CN111187346A

CN111187346A - Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof

Info

Publication number: CN111187346A
Application number: CN202010042620.7A
Authority: CN
Inventors: 周兴华; 张彩芹; 张勋; 肖香; 王云
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2020-05-22
Anticipated expiration: 2040-01-15
Also published as: CN111187346B

Abstract

The invention provides a colloidal gold test strip for detecting fipronil and its metabolites and a preparation method thereof, comprising the following steps: preparing a hapten, preparing an immunogen and a coating antigen, preparing a monoclonal antibody, preparing a gold-labeled antibody, and preparing a colloidal gold test strip; the detection line is made on the nitrocellulose membrane by using the fipronil coupled protein coating antigen, the quality control line is made on the nitrocellulose membrane by using the secondary antibody, the sample extracting solution and the gold-labeled antibody are incubated, and the test strip is inserted for detection. According to the invention, the monoclonal antibody which is high in sensitivity and specificity and is resistant to fipronil and the metabolite 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 the fipronil and the metabolite thereof is established on the basis of the monoclonal antibody.

Description

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

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 a metabolite thereof and a preparation method thereof.

Background

Fipronil, the english name of which is fipronil (fipronil), the chemical name of which is (RS) -5-amino-1- (2, 6-dichloro-4-trifluoromethylphenyl) -4-trifluoromethylsulfinyl sulfinyl pyrazole-3-carbonitrile, is a phenylpyrazole pesticide developed in the eighties of the 20 th century, is one of substitutes for organophosphorus pesticides, and is widely applied to pest control of crops such as fruits and vegetables and to parasite and pest control of families and entertainment venues due to good bactericidal effect and high broad spectrum. Fipronil is a chloride ion channel that disrupts the gamma aminobutyric acid (GABA) control of pests, thereby disrupting their normal central nervous system to control them. Related researches show that fipronil has chronic neurotoxicity and is a C-class carcinogen, and can affect human health and cause symptoms such as conjunctivitis, dizziness, epileptic seizure, restlessness, sweating and the like. Fipronil is degraded into more toxic metabolic products of fluoroformonitrile, fipronil sulfone and fipronil sulfoxide through photolysis, oxidation and reduction in natural environment. Rat LD of Fluorocarbonitrile₅₀It is simply 1/6 in its mother's own body, which is far more toxic to rats than itself. The toxicity of fipronil sulfone to daphnia is about 6 times of that of the mother body, and the bioaccumulation of fipronil sulfoxide is 5 times of that of the mother body. In the No. 1157 bulletin issued in 2009 in China, fipronil is only limited to be used in coating of partial seeds such as corn, and in the pesticide residue limit (GB-2763-.

At present, the detection method of fipronil is mainly an instrumental 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 although the methods have high sensitivity and accuracy, the methods need expensive instruments and professional operators, and the pretreatment is complex and time-consuming, so that the methods are not suitable for rapid screening of a large number of samples on site. Immunoassay methods are a type of detection based on the specific reaction of antigen antibodies. Currently, the domestic authorized or announced immunoassay method for fipronil mainly comprises the following steps: the kit for detecting fipronil with the publication number of CN201710990416.6 and the preparation method and application thereof, and the chemiluminescence immunoassay kit for detecting fipronil in tea with the publication number of CN201711059643.3 are both the invention application of the ELISA kit, the detection process needs repeated plate washing and takes long time, results have certain difference due to different operators, and the results need to be read by an instrument and are not suitable for rapid screening of a large number of samples on site; the surface-enhanced Raman gold-labeled test strip for rapidly detecting micro-fipronil with the publication number of CN201810005898.X and the preparation method thereof are disclosed, the detection method of the invention has the advantages that Raman signals are unstable, false positive phenomenon easily occurs, the detection result needs to be assisted by a Raman instrument, the Raman instrument is expensive, and the detection cost is high; the method for rapidly detecting fipronil by utilizing illumination and surface enhanced Raman disclosed by the patent number 2018103135712.0 indirectly detects the existence of fipronil by detecting a main photolysis product of the fipronil, increases detection steps and time, has low result accuracy, expensive Raman instruments and high cost; the invention discloses a colloidal gold test strip for rapidly detecting trace fipronil with publication number 201810005478.1 and a preparation method thereof, and the detection method only aims at fipronil per se and does not relate to the detection of more toxic fipronil metabolites. Fipronil is very easy to degrade in natural environment, and the accuracy of the method is not high.

Disclosure of Invention

Aiming at the problems, the colloidal gold test strip for simultaneously and rapidly detecting fipronil and the metabolites thereof and the preparation method thereof are provided by the invention, the colloidal gold test strip which has high sensitivity and high specificity and can resist the fipronil and the metabolites thereof is established on the basis of the monoclonal antibody, has high speed, low cost and high sensitivity and can simultaneously detect the fipronil and the metabolites thereof, can simultaneously detect the fipronil and the metabolites thereof without the assistance of instruments, has low cost and simple operation, and is suitable for rapidly screening field samples.

A preparation method of a colloidal gold test strip for detecting fipronil and its metabolites comprises the following steps:

preparation of hapten: the method comprises the following steps of (1) carrying out an acid hydrolysis method on fipronil, adding acetic acid, and introducing carboxyl to prepare hapten;

preparation of immunogen and coatingen: coupling the hapten with protein by an activated ester method to respectively prepare immunogen and coating antigen;

preparation of monoclonal antibody: preparing a monoclonal antibody by using the immunogen, and detecting the sensitivity and specificity of the monoclonal antibody by using the coating antigen through ic-ELISA to obtain the monoclonal antibody resisting the fipronil and the metabolites thereof;

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

preparing the colloidal gold test strip: the detection line is made on the nitrocellulose membrane by using fipronil coupled protein coating antigen, the quality control line is made on the nitrocellulose membrane by using secondary antibody, and then the test strip is assembled.

In the above scheme, the hapten is specifically prepared by:

dissolving fipronil in concentrated sulfuric acid, acetic acid and water, heating and refluxing, then dropwise adding a sodium hydroxide aqueous solution until the pH value reaches alkalinity, adding pure water to dissolve precipitates, washing the aqueous solution with n-hexane, separating liquid and acidifying the aqueous layer solution until the pH value reaches acidity, then extracting with ethyl acetate, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, decompressing and evaporating, and passing through a silica gel column to obtain a white solid, namely the hapten.

In the scheme, the mass fraction of the concentrated sulfuric acid is 98%;

the using amount ratio of the fipronil, the concentrated sulfuric acid, the acetic acid and the 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:

activation of hapten: dissolving the hapten in dimethylformamide DMF, adding N-hydroxysuccinimide NHS and 1-ethyl carbodiimide hydrochloride EDC, and stirring and activating the mixture to obtain activated hapten;

preparation of immunogen: fully dissolving Bovine Serum Albumin (BSA) in a carbonate buffer solution CBS to obtain a BSA solution, dropwise adding an 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 buffered PBS;

preparation of coating antigen: sufficiently dissolving chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA solution, dropwise adding the activated hapten solution into the OVA solution under magnetic stirring, stirring the mixture for reaction to obtain coating antigen, and dialyzing the coating antigen with phosphate buffered PBS.

In the scheme, in the activation process of the hapten, the dosage ratio of the hapten to DMF to NHS to 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 scheme, 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 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 stirring reaction temperature is 4 ℃, and the stirring reaction time is 12-18 h;

the temperature of the PBS buffer solution during dialysis is 4 ℃, and the time is 36-78 h.

In the above scheme, the preparation of the gold-labeled antibody specifically comprises:

heating pure water to boil, adding chloroauric acid solution under stirring, and adding trisodium citrate solution to obtain colloidal gold solution;

adding a boric acid buffer solution into a colloidal gold solution, fully and uniformly mixing, then dropwise adding the monoclonal antibody solution for reaction, and then dropwise adding 10% BSA for blocking to obtain a mixed solution;

and centrifuging the mixed solution, discarding the supernatant to obtain a precipitate, and dissolving the precipitate in a resuspension solution to obtain the gold-labeled antibody.

In the scheme, the using amount 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 solution, the boric acid buffer solution and the 10% BSA is 3-6 mL, 18-48 mu g, 300-600 mu L and 200-800 mu L;

the dosage ratio of the precipitate to the resuspension is 0.1-1 mg: 300-500 mu L.

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

soaking the sample pad in 0.01M PBS (pH 7.4) solution containing 0.2% Tween-20, 1% sucrose and 1% BSA, and oven drying;

coating 0.5-1 mg/mL of coating antigen on a nitrocellulose membrane to serve as a detection line, and coating 0.2-0.6 mg/mL of secondary antibody on the nitrocellulose membrane to serve as a quality control line;

and assembling the sample pad, the nitrocellulose membrane, the absorbent pad and the bottom plate.

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

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, acetic acid is added by an acid hydrolysis method to introduce carboxyl, the reaction rate is increased, the hapten is prepared, and then the immunogen with stronger immunogenicity is prepared by coupling protein by an activated ester method, so that the 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 the colloidal gold labeled monoclonal antibody can simultaneously detect fipronil and metabolites thereof with stronger toxicity, so that 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 without the assistance of an instrument, the expense 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 and the gold-labeled antibody, inserting the sample extracting solution into a test strip, and judging the detection result after 5 min; tests 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 is suitable for people groups such as law enforcement departments, farmers, consumers and the like. In addition, the method can provide certain guarantee for the life safety of consumers.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1A is a schematic structural diagram of a colloidal gold test strip of the present invention; FIG. 1B is a schematic diagram of a colloidal gold test strip.

FIG. 2 is a colloidal gold UV chart; where the inset is a TEM image of colloidal gold.

FIG. 3 is an antibody indirect competitive inhibition curve.

FIG. 4 is a diagram showing the effect of detecting the residue of fipronil and its metabolites by a colloidal gold test strip.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, which are illustrative and are not to be construed as limiting the invention.

The preparation method of the colloidal gold test strip for detecting fipronil and the 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 a sodium hydroxide aqueous solution until the pH value reaches 9-10, adding pure water to dissolve precipitates, washing the aqueous solution with 5-15 mL of n-hexane, separating and acidifying the aqueous layer solution until the pH value reaches 1, then extracting with ethyl acetate, combining organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, and evaporating under reduced pressure. Passing through silica gel column to obtain white solid, i.e. hapten;

the mass fraction of the concentrated sulfuric acid is 98 percent;

the dosage ratio of the pure water to the precipitate is 2-3 mL: 200-600 mg.

(2) Preparation of immunogen and coatingen:

activation of hapten: dissolving the hapten obtained in the hapten preparation step in dimethyl formamide DMF, adding N-hydroxysuccinimide NHS and 1-ethyl carbodiimide hydrochloride EDC into the dimethyl formamide DMF, and stirring the mixture at room temperature to obtain activated hapten;

preparation of immunogen: fully dissolving Bovine Serum Albumin (BSA) in a Carbonate Buffer Solution (CBS) to obtain a BSA solution, then dropwise adding the activated hapten solution into the BSA solution under magnetic stirring, and stirring the mixture under a certain temperature condition for reaction to obtain an immunogen;

preparation of coating antigen: sufficiently dissolving the ovalbumin OVA in a carbonate buffer solution CBS to obtain an OVA solution, then dropwise adding the activated hapten solution into the OVA solution under magnetic stirring, and stirring and reacting the mixture at a certain temperature to obtain a coating antigen; and (3) dialyzing the immunogen and the coating antigen respectively with phosphate buffered PBS, replacing the dialyzate for 8 times in the dialysis process, subpackaging after the dialysis is finished, and storing at-20 ℃ for later use.

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 time is 6-8 h;

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;

the temperature of the PBS buffer solution during dialysis is 4 ℃, and the time is 36-78 h;

the BSA and OVA were purchased from Sigma-Aldrich chemical reagent, Shanghai, and fipronil was purchased from Bailingwei Bioreagent, Inc, Shanghai.

(3) Preparation of monoclonal antibody: diluting the prepared immunogen to 1-2 mg/mL by using normal saline, mixing the diluted immunogen with an equivalent volume of Freund's complete adjuvant, and emulsifying; immunizing mice (Balb/C) by back subcutaneous multi-point injection, wherein the immunization amount of each mouse is 75-100 mu g;

then, the mice are boosted every three weeks, the immunogen diluted to 2mg/mL and an equal volume of Freund's incomplete adjuvant are fully emulsified, and the mice (Balb/C) are immunized by back subcutaneous multi-point injection, wherein the immunization amount of each mouse is 25-50 mu g.

From the third immunization, carrying out tail blood sampling detection on the mice; screening out mice with high titer and high inhibition rate to inject 20 mug of immunogen into the abdominal cavity; three days after injection, the mouse is killed by pulling the neck, soaked in 75% alcohol for 10min, the spleen cells are taken out under the aseptic condition, the spleen is fully ground on a copper net, and then the spleen cells are washed for three times by using a PRMI-1640 culture medium to obtain a cell suspension which is centrifuged for 10min under the condition of 1200 r/min; discarding the supernatant, resuspending the precipitated cells in PRMI-1640 medium, centrifuging, repeating the above operation for three times, and counting splenocytes;

mixing the mouse spleen cell and myeloma cell at a ratio of 10:1, centrifuging at 1200r/min for 10min,shaking to disperse the cells at the bottom of the centrifuge tube, slowly adding PEG4000 in the first minute, standing for 1min, and adding PRMI-1640 culture medium to stop the action of PEG; the dosage ratio of the mixed solution, PEG4000 and PRMI-1640 culture medium is 0.5 mL: 10 mL. The fused cells were kept standing and cultured in an oven at 37 ℃ for 15min, and then centrifuged at 3500r/min for 10 min. Then suspending the cells in HAT complete medium containing 10% fetal calf serum, mixing well, adding to 96-well plate, and standing at 37 deg.C and 5% CO₂Culturing in an incubator. And screening the supernatant by using a coating antigen by adopting an indirect competitive ic-ELISA method, selecting a cell hole with high inhibition rate and strong positive, performing subcloning, then performing amplification culture, and performing subcloning for 2-3 times to obtain a positive hybridoma cell strain with high purity.

Preparation and purification of ascites: the above-selected hybridoma cells were diluted to 1X 10 per ml with PRMI-1640 medium⁶And (3) injecting hybridoma cells of the 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 with n-caprylic acid-ammonium sulfate to obtain the monoclonal antibody.

The specific operation of the n-octanoic acid-ammonium sulfate purification is as follows: taking 1mL of antiserum, diluting the antiserum by 2 times with 0.06mol/L, pH 5.0.0 acetic acid buffer solution, and adjusting the pH value to 4.5 with 1mol/L HCl solution; slowly adding n-octanoic acid dropwise under stirring at room temperature according to the proportion of 33 μ L/mL original serum, standing at 4 deg.C for more than 2h to fully precipitate heteroproteins, centrifuging at 4 deg.C for 30min at 9000r/min, discarding the precipitate, collecting supernatant, filtering with filter paper, adding 1/10 (0.1 mol/L), pH7.4PBS (pH7.4PBS), adjusting the pH of the solution to 7.4, adding ammonium sulfate (0.277 g per mL), stirring, and standing at 4 deg.C for more than 4 h. And centrifuging the solution at 4 ℃ and 9000r/min for 30min, removing the supernatant, collecting the precipitate, dissolving the precipitate in PBS (phosphate buffer solution) with a certain volume, dialyzing the solution in 1L of PBS at 4 ℃ for desalting, dialyzing for 3-4 days, and replacing the dialysate for 3 times every day.

(4) Preparation of gold-labeled antibody: heating and boiling 500-900 mL of ultrapure water in a flask at 102 ℃ for 30-50 min, balancing for 30-50 min, adding 10% chloroauric acid solution under magnetic stirring, adding 10% trisodium citrate solution after 10min, continuing stirring until the solution becomes dark red and does not change color to obtain colloidal gold solution, and cooling to room temperature and storing in the shade for later use. And (3-6 mL of colloidal gold solution is taken, boric acid buffer solution with the pH value of 8.5 is added, and the mixture is fully and uniformly mixed and then is dropwise added into the prepared monoclonal antibody solution for reaction for 30-60 min. Then, 10% BSA was added dropwise to the above solution to block the unbound sites, and incubated at room temperature for 0.5-1 h. Centrifuging the mixed solution at 4 ℃ and 10000r/min for 25min to remove the unbound antibodies and gold nanoparticles, discarding the supernatant, dissolving the precipitate in a resuspension solution containing 2% BSA, 1% sucrose and 0.02% sodium azide in PBS, and storing at 4 ℃ for later use. Diluting the prepared gold-labeled antibody by 20-50 times, and freeze-drying 50 mu L of the diluted gold-labeled antibody in a 96-well plate for later use. The secondary antibody and the prepared coating antigen were sprayed on a nitrocellulose membrane as a quality control line (C line) and a detection line (T line) by a membrane spraying instrument, respectively, and were dried overnight at 37 ℃ for use.

The using amount ratio of the pure water to the chloroauric acid solution to the trisodium citrate solution is 500-900 mL to 1-2 mL to 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 mu g, 300-600 mu L and 200-800 mu L;

the dosage ratio of the precipitate to the resuspension is 0.1-1 mg: 300-500 mu L;

the secondary antibody is HRP-goat anti-mouse IgG purchased from Shanghai biological engineering Co.

(5) Preparing the colloidal gold test strip: the detection line is made on the nitrocellulose membrane by using fipronil coupled protein coating antigen, the quality control line is made on the nitrocellulose membrane by using secondary antibody, and then the test strip is assembled. Specifically, this test paper strip includes sample pad, nitrocellulose membrane (NC membrane), water absorption pad and bottom plate, pastes the NC membrane in the centre of bottom plate, and water absorption pad and sample pad paste respectively in the upper and lower both sides of bottom plate, overlap 2 ~ 3mm with the NC membrane. And respectively spraying 0.3-1 mg/mL secondary antibody and 0.5-1 mg/mL coating source on an NC film by using a film spraying instrument to serve as a C line and a T line, then cutting the NC film into test strips with the length of 3.8-4 mm by using a strip cutting machine, and putting the test strips into a self-sealing bag and storing the test strips in a drying box for later use.

Detection of fipronil and its metabolites: and incubating a sample extracting solution and the gold-labeled antibody, and inserting the sample extracting solution and the gold-labeled antibody into the test strip for detection.

Example 1

As shown in figure 1A, the colloidal gold test strip capable of simultaneously detecting fipronil and its metabolites of the invention uses a bottom Plate (PVC) as a support, the middle part of the bottom plate and the bottom plate is a nitrocellulose membrane, the nitrocellulose membrane is provided with a quality control line and a detection line, and a sample pad on the left and a water absorption 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 synthesized coating antigen. The conjugate of the colloidal gold labeled fipronil monoclonal antibody is arranged in a 96-well plate.

The main component of the material of the sample pad in the test strip is a glass fiber membrane, and the bottom plate is made of polyvinyl chloride material. The left-most side of the sample pad has a sticker with an arrow pointing downward to indicate the direction of insertion of the test strip into the sample extraction solution.

Example 2

In order to prepare the colloidal gold test strip capable of simultaneously and rapidly detecting fipronil and its metabolites, firstly, immunogen with strong immunogenicity and monoclonal antibody against fipronil and its metabolites are prepared, and then the test strip is assembled and prepared. The specific operation steps are as follows:

(1) preparation of hapten: and hydrolyzing the cyano group on the fipronil into carboxyl by an acid hydrolysis method. 437mg of 1mmoL fipronil is weighed, 2.5mL of acetic acid, 1.25mL of 98% concentrated sulfuric acid and 1.5mL of water are added, and after uniform mixing, the mixture is heated and refluxed for 3 hours. Then, the mixture was 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 solution until pH reached 1, and then extracted with 3 × 15mL of ethyl acetate. All organic phases were combined, washed with 15mL of saturated brine, dried over anhydrous sodium sulfate, and the organic solvent was removed by evaporation under reduced pressure. Finally, the white solid is obtained by passing through a silica gel column.

(2) Preparation of immunogen and coatingen: 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 h. 14mg BSA and 10mg OVA were each dissolved well in 5mLCBS buffer, and then the activated hapten solution was added dropwise to the BSA solution and OVA solution, respectively, under magnetic stirring. Stirring the mixture at 4 deg.C for 12h to obtain immunogen and coating antigen, dialyzing the immunogen and coating antigen with PBS buffer solution at 4 deg.C for 72 hr, changing the dialyzate for 8 times during dialysis, subpackaging and storing at-20 deg.C for use.

(3) Preparation of monoclonal antibody: the first immunization (priming) of the mice was performed by diluting the prepared immunogen to 2mg/mL with physiological saline, mixing with an equal volume of Freund's complete adjuvant, and emulsifying thoroughly with an emulsifier. Balb/C mice were then immunized by back subcutaneous multiple injections, each at 100. mu.g. Mice were then boosted every three weeks, diluted to 2mg/mL of immunogen and an equal volume of Freund's incomplete adjuvant were thoroughly emulsified and immunized in the same manner as the prime, 50. mu.g per mouse. From the third immunization, mice were tail bled for testing. The mice with high titer and high inhibition rate are screened out for infusion, namely 20 mu g of immunogen is injected in the abdominal cavity. Three days later, the mouse is killed by pulling the neck, soaked in 75% alcohol for 10min, the spleen cells are taken out under the aseptic condition, the spleen is fully ground on a copper net by using an injector core, then the spleen is washed for three times by using PRMI-1640 culture medium, the cell suspension is mixed with 1200r/min in a 50mL centrifuge tube, and the centrifugation is carried out for 10 min. The supernatant was discarded, the precipitated cells were resuspended in PRMI-1640 medium, and the spleen cells were counted after repeating the above procedure three times. And (3) fully and uniformly mixing the mouse spleen cells and the myeloma cells according to the ratio of 10:1, placing the mixture in a 50mL centrifuge tube at 1200r/min, centrifuging for 10min, gently bouncing the bottom of the centrifuge tube to disperse the cells, slowly adding 1mL of PEG4000 in the first minute, standing for 1min, and adding 10mL of PRMI-1640 culture medium 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 10 min. Then with 15% fetal bovine serumHAT in complete culture medium suspended cells, mixed well, added to 96-well plates and placed at 37 deg.C in 5% CO₂Culturing in an incubator. Screening the supernatant by using an indirect competitive ELISA method, selecting a cell pore with high inhibition rate and strong positive, performing subcloning, then performing amplification culture, and performing subcloning for 2-3 times to obtain a positive hybridoma cell strain with high purity.

Preparation and purification of ascites: the above-selected hybridoma cells were diluted to 1X 10 per ml with PRMI-1640 medium⁶The hybridoma cell of the cell is injected into each mouse and is injected into abdominal cavity according to 800uL, the abdomen of the mouse is swollen, ascites is collected by a 5mL syringe and is centrifuged at 3500r/min for 10min, and the supernatant is collected and purified by caprylic acid-ammonium sulfate. Taking 1mL of antiserum, diluting the antiserum by 2 times with 0.06mol/L, pH 5.0.0 acetic acid buffer solution, and adjusting the pH value to 4.5 with 1mol/L HCl solution; slowly adding n-octanoic acid dropwise under stirring at room temperature according to the proportion of 33 μ L/mL of original serum, standing at 4 deg.C for more than 2h to fully precipitate impure protein, centrifuging at 4 deg.C for 30min at 9000r/min, discarding the precipitate, collecting supernatant, filtering with filter paper, adding 1/10 volume of 0.1mol/L, pH 7.4.4 volume of PBS, adjusting pH of the solution to 7.4, adding 0.277g ammonium sulfate per mL, stirring, and standing at 4 deg.C for more than 4 h. And centrifuging the solution at 4 ℃ and 9000r/min for 30min, removing the supernatant, collecting the precipitate, dissolving the precipitate in PBS (phosphate buffer solution) with a certain volume, dialyzing the solution in 1L of PBS at 4 ℃ for desalting, dialyzing for 3-4 days, and replacing the dialysate for 3 times every day.

(4) Determination of antibody sensitivity and cross-over rate: fipronil with concentrations of 0, 0.1, 0.3, 0.9, 2.7, 8.1ng/mL, fipronil with concentrations of 0, 0.05, 0.15, 0.45, 1.35, 4.05ng/mL, fipronil sulfone with concentrations of 0, 0.1, 0.3, 0.9, 2.7, 8.1ng/mL, fipronil sulfoxide with concentrations of 0, 0.1, 0.3, 0.9, 2.7, 8.1ng/mL were tested for half maximal Inhibitory Concentration (IC) by IC-ELISA method₅₀) Antibody crossover rates were then calculated. The calculation formula of the crossing rate is as follows: percent crossing (fipronil IC)₅₀Analog IC₅₀) X 100%. As shown in Table 1 and FIG. 3, IC of fipronil antibody₅₀Is 0.44ng/mL, against fipronil sulfoxide and fluorine wormThe cross-over rates of nitrile sulfone and fluoronitrile were 69.84, 75.86 and 112.82%, respectively. The sensitivity of the fipronil antibody prepared by the invention is 700 and 14 times higher than that of the antibodies in Development of an enzyme immunological sample for detection and Poly-and monoclonal antibody-based ELISAs for fipronil, and the antibodies prepared in the two articles only recognize fipronil per se. The conclusion shows that the antibody prepared by the invention can simultaneously recognize fipronil and three metabolites thereof, and has higher sensitivity.

TABLE 1 determination of the Cross-over Rate of the antibodies

(5) Preparation of gold-labeled antibody: 800mL of ultrapure water are taken into a flask, heated to boiling equilibrium at 102 ℃ for 30min, 1.6mL of 10% chloroauric acid solution is added under magnetic stirring, after 10min 2.08mL 10% trisodium citrate solution is added, and stirring is continued until the solution becomes dark red and does not change color. Cooling to room temperature and storing for later use. As shown in figure 2, the preparation of the colloidal gold is successful, the particle size of the colloidal gold is uniform, the calculated particle size is about 15nm, the dispersibility is good, and the aggregation phenomenon is avoided. 3mL of the colloidal gold solution was added to 300. mu.L of a pH 8.5 boric acid buffer solution, and after mixing well, 18. mu.g of the antibody solution was added dropwise to react for 1 hour. Then 300 μ L of 10% BSA was added dropwise to the above solution to block unbound sites to reduce nonspecific adsorption in the assay, and incubated at room temperature for 0.5 h. Centrifuging the solution at 4 ℃ and 10000r/min for 25min to remove unbound antibodies and colloidal gold, discarding the supernatant, dissolving the precipitate in 200 μ L of resuspension solution which is PBS solution containing 2% BSA, 1% sucrose and 0.02% sodium azide, and storing the resuspension solution at 4 ℃ for later use. The gold-labeled antibody prepared above was diluted 20-fold, and 50. mu.L of the diluted antibody was put into a 96-well plate and lyophilized for use.

(6) Preparing the test strip: 0.8mg/mL of the coating antigen and 0.4mg/mL of the secondary antibody were sprayed on an NC membrane as a T line and a C line respectively by a membrane spraying instrument, and were dried overnight at 37 ℃ for later use, and then assembled according to the structure of the test strip shown in FIG. 1A. Before assembly, the sample is padded in a PBS solution of 0.2% Tween-20, 1% sucrose and 1% BSA (bovine serum albumin) and 0.01M, pH 7.4.4 for 30min and then dried at 37 ℃, and the solution is favorable for upward climbing of a gold-labeled antibody and combination with a coating antigen of a T line and a secondary antibody of a C line. The detection method of the invention has the following rules: as shown in fig. 1B: adding 150 mu L of sample extracting solution into the freeze-dried gold-labeled antibody, uniformly mixing, incubating for 3min at room temperature, inserting the test strip into the hole, and visually judging the detection result after five minutes, so that the full combination of the substance 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 the capillary action, the mixture ascends and develops color due to the antigen-antibody specific reaction captured by the coating antigen of the secondary antibody of the C line and the T line. If the color depth of the T line is consistent with that of the C line, no object to be detected in the sample 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 containing the object to be detected is a positive result. If the line C has no color, the test strip is a failure product.

(7) And (3) testing the detection limit of the test strip: placing 5g of uniformly mixed eggs into a 50mL centrifuge tube, adding fipronil and standard substances of metabolites thereof with different concentrations, wherein fipronil, fipronil sulfone and fipronil sulfoxide are all as follows: 0.5, 10, 20 ng/g; the fluorocarbonitriles are: 0. 2.5, 5 and 10ng/g, adding 3g of neutral alumina and 5mL of acetonitrile, then violently shaking for 5min, centrifuging at 4000r/min for 3min at room temperature, discarding the precipitate, and collecting 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 to collect the lower layer solution. The limit of detection is defined 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 fluoroform are respectively 10, 10 and 5 ng/g.

FIG. 3 is an antibody indirect competitive inhibition curve, IC of fipronil antibody₅₀The concentration of the active ingredient is 0.44ng/mL, and the crossing rates of the active ingredient on fipronil sulfoxide, fipronil sulfone and fipronil are 69.84, 75.86 and 112.82 respectively. Illustrating the inventionThe prepared antibody is a monoclonal antibody capable of simultaneously recognizing fipronil and its metabolites, and has high sensitivity. Provides powerful conditions for preparing the colloidal gold test strip capable of simultaneously detecting fipronil and metabolites thereof.

Directly incubating the sample extracting solution and the gold-labeled antibody for 3min, inserting a test strip, and judging the detection result after 5 min; tests show that the detection method has high sensitivity, and the detection limits of fipronil, fipronil sulfone, fipronil sulfoxide and fluoroform nitrile are respectively 10ng/g, 10ng/g and 5 ng/g.

FIG. 4 is a diagram showing the effect of detecting the residue of fipronil and its metabolites by a colloidal gold test strip. Wherein A, B, C is fipronil, fipronil sulfone, and fipronil sulfoxide, and the concentration gradient of the three components is 0, 5, 10, and 20 ng/g; d is fluoroformonitrile, and the concentration gradient is 0, 2.5, 5 and 10 ng/g. The test paper strip test results in the figure show that the detection limits of fipronil, fipronil sulfoxide, fipronil sulfone and fluoroform are respectively 10ng/g, 10ng/g and 5 ng/g.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims

1. A preparation method of a colloidal gold test strip for detecting fipronil and its metabolites is characterized by comprising the following steps:

preparation of monoclonal antibody: preparing a monoclonal antibody by using the immunogen, and detecting the sensitivity and specificity of the monoclonal antibody by using the coating antigen through ic-ELISA to obtain the monoclonal antibody resisting fipronil and metabolites thereof;

2. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 1, wherein the hapten is prepared by:

3. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 2, wherein the mass fraction of the concentrated sulfuric acid is 98%;

the dosage ratio of the pure water to the precipitate is 2-3 mL: 200-600 mg.

4. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 2, wherein the preparation of the immunogen and the coating antigen comprises:

5. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 4, wherein the hapten, DMF, NHS and EDC are used in an amount of 2-4 mg: 0.5-1 mL: 1-4 mg: 2-7 mg; the stirring activation time is 6-8 h.

6. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 4, wherein in the process of preparing the immunogen, the dosage ratio of the BSA and CBS buffer solution is 7-15 mg: 3-5 mL;

7. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 4, wherein the preparation of the gold-labeled antibody specifically comprises:

8. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 7, wherein the ratio of the pure water, the chloroauric acid solution and the trisodium citrate solution is 500-900 mL: 1-2 mL: 1-3 mL;

9. The method for preparing a colloidal gold test strip for detecting fipronil and its metabolites as claimed in claim 1, wherein the preparation of the colloidal gold test strip specifically comprises the following steps:

10. A colloidal gold test strip for detecting fipronil and its metabolites, characterized by being prepared according to the preparation method of the colloidal gold test strip for detecting fipronil and its metabolites of any one of claims 1-9.