CN111187346B - Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof - Google Patents
Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof Download PDFInfo
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Abstract
The invention provides a colloidal gold test strip for detecting fipronil and metabolites thereof and a preparation method thereof, comprising the following steps: hapten preparation, immunogen and coating antigen preparation, monoclonal antibody preparation, gold-labeled antibody preparation and colloidal gold test strip preparation; the invention uses fipronil coupling protein coating antigen to make detection line on nitrocellulose membrane, uses secondary antibody to make quality control line on nitrocellulose membrane, incubates sample extracting solution with gold mark antibody, inserts test paper strip to detect. According to the invention, the high-sensitivity and high-specificity monoclonal antibody against fipronil and the metabolites thereof is prepared, and then the colloidal gold test strip which is fast in speed, low in cost, high in sensitivity and capable of simultaneously detecting fipronil and the metabolites thereof is established on the basis of the monoclonal antibody.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a colloidal gold test strip for simultaneously and rapidly detecting fipronil and metabolites thereof and a preparation method thereof.
Background
Fipronil, english name is Fenpunil (fipronil), chemical name isThe (RS) -5-amino-1- (2, 6-dichloro-4-trifluoromethylphenyl) -4-trifluoromethyl sulfinyl pyrazole-3-nitrile is a phenyl pyrazole pesticide developed in the eighty th of the 20 th century, is one of substitutes of organophosphorus pesticides, and is widely applied to pest control of crops such as fruits, vegetables and the like, and parasite and pest control in families and entertainment places because of good sterilization effect and high broad spectrum. Fipronil is a chloride ion channel controlled by destroying gamma-aminobutyric acid (GABA) of pests, thereby destroying their normal neural system to control pests. Related researches show that fipronil has chronic neurotoxicity and is already identified as a C-class cancerogenic substance, and the fipronil can influence the health of a human body and cause symptoms such as conjunctivitis, dizziness, epileptic seizure, agitation, sweating and the like. Fipronil is degraded into more toxic fipronil, fipronil sulfone and fipronil sulfoxide metabolites through photolysis, oxidation and reduction in the natural environment. Rat LD of fluoronitrile 50 Only 1/6 of the parent itself, which is far more toxic to rats than itself. Fipronil sulfone has about 6 times of toxicity to daphnia as its parent, and fipronil sulfoxide has 5 times of bioaccumulation to fipronil parent. In 1157 publication published in 2009, fipronil is limited to be used in partial seed coating of corn and the like, and in national standard food, pesticide residue limit (GB-2763-2016) is limited to be 0.02-0.1 mg/kg in the residual limit range of grains, oil and fat, vegetables, fruits, sugar materials and edible fungi.
At present, the fipronil detection method is mainly an instrument analysis method and mainly comprises gas chromatography-tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), high Performance Liquid Chromatography (HPLC) and high performance liquid diode array detection (HPLC-DAD), and the methods have high sensitivity and accuracy, but require expensive instruments, professional operators and complicated and time-consuming pretreatment, so that the method is not suitable for rapid screening of a large number of samples on site. Immunoassay methods are one detection format based on the specific reaction of antigen antibodies. The immunoassay detection method of fipronil which is authorized or announced in China at present mainly comprises the following steps: the invention discloses a kit for detecting fipronil with the publication number of CN201710990416.6, a preparation method and application thereof, and a chemiluminescent immunoassay kit for detecting fipronil in tea with the publication number of CN201711059643.3, wherein the two inventions are both application of ELISA kits, the detection process needs repeated plate washing and takes long time, and as a result, certain differences are caused by different operators, and the result needs instrument reading, thus being not suitable for rapid screening of a large number of samples on site; the invention discloses a surface-enhanced Raman gold-labeled test strip for rapidly detecting trace fipronil, which has the publication number of CN201810005898.X, and a preparation method thereof, wherein the detection method has unstable Raman signal and is easy to cause false positive phenomenon, the detection result is aided by a Raman instrument, the Raman instrument is expensive, and the detection cost is high; the invention discloses a method for rapidly detecting fipronil by utilizing illumination and surface enhanced Raman, which is disclosed by the invention, wherein the existence of fipronil is indirectly detected by detecting the main photolysis product fipronil of fipronil, so that the detection steps and time are increased, the accuracy of the result is low, a Raman instrument is expensive, and the cost is increased; the colloidal gold-labeled test strip for rapidly detecting trace fipronil and the preparation method thereof are disclosed as 201810005478.1, and the detection method only aims at fipronil and does not relate to the detection of fipronil metabolites with more toxicity. Fipronil is very easy to degrade in natural environment, and the accuracy of the method is not high.
Disclosure of Invention
According to the colloidal gold test strip for simultaneously and rapidly detecting fipronil and metabolites thereof and the preparation method thereof, the monoclonal antibody which is high in sensitivity and specificity and resistant to fipronil and metabolites thereof is prepared, and then the colloidal gold test strip which is high in speed, low in cost and high in sensitivity and can simultaneously detect fipronil and metabolites thereof is established on the basis of the antibody, and the test strip can simultaneously detect fipronil and metabolites thereof without instrument assistance, is low in cost and simple to operate and is suitable for rapid screening of on-site samples.
A preparation method of a colloidal gold test strip for detecting fipronil and metabolites thereof comprises the following steps:
preparation of hapten: the fipronil is prepared into hapten through acid hydrolysis method and adding acetic acid and carboxyl;
preparation of immunogens and coating precursors: preparing immunogen and coating antigen respectively by coupling the hapten with protein through an activated ester method;
preparation of monoclonal antibodies: preparing a monoclonal antibody by using the immunogen, and detecting the sensitivity and the specificity of the monoclonal antibody by using a coating antigen through ic-ELISA to obtain the monoclonal antibody of the fipronil and the metabolite thereof;
preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to obtain a gold-labeled antibody;
preparing a colloidal gold test strip: and (3) preparing a detection line on the nitrocellulose membrane by using a fipronil coupled protein coating antigen, preparing a quality control line on the nitrocellulose membrane by using a secondary antibody, and then assembling the test strip.
In the above scheme, the preparation of the hapten specifically comprises the following steps:
fipronil is dissolved in concentrated sulfuric acid, acetic acid and water, heated and refluxed, then sodium hydroxide aqueous solution is dripped until the pH value reaches alkalinity, pure water is added to dissolve precipitate, water solution is washed by normal hexane, aqueous layer solution is acidified until the pH value reaches acidity by separating liquid, then ethyl acetate is used for extraction, organic phases are combined, the organic phases are washed by saturated brine, dried by anhydrous sodium sulfate and evaporated under reduced pressure, and white solid is obtained through a silica gel column, namely hapten.
In the scheme, the mass fraction of the concentrated sulfuric acid is 98%;
the dosage ratio of fipronil, concentrated sulfuric acid, acetic acid and water is 300-500 mg:1-4 mL:1-3 mL:1-2 mL;
the dosage ratio of the pure water to the precipitate is 2-3 mL:200-600 mg.
In the above scheme, the preparation of the immunogen and the coating antigen specifically comprises the following steps:
activation of hapten: dissolving the hapten in dimethylformamide DMF, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC into the mixture, and stirring and activating the mixture to obtain an activated hapten;
preparation of the immunogen: fully dissolving Bovine Serum Albumin (BSA) in a Carbonate Buffer (CBS) to obtain a BSA solution, dropwise adding the activated hapten solution into the BSA solution under magnetic stirring, stirring the mixture for reaction to obtain an immunogen, and dialyzing the immunogen with Phosphate Buffer (PBS);
preparation of coating raw material: fully dissolving chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA mass solution, dropwise adding activated hapten solution into the OVA mass solution under magnetic stirring, stirring the mixture for reaction to obtain coating raw material, and dialyzing the coating raw material with phosphate buffer PBS.
In the above scheme, in the activation process of the hapten, the dosage ratio of the hapten, DMF, NHS and EDC is 2-4 mg: 0.5-1 mL: 1-4 mg: 2-7 mg; the stirring activation time is 6-8 h.
In the above scheme, in the preparation process of the immunogen, the dosage ratio of the BSA to the CBS buffer is 7-15 mg: 3-5 mL;
in the preparation process of the coating antigen, the dosage ratio of the OVA to the CBS buffer solution is 5-10 mg: 2-5 mL;
the molar ratio of the hapten in the activated hapten solution to the protein in the BSA solution and the molar ratio of the hapten in the activated hapten solution to the protein in the OVA solution are both 20:1-40:1;
the temperature of the stirring reaction is 4 ℃, and the stirring reaction time is 12-18 h;
the temperature during the PBS buffer solution dialysis is 4 ℃ and the time is 36-78 hours.
In the above scheme, the preparation of the gold-labeled antibody specifically comprises the following steps:
heating pure water to boil, adding chloroauric acid solution under stirring, and adding trisodium citrate solution to obtain colloidal gold solution;
taking a colloidal gold solution, adding a boric acid buffer solution, fully and uniformly mixing, dropwise adding the monoclonal antibody solution for reaction, and then dropwise adding 10% BSA for blocking to obtain a mixed solution;
centrifuging the mixed solution, discarding the supernatant to obtain a precipitate, and dissolving the precipitate in the heavy suspension to obtain the gold-labeled antibody.
In the scheme, the dosage ratio of the pure water to the chloroauric acid solution to the trisodium citrate solution is 500-900 mL:1-2 mL:1-3 mL;
the dosage ratio of the colloidal gold solution, the monoclonal antibody solution, the boric acid buffer solution and the 10% BSA is 3-6 mL, 18-48 mug, 300-600 mug and 200-800 mug;
the dosage ratio of the sediment to the heavy suspension is 0.1-1 mg/300-500 mu L.
In the above scheme, the preparation of the colloidal gold test strip specifically includes the following steps:
the sample pad was soaked in 0.01M pH 7.4PBS solution containing 0.2% Tween-20,1% sucrose and 1% BSA and then dried;
spraying 0.5-1 mg/mL of coating antigen on the nitrocellulose membrane to serve as a detection line, and spraying 0.2-0.6 mg/mL of secondary antibody on the nitrocellulose membrane to serve as a quality control line;
the sample pad, nitrocellulose membrane, absorbent pad and base plate were assembled.
A colloidal gold test strip for detecting fipronil and metabolites thereof is prepared according to the detection method of the colloidal gold test strip for detecting fipronil and metabolites thereof.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, acetic acid is added to introduce carboxyl by an acid hydrolysis method, the reaction rate is increased, hapten is prepared, and then the protein is coupled by an activated ester method to prepare immunogen with stronger immunogenicity, so that monoclonal antibody capable of simultaneously recognizing fipronil and metabolites thereof is prepared, and the sensitivity of the antibody is higher.
2. The test strip prepared by adopting the colloidal gold labeled monoclonal antibody can detect fipronil and metabolites with stronger toxicity, so the method has more significance and value in the aspect of guaranteeing food safety.
3. The colloidal gold test strip prepared by the invention has low cost, high detection speed and high sensitivity; the detection result of the method can be directly visually interpreted, the auxiliary of an instrument is not needed, the cost of an expensive instrument is saved, and the method is suitable for detecting a large number of samples on site; directly incubating the sample extracting solution with the gold-labeled antibody, inserting a test strip, and judging a detection result after 5 min; experiments show that the detection method has high sensitivity.
4. The colloidal gold test strip prepared by the invention is simple to operate and has wide applicability; the detection method is simple and easy to understand, does not need professional operation, and can be suitable for law enforcement departments, farmers, consumers and the like. In addition, the method can provide a certain guarantee for the life safety of consumers.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1A is a schematic diagram of a colloidal gold test strip according to the present invention; fig. 1B is a schematic diagram of a colloidal gold test strip.
FIG. 2 is a UV diagram of colloidal gold; wherein the inset is a TEM image of colloidal gold.
FIG. 3 is an indirect competitive inhibition curve of antibodies.
FIG. 4 is a graph showing the effect of the colloidal gold test strip on detecting fipronil and the residues of the metabolites thereof.
Detailed Description
The embodiments of the present invention are described in detail below with reference to the accompanying drawings, which are exemplary and intended to be illustrative of the present invention and not to be construed as limiting the invention.
The preparation method of the colloidal gold test strip for detecting fipronil and metabolites thereof comprises the following steps:
(1) Preparation of hapten: dissolving a proper amount of fipronil in concentrated sulfuric acid, acetic acid and water, heating and refluxing for 2-3 h, then dropwise adding sodium hydroxide aqueous solution until the pH reaches 9-10, adding pure water to dissolve precipitate, washing the aqueous solution with 5-15 mL of n-hexane, separating and acidifying the aqueous layer solution until the pH reaches 1, then extracting with ethyl acetate, combining organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, and evaporating under reduced pressure. Passing through silica gel column to obtain white solid as hapten;
the mass fraction of the concentrated sulfuric acid is 98%;
the dosage ratio of fipronil, concentrated sulfuric acid, acetic acid and water is 300-500 mg:1-4 mL:1-3 mL:1-2 mL;
the dosage ratio of the pure water to the precipitate is 2-3 mL:200-600 mg.
(2) Preparation of immunogens and coating precursors:
activation of hapten: dissolving hapten obtained in the hapten preparation step in dimethylformamide DMF, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC into the hapten, and stirring the mixture at room temperature to obtain activated hapten;
preparation of the immunogen: fully dissolving Bovine Serum Albumin (BSA) in a Carbonate Buffer (CBS) to obtain a BSA solution, dropwise adding the activated hapten solution into the BSA solution under magnetic stirring, and stirring the mixture at a certain temperature for reaction to obtain an immunogen;
preparation of coating raw material: fully dissolving chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA solution, then dropwise adding the activated hapten solution into the OVA solution under magnetic stirring, and stirring the mixture under a certain temperature condition to react to obtain a coating antigen; the immunogen and the coating antigen are dialyzed respectively by phosphate buffer PBS, the dialysate is replaced for 8 times in the dialysis process, and the subpackaged solution is stored at the temperature of minus 20 ℃ for standby after the dialysis is finished.
The dosage ratio of hapten, DMF, NHS and EDC is 2-4 mg: 0.5-1 mL: 1-4 mg: 2-7 mg; the stirring time is 6-8 h;
in the preparation process of the immunogen, the dosage ratio of the BSA to the CBS buffer is 7-15 mg: 3-5 mL;
in the preparation process of the coating antigen, the dosage ratio of the OVA to the CBS buffer solution is 5-10 mg: 2-5 mL;
the molar ratio of the hapten in the activated hapten solution to the protein in the BSA solution and the molar ratio of the hapten in the activated hapten solution to the protein in the OVA solution are both 20:1-40:1;
the temperature of the stirring reaction is 4 ℃, and the stirring reaction time is 12-18 h;
the temperature during the PBS buffer solution dialysis is 4 ℃ and the time is 36-78 hours;
the BSA and OVA were purchased from Shanghai Sigma-Aldrich chemical reagent company and fipronil was purchased from Shanghai carbofuran biological reagent Co.
(3) Preparation of monoclonal antibodies: diluting the prepared immunogen to 1-2 mg/mL by using normal saline, mixing the immunogen with Freund's complete adjuvant with the same volume, and then emulsifying the mixture separately; immunization of mice (Balb/C) by subcutaneous multipoint injection at the back, each mouse being immunized at 75-100 μg;
the mice were then boosted every three weeks, fully emulsified with immunogen diluted to 2mg/mL and an equal volume of Freund's incomplete adjuvant, and immunized by subcutaneous multipoint injection on the back (Balb/C) with an immunization dose of 25-50 μg per mouse.
Starting from the third immunization, tail blood sampling detection is carried out on the mice; screening out mice with high titer and high inhibition rate, and performing intraperitoneal injection of 20 mug of immunogen; the mice are killed by pulling necks after three days of injection, soaked in 75% alcohol for 10min, spleen cells are taken under the aseptic condition, the spleen is fully ground on a copper mesh, and then the mice are washed three times by PRMI-1640 culture medium, so that cell suspension is obtained and centrifuged for 10min under the condition of 1200 r/min; removing supernatant, re-suspending the precipitated cells with PRMI-1640 medium, centrifuging, repeating the above steps for three times, and counting spleen cells;
fully and uniformly mixing the spleen cells and the myeloma cells of the mice according to the ratio of 10:1, centrifuging the obtained mixed solution for 10min under the condition of 1200r/min, oscillating to disperse the cells at the bottom of a centrifuge tube, slowly adding PEG4000 in the first minute, and standing for 1min, and adding PRMI-1640 culture medium to terminate the action of the PEG; the dosage ratio of the mixed solution to the PEG4000 to the PRMI-1640 culture medium is 0.5mL:10mL. The fused cells were statically placed in an oven at 37℃for 15min, and then centrifuged at 3500r/min for 10min. Then using HAT containing 10% fetal bovine serumThe cells were resuspended in complete medium, mixed well and then placed in 96-well plates and incubated at 37℃in 5% CO 2 Culturing in an incubator. The supernatant is screened by adopting an indirect competition ic-ELISA method by using a coating antigen, a cell hole with high inhibition rate and strong positive is selected for subcloning, and then the subcloning is carried out for 2-3 times, so that a positive hybridoma cell strain with high purity is obtained.
Preparation and purification of ascites: the hybridoma cells selected above were diluted to 1X 10 per ml with PRMI-1640 medium 6 And (3) injecting hybridoma cells of cells into each mouse, performing intraperitoneal injection according to 500-800 uL, swelling the abdomen of the mouse, collecting ascites, centrifuging at 3500r/min for 10min, collecting supernatant, and purifying by using n-octanoic acid-ammonium sulfate to obtain the monoclonal antibody.
The specific operation of the purification of the n-octanoic acid-ammonium sulfate is as follows: taking 1mL of antiserum, diluting 2 times with 0.06mol/L, pH 5.0.0 acetic acid buffer solution, and adjusting the pH to 4.5 with 1mol/L HCl solution; slowly adding n-octanoic acid dropwise at a ratio of 33 mu L/mL of original serum under stirring at room temperature, standing at 4 ℃ for more than 2 hours to fully precipitate the hybrid protein, centrifuging at 4 ℃ for 30 minutes at 9000r/min, discarding the precipitate, collecting the supernatant, filtering with filter paper, adding 0.1mol/L, pH 7.4.4 PBS with a volume of 1/10 of the volume of the supernatant, adjusting the pH of the solution to 7.4, adding ammonium sulfate at 0.277g per milliliter, uniformly stirring, and standing at 4 ℃ for more than 4 hours. Centrifuging at 4 ℃ and 9000r/min for 30min, discarding the supernatant, collecting the precipitate, dissolving the precipitate in a certain volume of PBS, dialyzing in 1L of PBS at 4 ℃ to remove salt, dialyzing for 3-4 days, and replacing the dialysate for 3 times per day.
(4) Preparation of gold-labeled antibody: heating 500-900 mL of ultrapure water in a flask at 102 ℃ for boiling balance for 30-50 min, adding 10% chloroauric acid solution under magnetic stirring, adding 10% trisodium citrate solution after 10min, continuing stirring until the color of the solution becomes dark red and does not change color, obtaining colloidal gold solution, cooling to room temperature and preserving in dark place for standby. Taking 3-6 mL of colloidal gold solution, adding boric acid buffer solution with pH of 8.5, fully and uniformly mixing, and then dropwise adding the prepared monoclonal antibody solution to react for 30-60 min. Then 10% BSA was added dropwise to the above solution to block the site without binding, and incubated at room temperature for 0.5 to 1 hour. The mixed solution was centrifuged at 10000r/min at 4℃for 25min to remove unbound antibody and gold nanoparticles, the supernatant was discarded, the precipitate was dissolved in a heavy suspension containing 2% BSA, 1% sucrose, 0.02% sodium azide in PBS and stored at 4℃for further use. The prepared gold-labeled antibody is diluted by 20 to 50 times, 50 mu L of the gold-labeled antibody is taken out and is frozen and dried in a 96-well plate for standby. Spraying the secondary antibody and the prepared coating antigen on a nitrocellulose membrane by a film spraying instrument to serve as a quality control line (C line) and a detection line (T line), and drying the nitrocellulose membrane at 37 ℃ overnight for standby.
The dosage ratio of the pure water, the chloroauric acid solution and the trisodium citrate solution is 500-900 mL:1-2 mL:1-3 mL;
the dosage ratio of the colloidal gold solution, the monoclonal antibody, the boric acid buffer solution and the 10% BSA is 3-6 mL, 18-48 mug, 300-600 mug and 200-800 mug;
the dosage ratio of the sediment to the heavy suspension is 0.1-1 mg:300-500 mu L;
the secondary antibody is HRP-goat anti-mouse IgG purchased from Shanghai Biotechnology Co.
(5) Preparing a colloidal gold test strip: and (3) preparing a detection line on the nitrocellulose membrane by using a fipronil coupled protein coating antigen, preparing a quality control line on the nitrocellulose membrane by using a secondary antibody, and then assembling the test strip. Specifically, the test strip comprises a sample pad, a nitrocellulose membrane (NC membrane), a water absorption pad and a bottom plate, wherein the NC membrane is stuck in the middle of the bottom plate, and the water absorption pad and the sample pad are respectively stuck on the upper side and the lower side of the bottom plate and overlapped with the NC membrane by 2-3 mm. Spraying the secondary antibody with the concentration of 0.3-1 mg/mL and the coating source with the concentration of 0.5-1 mg/mL on an NC film by using a film spraying instrument to serve as a C line and a T line, cutting the NC film into test strips with the length of 3.8-4 mm by using a strip cutting machine, and placing the test strips into a self-sealing bag to be stored in a drying box for standby.
Detection of fipronil and its metabolites: and incubating the sample extracting solution with the gold-labeled antibody, and inserting the test strip for detection.
Example 1
As shown in FIG. 1A, the colloidal gold test strip capable of simultaneously detecting fipronil and metabolites thereof is supported by a bottom Plate (PVC), the middle part of the colloidal gold test strip and the bottom plate is a nitrocellulose membrane, a quality control line and a detection line are arranged on the nitrocellulose membrane, and a left sample pad and a right water absorption pad are respectively overlapped with the nitrocellulose membrane. The quality control line is sprayed with a secondary antibody marked by horseradish peroxidase, and the detection line is sprayed with a synthetic coating antigen. In the 96-well plate is a conjugate of a fipronil monoclonal antibody marked by colloidal gold.
The main component of the sample pad material in the test strip is a glass fiber film, and the bottom plate is a polyvinyl chloride material. The leftmost side of the sample pad has a layer of adhesive paper with an arrow pointing downwards to indicate the direction of insertion of the test strip into the sample extraction solution.
Example 2
To prepare a colloidal gold test strip capable of simultaneously and rapidly detecting fipronil and metabolites thereof, an immunogen with strong immunogenicity and a monoclonal antibody for resisting fipronil and metabolites thereof are prepared, and then the test strip is assembled and prepared. The specific operation steps are as follows:
(1) Preparation of hapten: and hydrolyzing cyano groups on the fipronil into carboxyl groups by an acid hydrolysis method. 437mg of fipronil with the concentration of 1mmoL is weighed, 2.5mL of acetic acid, 1.25mL of 98% concentrated sulfuric acid and 1.5mL of water are added, and after the mixture is fully and uniformly mixed, the mixture is heated and refluxed for 3 hours. Then cooled to room temperature, 1M sodium hydroxide solution was slowly added dropwise at 0℃to adjust the pH of the solution to 10, 3mL of water was added to dissolve the precipitate, and the aqueous solution was washed with 10mL of n-hexane. After separation, the aqueous layer solution was collected, acidified with 1M hydrochloric acid until pH 1 was reached, and then extracted with 3X 15mL ethyl acetate. All organic phases were combined, washed with 15mL of saturated brine, dried over anhydrous sodium sulfate and evaporated under reduced pressure to remove the organic solvent. Finally, the mixture is passed through a silica gel column to obtain white solid.
(2) Preparation of immunogens and coating precursors: 3mg of hapten was dissolved in 1mL of DMF, 2.2mg of NHS and 4mg of EDC were added thereto, and the mixture was stirred at room temperature for 6 hours. 14mg BSA and 10mg OVA were each well dissolved in 5mL CBS buffer, and the activated hapten solutions were then added drop wise to the BSA solution and OVA solution, respectively, with magnetic stirring. Stirring the mixture at 4deg.C for reacting for 12 hr to obtain immunogen and coating antigen, dialyzing the immunogen and coating antigen with PBS buffer solution at 4deg.C for 72 hr, changing the dialysate for 8 times during dialysis, and packaging at-20deg.C.
(3) Preparation of monoclonal antibodies: the first immunization (priming) of the mice was performed by diluting the prepared immunogen to 2mg/mL with physiological saline, then mixing with an equal volume of Freund's complete adjuvant, and fully emulsifying with an emulsifier. Balb/C mice were then immunized by subcutaneous multi-point injection on the back, with 100. Mu.g of each mouse. The mice were then boosted every three weeks, fully emulsified with immunogen diluted to 2mg/mL and an equal volume of Freund's incomplete adjuvant, and immunized in the same manner as the first immunization, with an immunization dose of 50 μg per mouse. From the third immunization, mice were tested for tail blood sampling. Mice with high titers and high inhibition rates were screened for flushing-free i.e. intraperitoneal injection of 20 μg of immunogen. Three days later, the mice were sacrificed by pulling the neck, immersing in 75% alcohol for 10min, taking spleen cells under aseptic conditions, fully grinding the spleen on a copper mesh with a syringe core, then flushing three times with PRMI-1640 medium, and centrifuging the cell suspension with 1200r/min in a 50mL centrifuge tube for 10min. The supernatant was discarded, and the precipitated cells were resuspended in PRMI-1640 medium and the spleen cells were counted after three replicates according to the procedure described above. The spleen cells and myeloma cells of the mice are fully and evenly mixed according to the ratio of 10:1, the mixture is placed in a 50mL centrifuge tube for 1200r/min, the mixture is centrifuged for 10min, the cells are gently flicked from the bottom of the centrifuge tube, 1mL of PEG4000 is slowly added in the first minute, and 10mL of PRMI-1640 culture medium is added after the mixture is kept stand for 1min to stop the action of the PEG. The fused cells were placed in an oven at 37℃for 15min and then centrifuged at 800r/min for 10min. Then, the cells were resuspended in complete medium containing 15% fetal bovine serum HAT, mixed well and then placed in 96-well plates and incubated at 37℃in 5% CO 2 Culturing in an incubator. The supernatant is screened by an indirect competition ELISA method, the cell holes with high inhibition rate and strong positive are selected for subcloning, and then the subcloning is carried out for 2-3 times to obtain the positive hybridoma cell strain with high purity.
Preparation and purification of ascites: the hybridoma cells selected above were diluted to 1X 10 per ml with PRMI-1640 medium 6 Hybridoma cells of cells, each mouse was injected by intraperitoneal injection according to 800uL, the abdomen of the mouse was swelled, ascites were collected by a 5mL syringe and centrifuged at 3500r/min for 10min, and the supernatant was collected for purification of n-octanoic acid-ammonium sulfate. Taking 1mL of antiserum, diluting 2 times with 0.06mol/L, pH 5.0.0 acetic acid buffer solution, and adjusting the pH to 4.5 with 1mol/L HCl solution; slowly adding n-octanoic acid dropwise at a ratio of 33 mu L/mL of original serum under stirring at room temperature, standing at 4 ℃ for more than 2 hours to fully precipitate the hybrid protein, centrifuging at 4 ℃ for 30 minutes at 9000r/min, discarding the precipitate, collecting the supernatant, filtering with filter paper, adding PBS with a volume of 0.1mol/L, pH 7.4.4 which is 1/10 of the volume of the supernatant, adjusting the pH of the solution to 7.4, adding ammonium sulfate according to 0.277g per milliliter, uniformly stirring, and standing at 4 ℃ for more than 4 hours. Centrifuging at 4 ℃ and 9000r/min for 30min, discarding the supernatant, collecting the precipitate, dissolving the precipitate in a certain volume of PBS, dialyzing in 1L of PBS at 4 ℃ to remove salt, dialyzing for 3-4 days, and replacing 3 times of dialyzate every day.
(4) Determination of antibody sensitivity and crossover rate: fipronil with concentrations of 0, 0.1, 0.3, 0.9, 2.7 and 8.1ng/mL respectively, fipronil with concentrations of 0, 0.05, 0.15, 0.45, 1.35 and 4.05ng/mL respectively, fipronil sulfone with concentrations of 0, 0.1, 0.3, 0.9, 2.7 and 8.1ng/mL respectively, fipronil sulfoxide with concentrations of 0, 0.1, 0.3, 0.9, 2.7 and 8.1ng/mL respectively, and half Inhibition Concentration (IC) thereof was measured by IC-ELISA method 50 ) The antibody crossover rate was then calculated. The calculation formula of the crossing rate is as follows: cross ratio (%) = (fipronil IC) 50 Analog IC 50 ) X 100%. The results are shown in Table 1 and FIG. 3, which show the IC of fipronil antibody 50 The cross rates of the fipronil sulfoxide, fipronil sulfone and fipronil are respectively 69.84%, 75.86 and 112.82% at 0.44 ng/mL. The sensitivity of the fipronil antibody prepared by the invention is respectively improved by 700 and 14 times compared with the sensitivity of antibodies in Development of an enzyme immunoassay for detection of fipronil in environmental samples and Poly-and monoclonal antibody-based ELISAs for fipronil, and the antibodies prepared in the above two papers only recognize fipronilItself. In conclusion, the antibody prepared by the invention can simultaneously recognize fipronil and three metabolites thereof, and has higher sensitivity.
TABLE 1 determination of antibody Cross Rate
(5) Preparation of gold-labeled antibody: 800mL of ultrapure water was taken in a flask, heated at 102℃and boiled for 30min, 1.6mL of 10% chloroauric acid solution was added under magnetic stirring, after 10min, 2.08mL of 10% trisodium citrate solution was added, and stirring was continued until the solution became dark red in color and did not change color. Cooling to room temperature and preserving for standby. As shown in FIG. 2, the colloidal gold has the advantages of successful preparation, uniform particle size, good dispersibility and no aggregation phenomenon, and the particle size of the colloidal gold is calculated to be about 15 nm. 3mL of the colloidal gold solution was taken, 300. Mu.L of boric acid buffer with pH 8.5 was added thereto, and after thorough mixing, 18. Mu.g of the antibody solution was added dropwise thereto for reaction for 1 hour. Then 300 μl of 10% BSA was added dropwise to the above solution to block the sites without binding to reduce nonspecific adsorption in the assay, and incubated at room temperature for 0.5h. The solution was centrifuged at 10000r/min at 4℃for 25min to remove unbound antibody and colloidal gold, the supernatant was discarded, and the pellet was dissolved in 200. Mu.L of a heavy suspension in PBS containing 2% BSA, 1% sucrose, 0.02% sodium azide, which was favorable for protection of the gold-labeled antibody, and then stored at 4℃for further use. The prepared gold-labeled antibody is diluted 20 times, 50 mu L of the gold-labeled antibody is taken out and is frozen and dried in a 96-well plate for standby.
(6) Preparation of test strips: coating antigen 0.8mg/mL and secondary antibody 0.4mg/mL were sprayed on NC film with a film spraying instrument as T line and C line, and oven at 37deg.C overnight for use, and then assembled according to the structure of the test strip shown in FIG. 1A. Before assembly, the sample pad is soaked in PBS solution of 0.2% Tween-20,1% sucrose and 1% BSA of 0.01M, pH 7.4.4 for 30min and then dried at 37 ℃, and the solution is favorable for the upward climbing of the gold-labeled antibody and the combination of the gold-labeled antibody with the coating antigen of the T line and the secondary antibody of the C line. The detection method of the invention has the following rule: as shown in fig. 1B: and (3) adding 150 mu L of sample extracting solution into the lyophilized gold-labeled antibody, uniformly mixing, incubating for 3min at room temperature, inserting a test strip into the hole, and visually judging the detection result after five minutes, so that the full combination of the object to be detected, the gold-labeled antibody, the coating source and the secondary antibody is facilitated, and the sensitivity of the detection method is improved. According to capillary action, the mixture develops color when climbing up due to the capture of antigen-antibody specific reaction by the secondary antibody of the C-line and the coating antigen of the T-line. If the color depth of the T line is consistent with that of the C line, the sample is a negative result without the to-be-detected object. If the color depth of the T line is lighter than that of the C line or the T line is not colored, the sample contains the object to be detected, and the positive result is obtained. If the line C has no color, the test strip is indicated to be a failure product.
(7) Test strip detection limit determination: taking 5g of evenly mixed eggs, placing the eggs into a 50mL centrifuge tube, and adding standards of fipronil and metabolites thereof with different concentrations, wherein fipronil, fipronil sulfone and fipronil sulfoxide are as follows: 0.5, 10, 20ng/g; the fluoronitrile is as follows: 0. 2.5, 5, 10ng/g, 3g of neutral alumina and 5mL of acetonitrile are added, followed by vigorous shaking for 5min, centrifugation at 4000r/min for 3min at room temperature, and the precipitate is discarded to collect the supernatant. 1mL of the supernatant was taken in a 2mL centrifuge tube and nitrogen was blown at 60℃until the organic reagent was completely evaporated. The residue was redissolved in 1mL of n-hexane, 1mL of 0.01M PBS was added, and the mixture was centrifuged at 4000r/min for 3min, and the lower layer solution was collected. The limit of detection is defined as the limit of detection as the lowest concentration at which the T line is lighter in color than the C line compared to the negative control. The detection results are shown in fig. 4: the detection limits of fipronil, fipronil sulfoxide, fipronil sulfone and fipronil are 10, 10 and 5ng/g respectively.
FIG. 3 is an indirect antibody competition inhibition curve, IC of fipronil antibody 50 The cross rates of the fipronil sulfoxide, fipronil sulfone and fipronil are respectively 69.84%, 75.86 and 112.82% at 0.44 ng/mL. The antibody prepared by the invention is a monoclonal antibody capable of simultaneously recognizing fipronil and metabolites thereof, and has higher sensitivity. Provides a powerful condition for preparing the colloidal gold test strip capable of simultaneously detecting fipronil and the metabolites thereof.
Directly incubating the sample extracting solution with the gold-labeled antibody for 3min, inserting a test strip, and judging a detection result after 5 min; the test shows that the detection method has high sensitivity, and the detection limits of fipronil, fipronil sulfone, fipronil sulfoxide and fipronil are respectively 10, 10 and 5ng/g.
FIG. 4 is a graph showing the effect of the colloidal gold test strip on detecting fipronil and the residues of the metabolites thereof. Wherein A, B, C is fipronil, fipronil sulfone and fipronil sulfoxide, and the concentration gradients of the fipronil, the fipronil sulfone and the fipronil sulfoxide are 0, 5, 10 and 20ng/g; d is fluoronitrile with concentration gradient of 0, 2.5, 5 and 10ng/g. The test results of the test strips in the figure show that the detection limits of fipronil, fipronil sulfoxide, fipronil sulfone and fipronil are respectively 10, 10 and 5ng/g.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (6)
1. The preparation method of the colloidal gold test strip for detecting fipronil and metabolites thereof is characterized by comprising the following steps:
preparation of hapten: the fipronil is prepared into hapten by acid hydrolysis method and adding acetic acid and introducing carboxyl, and the hapten is prepared specifically as follows:
dissolving fipronil in concentrated sulfuric acid, acetic acid and water, heating and refluxing, then dropwise adding sodium hydroxide aqueous solution until the pH value reaches alkalinity, adding pure water to dissolve precipitate, washing aqueous solution with normal hexane, separating and acidifying aqueous layer solution until the pH value reaches acidity, then extracting with ethyl acetate, combining organic phases, washing with saturated saline water, drying with anhydrous sodium sulfate, decompressing and evaporating, and passing through a silica gel column to obtain white solid which is hapten; the mass fraction of the concentrated sulfuric acid is 98%; the dosage ratio of fipronil to concentrated sulfuric acid to acetic acid to water is 300-500 mg:1-4 mL:1-3 mL:1-2 mL; the dosage ratio of the pure water to the sediment is 2~3 mL:200~600 mg;
preparation of immunogens and coating precursors: the hapten is coupled with protein through an activated ester method to prepare an immunogen and a coating antigen respectively, wherein the preparation of the immunogen and the coating antigen is specifically as follows:
activation of hapten: dissolving the hapten in dimethylformamide DMF, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC into the mixture, and stirring and activating the mixture to obtain an activated hapten; the dosage ratio of hapten, DMF, NHS and EDC is 2-4 mg:0.5 to 1mL: 1-4 mg: 2-7 mg; the stirring activation time is 6-8 hours;
preparation of the immunogen: fully dissolving Bovine Serum Albumin (BSA) in a Carbonate Buffer (CBS) to obtain a BSA solution, dropwise adding the activated hapten solution into the BSA solution under magnetic stirring, stirring the mixture for reaction to obtain an immunogen, and dialyzing the immunogen with Phosphate Buffer (PBS);
preparation of coating raw material: fully dissolving chicken Ovalbumin (OVA) in Carbonate Buffer (CBS) to obtain an OVA solution, dropwise adding the activated hapten solution into the OVA solution under magnetic stirring, stirring the mixture for reaction to obtain a coating raw material, and dialyzing the coating raw material with Phosphate Buffer (PBS);
preparation of monoclonal antibodies: monoclonal antibodies were prepared using the immunogens and the coating antigen was used to detect the single antigen by ic-ELISA
The sensitivity and specificity of the clone antibody are used to obtain the monoclonal antibody of the fipronil and the metabolite thereof;
preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to obtain a gold-labeled antibody;
preparing a colloidal gold test strip: and (3) preparing a detection line on the nitrocellulose membrane by using a fipronil coupled protein coating antigen, preparing a quality control line on the nitrocellulose membrane by using a secondary antibody, and then assembling the test strip.
2. The method for preparing the colloidal gold test strip for detecting fipronil and metabolites thereof according to claim 1, wherein in the preparation process of the immunogen, the dosage ratio of the BSA buffer solution to the CBS buffer solution is 7-15 mg: 3-5 mL;
in the preparation process of the coating raw material, the dosage ratio of the OVA to the CBS buffer solution is 5-10 mg: 2-5 mL;
the molar ratio of the hapten in the activated hapten solution to the protein in the BSA solution and the molar ratio of the hapten in the activated hapten solution to the protein in the OVA solution are both 20:1-40:1;
the temperature of the stirring reaction is 4 ℃, and the stirring reaction time is 12-18 hours;
the temperature of the PBS buffer solution during dialysis is 4 ℃, and the time is 36-78 hours.
3. The method for preparing the colloidal gold test strip for detecting fipronil and the metabolites thereof according to claim 1, wherein the preparation of the gold-labeled antibody is specifically as follows:
heating pure water to boil, adding chloroauric acid solution under stirring, and adding trisodium citrate solution to obtain colloidal gold solution;
taking a colloidal gold solution, adding a boric acid buffer solution, fully and uniformly mixing, dropwise adding the monoclonal antibody solution for reaction, and dropwise adding 10% BSA for blocking to obtain a mixed solution;
centrifuging the mixed solution, discarding the supernatant to obtain a precipitate, and dissolving the precipitate in the heavy suspension to obtain the gold-labeled antibody.
4. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites according to claim 3, wherein the ratio of pure water, chloroauric acid solution and trisodium citrate solution is 500~900 mL:1~2mL:1~3 mL;
the dosage ratio of the colloidal gold solution, the monoclonal antibody solution, the boric acid buffer solution and the 10% BSA is 3-6 mL, 18-48 mug, 300-600 mug and 200-800 mug;
the dosage ratio of the sediment to the heavy suspension is 0.1-1 mg/300-500 mu L.
5. The method for preparing the colloidal gold test strip for detecting fipronil and the metabolites thereof according to claim 1, which is characterized in that the preparation of the colloidal gold test strip specifically comprises the following steps:
the sample pad was soaked in 0.01M pH 7.4PBS containing 0.2% Tween-20,1% sucrose and 1% BSA and then dried;
spraying 0.5-1 mg/mL of coating antigen on the nitrocellulose membrane to serve as a detection line, and spraying 0.2-0.6 mg/mL of secondary antibody on the nitrocellulose membrane to serve as a quality control line;
the sample pad, nitrocellulose membrane, absorbent pad and base plate were assembled.
6. A colloidal gold test strip for detecting fipronil and its metabolites, characterized in that it is prepared according to the method for preparing the colloidal gold test strip for detecting fipronil and its metabolites as set forth in any one of claims 1-5.
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