pendimethalin monoclonal antibody according to claim 1 for preparing the anti-pendimethalin monoclonal antibody.
4. The use of an anti-pendimethalin monoclonal antibody according to claim 2, characterized in that: the method is used for detecting the pendimethalin residue in food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061147A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting pendimethalin |
| CN109232286A (en) * | 2018-09-21 | 2019-01-18 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of pendimethalin haptens and antigen |
| CN109324182A (en) * | 2018-09-21 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting pendimethalin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061147A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting pendimethalin |
| CN109232286A (en) * | 2018-09-21 | 2019-01-18 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of pendimethalin haptens and antigen |
| CN109324182A (en) * | 2018-09-21 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of fluorescent micro-ball immune chromatography test paper strip and its preparation method and application detecting pendimethalin |
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