CN111748528B - Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain - Google Patents
Hybridoma cell strain secreting monoclonal antibody against fipronil and metabolite thereof and application of hybridoma cell strain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three 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
- C07D231/44—Oxygen and nitrogen or sulfur and nitrogen atoms
- C07D231/52—Oxygen atom in position 3 and nitrogen atom in position 5, or vice versa
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- 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
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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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 monoclonal antibodies against fipronil and its metabolites and application thereof belong to the technical field of food safety immunodetection. The invention discloses a hybridoma cell strain ABC10 secreting monoclonal antibodies against fipronil and its metabolites, which has been deposited in China general microbiological culture Collection center (CGMCC), is classified and named as a monoclonal cell strain, and has a preservation date of 2019, 11 and 28 months and a preservation number of CGMCC No. 19168. High potency, low IC50The mouse spleen cell is obtained by fusing a PEG method with a myeloma cell, and screening and three times of subcloning through an indirect competitive enzyme linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better affinity and detection sensitivity to fipronil and metabolites thereof500.46 mu g/L), can be used for immunodetection of the total residue of fipronil and metabolites thereof in food.
Description
Technical Field
The invention relates to a hybridoma cell strain secreting monoclonal antibodies against fipronil and its metabolites and application thereof, and belongs to the technical field of food safety immunodetection.
Background
Fipronil (FPN) is a benzopyrazole broad-spectrum high-efficiency pesticide and is widely applied to agricultural and non-agricultural fields and pet care products. Research shows that the kidney and the liver of a human being are damaged when the food containing a certain concentration of fipronil is eaten, meanwhile, the influence of the fipronil serving as a neurotoxic insecticide on the human being or other mammals is larger, and the metabolites of the fipronil, fipronil sulfone and fipronil thioether are proved to have far higher toxicity on the mammals than the fipronil per se. Fipronil is therefore classified by the world health organization as a "moderately toxic" chemical to humans. GB 2763-2019 food safety national standard food maximum residual limit of pesticide stipulates that the sum of fipronil and metabolites thereof is in eggs, the limit of vegetables is 0.02mg/kg, and the Maximum Residual Limit (MRL) in grains and oil is as low as 0.002 mg/kg.
An efficient and sensitive detection method for fipronil and its metabolites is an urgent problem to be solved. Current detection methods include instrumental assays and immunoassays. Instrumental analysis methods such as high performance liquid chromatography, high performance liquid chromatography tandem mass spectrometry and the like are not suitable for field detection due to the complex pretreatment of samples, more interferents, limitation on working conditions of instruments and higher technical requirements on operators. Compared with an instrument detection method, the immunoassay method has the characteristics of low cost, high flux, high sensitivity, low requirement on technical personnel and the like, so that the immunoassay method is suitable for rapid screening of a large number of samples. Therefore, the immunodetection method has important significance for fipronil and the metabolite thereof. The enzyme-linked immunosorbent assay (ELISA) is a low-cost, rapid and portable immunological detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for rapidly detecting results on the spot of a large number of samples. Establishing an efficient immunological detection method, and screening a monoclonal antibody with high specificity is an important prerequisite.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain secreting monoclonal antibodies against fipronil and its metabolites and application thereof.
According to the technical scheme, the hybridoma cell strain ABC10 secreting monoclonal antibodies against fipronil and its metabolites has been deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, No. 3, of Suzuolu No.1, North Chen West Lu, No. 3, of the Chaoyang district, Beijing, and is classified and named as a monoclonal cell strain, the preservation date is 2019, 11 months and 28 days, and the preservation number is CGMCC No. 19168.
The monoclonal antibody of fipronil and its metabolite is secreted by the hybridoma cell strain ABC10 with the preservation number of CGMCC number 19168.
The application of the monoclonal antibody for resisting fipronil and the metabolite thereof is used for analyzing and detecting the total residue of fipronil and the metabolite thereof in food safety detection.
The basic steps for preparing the hybridoma cell strain ABC10 secreting monoclonal antibodies against fipronil and its metabolites provided by the invention are as follows:
(1) preparation and identification of immunogen: hydrolyzing fipronil in NaOH solution to prepare corresponding hapten FPN-COOH, condensing with 6- (tert-butoxy) -6-oxohexanoic acid to prepare hapten FPN-HA, coupling the hapten with carrier protein by using a carbodiimide method to prepare immunogen and envelope antigen, and separating complete antigen and uncoupled micromolecular hapten by dialysis;
(2) immunization of mice: after the fipronil-BSA complete antigen is mixed and emulsified with an equal volume of Freund's adjuvant, BALB/c mice are injected subcutaneously through the back and the neck. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for multiple booster immunizations. The interval between the first immunization and the second boosting immunization is one month, and the interval between the boosting immunization is 21 days. The final puncture immunization with fipronil-BSA complete antigen (without adjuvant); by indirect competitive enzyme-linked immunization (icELISA) to detect serum titer and inhibition rate;
(3) cell fusion and cell line establishment: fusing mouse spleen cells and mouse myeloma cells by polyethylene glycol (PEG 4000) method, culturing in HAT medium, detecting positive cell pores by indirect ELISA, and further usingicThe inhibition effect of the positive cell hole is determined by an ELISA method, the positive cell hole with the best inhibition is subcloned for three times by a limiting dilution method, and finally a hybridoma cell strain ABC10 is obtained by screening;
(4) and (3) identification of the properties of hybridoma cell strains: the identification of antibody subtype adopts the method of mouse monoclonal antibody immune colloidal gold subtype kit, IC50Values, cross-reactivity and affinity constants were determined by ELISA.
The invention has the beneficial effects that: the monoclonal antibody secreted by the cell strain ABC10 has better specificity and detection sensitivity (IC) on fipronil and its metabolites50The values are 0.46, 1.76, 0.94 and 2.30 mu g/L), the detection of the total quantity of fipronil and the residual metabolites thereof in fruits, vegetables and grains can be realized, raw materials are provided for the immunodetection of the fipronil and the residual metabolites thereof in food, and the method has practical application value.
Biological material sample preservation: a hybridoma cell strain ABC10 secreting monoclonal antibodies against fipronil and its metabolites is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, No. 3, Xilu No.1, North Cheng, the south of the republic of Beijing, and is classified and named as monoclonal cell strain with the preservation date of 2019, 11 months and 28 days and the preservation number of CGMCC No. 19168.
Drawings
FIG. 1 is a diagram of the subtype identification of the ABC10 monoclonal antibody.
FIG. 2A standard curve for the inhibition of the ABC10 monoclonal antibody against fipronil and its metabolites.
Detailed Description
The following examples are intended to be illustrative of the scope of the present invention and are not intended to be a limitation or scope of the present invention. The invention is further illustrated by the following examples.
The invention immunizes mice with fipronil complete antigen, and then the mice are cultured by cell fusion, HAT selective medium andicand screening the cell supernatant by ELISA to finally obtain the monoclonal antibody hybridoma cell strain ABC10 with higher sensitivity to fipronil and its metabolites.
EXAMPLE 1 preparation of hybridoma cell line ABC10
1. Synthesis of hapten, the reaction equation is as follows:
the derivation procedure is briefly as follows:
(1) 200 mg of fipronil is dissolved in 1mL of acetone and placed in a round-bottom flask, 20 mL of 0.5 mol/L NaOH solution is added dropwise with stirring, and the mixture is reacted in a water bath at 60 ℃ for 24 hours. The reaction solution gradually changed from turbid to light yellow clear solution, cooled to room temperature, and the pH of the reaction solution was adjusted to about 5 with 1.0 mol/L hydrochloric acid in an ice bath. Light yellow solid is precipitated, filtered under reduced pressure, washed 3 times with pure water and dried. The product obtained is FPN-COOH and is stored at 4 ℃ for later use.
And (2) 200 mg of fipronil and 141.4 mg of 6- (tert-butoxy) -6-oxohexanoic acid were dissolved in 6 mL of dichloromethane, and 134.2 mg of 1-ethylcarbodiimide hydrochloride (EDC) and 80.6 mg of 1-ethylcarbodiimide hydrochloride (NHS) were added successively with stirring to react at room temperature for 3 d. Then, under ice-bath, 1.0 mL of trifluoroacetic acid (TFA) was slowly added dropwise and reacted at room temperature for 1 h. Washing with pure water for 3 times, collecting organic phase, and evaporating to dryness under reduced pressure to obtain product FPN-HA.
2. Preparation of complete antigen:
preparation of conjugate fipronil-BSA: 3.2 mg of the thus prepared FPN-COOH was weighed out and dissolved in 200. mu.L of DMF, and 1.6 mg of 1-ethylcarbodiimide hydrochloride and 1.0 mg of N-hydroxysuccinimide were added in this order with stirring and reacted at room temperature for 0.5 hour to obtain a mixture called solution A. Then, 6.0 mg of bovine serum albumin was dissolved in 2mL of a boric acid buffer solution, referred to as solution B. Slowly adding the solution A into the solution B, and reacting for 4 hours at room temperature under stirring. Dialyzing with 0.01 mol/L PBS for 3 d, separating complete antigen and unconjugated small molecule, and collecting supernatant to obtain conjugate fipronil-BSA, and freezing at-20 deg.C.
Preparation of coated fipronil-OVA: 6.0 mg of FPN-HA, 2.5 mg of EDC and 1.5 mg of NHS were weighed out, dissolved in 300. mu.L of anhydrous N, N-dimethylformamide and reacted for 6 hours with stirring at room temperature (referred to as solution C). Weighing 10 mg of chicken egg albumin OVA, dissolving the OVA in 2mL of boric acid buffer solution (called solution D), dropwise adding the solution C into the solution D at room temperature, stirring at room temperature for reacting overnight to obtain a conjugate fipronil-OVA mixed solution, and separating complete antigen and unconjugated hapten through dialysis.
3. Of miceImmunization: after the fipronil-BSA complete antigen is mixed and emulsified with an equal volume of Freund's adjuvant, BALB/c mice are injected subcutaneously through the back and the neck. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the multiple booster immunizations. The interval between the first immunization and the second boosting immunization is one month, and the interval between the boosting immunization is 21 days. The final puncture immunization with fipronil-BSA complete antigen (without adjuvant); by indirect competitive enzyme-linked immunization (icELISA) to detect serum titers and inhibition rates.
4. Cell fusion: after three days of spurting immunization, cell fusion is carried out according to a conventional PEG method, and the specific steps are as follows:
a. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleens of the mice through aseptic operation, properly grinding the spleens by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a splenocytes suspension, collecting, centrifuging (1200 rpm, 8 min), washing the splenocytes for three times by using an RPMI-1640 culture medium, diluting the splenocytes to a certain volume after the last centrifugation, and counting for later use;
b. collecting mouse myeloma SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO2Culturing in an incubator. Before fusion, the number of SP2/0 tumor cells is required to reach (1-4) x 107Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;
c. the fusion process is 7 min. 1min, 1mL of PEG4000 was added to the cells dropwise from slow to fast; standing for 2 min. Dropping 1mL of RPMI-1640 culture medium within 1min at 3min and 4 min; at 5min and 6min, 2mL of RPMI-1640 culture medium is added dropwise within 1 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5 min. Centrifuging (800 rpm, 8 min), discarding supernatant, resuspending in RPMI-1640 screening medium containing 20% fetal calf serum, 2% 50 × HAT, adding to 96-well cell plate at 200 μ L/well, standing at 37 deg.C and 5% CO2Culturing in an incubator.
5. Cell screeningAnd establishing a cell strain: on day 3 of cell fusion, 2% of half-change culture medium was selected from the fused cells in RPMI-1640 of 50 XHAT, on day 5, full-change culture was performed in RPMI-1640 containing 20% fetal bovine serum and 1% of 100 XHT, and on day 7, cell supernatants were collected and selected. The screening is divided into two steps: first step usingicELISA screening positive cell hole, the second step using fipronil as standard substanceicThe ELISA was used to determine the inhibitory effect of positive cells. Selecting cell pores which can well inhibit the fipronil and the metabolite standard substance thereof, adopting a limiting dilution method to perform subcloning, and detecting by using the same method. Repeating the steps for three times to obtain a monoclonal hybridoma cell strain ABC 10.
6. Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 106Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-ammonium sulfate method. The purified monoclonal antibody is finally obtained and stored 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 IgG2b type, and the light chain type is kappa type. The results of the subtype identification are shown in FIG. 1.
Use of indirect ELISA andicELISA, the affinity of the monoclonal antibody was determined to be 7.63X 109L/mol IC of p-fipronil, fipronil sulfone, fipronil sulfide50Respectively as follows: 0.46, 1.76, 0.94 and 2.30 mu g/L. The results show that the prepared monoclonal antibody has higher affinity, has better sensitivity to fipronil and metabolites thereof, and can be used for immunoassay detection of the total amount of the fipronil and the metabolites thereof and preparation of an affinity column.
7. The application of the antibody comprises the following steps: the monoclonal antibody prepared from the hybridoma cell strain ABC10 through in vivo ascites is applied to an addition recovery test of fipronil, and the method specifically comprises the following steps:
7.1 coating: 0.05 mol L of coated fipronil-OVA-1The carbonate buffer solution with the pH of 9.6 is diluted by a multiple ratio from 1 mu g/mL, 100 mu L/hole and reacted at 37 DEG C2h；
7.2 washing: the plate solution was decanted and washed 3 times for 3min each with washing solution;
7.3 sealing: add 200. mu.L/well blocking solution and react at 37 ℃ for 2 h. Drying for later use after washing;
7.4 sample adding: diluting antiserum from 1:1000 times, adding into coated wells of each dilution, reacting at 37 deg.C for 30min at 100 μ L/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 mu L/hole, and reacting for 30min at 37 ℃;
7.5 color development: taking out the ELISA plate, fully washing, adding 100 mu L of TMB color development solution into each hole, and reacting for 15min at 37 ℃ in a dark place;
7.6 termination and determination: the reaction was stopped by adding 50. mu.L of a stop solution to each well, and the OD of each well was measured by a microplate reader450The value is obtained.
By usingicIC of ELISA (enzyme-Linked immuno sorbent assay) determination monoclonal antibody on fipronil50The concentration is 0.46 mu g/L, which indicates that the kit has high sensitivity to fipronil and can be used for immunoassay detection of fipronil and its metabolites.
The standard curve for the inhibition of the ABC10 monoclonal antibody on fipronil is shown in FIG. 2. IC of ABC10 monoclonal antibody to fipronil, fipronil sulfone and fipronil thioether50And the cross-reactivity are shown in table 1.
TABLE 1
Solution preparation: carbonate Buffer (CBS): weighing Na2CO3 1.59 g，NaHCO32.93 g of the mixture is respectively dissolved in a small amount of double distilled water and then mixed, the double distilled water is added to about 800mL and mixed evenly, the pH value is adjusted to 9.6, the double distilled water is added to the volume of 1000mL, and the mixture is stored for later use at 4 ℃;
phosphate Buffered Saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH2PO4，2.9g Na2HPO4·12 H2Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and fixing the volume to 1000 mL;
PBST: PBS containing 0.05% tween-20;
TMB color development liquid: solution A: na (Na)2HPO4 .12H218.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. Mixing the liquid B according to the volume ratio of 1:5 to obtain the TMB color developing solution, and mixing the liquid B at the present time.
Claims (3)
1. A hybridoma cell strain ABC10 secreting monoclonal antibodies against fipronil and its metabolites is deposited in China general microbiological culture Collection center (CGMCC), China academy of sciences, institute of microbiology, No. 3, Xilu No.1, North Cheng, the south of the republic of Beijing, and is classified and named as monoclonal cell strain with the preservation date of 2019, 11 months and 28 days and the preservation number of CGMCC No. 19168.
2. The monoclonal antibody for resisting fipronil and its metabolite is characterized by that: the hybridoma cell strain ABC10 with the preservation number of CGMCC number 19168 as claimed in claim 1 secretes and produces the hybridoma cell strain ABC 10.
3. The use of monoclonal antibodies against fipronil and its metabolites according to claim 2, characterized in that: the method is used for analyzing and detecting the total residue of fipronil and metabolites thereof in food safety detection.
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