CN114317450B - Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof - Google Patents

Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof Download PDF

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CN114317450B
CN114317450B CN202210051794.9A CN202210051794A CN114317450B CN 114317450 B CN114317450 B CN 114317450B CN 202210051794 A CN202210051794 A CN 202210051794A CN 114317450 B CN114317450 B CN 114317450B
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fipronil
amide
compound
hapten
monoclonal antibody
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CN114317450A (en
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胥传来
潘晓敏
匡华
刘丽强
徐丽广
朱建平
郝昌龙
宋珊珊
胡拥明
吴爱红
郭玲玲
胥欣欣
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Jiangnan University
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Abstract

The application discloses a hybridoma cell strain secreting a fluorobenzamide monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection. The complete antigen of the fipronil amide is completely mixed and emulsified with the equivalent Freund's adjuvant, and BALB/c mice are immunized by subcutaneous injection on the back. The first immunization was performed with complete Freund's adjuvant, multiple booster immunization was performed with incomplete Freund's adjuvant, and the last immunization was performed with the complete antigen of fipronamide. High potency low IC 50 The spleen cells of the mice are fused with myeloma cells of the mice by a PEG method, and a hybridoma cell strain Ta 4C1 with a preservation number of CGMCC No.45020 is obtained through screening and three subcloning by an indirect competitive enzyme-linked immunosorbent assay. The monoclonal antibody secreted by the cell strain has better specificity and detection sensitivity (IC) 50 The value is 2.4 ng/mL), provides raw materials for immunodetection of the residual fipronil in food, and has practical application value.

Description

Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof
Technical Field
The application relates to a hybridoma cell strain secreting a fluorobenzamide monoclonal antibody and application thereof, and belongs to the field of food safety immunodetection.
Background
Flubendiamide (Flubendiamide), also known as fipronamide, fipronil, belongs to the class of bisamide pesticides, is the first commercial product in the class of pesticides, and belongs to a novel phthalic diamide pesticide. The flubendiamide has stomach toxicity and contact killing effects, has no systemic property, is efficient and broad-spectrum, and acts on the ryanodine receptor of insects. The main application crops thereof comprise: soybean, vegetable, fruit tree, corn, cotton, gourd, grape, nut tree, rice, tobacco, tea tree, etc.
However, fipronil increasingly exposes potential risks to environmental non-target organisms such as natural enemy insects, silkworm predatory mites, and aquatic organisms. For this reason, the department of agriculture of China has deregistered fipronil amide on rice crops, and the United states EPA also has paid attention to the risk of the product to aquatic invertebrates, deregistering it on more than 200 crops.
In order to effectively monitor the condition of using the fipronil in food, a determination method with good specificity and high sensitivity needs to be found, but the existing detection method mainly comprises high performance liquid chromatography-tandem mass spectrometry, liquid chromatography, gas chromatography and the like, has long separation and purification process, low sensitivity and more interferents in the food, and is difficult to obtain accurate results. Therefore, the establishment of a rapid and simple detection method for the flonicamid has important significance. An efficient immunological detection method is established, and screening of monoclonal antibodies with high specificity is an important precondition.
Disclosure of Invention
The application aims to provide a hybridoma cell strain secreting the Flubendiamide monoclonal antibody, and the antibody secreted by the cell strain has good specificity and detection sensitivity to the Flubendiamide, and can be used for establishing an immunological detection method of the Flubendiamide or establishing a colloidal gold immunochromatography test strip rapid detection method.
The technical scheme of the application is as follows:
the first object of the present application is to provide a hybridoma cell strain secreting the fluorobenzamide monoclonal antibody, which has been preserved in the China general microbiological culture Collection center, abbreviated as monoclonal cell strain Ta 4C1, with the preservation number of CGMCC No.45020, and the preservation address of North Chen West Lu No. 1, the institute of microbiology, china academy of sciences, at 12 months and 16 days of 2021. .
The second object of the application is to provide a fipronil amide monoclonal antibody which is secreted by a fipronil amide monoclonal antibody hybridoma cell strain Ta 4C1 with the preservation number of CGMCC No.45020.
The third object of the present application is to provide a fipronamide hapten, which is a fipronamide derivative with carboxyl, and has a structure as shown in the following formula (i):
the fourth object of the application is to provide a method for synthesizing the fipronil amide hapten, which comprises the following steps:
s1, 3-chlorophthalic anhydride is dissolved in methylene dichloride, triethanolamine and 4-aminobutyric acid tert-butyl ester hydrochloride are added, stirring is carried out at room temperature overnight, an organic phase is extracted, and the organic phase is dried and concentrated to obtain a compound 1, wherein the compound 1 is 2- (4- (tert-butyl) -4-oxybutyl) carbamoyl) -3-chlorobenzoic acid;
s2, dissolving the compound 1 in dichloromethane, dropwise adding triethanolamine under ice bath stirring, dropwise adding trifluoroacetic anhydride after stirring, and stirring under ice bath to obtain a compound 2 solution, wherein the compound 2 is tert-butyl (Z) -4- ((7-chloro-3-oxo isobenzofuran-1 (3H) -methylene) aminobutyrate;
s3, adding 2-methyl-4-heptafluoroisopropylaniline into the solution of the compound 2, stirring overnight at room temperature, extracting an organic phase, drying and concentrating to obtain a compound 3, wherein the compound 3 is tert-butyl-4- (2-chloro-6- ((2-methyl-4- (perfluoropropyl-2-yl) phenyl) carbamoyl) benzoylamino) butyrate;
s4, dissolving the compound 3 in dichloromethane, cooling to 0 ℃, adding trifluoroacetic acid, stirring at room temperature for reaction for 3 hours, and concentrating in vacuum and recrystallizing to obtain a white solid, namely the flonicamid hapten.
In one embodiment of the present application, in the step S1, the molar ratio of 3-chlorophthalic anhydride to t-butyl 4-aminobutyrate hydrochloride is 1:1. triethanolamine as a solvent provides a weakly alkaline environment.
In one embodiment of the present application, in the step S2, the molar ratio of the compound 1 to trifluoroacetic anhydride is 1 (1-2).
In one embodiment of the present application, in the step S2, the molar ratio of the compound 1 to trifluoroacetic anhydride is 1:1.07.
In one embodiment of the present application, in the step S3, the molar ratio of the compound 2, 2-methyl-4-heptafluoroisopropylaniline is 1 (1.5-2).
In one embodiment of the present application, in the step S3, the molar ratio of the compound 2, 2-methyl-4-heptafluoroisopropylaniline is 1:1.87.
The fifth object of the present application is to provide a method for synthesizing a complete antigen of fipronil amide, comprising the following steps: dissolving the fipronil amide hapten, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, stirring at room temperature, adding the activated liquid after 6-8h of activation into a carrier protein solution dropwise, stirring at room temperature, reacting, and dialyzing to obtain the fipronil amide complete antigen;
the fipronil amide hapten is the fipronil amide derivative with carboxyl, and the structure is shown as the following formula (I):
the carrier protein solution is prepared by dissolving carrier proteins in a borate buffer solution, wherein the carrier proteins comprise any one of KLH, BSA, OVA.
In one embodiment of the application, the molar ratio of the fipronil amide hapten to the carrier protein is (3000-6000): 1.
in one embodiment of the application, the molar ratio of the fipronil amide hapten to the carrier protein is 3000:1.
in one embodiment of the application, the molar ratio of the fipronil amide hapten, the N-hydroxysuccinimide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: (3-5): (3-5).
In one embodiment of the application, the molar ratio of the fipronil amide hapten, the N-hydroxysuccinimide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1:3:3.
in one embodiment of the application, the concentration of the carrier protein in the carrier protein solution is 2-3mg/mL.
The sixth object of the present application is to provide a complete antigen of fipronil amide prepared by the above method.
The preparation method of the florida monoclonal antibody hybridoma cell strain provided by one embodiment of the application comprises the following basic steps:
1) Structure of hapten:
2) Preparation of complete antigen:
complete antigens include immunogens for animal immunization and coating precursors for use in subsequent kit detection using plates, prior to antibody detection. The preparation methods of the fipronil amide immunogen and the fipronil amide coating antigen are respectively as follows:
preparation of the immunogen Flurobendiamide-KLH: 2.0mg of the fipronamide hapten (the molar ratio of the fipronamide hapten to the Keyhole Limpet Hemocyanin (KLH) is 3000:1), 1.27mg of N-hydroxysuccinimide (NHS), 2.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are weighed out, dissolved in 400 mu L of anhydrous N, N-dimethylformamide (referred to as solution A) and stirred at room temperature for 6-8h. 5mg of KLH was taken and diluted to 2mg/mL (referred to as solution B) with 0.01M borate buffer (BB, pH=8.6). And (3) dropwise adding the solution A into the solution B at room temperature, reacting overnight at room temperature to obtain a conjugate immunogen fipronil amide-KLH mixed solution, and separating the complete antigen and the unconjugated small molecule hapten by dialysis to obtain the fipronil amide immunogen.
Preparation of coated primordial fipronil amide-OVA: 3.6mg of the Fluofenamide hapten, 8mg of EDC and 2.3mg of N-hydroxysuccinimide are weighed out, dissolved in 400 mu L of anhydrous N, N-dimethylformamide (called A solution) and stirred at room temperature for 6 to 8 hours. Weighing 5mg chicken ovalbumin OVA, dissolving in 2mL boric acid buffer solution, dropwise adding the solution A into the solution B at room temperature, reacting overnight at room temperature to obtain a conjugate fipronil-OVA mixed solution, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain the fipronil amide coating antigen.
3) Immunization of mice: the complete antigen of fipronamide-KLH (namely fipronamide immunogen) is mixed and emulsified with the equivalent amount of Freund's adjuvant, and BALB/c mice are immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the multiple boosting. One month is separated between the first immunization and the second boosting, and 21 days is separated between the multiple boosting. The last time of impact immunization with the fipronil amide-KLH complete antigen (without adjuvant); serum titers and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);
4) Cell fusion and cell strain establishment: fusing the spleen cells of the mice with myeloma cells of the mice by a polyethylene glycol (PEG 4000) method, culturing the mice by a HAT culture medium, detecting positive cell holes by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), further measuring the inhibition effect of the positive cell holes by the ic-ELISA, subcloning the positive cell holes with the best inhibition by a limiting dilution method for three times, and finally screening to obtain a florbendiamide monoclonal antibody hybridoma cell strain Ta 4C1;
5) Identification of hybridoma cell line properties: sensitivity and specificity were determined by ic-ELISA.
High potency low IC 50 The spleen cells of the mice are fused with myeloma cells of the mice by a PEG method, and a hybridoma cell strain is obtained through screening and three subcloning by an indirect competitive enzyme-linked immunosorbent assay.
The seventh object of the present application is to provide an application of the obtained anti-fipronil monoclonal antibody, in particular to an immune detection method for establishing the content of fipronil, which is applied to the detection of fipronil in food.
In one embodiment of the application, the anti-fipronil monoclonal antibody is used for preparing a detection device for analyzing and detecting the residual quantity of fipronil in food safety detection, and the detection device is selected from one of a reagent, a detection plate and a kit.
In one embodiment of the present application, the detection device further includes a fipronil amide coating antigen, wherein the fipronil amide coating antigen is obtained by coupling a fipronil amide hapten with a carrier protein, and the carrier protein includes any one of KLH, BSA, OVA. Wherein, regarding the choice of carrier protein, the carrier protein used for preparing the coating antigen is different from the carrier protein used for preparing the immunogen.
The beneficial effects are that:
(1) The monoclonal antibody secreted by the cell strain provided by the application has better specificity and detection sensitivity (IC) 50 The value is 2.4 ng/mL), can realize the detection of the residual quantity of the florfenicol amide in water, fruits, vegetables and grains, provides a raw material for the immunodetection of the florfenicol amide residual in foods, and has practical application value.
(2) The anti-fipronil monoclonal antibody obtained by the application can be used for preparing an immune detection kit and a colloidal gold test strip of fipronil, and provides a powerful detection method and means for detecting the fipronil in food.
(3) The new method for synthesizing the fipronil amide hapten and the immunogen is provided, the synthesis steps are simplified and effective, and the thought and the method for synthesizing the immunogen are provided for the research of people in future.
Preservation of biological material samples: the Flubendiamide monoclonal antibody hybridoma cell strain Ta 4C1 is preserved in China general microbiological culture Collection center, and is called monoclonal cell strain Ta 4C1 for short, with the preservation number of CGMCC No.45020, and the address is: the collection date is 2021, 12 months and 16 days at the institute of microbiology, national academy of sciences, national institute of sciences, north Chen West Lu 1, the Korean region of Beijing.
Drawings
FIG. 1 shows the steps of synthesizing the fipronil amide hapten in the embodiment of the application.
FIG. 2 is a subtype identification of the Flurobendiamide monoclonal antibody in the examples of the present application.
FIG. 3 is a standard curve of inhibition of the sulfenamide monoclonal antibody to sulfenamide in the examples of the present application.
Detailed Description
The application will be further described with reference to the drawings and examples. The application 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 application and should not be construed as limiting the application described in detail in the claims.
The application obtains the monoclonal antibody hybridoma cell strain with better specificity and sensitivity to the flonicamid through immunizing a mouse with the full antigen of the flonicamid, cell fusion, culturing in a HAT selective culture medium and screening cell supernatant by ic-ELISA.
Example 1 preparation of hybridoma cell lines
(1) The hapten is synthesized by the steps shown in figure 1:
s1, 3-Chlorobenzenesulfonic anhydride (1 g,5.48 mmol) was dissolved in 20mL of Dichloromethane (DCM), triethanolamine (TEA, 1.52mL,11.5 mmol) was added, tert-butyl 4-aminobutyrate hydrochloride (1.07 g,5.48 mmol) and then stirred at room temperature overnight and the reaction was monitored by TLC plate. 1M HCl was added and extracted three times with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Elution with a mixed solution of ether and n-hexane gave the compound 1 (2- (4- (tert-butyl) -4-oxybutyl) carbamoyl) -3-chlorobenzoic acid, 1.4g,4.1mmol, as a pale yellow solid.
S2, compound 1 (0.5 g,1.5 mmol) was weighed into dichloromethane (DCM, 5 mL) and triethanolamine (TEA, 305. Mu.L, 2.19 mmol) was added dropwise with ice bath stirring. After stirring for 5min, trifluoroacetic anhydride (TFAA, 227. Mu.L, 1.61 mmol) was added dropwise. Stirring under ice bath, TLC plate monitoring reaction progress, compound 2 (tert-butyl (Z) -4- ((7-chloro-3-oxo isobenzofuran-1 (3H) -methylene) aminobutyrate) solution.
S3 then 2-methyl-4-heptafluoroisopropylaniline (772 mg,2.8 mmol) was added to a solution of the above compound 2 (tert-butyl (Z) -4- ((7-chloro-3-oxoisobenzofuran-1 (3H) -methylene) aminobutyrate, 500mg,1.5 mmol)Stirred overnight, extracted with dichloromethane DCM, extracted with 1M HCl, saturated NaHCO 3 Washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound 3 (tert-butyl-4- (2-chloro-6- ((2-methyl-4- (perfluoroprop-2-yl) phenyl) carbamoyl) benzoylamino) butanoate as a pale yellow solid, 350mg,0.6 mmol.
S4, compound 3 (tert-butyl-4- (2-chloro-6- ((2-methyl-4- (perfluoroprop-2 yl) phenyl) carbamoyl) benzoylamino) butyrate, 350mg,0.6 mmol) was dissolved in dichloromethane (DCM, 3 mL) and cooled to 0deg.C. Trifluoroacetic acid (TFA, 3mL,39 mmol) was added at 0deg.C. Then stirring and reacting for 3 hours at room temperature, concentrating in vacuum, and recrystallizing to obtain white solid, namely the fipronil amide hapten.
The structure of the obtained fipronil amide hapten is as follows:
(2) Preparation of complete antigen:
the complete antigen comprises immunogen and coating antigen, and the preparation methods of the fipronil amide immunogen and the fipronil amide coating antigen are as follows:
preparation of the immunogen Flurobendiamide-KLH: 2.0mg (3.7 mmol) of the Fluorobenzamide derivative (molar ratio of the Fluorobenzamide hapten to Keyhole Limpet Hemocyanin (KLH) 3000:1), 1.27mg (1.1 mmol) of N-hydroxysuccinimide (NHS), 2.1mg (1.1 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were weighed, dissolved in 400. Mu.L of anhydrous N, N-dimethylformamide (referred to as solution A), and stirred at room temperature for 6-8h. 5mg of KLH was taken and diluted to 2mg/mL (referred to as solution B) with 0.01M borate buffer (BB, pH=8.6). And (3) dropwise adding the solution A into the solution B at room temperature, reacting overnight at room temperature to obtain a conjugate immunogen fipronil amide-KLH mixed solution, and separating the complete antigen and the unconjugated small molecule hapten by dialysis to obtain the fipronil amide immunogen which is used for animal immunization in the subsequent step (3).
Preparation of coated primordial fipronil amide-OVA: 3.6mg (6.64 mmol) of the flonicamid hapten, 3.8mg (20 mmol) of EDC, 2.3mg (20 mmol) of N-hydroxysuccinimide and 400. Mu.L of anhydrous N, N-dimethylformamide are weighed out and dissolved (referred to as solution A) and stirred at room temperature for 6 to 8 hours. Weighing 5mg chicken ovalbumin OVA, dissolving in 2mL boric acid buffer solution, dropwise adding the solution A into the solution B at room temperature, reacting overnight at room temperature to obtain a conjugate fipronil-OVA mixed solution, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain fipronil coating antigen which is used for coating in the step (1) in the subsequent antibody detection application of the embodiment 3.
(3) Animal immunization: healthy BALB/c mice of 6-8 weeks of age were selected for immunization. Mixing and emulsifying the full antigen of the fipronil amide (namely the fipronil amide immunogen obtained in the step (2)) with the equivalent Freund adjuvant, and immunizing a BALB/c mouse through subcutaneous injection on the back. The first immunization was with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. One month is separated between the first immunization and the second boosting, and 21 days is separated between the multiple boosting. Blood was collected 7 days after the third immunization, the serum titer and inhibition of mice were determined using ic-ELISA, mice with high titer were selected for impact immunization 21 days after the fifth immunization, and intraperitoneal injection, requiring halving of the wash-out dose without any adjuvant.
(4) Cell fusion: three days after the impact immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, specifically as follows:
a. taking eyeball and blood, killing a mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out spleen of the mouse by aseptic operation, moderately grinding the spleen by a rubber head of a syringe, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume and counting for later use after the last centrifugation;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium in 5% CO 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, 1mL of PEG 1500 was added dropwise to the 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 cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full replacement with a 100 XHT RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection. Screening is carried out in two steps: the first step is to screen out positive cell holes by using ic-ELISA, and the second step is to select the fluorobenzamide as a standard substance, and to measure the inhibition effect of positive cells by using ic-ELISA. Selecting cell holes with better inhibition on the standard substance of the florfenicol amide, subcloning by adopting a limiting dilution method, and detecting by adopting the same method. Repeating for three times to obtain cell strain, and preserving in 2021 at 12 months and 16 days in China general microbiological culture Collection center, called monoclonal cell strain Ta 4C1 for short, with preservation number of CGMCC No.45020, and preservation address of North Chen West Lu No. 1, 3 of the Chaoyang district of Beijing city, china academy of sciences microbiological study. .
Example 2 preparation and identification of monoclonal antibodies:
taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the octanoic acid-ammonium sulfate method. Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating IgG type monoclonal antibody with equal amount of saturated ammonium sulfate solution, and isolatingThe supernatant was discarded, dissolved in 0.01M PBS (pH 7.4), and then dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.
Monoclonal antibodies obtained by ascites purification were subjected to immunoglobulin subtype identification using a mouse monoclonal antibody subtype identification kit, the subtype of which was IgG2b type, and the identification results are shown in fig. 2.
Determination of monoclonal antibody against Flubendiamide IC using indirect competition ELISA method 50 IC with 2.4. Mu.g/L and its analog measured 50 And the cross reaction rate (see table 1), the monoclonal antibody has good specificity and sensitivity.
Table 1 Cross-reaction of monoclonal antibodies with Flurobendiamide structural analogues
Example 3 antibody uses
The monoclonal antibody prepared in the example 2 is applied to a Flurobendiamide ELISA (enzyme-Linked immunosorbent assay) and is subjected to the following specific steps:
(1) Coating: the coated crude flubendiamide-OVA prepared in step (2) of example 1 was diluted in a 1. Mu.g/mL ratio with 0.05M carbonate buffer at pH9.6, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(2) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
(3) Closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
(4) Sample adding: the antisera was diluted from 1:1000 in a double ratio and added to the coated wells at each dilution, 100. Mu.L/well, and reacted at 37℃for 30min; after extensive washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100. Mu.L/well, and reacted at 37℃for 30 minutes.
(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 ℃.
(6) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD 450 value of each well was measured by using a microplate reader.
Sensitivity of detection of Parafricamide by ic-ELISA as shown in FIG. 3, according to the standard equation y=0.105+ (1.924-0.105)/(1+ (x/2.44) 0.952 ) IC for determining monoclonal antibody Flubendiamide by IC-ELISA 50 2.4ng/mL, the sensitivity to the flonicamid is very good, and the method can be used for the immunoassay detection of the flonicamid.
The configuration of the relevant solutions in the above embodiments of the present application is as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use.
Phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g 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. And mixing the solution B according to a ratio of 1:5 to obtain TMB.
The color development liquid is mixed in the prior art.
The anti-fipronil amide monoclonal antibody secreted by the hybridoma cell strain Ta 4C1 has better affinity and higher sensitivity to the fipronil amide, and particularly has 50% inhibition concentration IC of the fipronil amide 50 The method reaches 4.04ng/mL, can be used for preparing immune detection kits of the fipronil and colloidal gold test strips, detection plates, reagents and the like, is used for analyzing and detecting the residual fipronil in food safety detection, and particularly provides a powerful detection method and means for detecting the fipronil in food.
The foregoing is only a preferred embodiment of the application, 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 application, and such modifications and adaptations are intended to be comprehended within the scope of the application.

Claims (14)

1. Hybridoma cell lines secreting anti-fluorobenzamide monoclonal antibodies are preserved in China general microbiological culture Collection center, called monoclonal cell line Ta 4C1 for short, with the preservation number of CGMCC No.45020 and the preservation address of Beijing Kogyo-region North Chenxi Lu No. 1, national academy of sciences of China, and the culture Collection of microorganisms is carried out on 12 months and 16 days of 2021.
2. An anti-fipronil amide monoclonal antibody which is characterized by being secreted by a monoclonal cell strain Ta 4C1 with the preservation number of CGMCC No.45020 according to claim 1.
3. A fipronil amide hapten for preparing the hybridoma cell strain according to claim 1, wherein the fipronil amide hapten has a structure shown in the following formula (i):
4. a method of synthesizing the fipronil amide hapten as claimed in claim 3, comprising the steps of:
s1, 3-chlorophthalic anhydride is dissolved in methylene dichloride, triethanolamine and 4-aminobutyric acid tert-butyl ester hydrochloride are added, stirring is carried out at room temperature overnight, an organic phase is extracted, and the organic phase is dried and concentrated to obtain a compound 1, wherein the compound 1 is 2- (4- (tert-butyl) -4-oxybutyl) carbamoyl) -3-chlorobenzoic acid;
s2, dissolving the compound 1 in dichloromethane, dropwise adding triethanolamine under ice bath stirring, dropwise adding trifluoroacetic anhydride after stirring, and stirring under ice bath to obtain a compound 2 solution, wherein the compound 2 is tert-butyl (Z) -4- ((7-chloro-3-oxo isobenzofuran-1 (3H) -methylene) aminobutyrate;
s3, adding 2-methyl-4-heptafluoroisopropylaniline into the solution of the compound 2, stirring overnight at room temperature, extracting an organic phase, drying and concentrating to obtain a compound 3, wherein the compound 3 is tert-butyl-4- (2-chloro-6- ((2-methyl-4- (perfluoropropyl-2-yl) phenyl) carbamoyl) benzoylamino) butyrate;
s4, dissolving the compound 3 in dichloromethane, cooling to 0 ℃, adding trifluoroacetic acid, stirring at room temperature for reaction for 3 hours, and concentrating in vacuum and recrystallizing to obtain a white solid, namely the flonicamid hapten.
5. The method according to claim 4, wherein in the step S1, the molar ratio of 3-chlorophthalic anhydride to t-butyl 4-aminobutyrate hydrochloride is 1:1.
6. a process according to claim 4, wherein in step S2, the molar ratio of compound 1 to trifluoroacetic anhydride is 1 (1-2).
7. The process according to claim 4, wherein in step S3, the molar ratio of the compound 2, 2-methyl-4-heptafluoroisopropylaniline is 1 (1.5-2).
8. The method for synthesizing the complete antigen of the fipronil amide is characterized by comprising the following steps of: dissolving the fipronil amide hapten, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in N, N-dimethylformamide, stirring at room temperature, adding the activated liquid after activation for 6-8 hours into a carrier protein solution dropwise, stirring at room temperature, reacting, and dialyzing to obtain the fipronil amide complete antigen;
the carrier protein solution is prepared by dissolving carrier protein in borate buffer solution, and the carrier protein is KLH.
9. The method of claim 8, wherein the molar ratio of fipronil amide hapten to carrier protein is (3000-6000): 1.
10. the method of claim 8, wherein the molar ratio of fipronil amide hapten, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: (3-5): (3-5).
11. A complete antigen, characterized in that it is prepared according to the method of any one of claims 8-10.
12. The application of the anti-fipronil amide monoclonal antibody as claimed in claim 2, wherein an immunodetection method of the fipronil amide content is established and is applied to the detection of the fipronil amide in food.
13. The use of the anti-fipronil amide monoclonal antibody according to claim 12, wherein the anti-fipronil amide monoclonal antibody is used for preparing a detection device for analysis and detection of residual amount of fipronil amide in food safety detection, and the detection device is selected from any one of a reagent, a detection plate and a kit.
14. The use of an anti-fipronil monoclonal antibody according to claim 13, wherein the detection device further comprises a fipronil coating antigen, wherein the fipronil coating antigen is obtained by coupling a fipronil amide hapten with a carrier protein, the carrier protein is OVA, and the structure of the fipronil amide hapten is shown as the following formula (i):
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Citations (3)

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CN102627628A (en) * 2012-03-08 2012-08-08 中国农业大学 Chlorantraniliprole antigen, its preparation method and application
CN108640866A (en) * 2018-06-01 2018-10-12 中国农业大学 Fluorobenzene insect amide antigen and the preparation method and application thereof
CN113736743A (en) * 2021-09-15 2021-12-03 江南大学 Hybridoma cell strain secreting monoclonal antibody against bisamide compounds and application thereof

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CN108640866A (en) * 2018-06-01 2018-10-12 中国农业大学 Fluorobenzene insect amide antigen and the preparation method and application thereof
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