CN113717948B - Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN113717948B CN113717948B CN202111096426.8A CN202111096426A CN113717948B CN 113717948 B CN113717948 B CN 113717948B CN 202111096426 A CN202111096426 A CN 202111096426A CN 113717948 B CN113717948 B CN 113717948B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- cell strain
- hybridoma cell
- sclerostin
- hapten
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/36—Oxygen or sulfur atoms
- C07D207/40—2,5-Pyrrolidine-diones
- C07D207/416—2,5-Pyrrolidine-diones with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to other ring carbon atoms
-
- 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
- G01N33/48—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
-
- 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
-
- 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
Abstract
The invention discloses a hybridoma cell strain secreting an anti-sclerotium net monoclonal antibody and application thereof, wherein the preservation name of the monoclonal antibody hybridoma cell strain is monoclonal cell strain SLS 2G5, and the preservation number is CGMCC No.20792. The immunogen PD-BSA is prepared by coupling hapten and carrier protein by taking the addition reaction hydrolysate PD of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione (Done) and 3-ethyl mercaptopropionate as hapten. After the mice are subjected to injection immunization by an immunogen PD-BSA, the mice are fused with myeloma cells by a PEG method, and the monoclonal antibody hybridoma cell strain is obtained through screening and five subcloning by an indirect competition enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain can be prepared into a sclerotinia sclerotiorum detection box, has better affinity and detection sensitivity for sclerotinia sclerotiorum, and can be used for immunodetection of the residue of sclerotinia sclerotiorum in food.
Description
Technical Field
The invention belongs to the technical field of food safety immunodetection, and particularly relates to a hybridoma cell strain secreting an anti-sclerostin monoclonal antibody and application thereof.
Background
Dimethachlon (DMT) belongs to a dicarbonyl imine broad-spectrum bactericide, has the advantages of low toxicity, high efficiency, long lasting period and the like, and is widely applied to preventing and treating rice sheath blight, rape, sunflower, soybean sclerotinia and tobacco brown spot. The increase in the amount of pesticide used not only increases the risk of humans contacting the pesticide through the food chain, but also creates toxic effects on the environment and non-target organisms. The human body may be exposed to the sclerotium-based bactericide by ingestion of foods, fruits and vegetables, absorption of skin chemicals, and inhalation of dust particles. Therefore, the establishment of a method for rapidly and effectively detecting the net sclerotium content has important significance and market value.
Current detection methods include instrumental and immunoassay. Instrumental analysis methods such as high performance liquid chromatography ([ 1)]East-guard column Wang Zhen, jing Lan, zhou Hongyou HPLC method for simultaneously detecting 4 bactericides of carbendazim, dimethachlon, prochloraz [ J ]]Pesticide, 2016, 55 (03): 205-207.), gas chromatography tandem mass spectrometry ([ 2 ]]Liu Jianping, luo Hong, lianhaifei, cui Yan, nie Jing analysis method for detecting 6 bactericides in bulb vegetables such as green Chinese onion by QuEChERS extraction-gas chromatography-triple four-level rod tandem mass spectrometry [ J]Analytical instrument 2020 (06): 40-45.) detection range is 0.04-10.0 mug mL -1 . Because of complicated pretreatment of the sample, more interferents and limitation of the working conditions of the instrument, and higher technical requirements on operators, the instrument method is not suitable for on-site detection.
Compared with an instrument detection method, the immunoassay method has the characteristics of low cost, high flux, high sensitivity, low requirements on technicians and the like, and is suitable for rapid screening of a large number of samples. Thus, the immunoassay method has important significance for the detection of the sclerotinia sclerotiorumMeaning. Liu Rongrong in 2009 to 2012 the preparation of polyclonal antibodies against dimethachlon was successively reported ([ 3]Liu Yuan, liu Xianjin, zhang Cunzheng, liu Rongrong polyclonal antibodies to sclerostin, their preparation and use [ P ]]Jiangsu: CN101691404a, 2010-04-07.) time-resolved fluoroimmunoassay ([ 4 ]]Liu Yuan A, liu Rongrong A, liu Xianjin A, fanghuang, song Chunman A, huovinen Tuomas, vehni inen Markus A, L vgren Timo. Time resolved fluoroimmunoassay method for detecting sclerotin residue in tobacco [ J]Analytical chemistry 2012, 40 (07): 1114-1117; [5]Liu Yuan, liu Xianjin, zhang Xiao, wang Donglan method for detecting sclerotinia residue in tobacco by indirect competitive time resolved fluoroimmunoassay system [ P]Jiangsu: CN102590499a, 2012-07-18.), but the detection sensitivity is not high, wherein the sensitivity is optimal for indirect competition time-resolved fluoroimmunoassay, IC 50 Is 32ng mL -1 。
The enzyme-linked immunosorbent assay (ELISA) is a low-cost, rapid and portable immunological detection method, has low requirements on the purity of samples during detection, is simple and convenient to operate, and is suitable for rapidly detecting results on site of a large number of samples. The establishment of an efficient immunological detection method and the screening of a monoclonal antibody with high specificity are important preconditions, so that the development of an efficient and sensitive detection method aiming at the sclerotium is a problem to be solved urgently.
Disclosure of Invention
The invention aims to: in order to overcome the defects in the prior art, the invention provides a hybridoma cell strain secreting a sclerostin monoclonal antibody and application thereof, and the antibody secreted by the cell strain has higher detection sensitivity to the sclerostin and can be used for establishing an immunological detection method to the sclerostin.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the following technical scheme:
the invention aims to provide a hybridoma cell strain secreting a sclerotium net monoclonal antibody, which is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) and has the preservation name of monoclonal cell strain SLS 2G5, the preservation number of which is CGMCC No.20792, and the address of which is: the national institute of microbiology, national academy of sciences, national institute of sciences, and date of preservation 2020, 09, 27.
The second object of the present invention is to provide a sclerostin monoclonal antibody which is secreted by the hybridoma cell line SLS 2G5 of the sclerostin monoclonal antibody with the preservation number of CGMCC No.20792.
The third object of the present invention is to provide a dimethachlon hapten, which has the following structural formula:
the fourth object of the present invention is to provide a method for synthesizing a dimethachlon hapten, wherein the dimethachlon hapten is obtained by hydrolyzing an addition reaction product of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione and ethyl 3-mercaptopropionate, and the method comprises the following steps:
1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione was dissolved in dimethyl sulfoxide and Na was added 2 CO 3 Adding 3-ethyl mercaptopropionate under stirring, heating to 50-70 ℃, stirring and reacting for 20-30 h, cooling to room temperature after the reaction is complete, and concentrating; adding NaOH solution, heating to 70-90 ℃ and stirring for reaction for 0.5-2 h; adding proper amount of pure water, regulating pH to acidity, extracting, purifying and drying to obtain brown solid which is hapten.
In an embodiment of the invention, the 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione is reacted with Na in a ratio by weight of the material 2 CO 3 The ratio of the 3-mercaptopropionic acid ethyl ester is 1:1-3:1-2.
In an embodiment of the present invention, the 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione is reacted with Na 2 CO 3 The ratio of the ethyl 3-mercaptopropionate is 1:1.2:1.2.
In an embodiment of the present invention, the concentration of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione dissolved in the dimethylsulfoxide is 0.01 to 0.05g/mL.
In an embodiment of the invention, the concentration of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione dissolved in the dimethyl sulfoxide is 0.02g/mL.
In an embodiment of the invention, after the addition of ethyl 3-mercaptopropionate, the mixture is heated to 60℃and stirred for 24 hours.
In the embodiment of the invention, the NaOH solution is added to be heated and stirred for reaction, and 1mol L of the NaOH solution is added -1 The NaOH solution was heated to 80℃and reacted with stirring for 1h.
In an embodiment of the present invention, the mass ratio of NaOH to the 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione is 1 to 6:1.
In an embodiment of the invention, the mass ratio of NaOH to the 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione is 5:1.
In an embodiment of the present invention, the pH is adjusted to be acidic, and the pH is adjusted to 4 to 5 with HCl solution.
In an embodiment of the present invention, the hapten is prepared by dissolving 1equ.1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione in dimethyl sulfoxide and adding 1.2equ.Na 2 CO 3 Adding 1.2 equ.3-ethyl mercaptopropionate under stirring, heating to about 60 ℃, stirring, cooling to room temperature after the reaction is complete, and concentrating; adding NaOH solution with the volume of 5 times of that of dimethyl sulfoxide, heating to 80 ℃, and stirring for reaction for 1h; adding proper amount of pure water, regulating the PH to 4-5 by using HCl solution, extracting, purifying and drying to obtain brown solid which is hapten.
A fifth object of the present invention is to provide a sclerostin complete antigen having the structural formula:
wherein carrier protein is a carrier protein.
The sixth object of the present invention is to provide a method for preparing a complete antigen of sclerostin, wherein the complete antigen comprises a complete immunogen of sclerostin and a complete antigen of sclerostin, the complete antigen is obtained by coupling a PD hapten with a carrier protein, wherein the carrier protein comprises bovine serum albumin BSA and ovalbumin OVA, and the PD hapten has the structural formula of
In an embodiment of the invention, the method comprises the steps of: hapten PD is dissolved in anhydrous N, N-dimethylformamide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDCI and N-hydroxysuccinimide NHS are sequentially added, and stirring reaction is carried out at room temperature for 0.3-1 h, which is called solution A; weighing bovine serum albumin BSA, and adding boric acid buffer solution called solution B; slowly dropwise adding the solution A into the solution B under stirring, and reacting for 1-3 hours at room temperature to obtain a mixed solution containing PD-BSA; dialyzing to obtain the sclerostin complete antigen.
In the embodiment of the invention, the mass ratio of the hapten PD to the EDCI and the NHS is 1:1-1.5:1-1.5, and the concentration of the hapten PD in anhydrous N, N-dimethylformamide is 10-15 g L -1 。
In an embodiment of the present invention, the ratio of the hapten PD to the EDCI and NHS is 1:1.2:1.2, and the concentration of hapten PD in anhydrous N, N-dimethylformamide is 13g L -1 。
In an embodiment of the invention, the ratio of the hapten PD to the carrier protein is in the amount of 30-80:1.
In an embodiment of the invention, the ratio of the hapten PD to the carrier protein is 50:1.
In an embodiment of the present invention, the volume ratio of the anhydrous N, N-dimethylformamide to the boric acid buffer solution is 1:5-15.
In an embodiment of the invention, the volume ratio of the anhydrous N, N-dimethylformamide to the boric acid buffer solution is 1:10.
According to the preparation method of the sclerostin monoclonal antibody hybridoma cell strain, after the sclerostin complete antigen immunized mice are cultured by a cell fusion and HAT selective medium, the sclerostin monoclonal antibody hybridoma cell strain is established. Wherein the sclerostin complete antigen comprises an immunogen and a coating antigen, and is obtained by coupling hapten PD and carrier protein, wherein the carrier protein comprises Bovine Serum Albumin (BSA) and Ovalbumin (OVA), and the corresponding sclerostin immunogen comprises PD-BSA and PD-OVA; the hapten PD is obtained by purifying an addition reaction product of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione and 3-mercaptopropionic acid ethyl ester.
Taking PD-BSA immunogens as an example, the basic steps are:
(1) Preparation and identification of immunogens: hydrolyzing an addition reaction product of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione and ethyl 3-mercaptopropionate to obtain PD, coupling the hapten with carrier proteins comprising BSA and OVA respectively by adopting a carbodiimide method to prepare immunogen and coating antigen, separating complete antigen, unconjugated hapten, NHS and urea generated by reaction by dialysis, wherein the separated complete antigen is sclerotium net immunogen and coating antigen, the immunogen PD-BSA is used for preparing hybridoma cell strains by mouse immunization, and the coating antigen PD-OVA is used for subsequent kit detection;
(2) Immunization of mice: immunization of mice was performed by subcutaneous injection of white laboratory mice, i.e., BALB/c mice, via the nuchal back using Freund's adjuvant, freund's incomplete adjuvant, PD-BSA immunogen to obtain dimethachlon immunized mice. First immunization (100 mug/dose) uses an equal volume mixed emulsion of Freund's complete adjuvant and dimethachlon immunogen as an injection, and several times of boosting uses an equal volume mixed emulsion of Freund's incomplete adjuvant and dimethachlon immunogen as an injection, wherein the interval between the first immunization and the first boosting is one month, and the interval between the multiple boosting is 21 days; the final immunization was performed by sprinting with 50. Mu.L of PD-BSA immunogen (25. Mu.g/dose, without adjuvant) diluted to 0.5mg/mL with physiological saline; serum titers and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (iceelisa); the number of boosting is 4-6, preferably 5;
(3) Cell fusion and cell strain establishment: fusing spleen cells and myeloma cells of a sclerotium-immunized mouse by a polyethylene glycol (PEG 4000) 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 icELISA method, subcloning the positive cell holes with the best inhibition for three times by using a limiting dilution method, and finally screening to obtain a hybridoma cell strain SLS 2G5;
(4) Identification of hybridoma cell line properties: identification of antibody subtype adopts mouse monoclonal antibody immune colloid Jin Yaxing kit method, IC 50 Values, cross-reactivity and affinity constants were determined by ELISA.
It is another object of the present invention to provide the use of the above-described sclerostin monoclonal antibodies for analytical detection of sclerostin residues in food safety detection.
In an embodiment of the present invention, the dimethachlon monoclonal antibody is used for preparing a detection subject for analysis and detection of dimethachlon residual quantity in food safety detection.
In an embodiment of the present invention, the detection body includes a reagent, a detection plate, and a kit.
In the embodiment of the invention, when the dimethachlon immunogen is PD-BSA, the corresponding dimethachlon coating immunogen is preferably PD-OVA, and the dimethachlon immunogen has better specificity and detection sensitivity.
The invention has the beneficial effects that: firstly, the invention makes up the technical blank of the field of the detection of the sclerostin, and firstly provides a preparation method of a cell strain capable of secreting the sclerostin monoclonal antibody, in particular to a hapten used and a preparation method of the hapten. Secondly, the monoclonal antibody secreted by the cell strain SLS 2G5 provided by the invention has better specificity and detection sensitivity (IC) to the sclerotin 50 The value was 3.2. Mu. g L -1 ) The method can realize the detection of the dimethachlon residue, provides raw materials for the immunodetection of the dimethachlon residue in food, and has practical application value.
Drawings
FIG. 1 is a subtype identification of a sclerostin monoclonal antibody;
FIG. 2 is a monoclonal antibody affinity assay;
FIG. 3 is a standard curve of inhibition of dimethachlon by monoclonal antibodies.
Detailed Description
The invention will be further described with reference to the drawings and examples.
Reagents used in the examples of the present invention:
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): 8.0g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 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. Mixing the solution B according to the ratio of 1:5 to obtain TMB color development liquid, and mixing the solution B immediately before use.
Examples
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
The monoclonal antibody hybridoma cell strain with higher sensitivity to the sclerotinia sclerotiorum is finally obtained by immunizing a mouse with the sclerotinia sclerotiorum complete antigen, performing cell fusion, culturing in a HAT selective medium and screening cell supernatant by using an ICELISA.
1. Synthesis of hapten
The synthesis route of hapten PD is as follows:
0.10g (0.4 mmol) of 1- (3, 5-dichlorophenyl) -1H-pyrrole-2, 5-dione are dissolved in 5mL of dimethyl sulfoxide and 0.05g (0.48 mmol) of Na are added 2 CO 3 Stirring for 0.5h. Then, 0.065g (0.48 mmol) of 3-mercaptoethyl ester was added with stirring, heated to 60℃and stirred for 24h, monitored by TLC. Stopping heating until the reaction liquid is cooled to room temperature and concentrating; 2mL of 1mol L was added -1 Heating NaOH solution to 80 ℃ and stirring for reaction for 1h; after the reaction mixture had cooled to room temperature, 3mL of pure water was added thereto, and 1mol L of the mixture was used -1 HCl is adjusted to pH 4, extracted with ethyl acetate, and anhydrous Na 2 SO 4 Drying and concentrating; purifying with silica gel column, concentrating, and drying to obtain brown solid, i.e. hapten PD.
2. Preparation of complete antigen: and (3) coupling hapten PD prepared in the step (1) with Bovine Serum Albumin (BSA) to obtain complete antigen PD-BSA.
The preparation method of the complete antigen PD-BSA comprises the following steps:
a. 2.6mg (0.0075 mmol) of hapten PD prepared in step 1 is weighed and dissolved in 200 mu L of anhydrous N, N-dimethylformamide, 1.7mg (0.009 mmol) of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1.0mg (0.009 mmol) of N-hydroxysuccinimide (NHS) are sequentially added, and the mixture is stirred and reacted for 0.5h at room temperature, which is called solution A; 10mg (0.00015 mmol) of bovine serum albumin BSA was weighed and 2mL of boric acid buffer solution, designated as solution B, was added; slowly dropwise adding the solution A into the solution B under stirring, and reacting for 2 hours at room temperature to obtain a mixed solution containing PD-BSA;
b. and (3) dialysis: taking a 10cm dialysis bag, boiling in boiling water for 5min, washing with deionized water at 60 ℃ for 3min, and preserving in deionized water at 4 ℃ for later use; placing the PD-BSA-containing mixture obtained in step (a) in a dialysis bag with 0.01mol L -1 PBS is used as a dialysate for 3d dialysis at 4 ℃, and the dialysate is changed three times a day to separate complete antigen from unconjugated hapten and other small molecular substances, so that the complete antigen is obtained, and the structure is as follows:
complete antigens were prepared including immunogen and coating antigen, wherein the immunogen PD-BSA was used for the next step of mouse immunization.
The preparation method of the coating original PD-OVA is the same as that of PD-BSA, and is used for detecting in the subsequent step 7.
3. Immunization of mice: PD-BSA immunogen was mixed with an equal volume of Freund's adjuvant and emulsified to obtain an injection, and BALB/c mice were injected subcutaneously through the nape of the neck.
First immunization (100. Mu.g/dose) with equal volume of mixed emulsion of Freund's complete adjuvant and PD-BSA immunogen as injection, and 5 booster immunization (50. Mu.g/dose) with equal volume of mixed emulsion of Freund's incomplete adjuvant and PD-BSA immunogen as injection. One month is separated from the first immunization and the first boosting, and 21 days is separated from the multiple boosting. The final immunization was performed by sprinting with PD-BSA immunogen (25. Mu.g/dose, without adjuvant) diluted to a concentration of 0.5mg/mL with physiological saline; serum titers and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (iceelisa).
4. Cell fusion
After three days of sprint immunization, cell fusion was performed according to the conventional PEG method, specifically as follows:
a. collecting eyeball to remove blood, killing a sclerotium-free immunized mouse by a cervical dislocation method, immediately putting the immunized mouse into 75% alcohol for sterilization, soaking for about 5min, taking out spleen of the mouse by aseptic operation, moderately grinding the spleen with a rubber head of a syringe, obtaining spleen cell suspension through a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells with RPMI-1640 culture medium for three times, and diluting the spleen cells to a certain volume after the last centrifuging, and counting for later use;
b. collecting murine myeloma SP2/0 cells: 7-10 days prior to fusion, SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) in RPMI-1640 medium at 5% C0 2 In an incubator. The number of SP2/0 tumor cells before fusion reaches 1 to 4 multiplied by 10 7 Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. the fusion process was 7min. 1min, 1mPEG 1500 of L was added dropwise to cells from slow to fast; and (2) 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. Then, the mixture was incubated 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.
5. Cell screening and cell strain establishment
The fused cells were subjected to a half-change of 2% of 50 XHAT RPMI-1640 medium on day 3 of cell fusion, a full-change of 20% fetal bovine serum and 1% of 100XHT RPMI-1640 medium 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 iceELISA, and the second step is to use the dimethachlon as a standard substance, and to measure the inhibition effect of positive cells by using the iceELISA. Cell holes with better inhibition on the sclerotium net standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. The monoclonal hybridoma cell line SLS 2G5 was obtained by repeating the above steps three times.
6. Preparation and identification of sclerostin monoclonal antibodies
Taking 8-10 week old sclerotium net immunized BALB/c mice, and injecting 1mL of sterile paraffin oil into each mouse intraperitoneally; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the octanoic acid-ammonium sulfate method. Finally, the purified sclerotium-purified monoclonal antibody is obtained and stored at the temperature of minus 20 ℃.
Immunoglobulin subtype identification is carried out on the sclerotium net monoclonal antibody obtained by ascites purification by using a mouse monoclonal antibody subtype identification kit, the subtype is IgG2b type, and the light chain type is kappa type detected by the mouse monoclonal antibody subtype identification kit, as shown in figure 1.
Calculation of affinity constants using indirect ELISA assay: the concentration of the antibody corresponding to OD450nm at different antigen concentrations was calculated by plotting the concentration of the antibody against OD450nm, and when the concentration of the antibody at OD450nm was 50%The concentration of the monoclonal antibody was substituted into the formula Ka= (n-1)/2 (nAb' -Ab) to calculate the affinity constant Ka (Lmol) -1 ). Ab and Ab 'represent the antibody concentrations (mol L) corresponding to OD450nm when the antigen concentrations are Ag and Ag', respectively -1 ) n=ag/Ag'. As shown in FIG. 2, the affinity of the sclerotium net monoclonal antibody was 9.73X10 8 L mol -1 。
The results show that the prepared dimethachlon monoclonal antibody has higher affinity and sensitivity, and can be used for dimethachlon immunoassay detection and preparation of an affinity column.
7. Antibody application
The monoclonal antibody of the sclerostin prepared by the hybridoma cell strain SLS 2G5 through in vivo ascites is applied to an additive recovery test of the sclerostin, and the specific steps are as follows:
7.1 coating: the coating antigen PD-OVA obtained in the step 2 is used for 0.05mol L -1 The pH 9.6 carbonate buffer was diluted to 0.1. Mu.g/mL, 100. Mu.L/well, and reacted at 37℃for 2 hours.
7.2 washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
7.3 closing: 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
7.4 sample addition: 100 μl PBS was added to the positive control wells; 100 mu L of the detection hole is added with 0.3 to 50 mu gL of concentration -1 Sclerotium solution standard. Then, the sclerotium-purifying monoclonal antibody was used in an amount of 0.01mol L -1 PBS was diluted to 0.10.1. Mu.g/mL and added to each dilution of the coated wells, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, HRP-goat anti-mouse IgG was added at 1:3000 dilution, 100. Mu.L/well, and reacted at 37℃for 30min.
7.5 color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
7.6 termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD of each well was measured with a microplate reader 450 Values.
Sensitivity to sclerotium net was detected using an iceelisa as shown in fig. 3 according to standard equation y= 0.01424+ (1.6311-0.01424) (1+x/2.8444) 0.96092 (R 2 = 0.9981), calculate IC 50 Is 3.2 mu g L -1 。
IC for determining sclerotium net monoclonal antibody by using ICELISA 50 Is 3.2 mu g L -1 The method has high sensitivity to the dimethachlon and can be used for the dimethachlon immunoassay detection.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (5)
1. A hybridoma cell strain secreting a sclerostin monoclonal antibody, characterized in that: the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), and has a preservation name of a monoclonal cell strain SLS 2G5, a preservation number of CGMCC No.20792 and a preservation date of 2020, 09 and 27 days.
2. A sclerostin monoclonal antibody, which is secreted by a hybridoma cell line SLS 2G5 having a collection number of CGMCC No.20792 according to claim 1.
3. The use of a monoclonal antibody to dimethachlon as claimed in claim 2, for analytical detection of dimethachlon residues in food safety tests.
4. The use of a monoclonal antibody to sclerostin according to claim 3, for preparing a test body for analytical detection of the amount of sclerostin residue in food safety tests.
5. The use of the monoclonal antibody of claim 4, wherein the test body comprises a reagent, a test plate, and a kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111096426.8A CN113717948B (en) | 2021-09-16 | 2021-09-16 | Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111096426.8A CN113717948B (en) | 2021-09-16 | 2021-09-16 | Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113717948A CN113717948A (en) | 2021-11-30 |
| CN113717948B true CN113717948B (en) | 2023-09-15 |
Family
ID=78684249
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111096426.8A Active CN113717948B (en) | 2021-09-16 | 2021-09-16 | Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113717948B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691404A (en) * | 2009-10-20 | 2010-04-07 | 江苏省农业科学院 | Polyclonal antibody of dimethachlon and preparation and application thereof |
| CN109265401A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of iprodione haptens and antigen |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
| CN112877296A (en) * | 2021-03-01 | 2021-06-01 | 江南大学 | Anti-phenacetin monoclonal antibody hybridoma cell strain AD and preparation method and application thereof |
-
2021
- 2021-09-16 CN CN202111096426.8A patent/CN113717948B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101691404A (en) * | 2009-10-20 | 2010-04-07 | 江苏省农业科学院 | Polyclonal antibody of dimethachlon and preparation and application thereof |
| CN109265401A (en) * | 2018-09-21 | 2019-01-25 | 中国烟草总公司郑州烟草研究院 | A kind of preparation method and application of iprodione haptens and antigen |
| CN112375744A (en) * | 2020-12-04 | 2021-02-19 | 江南大学 | Dihydropyridine monoclonal antibody hybridoma cell strain and application thereof |
| CN112877296A (en) * | 2021-03-01 | 2021-06-01 | 江南大学 | Anti-phenacetin monoclonal antibody hybridoma cell strain AD and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113717948A (en) | 2021-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111304174A (en) | Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof | |
| CN112877296B (en) | Anti-phenacetin monoclonal antibody hybridoma cell strain AD and preparation method and application thereof | |
| CN110607283A (en) | Hybridoma cell strain CBC for secreting monoclonal antibody of dicofol and application thereof | |
| CN107119022B (en) | Isobacteriumurea monoclonal antibody hybridoma cell strain ZXL-2 and application thereof | |
| CN110117575B (en) | Pyrimidinemonoclonal antibody hybridoma cell strain HFG and application thereof | |
| CN112574956B (en) | Hybridoma cell strain secreting propamocarb monoclonal antibody and application thereof | |
| CN112458062B (en) | Ethyl maltol monoclonal antibody hybridoma cell strain and application thereof | |
| CN110343669B (en) | Hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof | |
| CN108456661A (en) | One plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its application | |
| CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
| CN109438424B (en) | Ribavirin hapten and artificial antigen as well as preparation method and application thereof | |
| CN113717948B (en) | Hybridoma cell strain secreting anti-sclerotinia monoclonal antibody and application thereof | |
| CN113637642B (en) | Hybridoma cell strain secreting chlorfenapyr monoclonal antibody and application thereof | |
| CN116675696A (en) | Saxitoxin monoclonal antibody, and preparation method and application thereof | |
| CN110927382A (en) | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof | |
| CN111748528B (en) | Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain | |
| CN113005097B (en) | Hybridoma cell strain secreting anti-carbamazepine monoclonal antibody and application thereof | |
| CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN110616193B (en) | Hybridoma cell strain BCB secreting anti-sodium cyclamate monoclonal antibody and application thereof | |
| CN108330102B (en) | Tiamulin monoclonal antibody hybridoma cell strain and application thereof | |
| CN114989144B (en) | Difenoconazole hapten, complete antigen, antibody and preparation method and application thereof | |
| CN114807051B (en) | Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof | |
| CN106929477B (en) | Anti-prostaglandin F2αSpecific monoclonal antibody hybridoma cell strain WXX-2 and application thereof | |
| CN110616194A (en) | Aconitine monoclonal antibody cell strain SJ and application thereof | |
| CN114317450B (en) | Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |