CN113637081A - Hybridoma cell strain secreting pendimethalin-resistant monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting pendimethalin-resistant monoclonal antibody and application thereof Download PDFInfo
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- CN113637081A CN113637081A CN202110905029.4A CN202110905029A CN113637081A CN 113637081 A CN113637081 A CN 113637081A CN 202110905029 A CN202110905029 A CN 202110905029A CN 113637081 A CN113637081 A CN 113637081A
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- pendimethalin
- monoclonal antibody
- hybridoma cell
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- 239000005591 Pendimethalin Substances 0.000 title claims abstract description 89
- CHIFOSRWCNZCFN-UHFFFAOYSA-N pendimethalin Chemical compound CCC(CC)NC1=C([N+]([O-])=O)C=C(C)C(C)=C1[N+]([O-])=O CHIFOSRWCNZCFN-UHFFFAOYSA-N 0.000 title claims abstract description 81
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- 230000003248 secreting effect Effects 0.000 title claims abstract description 15
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
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Classifications
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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
-
- 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
-
- 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
-
- 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—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- 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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- 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
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- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
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- General Physics & Mathematics (AREA)
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- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
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Abstract
A hybridoma cell strain secreting pendimethalin-resistant monoclonal antibody and application thereof belong to the field of food safety immunodetection. The invention provides a hybridoma cell strain secreting pendimethalin-resistant monoclonal antibody, which is classified and named as monoclonal cell strain, the preservation date is 2020, 9 and 27 days, and the preservation number is CGMCC No. 20791. The invention mixes and emulsifies pendimethalin complete antigen and equivalent Freund's adjuvant, and immunizes BALB/c mice through subcutaneous multipoint injection on the back of the neck. High potency, low IC50Spleen cells of a mouse are fused with myeloma cells of the mouse by a PEG method,adopting a selective culture medium to screen hybrid cells after the fusion of the two cells; and screening cells by an indirect competitive enzyme-linked immunosorbent assay and subcloning for three times to finally obtain the monoclonal antibody hybridoma cell strain ACE. The monoclonal antibody secreted by the hybridoma cell strain ACE has good affinity and high sensitivity to pendimethalin, and provides a powerful detection method and means for detecting pendimethalin in animal-derived food.
Description
Technical Field
The invention relates to a hybridoma cell strain secreting an anti-pendimethalin monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.
Background
Pendimethalin belongs to dinitroaniline herbicides, has a chemical name of N- (1-ethyl propyl) -2, 6-dinitro-3, 4-dimethylaniline, is pendimethalin, is used for field application and tonification, is used for removing buds and the like. Pendimethalin is a high-efficiency and low-toxicity herbicide, and the action mechanism of the pendimethalin is that pendimethalin enters a plant body and is combined with tubulin to inhibit division and elongation of plant cells. In recent years, the herbicide is widely applied to weeding of various crops such as corn, potato, rice, soybean, tobacco and vegetables. Its widespread use has resulted in detection in soil, groundwater, surface water and air, creating significant damage to ecosystem and human health.
A few reports about the detection method of pendimethalin remained in fruits and vegetables at home and abroad mainly use a high performance liquid chromatography-fluorescence detector detection method and a liquid chromatography-tandem mass spectrometry method. The extraction methods are also different, and there are liquid-liquid extraction, liquid-solid extraction, etc. The instrumental detection method can carry out quantitative analysis and has lower detection limit, but generally needs expensive instruments and complex operation, has long pretreatment and detection time, and seriously restricts the popularization of the detection methods. The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirement on technical personnel and the like, so that the immunoassay method is suitable for rapid screening of a large number of samples.
CN 10961147A discloses a test strip for detecting pendimethalin and a preparation method and application thereof, wherein an immunoassay method is also adopted for detection, and the pendimethalin hapten adopted by the test strip is obtained by reacting N- (1-ethyl) -3, 4-dimethylaniline with 4-ethyl bromobutyrate to generate alkylated N- (1-ethyl propyl) -3, 4-dimethylaniline, then reacting with concentrated nitric acid to generate nitro-alkylated N- (1-ethyl propyl) -3, 4-dimethylaniline, and then reacting with potassium hydroxide; the hapten is derived from the site of imino in the middle of pendimethalin, while the structural analogue 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid of pendimethalin is directly used as the hapten, the analogue is extremely similar to the pendimethalin in structure, and on the basis of completely retaining the chemical structure of pendimethalin, one carboxyl is added on the side of pendimethalin for binding protein so as to prepare immunogen. Compared with two hapten structures, the hapten in the invention retains the original structure of pendimethalin to a greater extent, and after carboxyl coupled protein on the side, the specific recognition site of pendimethalin is easier to be exposed, thus being beneficial to generating antibodies with higher sensitivity to pendimethalin. The hapten can be directly obtained from the existing compound, so that complicated chemical synthesis steps are not needed, and the cost is saved.
CN 109324182A discloses a fluorescent microsphere immunochromatographic test strip for detecting pendimethalin, and a preparation method and application thereof, wherein pendimethalin hapten adopted is obtained by reacting 3- (1-ethyl propylamine) -6-methyl-2, 4-dinitrobenzaldehyde and 3-hydrazino propionic acid. However, compared with pendimethalin, the final hapten structure has the unnecessary structures such as double bonds, imino groups and the like, and the design principle of the hapten is that the original structure is kept to the greatest extent possible, so that the antibody with high sensitivity can be obtained. Thus, the haptens of the invention have better concordance with pendimethalin than do the haptens. The hapten of the invention is therefore more favorable for the production of highly sensitive antibodies.
CN 109232286A discloses a preparation method and application of pendimethalin hapten and antigen, N- (1-ethyl propyl) -3, 4-dimethylaniline reacts with 4-ethyl bromobutyrate to generate alkylated N- (1-ethyl propyl) -3, 4-dimethylaniline which reacts with concentrated nitric acid to generate nitro alkylated N- (1-ethyl propyl) -3, 4-dimethylaniline which then reacts with potassium hydroxide, wherein the hapten is obtained by deriving carboxyl from the site of imino in the middle of pendimethalin; the structural analogue 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid of pendimethalin is directly used as hapten, the analogue is extremely similar to the pendimethalin in structure, and on the basis of completely reserving the chemical structure of the pendimethalin, a carboxyl is added on the side of the pendimethalin for binding protein so as to prepare immunogen. Compared with two hapten structures, the hapten in the invention retains the original structure of pendimethalin to a greater extent, and after carboxyl coupled protein on the side, the specific recognition site of pendimethalin is easier to be exposed, thus being beneficial to generating antibodies with higher sensitivity to pendimethalin. The hapten can be directly obtained from the existing compound, so that complicated chemical synthesis steps are not needed, and the cost is saved.
Disclosure of Invention
The invention aims to overcome the defects and provide a hybridoma cell strain secreting the pendimethalin-resistant monoclonal antibody and application thereof.
The technical scheme of the invention is that a hybridoma cell strain ACE for secreting a pendimethalin monoclonal antibody has been preserved in China general microbiological culture Collection center (CGMCC), the institute of microbiology of China academy of sciences No. 3, Xilu No. 1, North Chen, south China, Kyoho, the address Beijing, is classified and named as a monoclonal cell strain, the preservation date is 2020, 9 months and 27 days, and the preservation number is CGMCC No. 20791.
The pendimethalin monoclonal antibody is secreted and produced by the hybridoma cell strain ACE with the preservation number of CGMCC number 20791.
Pendimethalin hapten, 4- [ (1-ethylpropyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid is used as pendimethalin hapten; the structure is simple 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 the amino groups of a protein carrier by an activated ester method, and separating the complete antigen and an uncoupled small molecular hapten by dialysis after the reaction is finished to obtain the pendimethalin complete antigen.
The preparation method of the pendimethalin complete antigen comprises the following steps: taking 4-6 mg of 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid, adding 5-10 mg of EDC 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 3-5 mg of NHS N-hydroxysuccinimide, dissolving with DMF N, N-dimethylformamide, stirring at room temperature, and activating for 6-8 h; dissolving 10-15mg of BSA bovine serum albumin in 3-5mL of CB carbonate buffer solution with the pH of 0.05M and 9.6; dropwise adding the activating solution into BSA solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 2-3 days, and subpackaging at-20 ℃.
The preparation method of the hybridoma cell strain ACE for secreting the pendimethalin monoclonal antibody comprises the following steps:
(1) immunization of mice: selecting a BALB/c mouse with the age of 6-8 weeks for immunization; taking pendimethalin complete antigen as immunogen, emulsifying the immunogen and Freund's adjuvant completely, and injecting the mixture to immunize a mouse through subcutaneous multipoint injection; the Freund complete adjuvant is adopted for the first immunization, the Freund incomplete adjuvant is used for strengthening the immunization, the immune dose is half of that of the former immune dose during the spurting immunization, and the mixture is directly injected into the abdominal cavity after being uniformly mixed with the normal saline; each immunization interval was three weeks; after the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition;
(2) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol method, culturing by a HAT culture medium, detecting positive cell holes by indirect ELISA, further determining the inhibition effect of the positive cell holes by an indirect competitive ELISA method, carrying out three times of subcloning on the positive cell holes with the best inhibition by a limiting dilution method, and finally screening to obtain a hybridoma cell strain ACE;
(3) and (3) identification of the properties of hybridoma cell strains: determining by using an enzyme-labeled secondary antibody kit for identifying mouse monoclonal antibody Ig class/subclass; IC (integrated circuit)50Values, 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 preparation of the pendimethalin monoclonal antibody.
The application of the pendimethalin-resistant monoclonal antibody is used for detecting pendimethalin residues in food.
The preparation method of the fluazinam-coated antigen is applied to identification of fluazinam.
The invention has the beneficial effects that: the anti-pendimethalin monocke prepared by the method of the inventionThe monoclonal antibody has good detection sensitivity and affinity to pendimethalin, and 50% inhibition concentration IC of pendimethalin50The detection reagent is 2.05 ng/mL, can be used for preparing an immunodetection kit of pendimethalin and a colloidal gold test strip, and provides a powerful detection method and means for detecting the pendimethalin in animal-derived food;
the invention provides a novel method for synthesizing the pendimethalin immunogen, which simplifies and is effective in synthesis steps and provides an idea and a method for synthesizing the immunogen for the research of people in the future.
Biological material sample preservation: a hybridoma cell strain ACE for secreting pendimethalin-resistant monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute of China institute of sciences No. 3, West Lu No. 1, North Kyoho, Beijing, and has been classified and named as monoclonal cell strain with the preservation date of 2020, 9 months and 27 days and the preservation number of CGMCC No. 20791.
Drawings
FIG. 1 is a standard inhibition curve for pendimethalin 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 mouse is immunized by a pendimethalin complete antigen, cell fusion and HAT selective culture medium culture are carried out, and cell supernatant is screened by indirect ELISA and indirect competitive ELISA, so that the monoclonal antibody hybridoma cell strain ACE with good affinity and sensitivity to pendimethalin is finally obtained.
Example 1: preparation of anti-pendimethalin monoclonal antibody hybridoma cell strain ACE
1. Synthesis of complete antigen: 4.5 mg of 4- [ (1-ethylpropyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid was taken, and 5.0 mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 3.7 mg of NHS (N-hydroxysuccinimide) were added, dissolved in DMF (N, N-dimethylformamide), stirred at room temperature and activated for 6 hours; dissolving 15mg BSA (bovine serum albumin) in 3mL CB (carbonate buffer solution) solution with pH of 0.05M and 9.6; and dropwise adding the activating solution into a BSA solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 3 days, and subpackaging and storing at-20 ℃.
2. Animal immunization: healthy 608 week old BALB/c mice were selected for immunization. After a pendimethalin complete antigen (1 mg/mL) was emulsified with an equal amount of Freund's adjuvant, BALB/c mice were immunized by subcutaneous multi-site injection, each at 100. mu.L. The Freund complete adjuvant is adopted for the first immunization, the Freund incomplete adjuvant is used for strengthening the immunization, the immune dose is half of that of the former immune dose during the spurting immunization, and the mixture is directly injected into the abdominal cavity after being uniformly mixed with the normal saline; the intervals between immunizations were three weeks. After the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition; the best-suppressed mice were selected, immunized by boosting 18 days after five immunizations, and prepared for fusion.
3. Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to the conventional PEG (polyethylene glycol, molecular weight 2000-4000) method, and the specific steps are as follows:
(1) taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells;
(2) collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells;
(3) mixing splenocytes and SP2/0 cells at a ratio of 2-0:1, centrifuging, fusing with PEG for 1 min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 × HAT, adding to 96-well cell culture plate, standing at 37 deg.C and 5% CO2Cultured in an incubator.
4. 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 and screened. The screening is divided into two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, pendimethalin is selected as a standard substance, and inhibition effect determination is carried out on positive cells by indirect competition ELISA. And selecting a cell hole with good inhibition to pendimethalin, subcloning by using a limiting dilution method, and detecting by using the same method. Repeating the steps for three times to obtain a cell strain ACE.
5. Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106And (3) collecting ascites from the 7 th day, purifying the ascites by an octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.
And (3) carrying out immunoglobulin subtype identification on the monoclonal antibody obtained by ascites purification by using a mouse monoclonal antibody subtype identification kit, wherein the subtype is IgG2a type, and is specifically shown in Table 1.
TABLE 1 subtype identification of pendimethalin monoclonal antibodies
| Subclass of antibody | OD value |
| IgA | 0.204 |
| IgG1 | 0.203 |
| IgG2a | 0.533 |
| IgG2b | 2.221 |
| IgG3 | 0.212 |
| IgM | 0.053 |
Determination of IC of monoclonal antibody p-pendimethalin Using Indirect competitive ELISA502.05 ng/mL, and verified its IC for diclazuril, etc50And the cross-reactivity ratio are shown in Table 2.
TABLE 2 IC of pendimethalin monoclonal antibodies for pendimethalin, trifluralin, butralin, flumetralin50And cross reaction rate
| IC50 (ng/mL) | Rate of cross reaction | |
| Pendimethalin | 2.05 | 100% |
| Trifluralin | ＞500 | ＜5% |
| Butralin | ＞500 | ＜5% |
| Flumetralin | ＞500 | ＜5% |
6. The application of the antibody comprises the following steps: the monoclonal antibody prepared from hybridoma cell strain ACE through in-vivo ascites is applied to pendimethalin ELISA addition recovery test, and the method specifically comprises the following steps:
(1) coating 0.1 mu g/mL pendimethalin diluted by Carbonate Buffer Solution (CBS) as a coating source for coating a 96-well enzyme label plate, wherein each well is 100 mu L, after coating at 37 ℃ for 2h, washing the plate with PBST washing solution three times, each time is 200 mu L, each time is 3min, and then patting to dry;
(2) sealing with CBS containing 0.2% gelatin, sealing at 37 deg.C for 2 hr with 200 μ L per well, washing the plate with PBST lotion for three times, each time with 200 μ L per well, each time for 3min, and drying;
(3) phosphate Buffered Saline (PBS) was used to prepare 0, 0.03, 0.1, 0.3, 1, 3, 10, 30, 90ng/mL pendimethalin standard solutions. Respectively adding the standard solution and the extract of the sample to be detected into the closed enzyme label plate, wherein each hole is 50 mu L, each sample is repeatedly provided with 3 holes, and each hole is added with 50 mu L1: 16000 diluting anti-pendimethalin monoclonal antibody, reacting at 37 deg.C for half an hour, washing and drying;
(4) add 100 μ L per well of PBS 1: reacting a goat anti-mouse IgG secondary antibody marked by HRP and diluted by 3000 at 37 ℃ for half an hour, washing the plate and drying the plate;
(5) adding 100 μ L of TMB color developing solution into each well, developing at 37 deg.C for 15min, and adding 50 μ L of 2M H into each well2SO4Stopping solution, measuring the light absorption value at 450 nm;
(6) adding and recovering and sample pretreatment: taking 5g of a fresh cucumber sample, and adding three different dosages of oxadixyl standard substances, namely 5ng, 10ng and 20 ng. Placing the mixture into a 50 mL centrifuge tube, slowly dropping 1mL of 50% potassium hydroxide solution, fully oscillating on a vortex mixer, slowly dropping 20mL of ethyl acetate, oscillating on the vortex mixer for 10 min, and then placing the mixture into a centrifuge to centrifuge for 5min at 3000 r/min. 4 mL of the supernatant was removed from the other centrifuge tube, blown dry with nitrogen, and 1mL of 10% methanol in PBS was added for reconstitution, and 50. mu.L was used for detection. The additive recovery tests were performed by indirect competitive ELISA with recoveries of 92.2%, 101.1%, 95.7%, respectively.
Solution preparation:
carbonate Buffer (CBS): weighing Na2CO3 1.59g，NaHCO32.93g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH2PO4，3.62 g 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: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A. The volume ratio of the solution B to the solution B is 1: 5, mixing to obtain the TMB color developing solution which is mixed at present.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. That is, all equivalent changes and modifications made within the scope of the present application are within the technical scope of the present invention.
Claims (10)
1. A hybridoma cell strain ACE for secreting pendimethalin-resistant monoclonal antibody has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences microorganism institute of China institute of sciences No. 3, West Lu No. 1, North Kyoho, Beijing, and has been classified and named as monoclonal cell strain with the preservation date of 2020, 9 months and 27 days and the preservation number of CGMCC No. 20791.
2. Pendimethalin monoclonal antibody, characterized in that: it is secreted and produced by hybridoma cell strain ACE with the preservation number of CGMCC number 20791 as claimed in claim 1.
3. Pendimethalin hapten, characterized in that: 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid is adopted as pendimethalin hapten; the structure is simple as follows:
4. the pendimethalin complete antigen is characterized by having the following structural formula:
5. the preparation method of the pendimethalin complete antigen is characterized by comprising the following steps: 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid is used as a raw material, the amino groups of the protein carrier are connected through an activated ester method, and after the reaction is finished, the complete antigen and the uncoupled small molecular hapten are separated through dialysis, so that the pendimethalin complete antigen is obtained.
6. The method for preparing pendimethalin complete antigen according to claim 5, which comprises the steps of: taking 4-6 mg of 4- [ (1-ethyl propyl) amino ] -2-methyl-3, 5-dinitrobenzoic acid, adding 5-10 mg of EDC 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride and 3-5 mg of NHS N-hydroxysuccinimide, dissolving with DMF N, N-dimethylformamide, stirring at room temperature, and activating for 6-8 h; dissolving 10-15mg of BSA bovine serum albumin in 3-5mL of CB carbonate buffer solution with the pH of 0.05M and 9.6; dropwise adding the activating solution into BSA solution, stirring at room temperature for overnight reaction, taking out immunogen PBS, dialyzing for 2-3 days, and subpackaging at-20 ℃.
7. The preparation method of the hybridoma cell strain ACE for secreting the pendimethalin monoclonal antibody is characterized by comprising the following steps:
(1) immunization of mice: selecting a BALB/c mouse with the age of 6-8 weeks for immunization; taking pendimethalin complete antigen as immunogen, emulsifying the immunogen and Freund's adjuvant completely, and injecting the mixture to immunize a mouse through subcutaneous multipoint injection; the Freund complete adjuvant is adopted for the first immunization, the Freund incomplete adjuvant is used for strengthening the immunization, the immune dose is half of that of the former immune dose during the spurting immunization, and the mixture is directly injected into the abdominal cavity after being uniformly mixed with the normal saline; each immunization interval was three weeks; after the third immunization, blood is collected at intervals of one week to detect serum titer and inhibition;
(2) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by a polyethylene glycol method, culturing by a HAT culture medium, detecting positive cell holes by indirect ELISA, further determining the inhibition effect of the positive cell holes by an indirect competitive ELISA method, carrying out three times of subcloning on the positive cell holes with the best inhibition by a limiting dilution method, and finally screening to obtain a hybridoma cell strain ACE;
(3) and (3) identification of the properties of hybridoma cell strains: determining by using an enzyme-labeled secondary antibody kit for identifying mouse monoclonal antibody Ig class/subclass; IC (integrated circuit)50Values, cross-reactivity and affinity were determined by ELISA.
8. The application of the hybridoma cell line ACE for secreting anti-pendimethalin monoclonal antibody according to claim 1 or the hybridoma cell line ACE for secreting anti-pendimethalin monoclonal antibody according to claim 7 in preparing pendimethalin monoclonal antibody.
9. The use of the anti-pendimethalin monoclonal antibody of claim 2, wherein: the method is used for detecting pendimethalin residues in food.
10. Use of the method for preparing a fluazinam-coated antigen according to claim 8 for the identification of fluazinam.
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