CN111273018A

CN111273018A - Enzyme linked immunosorbent assay kit for detecting bromadiolone and preparation and application thereof

Info

Publication number: CN111273018A
Application number: CN202010285672.7A
Authority: CN
Inventors: 温凯; 马立才; 刘河冰; 聂靖东; 邢维维; 刘薇; 杨柳; 丁亚芳; 崔乃元
Original assignee: Beijing Wdwk Biotechnology Co ltd
Current assignee: Beijing Wdwk Biotechnology Co ltd
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2020-06-12
Anticipated expiration: 2040-04-13
Also published as: CN111273018B

Abstract

The invention discloses an ELISA method for detecting bromadiolone and a kit thereof, wherein the ELISA kit for detecting bromadiolone comprises an ELISA plate (coated with a conjugate of bromadiolone drug hapten and carrier protein shown in formula I), an antibody working solution (containing bromadiolone drug monoclonal antibody), an enzyme marker working solution (containing an anti-bromadiolone drug monoclonal antibody labeled by horseradish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a standard solution containing bromadiolone with different gradient concentrations, a substrate developing solution and a stop solution. The bromadiolone detection kit provided by the invention can be used for detecting toxicants in food and human serum, the detection limit of the bromadiolone in pork, milk and serum is 5 mug/kg, the inter-batch variation coefficient is less than 10 percent, and the intra-batch variation coefficient is less than 15 percent, and the bromadiolone detection kit has the advantages of simple and rapid operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Description

Enzyme linked immunosorbent assay kit for detecting bromadiolone and preparation and application thereof

Technical Field

The invention belongs to the field of rapid detection of drug residues, and relates to a kit for detecting bromadiolone, and a preparation method and application thereof.

Background

The chemical name of bromadiolone is 3- (4-hydroxy-3-coumarin) -3-phenyl-1- (p-bromobiphenyl) -propanol-1, and the bromadiolone belongs to 4-hydroxy dicoumarol anticoagulant raticide. It inhibits the liver from producing prothrombin and blood coagulation factors by competitively inhibiting the action of vitamin K, improves the permeability and fragility of capillary vessels and causes bleeding and death in mice. With the large amount of administration of rodenticides, rodenticides inevitably remain in the food, affecting human health. In recent years, the rodenticide causes poisoning, which seriously harms the health and life safety of people.

At present, the detection method of bromadiolone mainly comprises a high performance liquid chromatography and a liquid chromatography-mass spectrometry method, which have the advantages of strong specificity and high sensitivity, but have the disadvantages of complex operation and expensive instrument, and are not suitable for screening and detecting mass samples. The rapid detection method is mainly a chemical colorimetric detection method, has poor specificity and sensitivity, is easy to generate misjudgment, and cannot meet the field detection requirement.

Disclosure of Invention

The invention aims to provide a bromadiolone enzyme-linked immunoassay kit and a detection method which has high sensitivity, strong specificity and low detection cost and is suitable for screening large-batch samples.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an enzyme-linked immunoassay kit for detecting bromadiolone comprises a bromadiolone detection enzyme label plate, bromadiolone series standard substance working solution, bromadiolone antibody working solution, enzyme marker working solution, sample diluent, sample extracting solution, washing solution, substrate developing solution and stop solution.

The bromadiolone enzyme label plate is prepared by coating a conjugate of bromadiolone drug hapten and carrier protein, wherein the bromadiolone hapten is a compound shown in a formula I:

formula I

The bromadiolone hapten is obtained by reacting bromadiolone with phthalic anhydride, and the specific preparation method comprises the following steps: weighing 500mg of bromadiolone, dissolving the bromadiolone in 6 mL of pyridine, adding 154.45 mg of phthalic anhydride, heating to 60 ℃, stirring, purifying, spin-drying and terminating the reaction to obtain the compound shown in the formula I.

The carrier protein is Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), mouse serum protein (MSA), thyroxine (BCG), rabbit serum protein (RSA), hemocyanin (KLH) or Ovalbumin (OVA).

The conjugate of the bromadiolone hapten and carrier protein is a product obtained by the bromadiolone hapten and the carrier protein through an active ester method, and specifically comprises the following steps:

1) dissolving the bromadiolone hapten (shown in the formula I) in Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and reacting for 2-3h at 20-25 ℃ by magnetic stirring to obtain a solution I;

wherein the ratio of the bromadiolone hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 30.22 mg: 1.5 mL: 25.75 mg: 15.46 mg;

2) putting the carrier protein into 0.1M sodium bicarbonate buffer solution, stirring at 200 rpm for 10 min, and fully dissolving to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer solution is 50 mg: 3.5 mL;

wherein, if the carrier protein is Bovine Serum Albumin (BSA), the ratio of the Bovine Serum Albumin (BSA) to the 0.1M sodium bicarbonate buffer solution is 50 mg: 3.5 mL; if the carrier protein is Ovalbumin (OVA), the ratio of the Ovalbumin (OVA) to the 0.1M carbonic acid buffer solution is 33.6 mg: 3.5 mL;

3) mixing the solution I and the solution II, specifically, dropwise adding the solution I into the solution II under the condition of 0-4 ℃ and stirring at 1000 rpm, and stirring at 500 rpm for 24 hours to obtain a solution III;

4) the solution III was dialyzed against PBS buffer (0.01M PBS, pH 7.2) at 4 ℃ for 3 days with stirring to give the bromodiuron-coated antigen.

The bromadiolone antibody is a specific antibody of a bromadiolone drug.

The bromadiolone antibody working solution is prepared by diluting a monoclonal antibody of bromadiolone by 1000 times with an antibody diluent to obtain the monoclonal antibody working solution containing the bromadiolone drug.

The antibody diluent is a PBS buffer solution containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, Proclin300 and Triton X-100, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 muL/L and 500 muL/L respectively.

The enzyme marker working solution is obtained by diluting an anti-antibody of an anti-bromadiolone drug monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent.

The anti-antibody diluent is a PBS (phosphate buffer solution) containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L respectively. In the kit, the conjugate of the bromadiolone drug hapten and the carrier protein is coated on an enzyme label plate.

In the kit, the antibody specific to the bromadiolone drug is the bromadiolone monoclonal antibody.

In the kit, the bromodiuron monoclonal antibody is composed of a heavy chain and a light chain. The amino acid sequence of the variable region of the heavy chain can be shown as a sequence 1 in a sequence table. The amino acid sequence of the variable region of the light chain can be shown as a sequence 2 in a sequence table.

In the kit, the solvent of the 6 standard substance working solutions is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is bromadiolone; the concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L and 0.1g/L, and the pH value is 7.2.

In the kit, the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.1g/L, and the pH value is 7.2.

In the kit, the sample extracting solution is PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.2 g/L, and the pH value is 7.2.

In the kit, the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

In the kit, the substrate color development liquid is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

In the kit, the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

Another object of the present invention is to provide a method for detecting bromadiolone in a sample, comprising the steps of:

1) pretreating a sample to obtain a solution of the sample;

2) detecting with any one of the above-mentioned kits;

3) and analyzing the detection result.

In the above method, the detection with the kit comprises the following steps: adding standard working solution or the solution of the sample into an ELISA plate coated with the conjugate of the bromadiolone drug hapten and the carrier protein; adding a specific antibody solution containing a bromadiolone drug; after incubation, the swatches were washed dry, the enzyme-labeled anti-antibody was added, the substrate was developed, stopped and the absorbance was measured with an enzyme-linked plate reader.

In the above method, the method for pretreating the sample specifically comprises the following steps:

accurately weighing 1+ -0.01 g (or mL) of sample, adding 5 mL of sample extract, whirling at high speed for 1min at room temperature (25 + -2 deg.C), and centrifuging at 4000 g for 5 min to obtain sample solution. And adding 200 mu L of sample solution into 200 mu L of sample diluent, whirling at a high speed for 1min, and taking 50 mu L of supernatant to analyze.

The detection principle of the kit of the invention is as follows: an indirect competitive ELISA method for detecting bromadiolone features that the conjugate of hapten and carrier protein of bromadiolone is used to coat the ELISA plate and the bromadiolone is usedThe monoclonal antibody of the substance is used as a first antibody, an anti-antibody of an anti-bromadiolone drug monoclonal antibody marked with horseradish peroxidase (HRP) is used as a second antibody, a substrate solution is added for color development reaction, and the concentration of the antibody is 0.05 mol/L H₂SO₄The reaction was terminated and absorbance at 450 nm was measured to detect the bromadiolone drug.

The analysis process of the detection result provided by the invention comprises the following steps:

the absorbance average (B) of the standard working solution of each concentration obtained was divided by the absorbance value (B0) of the first standard solution (0 standard) and multiplied by 100%, i.e., the percent absorbance value. The calculation formula is as follows: percent absorbance value (%) - (B/B0). times.100%

And drawing a standard curve chart by taking a half logarithmic value of the concentration (mug/L) of the working solution of the bromadiolone standard as an X axis and the percent absorbance value as a Y axis. The percent absorbance of the sample solution is calculated by the same method, and the content of bromadiolone in the sample can be read from the standard curve corresponding to the concentration of each sample.

The analysis of the detection result in the invention can also adopt a regression equation method to calculate the concentration of the sample solution.

The analysis of the detection result can also utilize computer professional software, the method is more convenient for the rapid analysis of a large number of samples, and the whole detection process can be completed within 1.5 h in a short time.

Experiments prove that the kit can detect bromadiolone in pork, milk and serum; the detection limit of bromadiolone in pork, milk and serum is 5 mug/kg, the inter-batch variation coefficient is less than 10 percent, the intra-batch variation coefficient is less than 15 percent, and the stability is good.

The bromadiolone detection kit provided by the invention can be used for detecting toxicants in food and human serum, has the advantages of simple and rapid operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Drawings

FIG. 1 identification mass spectrum of bromadiolone hapten.

Figure 2 bromadiolone standard graph.

FIG. 3 is a graph comparing the results of the detection by the kit with those by LC-MS/MS.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of enzyme-linked immunosorbent assay kit for detecting bromadiolone

The kit comprises the following components: an enzyme label plate (a conjugate coated with bromadiolone drugs and carrier protein), an antibody working solution (containing bromadiolone drug monoclonal antibodies), an enzyme marker working solution (containing horseradish peroxidase-labeled anti-bromadiolone drug monoclonal antibodies), a washing solution, a sample diluent, a sample extracting solution, a standard working solution containing bromadiolone with different gradient concentrations, a substrate developing solution and a stop solution.

The preparation method comprises the following steps:

firstly, preparation of enzyme label plate

1. Preparation of bromadiolone coating antigen

(1) Preparation of bromadiolone hapten

500mg of bromadiolone was dissolved in 6 mL of pyridine, and 154.45 mg of phthalic anhydride was weighed and added thereto, stirred, and heated to 60 ℃. And when the reaction is not changed through TLC, processing, spin-drying pyridine, dissolving with methanol, adding silica gel for sample mixing, performing column chromatography on the spin-dried sample, collecting the required solution, spin-drying and collecting to obtain the bromadiolone hapten. The mass spectrum structure is confirmed, and the structural formula is shown as formula I.

Formula I

(2) Preparation of bromadiolone coating antigen

Dissolving 30.22 mg of bromadiolone hapten in 1.5 mL of DMF, stirring at 200 rpm for 10 min, adding 25.75 mg of EDC and 15.46 mg of NHS to dissolve, and stirring at room temperature (500 rpm) to activate for 2-3 h; weighing OVA 33.6 mg, dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve the OVA, cooling at 0-4 ℃ in an ice bath, stirring at 1000 rpm, dropwise adding the reaction solution obtained in the step 1 (1 mL/min), and stirring at 500 rpm for reaction for 24 h; and filling the reaction product into a distilled water washing dialysis bag (10 cm), stirring 1L 0.01M PBS (1 x, pH7.2) at 4 ℃ and dialyzing for 3 d at 100 rpm, changing the solution 3 times (once in the morning, at noon and at night) every day, changing the solution 9 times in total, and centrifuging the dialysis product at 5000 rpm for 6 min to obtain the bromadiolone coating antigen.

2. Preparation of ELISA plates

Diluting the obtained bromadiolone coating source to 10.0 mu g/mL by using a coating buffer solution, adding 100 mu L of the coating source into each hole, incubating for 2 h at 37 ℃, pouring out the coating solution, washing for 2 times by using a washing solution diluted by 20 times, drying by beating for 30 seconds each time, then adding 150 mu L of a sealing solution into each hole, incubating for 1 h at 37 ℃, pouring out the liquid in the hole, drying to obtain an ELISA plate coated with the coating source (conjugate of bromadiolone drug hapten and carrier protein), and preserving in a vacuum sealing manner by using an aluminum film.

Coating buffer solution: 0.03 mol/L sodium carbonate buffer solution with the pH value of 9.6;

sealing liquid: contains 50 g/L sucrose, 2.5 g/L casein, 0.5% calf serum, and 3 ‰ sodium azide in 0.2 mol/LpH7.7 phosphate buffer solution.

Preparation of antibody working solution

1. Preparation of bromadiolone monoclonal antibody

(1) Preparation of bromadiolone immunogen

Dissolving 30.22 mg of bromadiolone hapten in 1.5 mL of DMF, stirring at 200 rpm for 10 min, adding 25.75 mg of EDC and 15.46 mg of NHS to dissolve, and stirring at room temperature (500 rpm) to activate for 2-3 h; weighing 50 mg of BSA, dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve the BSA, cooling in an ice bath at 0-4 ℃, dropwise adding the reaction solution obtained in the step 1 (1 mL/min) while stirring at 1000 rpm, and stirring at 500 rpm for reaction for 24 h; and filling the reaction product into a distilled water washing dialysis bag (10 cm), stirring 1L 0.01M PBS (1 x, pH7.2) at 4 ℃ and dialyzing for 3 d at 100 rpm, changing the solution 3 times (once in the morning, at noon and at night) every day, changing the solution 9 times in total, and centrifuging the dialysis product at 5000 rpm for 6 min to obtain the bromadiolone immunogen.

(2) Animal immunization

Dissolving the prepared bromadiolone immunogen by using normal saline according to 100 mu g/mouse, uniformly mixing the dissolved immunogen with Freund's complete adjuvant in equal volume, injecting and immunizing Balb/c female mice with 6-8 weeks old by subcutaneous injection at the neck and the back, uniformly mixing the immunogen with Freund's incomplete adjuvant in equal volume on 7 th, 14 th and 28 th days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g/mouse of immune complex 3 days before fusion without adding Freund's adjuvant.

(3) Cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells of mice (SP 2/0) in logarithmic growth phase, slowly adding preheated fusion agent (PEG 4000) within 45s for fusion, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37 deg.C and 5% CO₂Culturing in an incubator, half-changing the culture medium with HT after 5 days, and completely changing the culture medium after 9 days.

After cell fusion, when the cells grow to 1/4 of the culture hole area, hybridoma cells are screened by a step screening method. The primary selection adopts an indirect ELISA method, an enzyme label plate is coated with coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), culture supernatant of a detected hole is added, incubation and washing are carried out, and then goat anti-mouse IgG-HRP, IgM-HRP and OPD are added for color reaction. The screened positive hole is screened by an indirect competitive ELISA method, the cell supernatant is mixed with 100 mu g/mL bromadiolone with equal volume, the mixture is taken in water bath at 37 ℃ for 30 min, and then the mixture is added into a coated enzyme label plate. PBS was also used as a control in place of bromadiolone, and the rest of the procedure was as above. OD if after Bromodiron blocking₄₅₀And (3) judging the wells to be positive when the nm value is reduced to below 50% of the control wells, and subcloning the wells which are positive after 2-3 detections by using a limiting dilution method immediately.

(4) Preparation and purification of monoclonal antibodies

Performing expanded culture on the hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, and measuring by indirect ELISAFixing the potency, and freezing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, injecting 1-2 × 10 hybridoma cells into the abdominal cavity after 7-10 days⁵Ascites was extracted 7 to 10 days later. Collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 of the cell supernatant or ascites, expressed as the maximum dilution factor), the results showed that the titer of the cell supernatant was 1: 10000, ascites titer 1: 60000. then, the resulting mixture was purified by an octanoic acid-saturated ammonium sulfate method, and the supernatant was collected to obtain a purified monoclonal antibody against bromadiolone.

The chessboard method is utilized to measure the titer of the monoclonal antibody, and the result shows that: the potency of the bromadiolone drug monoclonal antibody is 1: 128000 half maximal inhibitory amount (IC)₅₀) It was 0.35. mu.g/L.

Through detection, the amino acid sequence of the variable region of the heavy chain of the bromadiolone monoclonal antibody is shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain of the bromadiolone monoclonal antibody is shown as the sequence 2 in the sequence table.

2. Preparation of antibody working solution

Diluting the obtained monoclonal antibody of the bromadiolone drug by 1000 times with an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the bromadiolone drug.

Thirdly, preparation of enzyme marker working solution

1. Preparation of anti-antibodies

The obtained monoclonal antibody of the bromadiolone drug is taken as immunogen, and the goat is taken as immune animal, so as to obtain the goat anti-mouse anti-antibody of the monoclonal antibody of the anti-bromadiolone drug.

2. Preparation of horse radish peroxidase-labeled anti-antibody

Coupling the anti-antibody of the monoclonal antibody of the anti-bromadiolone drug obtained in the step 1 with horseradish peroxidase (HRP) by adopting a modified sodium periodate method, and comprising the following steps of:

dissolving 8 mg of horseradish peroxidase in 2 mL of distilled water; adding 100 mmol/L NaIO prepared now₄Stirring and reacting the solution for 20 min at room temperature, wherein the solution is 0.4 mL; dialyzing with 1 mmol/L acetate buffer solution at 4 deg.C overnight; removing excess NaIO₄Simultaneously reducing the enzyme coupled with the enzyme; adding 40. mu.L of LPBS buffer (pH 8.6, 0.5 mol/L) and 2.0mL of PBS buffer (pH 8.6, 5 mol/L) containing 16mg of an anti-antibody against a monoclonal antibody to a bromadiolone drug, and reacting at room temperature with stirring for 4 hours; 0.1 mL of 1 mol/L NaBH prepared now is added₄Reacting the aqueous solution at 4 ℃ for 4 hours, purifying and storing.

3. Preparation of enzyme-labeled working solution

And (3) diluting the anti-antibody of the anti-bromadiolone drug monoclonal antibody marked by the horseradish peroxidase by 500 times by using an anti-antibody diluent to obtain an enzyme marker working solution of the anti-antibody of the anti-bromadiolone drug monoclonal antibody marked by the horseradish peroxidase.

The anti-antibody diluent is a PBS (phosphate buffer solution) containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, the solutes are anhydrous disodium hydrogen phosphate, dihydrate sodium dihydrogen phosphate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072 g/L, 0.6 g/L, 16 g/L, 0.4g/L, 50 mL/L and 200 muL/L respectively.

Fourth, preparation of standard substance working solution

The kit also comprises 6 standard working solutions, wherein the concentrations of the solutes in the 6 standard working solutions are respectively 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L and 0.1g/L, and the pH value is 7.2.

Preparation of five, other reagents

The kit may further contain a sample diluent and/or a sample extract and/or a washing solution and/or a substrate developing solution and/or a stop solution.

The sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.1g/L, and the pH value is 7.2.

The sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.2 g/L, and the pH value is 7.2.

The washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively.

The substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water.

The stop solution is 0.05 mol/L sulfuric acid aqueous solution.

Example 2 and example 1 methods of Using the kits

First, pretreatment of sample

1. Pretreatment of meat sample, dairy product and serum sample

Second, detection Using the kit of example 1

1. Inserting an enzyme label plate into an enzyme label plate frame, recording the positions of each standard product and each sample, making 3 samples in parallel, sealing unused enzyme label plates by using self-sealing bags, and immediately storing in an environment at 2-8 ℃;

2. respectively adding 50 mu L of each standard substance working solution or sample solution into the corresponding standard substance or sample hole;

3. adding 50 mu L of antibody working solution into each plate hole;

4. covering a cover plate membrane, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

5. uncovering the plate film, pouring out liquid in the plate holes, adding 260 mu L of washing working solution (the washing solution is diluted by 20 times by deionized water) into each hole, and fully washing for 3-4 times, wherein each time of soaking is 15-30 s; (ii) a

6. Pouring out liquid in the plate hole, pouring the enzyme label plate on absorbent paper, and patting dry;

7. adding 100 mu L of enzyme marker working solution into each plate hole; covering a cover plate film, slightly oscillating the ELISA plate for 10 s, fully and uniformly mixing, and reacting for 30 min at room temperature (25 +/-2 ℃) in a dark place;

8. repeating the step 5-6;

9. immediately adding 100 μ L of substrate color developing solution A, B mixed solution (substrate color developing solution A and substrate color developing solution B are mixed according to volume ratio of 1: 1) into each well, covering with a cover plate membrane, and reacting for 15 min in dark place;

10. uncovering the microplate membrane, adding 50 mu L of stop solution into each plate hole, slightly oscillating the ELISA plate for 10 s, and fully and uniformly mixing;

11. and reading the absorbance values of the ELISA plate at the dual wavelengths of 405 nm and 630 nm by using an ELISA reader within 5 min after termination.

Thirdly, analyzing the detection result

1. Calculating percent absorbance value

The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of zero standard (standard with concentration of 0 mug/L) and multiplied by 100 percent, so as to obtain the percentage of absorbance corresponding to each standard (or sample to be measured), namely the percent absorbance value.

Percent absorbance = B/B₀×100%

Wherein: b-mean absorbance value of standard (or sample); b is₀Average absorbance values of standards at a concentration of 0 ppb.

2. Making a standard curve

Taking the percent absorbance value of each standard as a vertical coordinate, taking the concentration (mu g/L) of bromadiolone in the working solution of each standard as a horizontal coordinate to draw a standard curve graph, and carrying out nonlinear fitting analysis by using origin8.0 (originLab Corp., Northamapton, MA, USA) to form a four-parameter fitting curve:

y=(A-D)/[1+(x/C)B]+D

wherein y is the percentage of absorbance; x is the concentration of the substance to be detected; a, B, C and D are the four parameters of the standard curve.

The fitting of the test data shows that the standard curve of bromadiolone is: y = -0.141+ (2.304+0141) (1+ (x1.007) ^0.822), linear correlation R²Is 0.997.

The standard graph is shown in fig. 2.

3. Calculating the content of bromadiolone in the sample

Substituting the percent absorbance value of the sample to be detected into the standard curve to obtain the corresponding residual concentration of the sample to be detected, and multiplying the residual concentration by the dilution times of the corresponding samples to obtain the actual content of the bromadiolone in the original sample to be detected.

Example 3, example 1 kit specificity, accuracy, precision detection

Firstly, specific test of the kit:

the specificity of the bromadiolone enzyme-linked immunosorbent assay kit is determined by carrying out a cross reaction test with a corresponding substance.

Respectively diluting bromadiolone and common raticide (dazomet, difenac, warfarin and rodenticide) in series, respectively operating according to example 2, replacing 'bromadiolone standard working solution' with serial dilutions of bromadiolone and analogs thereof, making standard curve, and finding out respective 50% Inhibition Concentration (IC) on the curve₅₀) The specific method comprises the following steps: the corresponding concentration of bromadiolone (mug/L), namely IC, with the value of the ordinate equal to 50 percent is obtained₅₀The value is obtained. The cross-reactivity of the kit to bromadiolone and each of the commonly used muriatics was calculated using the following formula:

the cross-reactivity ratio (%) × (concentration of bromadiolone causing 50% inhibition/concentration of bromadiolone analogue causing 50% inhibition) × 100%.

The results are shown in Table 1.

TABLE 1 specificity of the kit

Experiments show that the kit has good specificity to bromadiolone, and the kit can detect the raticide bromadiolone.

Second, minimum detection Limit determination

A blank sample (LC-MS/MS negative test) of pork, milk and serum is taken and tested according to the method of the embodiment 2, the measured value is obtained according to the standard curve, the average value is calculated, and the lowest limit of detection (LOD) is obtained by adding 3 times of standard deviation. The results are shown in Table 2.

TABLE 2 statistical table of blank sample measurement results (μ g/L)

The result shows that the lowest detection limit of the bromadiolone in the pork is 4.87 mu g/L, the lowest detection limit of the bromadiolone in the milk is 4.86 mu g/L, and the lowest detection limit of the bromadiolone in the serum is 4.09 mu g/L, so that the lowest detection limit of the bromadiolone detection kit in the pork, the milk and the serum can be considered to be 5.0 mu g/L in order to ensure the stability of the kit.

Thirdly, testing the accuracy and precision of the kit

Pork, milk and a serum blank sample (negative LC-MS/MS) are respectively pretreated according to the method in the first step of the embodiment 2, and then bromadiolone is added to obtain a detection sample solution, wherein the final concentrations of the bromadiolone in the sample are respectively 5.0 mug/L and 10.0 mug/L.

The detection is carried out by using 3 kits of different batches, each experiment is repeated for 5 times, and the coefficient of variation is calculated respectively. The results are shown in Table 3, respectively.

The method for calculating the intra-batch variation coefficient comprises the following steps: intra-batch coefficient of variation = coefficient of variation for each parallel sample in the same assay.

The method for calculating the inter-batch variation coefficient comprises the following steps: the inter-batch coefficient of variation = coefficient of variation of measurement results of the same sample in different batches, and the average value thereof is taken.

TABLE 3 accuracy and precision

The result shows that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The variation coefficient in batch of each addition concentration is lower than 10%, and the variation coefficient between batches is lower than 15%.

Fourthly, the kit is compared with the detection result of LC-MS/MS

The method of example 2 is used for detecting pork, milk and serum samples, and the detection results of LC-MS/MS are respectively used for confirmation and comparison.

A scatter diagram is drawn by taking the concentration of the bromadiolone drug measured by the kit as an X axis and the concentration of the bromadiolone drug measured by LC-MS/MS as a Y axis. The measurement results of the two methods were subjected to linear analysis, and the results are shown in fig. 3, and the regression equation is: y =0.916x +0.056, which shows that the method established by the invention has good consistency with the detection result of LC-MS/MS.

Fifth, shelf life test of kit

The storage conditions of the kit in example 1 were 2-8 ℃, and the maximum absorbance (zero standard), the 50% inhibition concentration, and the actual measurement value of bromadiolone addition of the kit were within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed at 37 ℃ for 8 days for accelerated aging test, and the result shows that all indexes of the first step to the fourth step of the kit completely meet the requirements. And (3) considering the occurrence of the freezing condition of the kit, placing the kit at the temperature of-20 ℃ for 8 days, wherein all indexes of the first step to the fourth step completely meet the requirements. As can be seen from the above results, the kit of example 1 can be stored at 2-8 ℃ for at least 12 months.

Sequence listing

<110> Beijing Weideweikang Biotech Ltd

<120> enzyme linked immunosorbent assay kit for detecting bromadiolone and preparation and application thereof

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Ala Glu Ile Arg Leu Ile Ser Asn Asn Tyr Val Pro Tyr Ala His Glu

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Claims

1. An enzyme linked immunosorbent assay kit for detecting bromadiolone, comprising: the bromadiolone detection enzyme label plate comprises a bromadiolone detection enzyme label plate, bromadiolone series standard substance working solution, bromadiolone antibody working solution, enzyme marker working solution, sample diluent, sample extracting solution, washing solution, substrate developing solution and stop solution, and is characterized in that:

formula I

The bromadiolone antibody is a specific antibody of a bromadiolone drug.

2. The ELISA kit for detecting bromadiolone according to claim 1, wherein: the preparation method of the bromadiolone hapten comprises the following steps: weighing 500mg of bromadiolone, dissolving the bromadiolone in 6 mL of pyridine, adding 154.45 mg of phthalic anhydride, heating to 60 ℃, stirring, purifying, spin-drying and terminating the reaction to obtain the compound shown in the formula I.

3. The ELISA kit for detecting bromadiolone according to claim 1, wherein: the specific antibody of the bromadiolone drug is the bromadiolone monoclonal antibody, the bromadiolone monoclonal antibody consists of a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain can be shown as the sequence 1 in the sequence table, and the amino acid sequence of the variable region of the light chain can be shown as the sequence 2 in the sequence table.

4. The ELISA kit for detecting bromadiolone according to claim 1 or 3, wherein the kit comprises: the bromadiolone antibody working solution is prepared by diluting a monoclonal antibody of bromadiolone by 1000 times with an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of a bromadiolone medicament;

5. The ELISA kit for detecting bromadiolone according to claim 1, wherein: the enzyme marker working solution is obtained by diluting an anti-antibody of an anti-bromadiolone drug monoclonal antibody marked by horseradish peroxidase by 500 times with an anti-antibody diluent;

6. The ELISA kit for detecting bromadiolone according to claim 1, wherein: the bromadiolone series standard working solution is 6 bromadiolone standard working solutions, the solvent of the solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is bromadiolone; the concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L and 0.1g/L, and the pH value is 7.2.

7. The ELISA kit for detecting bromadiolone according to claim 1, wherein: the kit also contains a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution;

the sample diluent is a PBS buffer solution containing a light stabilizer, bovine serum albumin and a surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.1g/L, and the pH value is 7.2;

the sample extracting solution is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2 g/L, 0.1g/L and 0.2 g/L, and the pH value is 7.2;

the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing solution is deionized water, the solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentrations of the solutes in the PBS buffer solution are 23.2 g/L, 2.0 g/L, 64 g/L, 0.036 g/L, 20 mL/L and 300 muL/L respectively;

the substrate color developing solution is a mixed aqueous solution of 1.0 g/L carbamide peroxide, 5.0 g/L sodium acetate, 0.5 g/L light stabilizer, 2.5 mL/L phosphoric acid and 5.0 g/L tetramethyl benzidine, and the solvent is deionized water;

the stop solution is 0.05 mol/L sulfuric acid aqueous solution.

8. A method for detecting bromadiolone residues in a sample, comprising the following steps:

1) pretreating a sample;

2) detecting with the kit of claim 1;

3) and analyzing the detection result.

9. The method of detecting bromadiolone residues in a sample of claim 8, wherein: the detection by using the kit comprises the following steps: adding standard working solution or the solution of the sample into an ELISA plate coated with the conjugate of the bromadiolone drug hapten and the carrier protein; adding an antibody working solution of a bromadiolone medicament; after incubation, washing and drying, adding the enzyme marker working solution, developing by a substrate, stopping, and measuring the light absorption value by an enzyme marker.

10. The method of detecting bromadiolone residues in a sample of claim 8, wherein: the bromadiolone in pork, milk and serum can be detected by using the kit; the detection limit of bromadiolone in pork, milk and serum is 5 mug/kg, the inter-batch variation coefficient is less than 10 percent, and the intra-batch variation coefficient is less than 15 percent.