CN111187346B - Colloidal gold test strip for detecting fipronil and its metabolites and preparation method thereof - Google Patents

Abstract

The invention provides a colloidal gold test strip for detecting fipronil and its metabolites and a preparation method thereof, comprising the following steps: preparation of hapten, preparation of immunogen and coating original, preparation of monoclonal antibody, preparation of gold-labeled antibody Preparation, preparation of colloidal gold test strips; the present invention uses the fipronil-coupled protein coating to make a detection line on the nitrocellulose membrane, uses the secondary antibody to make a quality control line on the nitrocellulose membrane, and extracts the sample The solution was incubated with the gold-labeled antibody, and a test strip was inserted for detection. The present invention prepares high-sensitivity and high-specificity monoclonal antibodies against fipronil and its metabolites, and then builds on the monoclonal antibodies with fast speed, low cost, high sensitivity and can simultaneously detect fipronil and its metabolites. A colloidal gold test strip for metabolites, the test strip of the invention can simultaneously detect fipronil and its metabolites without instrument assistance, has low cost, simple operation, and is suitable for rapid screening of on-site samples.

Description

Colloidal gold test strip for detecting fipronil and its metabolites and preparation method thereof

technical field

The invention belongs to the technical field of food safety detection, and in particular relates to a colloidal gold test strip for simultaneous rapid detection of fipronil and its metabolites and a preparation method thereof.

Background technique

Fipronil, the English name is fipronil (fipronil), the chemical name is (RS)-5-amino-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylidene Sulfonylpyrazole-3-carbonitrile is a phenylpyrazole insecticide developed in the 1980s. It is one of the substitutes for organophosphorus pesticides. Due to its good bactericidal effect and high broad-spectrum , Widely used in the pest control of fruits, vegetables and other crops, the control of parasites and pests in homes and entertainment places. Fipronil controls pests by destroying the chloride channel controlled by γ-aminobutyric acid (GABA) in pests, thereby destroying their normal nervous system. Relevant studies have shown that fipronil has chronic neurotoxicity and has been classified as a class C carcinogen. Fipronil can affect human health and cause symptoms such as conjunctivitis, dizziness, seizures, restlessness, and sweating. Fipronil is degraded into more toxic metabolites of flupronil, fipronil sulfone and fipronil sulfoxide through photolysis, oxidation, and reduction in the natural environment. The LD ₅₀ of fluorocarbonitrile in rats is only 1/6 of that of the parent itself, and its toxicity to rats is far greater than that of itself. The toxicity of fipronil sulfone to daphnia is about 6 times that of its parent itself, and the bioaccumulation of fipronil sulfoxide is 5 times that of fipronil parent. In my country's Announcement No. 1157 issued in 2009, it is stipulated that fipronil is only limited to the use of some seed coatings such as corn, and fipronil and its metabolism are specified in the national standard food pesticide residue limit (GB-2763-2016). The residue limit range of food in grains, oilseeds and oils, vegetables, fruits, sugar crops and edible fungi is 0.02-0.1mg/kg.

At present, the detection methods of fipronil are mainly instrumental analysis methods, mainly including gas chromatography-tandem mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), high performance liquid chromatography ( HPLC), high-performance liquid phase diode array detection (HPLC-DAD), although these methods have high sensitivity and accuracy, they require expensive instruments, professional operators, complicated and time-consuming pre-treatment, therefore, they are not suitable for the detection of a large number of samples on site. Quick filter. The immunoassay method is a detection method based on the specific reaction of antigen and antibody. At present, the immunoassay and detection methods for fipronil that have been authorized or announced in China mainly include: a kit for detecting fipronil with a publication number of CN201710990416.6 and its preparation method and application, a publication number of CN201711059643.3 A chemiluminescent immunoassay kit for detecting fipronil in tea. The above two inventions are both inventions and applications of ELISA kits. The detection process needs to be washed repeatedly and takes a long time. The results will vary due to different operators. The result requires an instrument for reading, which is not suitable for rapid screening of a large number of samples on site; the publication number is CN201810005898.X, a surface-enhanced Raman gold standard test strip for rapid detection of trace amounts of fipronil and its preparation method, the detection method of the invention The Raman signal is unstable and prone to false positives, and the detection results need the assistance of Raman instruments, which are expensive and costly; the publication number is 2018103135712.0, which uses light and surface-enhanced Raman to quickly detect fipronil Method, the method of this invention is to indirectly detect the existence of fipronil by detecting the main photolysis product of fipronil, flupronil, which increases the detection steps and time, the accuracy of the result is not high, the Raman instrument is expensive, and the cost rises. Gao; Publication No. 201810005478.1 A colloidal gold standard test strip for rapid detection of trace amounts of fipronil and its preparation method. The detection method of the invention is only for fipronil itself, and for the more toxic fipronil metabolites Testing is not involved. Fipronil is easily degraded in the natural environment, and the accuracy of this method is not high.

Contents of the invention

In view of the above problems, the present invention provides a colloidal gold test strip and a preparation method thereof for rapid detection of fipronil and its metabolites at the same time. Antibodies, and then based on antibodies, the establishment of fast, low cost, high sensitivity and can simultaneously detect fipronil and its metabolites colloidal gold test strips, the test strips can simultaneously detect fipronil and its metabolites It does not require instrument assistance, is low in cost, simple in operation, and is suitable for rapid screening of on-site samples.

A method for preparing a colloidal gold test strip for detecting fipronil and its metabolites, comprising the following steps:

Preparation of hapten: fipronil is hydrolyzed by acid and acetic acid is added to introduce carboxyl group to prepare hapten;

Preparation of the immunogen and the coating source: the hapten is coupled to the protein by the activated ester method to prepare the immunogen and the coating source respectively;

Preparation of monoclonal antibody: use the immunogen to prepare monoclonal antibody, use the coating source to detect the sensitivity and specificity of the monoclonal antibody by ic-ELISA, and obtain the monoclonal antibody against fipronil and its metabolites;

Preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to prepare gold-labeled antibody;

Preparation of colloidal gold test strips: use fipronil-coupled protein-coated originals to make detection lines on nitrocellulose membranes, use secondary antibodies to make quality control lines on nitrocellulose membranes, and then assemble them into test strips.

In the above scheme, the preparation of the hapten is specifically:

Dissolve fipronil in concentrated sulfuric acid, acetic acid and water, heat to reflux, then add aqueous sodium hydroxide solution dropwise until the pH reaches alkalinity, add pure water to dissolve the precipitate, wash the aqueous solution with n-hexane, separate and acidify the aqueous layer solution, Until the pH becomes acidic, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, evaporate under reduced pressure, and pass through a silica gel column to obtain a white solid, which is the hapten.

In such scheme, the massfraction of described concentrated sulfuric acid is 98%;

The dosage ratio of said fipronil, concentrated sulfuric acid, acetic acid and water is 300～500mg:1～4mL:1～3mL:1～2mL;

The usage ratio of the pure water and the precipitate is 2-3mL:200-600mg.

In the above scheme, the preparation of the immunogen and the coating agent is specifically:

Activation of hapten: Dissolve the hapten in dimethylformamide DMF, add N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC to it, and stir the mixture to activate , to obtain the activated hapten;

Preparation of immunogen: fully dissolve bovine serum albumin BSA in carbonate buffer CBS to obtain a BSA solution, add the activated hapten solution dropwise to the BSA solution under magnetic stirring, and stir the mixture React to obtain the immunogen, and the immunogen is dialyzed with phosphate-buffered PBS;

Preparation of coating source: Fully dissolve chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA substance solution, add the activated hapten solution dropwise to the OVA solution under magnetic stirring, and put The mixture was reacted with stirring to obtain the coated original, which was dialyzed against phosphate buffered PBS.

In the above scheme, during the activation process of the hapten, the dosage ratio of the hapten, DMF, NHS and EDC is 2-4mg: 0.5-1mL: 1-4mg: 2-7mg; the stirring activation time is 6 ~8h.

In the above scheme, during the preparation of the immunogen, the dosage ratio of the BSA and CBS buffer is 7-15mg: 3-5mL;

During the preparation of the coating agent, the dosage ratio of the OVA and CBS buffer solution is 5-10 mg:2-5 mL;

The mol ratio of the hapten in the activated hapten solution to the protein in the BSA solution, and the mol ratio of the hapten in the activated hapten solution to the protein in the OVA solution are 20:1～40: 1;

The temperature of the stirring reaction is 4° C., and the time of the stirring reaction is 12 to 18 hours;

The temperature of the PBS buffer dialysis is 4° C., and the time is 36-78 hours.

In the above scheme, the preparation of the gold-labeled antibody is specifically as follows:

Take pure water and heat to boil, add chloroauric acid solution under stirring condition, then add trisodium citrate solution to obtain colloidal gold solution;

Take the colloidal gold solution, add boric acid buffer solution, mix well, then add the monoclonal antibody solution dropwise for reaction, and then add 10% BSA dropwise to block to obtain a mixed solution;

The mixed solution is centrifuged, and the supernatant is discarded to obtain a precipitate, which is dissolved in the resuspension to obtain the gold-labeled antibody.

In the above-mentioned scheme, the consumption ratio of described pure water, chloroauric acid solution and trisodium citrate solution is 500～900mL:1～2mL:1～3 mL;

The dosage ratio of the colloidal gold solution, monoclonal antibody solution, boric acid buffer solution and 10% BSA is 3～6mL:18～48μg: 300～600μL:200～800μL;

The dosage ratio of the precipitate and the resuspension is 0.1-1 mg:300-500 μL.

In the foregoing scheme, the preparation of the colloidal gold test strip specifically comprises the following steps:

Soak the sample pad in a 0.01M pH 7.4 PBS solution containing 0.2% Tween-20, 1% sucrose and 1% BSA and then dry it;

Spray 0.5-1 mg/mL of the original coating on the nitrocellulose membrane as the detection line, and spray 0.2-0.6 mg/mL of the secondary antibody on the nitrocellulose membrane as the quality control line;

The sample pad, nitrocellulose membrane, absorbent pad and bottom plate were assembled.

A colloidal gold test strip for detecting fipronil and its metabolites is prepared according to the detection method for the colloidal gold test strip for detecting fipronil and its metabolites.

Compared with prior art, the beneficial effect of the present invention is:

1. The present invention adds acetic acid to introduce carboxyl groups through acid hydrolysis, increases the reaction rate, and prepares haptens, and then prepares immunogens with strong immunogenicity by coupling proteins with activated ester method, and prepares a compound that can simultaneously recognize fipronil and its Metabolite monoclonal antibody, and the sensitivity of the antibody is high.

2. The present invention can simultaneously detect fipronil and its more toxic metabolites by using the test strip prepared by colloidal gold-labeled monoclonal antibody, so this method will be more meaningful and valuable in ensuring food safety.

3. The colloidal gold test strip prepared by the present invention is low in cost, fast in detection speed and high in sensitivity; the detection result of the method can be directly visually interpreted without the assistance of an instrument, saves the cost of expensive instruments, and is suitable for the detection of a large number of samples on site; Directly incubate the sample extract with the gold-labeled antibody, insert the test strip, and the test result can be interpreted after 5 minutes; the test shows that the detection method has high sensitivity.

4. The colloidal gold test strip prepared by the present invention is simple to operate and has wide applicability; the detection method is simple and easy to understand, does not require professional operation, and can be used by law enforcement agencies, farmers, consumers and other groups. In addition, this method can provide a certain guarantee for the life safety of consumers.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and understandable from the description of the embodiments in conjunction with the following drawings, wherein:

In Fig. 1, Fig. 1A is a schematic structural view of a colloidal gold test strip of the present invention; Fig. 1B is a schematic diagram of a colloidal gold test strip.

Figure 2 is the UV image of colloidal gold; the inset is the TEM image of colloidal gold.

Figure 3 is the antibody indirect competition inhibition curve.

Fig. 4 is an effect diagram of detecting fipronil and its metabolite residues by colloidal gold test strips.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

The preparation method of the colloidal gold test strip for detecting fipronil and its metabolites of the present invention comprises the following steps:

(1) Preparation of hapten: Dissolve an appropriate amount of fipronil in concentrated sulfuric acid, acetic acid and water, heat and reflux for 2-3 hours, then add aqueous sodium hydroxide solution dropwise until the pH reaches 9-10, add pure water to dissolve the precipitate , wash the aqueous solution with 5-15mL of n-hexane, separate and acidify the aqueous solution until the pH reaches 1, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, and evaporate under reduced pressure. Pass through a silica gel column to obtain a white solid, which is the hapten;

The massfraction of described concentrated sulfuric acid is 98%;

The dosage ratio of said fipronil, concentrated sulfuric acid, acetic acid and water is 300～500mg:1～4mL:1～3mL:1～2mL;

The usage ratio of the pure water and the precipitate is 2-3mL:200-600mg.

(2) Preparation of immunogen and coating agent:

Hapten activation: The hapten obtained in the hapten preparation step is dissolved in dimethylformamide DMF, and N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC are added thereto , and the mixture was stirred at room temperature to obtain an activated hapten;

Preparation of immunogen: fully dissolve bovine serum albumin BSA in carbonate buffer CBS to obtain a BSA solution, then add the activated hapten solution to the BSA solution drop by drop under magnetic stirring, and the mixture Perform stirring reaction under certain temperature conditions to obtain the immunogen;

Preparation of coating source: fully dissolve chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA substance solution, and then add the activated hapten solution to the OVA solution drop by drop under magnetic stirring, The mixture was stirred and reacted under certain temperature conditions to obtain the coating source; the immunogen and the coating source were dialyzed with phosphate-buffered PBS respectively, and the dialysate was changed 8 times during the dialysis process. Store at 20°C for later use.

The dosage ratio of the hapten, DMF, NHS and EDC is 2-4mg: 0.5-1mL: 1-4mg: 2-7mg; the stirring time is 6-8h;

During the preparation of the immunogen, the dosage ratio of the BSA and CBS buffer is 7-15 mg: 3-5 mL;

During the preparation of the coating agent, the dosage ratio of the OVA and CBS buffer solution is 5-10 mg:2-5 mL;

The mol ratio of the hapten in the activated hapten solution to the protein in the BSA solution, and the mol ratio of the hapten in the activated hapten solution to the protein in the OVA solution are 20:1～40: 1;

The temperature of the stirring reaction is 4° C., and the time of the stirring reaction is 12 to 18 hours;

The temperature during dialysis of the PBS buffer solution is 4°C, 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 Bailingwei Biological Reagent Co., Ltd.

(3) Preparation of monoclonal antibody: Dilute the prepared immunogen to 1-2 mg/mL with normal saline, then mix it with an equal volume of Freund's complete adjuvant and emulsify it; Mice (Balb/C) were immunized, and the amount of immunization for each mouse was 75-100 μg;

Afterwards, the mice were boosted every three weeks, and the immunogen diluted to 2 mg/mL and an equal volume of Freund's incomplete adjuvant were fully emulsified, and the mice were injected subcutaneously at multiple points on the back (Balb/C) For immunization, the immune dose per mouse was 25-50 μg.

From the third immunization, the mice were tested by tail blood sampling; the mice with high titer and high inhibition rate were screened out and 20 μg of immunogen was injected intraperitoneally; three days after the injection, the mice were killed by pulling the neck and soaked in 75% After soaking in alcohol for 10 minutes, take the spleen cells under sterile conditions, grind the spleen well on a copper grid, and then wash it three times with PRMI-1640 medium, and centrifuge the cell suspension at 1200r/min for 10 minutes; Supernatant, and then use PRMI-1640 medium to resuspend the precipitated cells, centrifuge, and count the spleen cells after repeating the above operation three times;

According to the 10:1 ratio of mouse splenocytes and myeloma cells, the two were fully mixed, and the mixed solution was centrifuged at 1200r/min for 10 minutes, and the cells were dispersed at the bottom of the centrifuge tube. Add PEG4000 slowly, and add PRMI-1640 medium after standing for 1min to terminate the effect of PEG; the dosage ratio of the mixed solution, PEG4000, and PRMI-1640 medium is 0.5mL: 10mL. The fused cells were cultured in a 37°C oven for 15 minutes, and then centrifuged at 3500 r/min for 10 minutes. Then the cells were resuspended with the complete medium of HAT containing 10% fetal bovine serum, mixed evenly and added to the 96-well plate, and placed in a 37°C, 5% CO ₂ incubator for cultivation. The supernatant was screened by the indirect competitive ic-ELISA method with the coating original, and the cell wells with high inhibition rate and strong positive were selected for subcloning, and then expanded culture. After 2 to 3 times of subcloning, high purity cells were obtained Positive hybridoma cell lines.

Preparation and purification of ascites: Dilute the above-screened hybridoma cells with PRMI-1640 medium to 1×10 ⁶ hybridoma cells per milliliter, and inject 500-800uL into each mouse intraperitoneally. Swelling, ascites was collected and centrifuged at 3500r/min for 10min, and the supernatant was collected for n-octanoic acid-ammonium sulfate purification to obtain monoclonal antibodies.

The specific operation for the purification of n-octanoic acid-ammonium sulfate is as follows: take 1 mL of antiserum, dilute it twice with 0.06 mol/L, pH 5.0 acetate buffer, adjust the pH to 4.5 with 1 mol/L HCl solution; For the ratio of serum, slowly add n-octanoic acid drop by drop under stirring at room temperature, let stand at 4°C for more than 2 hours to fully precipitate the miscellaneous proteins, centrifuge at 4°C, 9000r/min for 30min, discard the precipitate, collect the supernatant and filter it with filter paper, add volume 0.1mol/L, pH7.4PBS, which is 1/10 of the volume of the supernatant, and adjust the pH of the solution to 7.4, add ammonium sulfate at 0.277g per ml, stir well, and stand at 4°C for more than 4h. Then centrifuge at 4°C and 9000r/min for 30min, discard the supernatant, collect the precipitate, dissolve the precipitate in a certain volume of PBS, and dialyze in 1L of PBS at 4°C to remove salt, and dialyze for 3 to 4 days , changing the dialysate 3 times a day.

(4) Preparation of gold-labeled antibody: Take 500-900mL of ultrapure water in a flask, heat and boil at 102°C for 30-50 minutes, add 10% chloroauric acid solution under magnetic stirring, and add 10% lemon after 10 minutes Trisodium acid solution, continue to stir until the color of the solution turns dark red and does not change color, to obtain a colloidal gold solution, cool to room temperature and store in a dark place for later use. Take 3-6mL of colloidal gold solution, add boric acid buffer solution with pH 8.5, mix well, then add the prepared monoclonal antibody solution drop by drop to react for 30-60min. Then 10% BSA was added dropwise to the above solution to block unbound sites, and incubated at room temperature for 0.5-1 h. Centrifuge the mixed solution at 10000r/min for 25min at 4°C to remove unbound antibodies and gold nanoparticles, discard the supernatant, and dissolve the precipitate in a resuspension containing 2% BSA, 1% Sucrose, 0.02% sodium azide in PBS solution, and stored at 4°C for later use. The gold-labeled antibody prepared above was diluted 20-50 times, and 50 μL was taken in a 96-well plate for freeze-drying for later use. The secondary antibody and the prepared coating source were sprayed on the nitrocellulose membrane as the quality control line (C line) and the detection line (T line) with a film sprayer, and were kept in an oven at 37°C overnight for later use.

The consumption ratio of described pure water, chloroauric acid solution and trisodium citrate solution is 500～900mL:1～2mL:1～3mL;

The dosage ratio of the colloidal gold solution, monoclonal antibody, boric acid buffer and 10% BSA is 3～6mL:18～48μg:300～600μL:200～800μL;

The dosage ratio of the precipitate and the resuspension is 0.1-1 mg:300-500 μL;

The secondary antibody is HRP-goat anti-mouse IgG purchased from Shanghai Sangon Bioengineering Co., Ltd.

(5) Preparation of colloidal gold test strips: use fipronil-coupled protein coatings to make detection lines on nitrocellulose membranes, use secondary antibodies to make quality control lines on nitrocellulose membranes, and then assemble them into test strips. note. Specifically, the test strip includes a sample pad, a nitrocellulose membrane (NC membrane), an absorbent pad and a bottom plate, and the NC membrane is pasted in the middle of the bottom plate, and the water-absorbent pad and the sample pad are respectively pasted on the upper and lower sides of the bottom plate. Overlap 2-3mm. Spray 0.3-1 mg/mL of secondary antibody and 0.5-1 mg/mL of the coating source on the NC film as C line and T line with a film sprayer, and then cut it into 3.8-4 mm long with a strip cutter The test strips were put into a ziplock bag and stored in a dry box for later use.

Detection of fipronil and its metabolites: incubate the sample extract with the gold-labeled antibody, insert the test strip for detection.

Example 1

As shown in Figure 1A, the colloidal gold test strip of the present invention that can detect fipronil and its metabolites simultaneously is supported by a base plate (PVC), and the middle part of the two is a nitrocellulose membrane, and on the membrane There are quality control lines and detection lines, the sample pad on the left and the absorbent pad on the right are respectively lapped with the nitrocellulose membrane. The quality control line is sprayed with horseradish peroxidase-labeled secondary antibody, and the detection line is sprayed with a synthetic coating agent. The 96-well plate is a conjugate of colloidal gold-labeled fipronil monoclonal antibody.

The main component of the material of the sample pad in the test strip is glass fiber membrane, and the bottom plate is polyvinyl chloride material. The leftmost side of the sample pad has a layer of stickers with an arrow pointing down to indicate the direction in which the test strip is inserted into the sample extraction solution.

Example 2

To prepare colloidal gold test strips that can rapidly detect fipronil and its metabolites at the same time, it is first necessary to prepare immunogens with strong immunogenicity and monoclonal antibodies against fipronil and its metabolites, and then assemble the test strips and preparation. The specific operation steps are as follows:

(1) Preparation of hapten: the cyano group on fipronil was hydrolyzed into carboxyl group by acid hydrolysis method. Weigh 437mg, 1mmoL of fipronil, add 2.5mL acetic acid, 1.25mL, 98% concentrated sulfuric acid, 1.5mL water, mix thoroughly and heat the mixture to reflux for 3h. Then cool to room temperature, slowly add 1M sodium hydroxide solution dropwise at 0°C, adjust the pH of the solution to 10, add 3 mL of water to dissolve the precipitate, and wash the aqueous solution with 10 mL of n-hexane. After separation, the aqueous layer solution was collected, acidified with 1M hydrochloric acid solution until the pH reached 1, and then extracted with 3×15 mL ethyl acetate. All organic phases were combined, washed with 15 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to remove the organic solvent. Finally, it was passed through a silica gel column to obtain a white solid.

(2) Preparation of immunogen and coating original: 3 mg of hapten was dissolved in 1 mL of DMF, 2.2 mg of NHS and 4 mg of EDC were added thereto, and the mixture was stirred at room temperature for 6 h. 14mg BSA and 10mg OVA were fully dissolved in 5mL CBS buffer, respectively, and then the activated hapten solution was added dropwise to the BSA solution and the OVA solution under magnetic stirring. The mixture was stirred and reacted at 4°C for 12 hours to obtain the immunogen and the coating agent, which were dialyzed against PBS buffer at 4°C for 72 hours, and the dialysate was changed 8 times during the dialysis process. After dialysis, aliquots were stored at -20°C for future use.

(3) Preparation of monoclonal antibody: The first immunization (first immunization) of mice is to dilute the prepared immunogen to 2mg/mL with normal saline, then mix it with an equal volume of Freund's complete adjuvant, and use emulsifying fully emulsified. Then Balb/C mice were immunized by multi-point subcutaneous injection on the back, and the immune dose of each mouse was 100 μg. Afterwards, the mice were boosted every three weeks, and the immunogen diluted to 2 mg/mL and an equal volume of Freund's incomplete adjuvant were fully emulsified, and the same method as the first immunization was used for immunization. The immunization of each mouse The amount is 50 μg. From the third immunization, mice were tested by tail blood sampling. Mice with high titer and high inhibition rate were screened out for flushing, that is, 20 μg of immunogen was injected intraperitoneally. Three days later, the mouse was killed by pulling the neck, soaked in 75% alcohol for 10 minutes, and its spleen cells were taken under sterile conditions, and the spleen was fully ground on the copper grid with a syringe core, and then washed with PRMI-1640 medium for three times. For each time, the cell suspension was centrifuged at 1200r/min in a 50mL centrifuge tube for 10min. Discard the supernatant, resuspend the precipitated cells with PRMI-1640 medium, and count the splenocytes after repeating the above operation three times. According to the 10:1 ratio of mouse splenocytes and myeloma cells, the two were fully mixed, then centrifuged at 1200r/min in a 50mL centrifuge tube for 10min, and the cells were dispersed by flicking the bottom of the centrifuge tube gently. Slowly add 1 mL of PEG4000 within 1 minute, and add 10 mL of PRMI-1640 medium after standing for 1 minute to terminate the effect of PEG. The fused cells were placed in an oven at 37°C for 15 minutes, and then centrifuged at 800 r/min for 10 minutes. Then the cells were resuspended with the complete HAT medium containing 15% fetal bovine serum, mixed evenly, added to the 96-well plate, and placed in a 37°C, 5% _CO2 incubator for cultivation. The supernatant was screened by indirect competitive ELISA method, and the cell wells with high inhibition rate and strong positivity were selected for subcloning, and then expanded and cultured. Two to three times of subcloning were required to obtain high-purity positive hybridoma cell lines.

Preparation and purification of ascites: Dilute the above-screened hybridoma cells with PRMI-1640 medium to 1×10 ⁶ hybridoma cells per milliliter, inject 800uL intraperitoneally into each mouse, and the abdomen of the mouse is enlarged , collected ascitic fluid with a 5mL syringe, centrifuged at 3500r/min for 10min, and collected the supernatant for n-octanoic acid-ammonium sulfate purification. Take 1 mL of antiserum, dilute it twice with 0.06 mol/L, pH 5.0 acetate buffer, adjust the pH to 4.5 with 1 mol/L HCl solution; add n-octanoic acid drop by drop slowly under stirring at room temperature according to the ratio of 33 μL/mL of original serum , let stand at 4°C for more than 2 hours to fully precipitate the miscellaneous proteins, centrifuge at 4°C, 9000r/min for 30min, discard the precipitate, collect the supernatant and filter it with filter paper, add 0.1mol/ L. PBS with pH 7.4, adjust the pH of the solution to 7.4, add ammonium sulfate at 0.277g per ml, stir evenly, and stand at 4°C for more than 4h. Then centrifuge at 4°C and 9000r/min for 30min, discard the supernatant, collect the precipitate, dissolve the precipitate in a certain volume of PBS, and dialyze in 1L of PBS at 4°C to remove salt, and dialyze for 3 to 4 days. The dialysate was changed 3 times a day.

(4) Determination of antibody sensitivity and crossover rate: the concentrations of fipronil were 0, 0.1, 0.3, 0.9, 2.7, 8.1ng/mL, respectively, and the concentrations were 0, 0.05, 0.15, 0.45, 1.35, 4.05ng/mL mL of fluformil at concentrations of 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/mL of fipronil sulfone, and concentrations of 0, 0.1, 0.3, 0.9, 2.7, 8.1 ng/mL of fipronil For sulfoxide, the half maximal inhibitory concentration (IC ₅₀ ) was measured by ic-ELISA method, and then the antibody crossover rate was calculated. The formula for calculating the crossover rate is as follows: crossover rate (%)=(fipronil IC ₅₀ /similar IC ₅₀ )×100%. The results are shown in Table 1 and Figure 3. The IC ₅₀ of the fipronil antibody was 0.44ng/mL, and the crossover rates to fipronil sulfoxide, fipronil sulfone, and fluformil were 69.84, 75.86, and 112.82%, respectively. The sensitivity of the fipronil antibody prepared by the present invention is 700 and 14 times higher than that of the antibody in Development of an enzyme immunoassay for detection of fipronil in environmental samples and Poly-and monoclonal antibody-based ELISAs for fipronil, respectively, and in the above two articles The prepared antibody only recognizes fipronil itself. In summary, the antibody prepared by the present invention recognizes fipronil and its three metabolites simultaneously, and has high sensitivity.

Table 1 Determination of Antibody Crossover Rate

(5) Preparation of gold-labeled antibody: Take 800mL of ultrapure water in a flask and heat to boil at 102°C for 30min, add 1.6mL of 10% chloroauric acid solution under magnetic stirring, and add 2.08mL of 10% citric acid after 10min Trisodium solution, continue to stir until the color of the solution turns dark red and does not change color. Cool to room temperature and store for later use. As shown in Figure 2, the colloidal gold was successfully prepared, and the colloidal gold particles were uniform in size, and the calculated particle size was about 15nm, with good dispersion and no aggregation. Take 3mL of colloidal gold solution, add 300μL of pH 8.5 borate buffer, mix well, then add 18μg antibody solution drop by drop to react for 1h. Then, 300 μL of 10% BSA was added dropwise to the above solution to block unbound sites to reduce non-specific adsorption in the test, and incubated at room temperature for 0.5 h. Centrifuge the liquid at 4°C and 10000r/min for 25min to remove unbound antibody and colloidal gold, discard the supernatant, and dissolve the precipitate in 200μL of resuspension containing 2% BSA, 1% sucrose, 0.02% sodium azide in PBS solution, the resuspension is conducive to the protection of the gold-labeled antibody, and then stored at 4 ° C for use. The gold-labeled antibody prepared above was diluted 20 times, and 50 μL was taken in a 96-well plate for freeze-drying.

(6) Preparation of test strips: Spray 0.8mg/mL of the original coating material and 0.4mg/mL of the secondary antibody on the NC film as T-line and C-line with a film sprayer, and put them in an oven at 37°C overnight for later use. The test strip configuration shown in Figure 1A was assembled. Before assembly, soak the sample mat in 0.2% Tween-20, 1% sucrose, 1% BSA, 0.01M, pH 7.4 PBS solution for 30 minutes and then dry it at 37°C. This solution is conducive to the upward climbing of the gold-labeled antibody and the T line The coating primary and C-line secondary antibody binding. The rules of the detection method of the present invention: as shown in Figure 1B: Take 150 μL of the sample extract and add it to the lyophilized gold-labeled antibody, mix it evenly and incubate at room temperature for 3 minutes, insert the test strip into the hole, and wait for five minutes Post-visual interpretation of the test results is conducive to the full combination of the analyte, gold-labeled antibody, coating source, and secondary antibody, and improves the sensitivity of the detection method. According to capillary action, when the mixture rises upwards, it will develop color due to the specific reaction of the antigen and antibody being captured by the secondary antibody of the C line and the coating source of the T line. If the T line and C line have the same color depth, then there is no analyte in the sample, which is a negative result. If the color depth of the T line is lighter than that of the C line or the T line has no color, the sample contains the analyte, which is a positive result. If the C line has no color, it indicates that the test strip is an invalid product.

(7) Determination of the detection limit of test strips: Take 5g of eggs mixed evenly and place them in a 50mL centrifuge tube, add different concentrations of fipronil and its metabolites as standard products, fipronil, fipronil sulfone, fipronil Sulfoxide: 0, 5, 10, 20ng/g; fluoroformonitrile: 0, 2.5, 5, 10ng/g, add 3g of neutral alumina and 5mL of acetonitrile, shake vigorously for 5min, Centrifuge at 4000r/min for 3min, discard the precipitate and collect the supernatant. Take 1 mL of the supernatant in a 2 mL centrifuge tube and blow with nitrogen at 60°C until the organic reagent is completely evaporated. Redissolve the residue in 1mL of n-hexane, then add 1mL of 0.01M PBS, centrifuge at 4000r/min for 3min, and collect the lower layer solution. The limit of detection was defined as the lowest concentration at which the T line was lighter in color than the C line compared to the negative control. The detection results are shown in Figure 4: the detection limits of fipronil, fipronil sulfoxide, fipronil sulfone, and fluformil are 10, 10, 10, and 5 ng/g, respectively.

Figure 3 is the indirect competitive inhibition curve of the antibody. The IC ₅₀ of the fipronil antibody is 0.44ng/mL, and the crossover rates to fipronil sulfoxide, fipronil sulfone, and fluformil are 69.84, 75.86, and 112.82%, respectively. It shows that the antibody prepared by the present invention is a monoclonal antibody capable of simultaneously recognizing fipronil and its metabolites, and has high sensitivity. Provide powerful conditions for the preparation of colloidal gold test strips that can simultaneously detect fipronil and its metabolites.

Directly incubate the sample extract with the gold-labeled antibody for 3 minutes, insert the test strip, and the test result can be interpreted after 5 minutes; the test shows that the detection method has high sensitivity, and fipronil, fipronil sulfone, fipronil sulfoxide, The detection limits of nitriles were 10, 10, 10, 5 ng/g, respectively.

Fig. 4 is an effect diagram of detecting fipronil and its metabolite residues by colloidal gold test strips. Among them, A, B, and C are respectively fipronil, fipronil sulfone, and fipronil sulfoxide, and the concentration gradients of the three are 0, 5, 10, and 20 ng/g; D is fluformil, and the concentration gradient is 0 , 2.5, 5, 10 ng/g. The detection results of the test strips in the figure show that the detection limits of fipronil, fipronil sulfoxide, fipronil sulfone, and fluformil are 10, 10, 10, and 5 ng/g, respectively.

The series of detailed descriptions listed above are only specific descriptions for feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent embodiment or All changes should be included within the protection scope of the present invention.

Claims (6)

1. a colloidal gold test strip preparation method that detects fipronil and its metabolites, is characterized in that, comprises the following steps: Preparation of hapten: fipronil is hydrolyzed by acid and acetic acid is added to introduce carboxyl group to prepare hapten. The preparation of said hapten is as follows: Dissolve fipronil in concentrated sulfuric acid, acetic acid and water, heat to reflux, then add aqueous sodium hydroxide solution dropwise until the pH reaches alkaline, add pure water to dissolve the precipitate, wash the aqueous solution with n-hexane, separate and acidify the aqueous layer solution, until the pH becomes acidic, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, evaporate under reduced pressure, and pass through a silica gel column to obtain a white solid, which is a hapten; the concentrated sulfuric acid The mass fraction is 98%; said fipronil, concentrated sulfuric acid, acetic acid, water consumption ratio is 300～500mg:1～4mL:1～3mL:1～2mL; the consumption ratio of described pure water and deposit is 2 ~3 mL: 200~600mg; Preparation of the immunogen and the coating source: the hapten is coupled to the protein by the activated ester method to prepare the immunogen and the coating source respectively, and the preparation of the immunogen and the coating source is specifically as follows: Activation of hapten: Dissolve the hapten in dimethylformamide DMF, add N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC to it, and stir the mixture to activate , to obtain the activated hapten; the dosage ratio of the hapten, DMF, NHS and EDC is 2~4mg: 0.5~1mL: 1~4 mg: 2~7 mg; the stirring activation time is 6~8h; Preparation of immunogen: fully dissolve bovine serum albumin BSA in carbonate buffer CBS to obtain a BSA solution, add the activated hapten solution dropwise to the BSA solution under magnetic stirring, and stir the mixture React to obtain the immunogen, and the immunogen is dialyzed with phosphate-buffered PBS; Preparation of coating source: Fully dissolve chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA substance solution, add the activated hapten solution dropwise to the OVA solution under magnetic stirring, and put The mixture was stirred and reacted to obtain the coated original, which was dialyzed with phosphate buffered PBS; Preparation of monoclonal antibody: use the immunogen to prepare monoclonal antibody, and use the coating original to detect monoclonal antibody by ic-ELISA Sensitivity and specificity of cloned antibodies to obtain monoclonal antibodies against fipronil and its metabolites; Preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to prepare gold-labeled antibody; Preparation of colloidal gold test strips: use fipronil-coupled protein-coated originals to make detection lines on nitrocellulose membranes, use secondary antibodies to make quality control lines on nitrocellulose membranes, and then assemble them into test strips. 2. the colloidal gold test strip preparation method that detects fipronil and its metabolites according to claim 1, is characterized in that, in the preparation process of described immunogen, the consumption ratio of described BSA and CBS damping fluid is 7~15mg: 3~5mL; In the preparation process of the coating former, the dosage ratio of the OVA and the CBS buffer solution is 5-10 mg: 2-5 mL; The mol ratio of the hapten in the activated hapten solution to the protein in the BSA solution, and the mol ratio of the hapten in the activated hapten solution to the protein in the OVA solution are 20:1～40: 1; The temperature of the stirring reaction is 4° C., and the time of the stirring reaction is 12 to 18 h; The temperature of the PBS buffer dialysis was 4°C, and the time was 36-78 h. 3. the colloidal gold test strip preparation method that detects fipronil and its metabolites according to claim 1, is characterized in that, the preparation of described gold-labeled antibody is specifically: Take pure water and heat to boil, add chloroauric acid solution under stirring condition, then add trisodium citrate solution to obtain colloidal gold solution; Take the colloidal gold solution, add boric acid buffer solution, mix well, then add the monoclonal antibody solution drop by drop to react, then add 10% BSA drop by drop to block to obtain a mixed solution; The mixed solution is centrifuged, and the supernatant is discarded to obtain a precipitate, which is dissolved in the resuspension to obtain the gold-labeled antibody. 4. the colloidal gold test strip preparation method that detects fipronil and its metabolites according to claim 3 is characterized in that, the consumption ratio of described pure water, chloroauric acid solution and trisodium citrate solution is 500～ 900 mL: 1~2mL: 1~3 mL; Described colloidal gold solution, monoclonal antibody solution, boric acid buffer solution and 10%BSA dosage ratio are 3～6mL:18～48μg:300～600μL:200～800μL; The dosage ratio of the precipitate and the resuspension is 0.1-1 mg:300-500 μL. 5. the colloidal gold test strip preparation method that detects fipronil and its metabolites according to claim 1, is characterized in that, the preparation of described colloidal gold test strip specifically comprises the following steps: Soak the sample pad in a 0.01M pH 7.4 PBS solution containing 0.2% Tween-20, 1% sucrose and 1% BSA and then dry it; Spray 0.5~1 mg/mL of the original coating on the nitrocellulose membrane as the detection line, and spray 0.2~0.6 mg/mL of the secondary antibody on the nitrocellulose membrane as the quality control line; The sample pad, nitrocellulose membrane, absorbent pad and bottom plate were assembled. 6. A colloidal gold test strip for detecting fipronil and its metabolites, characterized in that, it is prepared according to the colloidal gold test strips for detecting fipronil and its metabolites described in any one of claims 1-5 Method preparation.

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