CN111289753B

CN111289753B - Enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticide and preparation and application thereof

Info

Publication number: CN111289753B
Application number: CN202010285655.3A
Authority: CN
Inventors: 刘河冰; 温凯; 马立才; 杨柳; 刘薇; 崔乃元; 聂靖东; 邢维维; 丁亚芳
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: 2023-04-11
Anticipated expiration: 2040-04-13
Also published as: CN111289753A

Abstract

The invention discloses an enzyme-linked immunoassay kit for detecting coumarin and indandione rodenticides, and preparation and application of the kit. The murek detection kit provided by the invention can be used for detecting coumarin and indandione rodenticide in food and human serum, the detection limits of murek, clomazone and diphacinone in pork, milk and serum are 5.0 mug/kg, the detection limit of chlorophacinone is 2.5 mug/kg, the detection limits of bromadiolone, dazomet and warfarin are 10.0 mug/kg, the detection limit of flocoumazone is 15.0 mug/kg, the batch variation coefficient is less than 10%, and the batch variation coefficient is less than 15%.

Description

Enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticide as well as 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 coumarin and indandione rodenticides, and a preparation method and application thereof.

Background

The raticide is various, and the anticoagulant raticide is the most widely used raticide, and comprises coumarins such as difenac, clomazone, flocmazone, rodenticide and the like, and indandiones such as dazomet, diphacinone, clomuride and the like, which inhibit the liver from generating prothrombin and coagulation factors by competitively inhibiting the action of vitamin K, improve the permeability and fragility of capillary vessels and cause bleeding and death in mice. With the large-scale administration of such rodenticides, rodenticides inevitably remain in the food, affecting human health. In recent years, the rodenticide causes poisoning, and seriously harms the health and life safety of people.

At present, the detection method for detecting the anticoagulant rodenticide mainly comprises a high performance liquid chromatography and liquid chromatography mass combination method, and the methods have the advantages of strong specificity and high sensitivity, but are complex to operate, expensive in instrument and not suitable for screening and detecting large-scale 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 an enzyme-linked immunoassay kit for coumarin and indandione rodenticide, and provides a detection method which is high in sensitivity, strong in specificity, low in detection cost and suitable for screening large-batch samples.

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

an enzyme linked immunosorbent assay kit for detecting anticoagulant rodenticide comprises an enzyme label plate, a standard substance working solution, an antibody working solution, an enzyme marker working solution, a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution.

The ELISA plate is prepared by coating conjugate of mouse Dekker drug hapten and carrier protein.

The preparation method of the mouse becker hapten comprises the following steps: weighing 100mg of murraya cordata, dissolving the murraya cordata in 10ml of methanol, adding 30mg of sodium hydride and 36mg of tetrabromobutyric acid to react at room temperature, adding 50ml of saturated salt water into a system after TLC detection reaction is completed, and filtering, washing and drying the mixture 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 rhabdoxin and the carrier protein refers to a product obtained by the active ester method of the rhabdoxin and the carrier protein, and specifically comprises the following steps:

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

wherein the ratio of the difenaker hapten (formula I), the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) is 36.4 mg:1.5 mL:38.3 mg:23mg;

2) Placing the carrier protein in 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;

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 to react for 24 h 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 obtain the paneth-coated antigen.

The murder antibody is a specific antibody of the murder drug.

The Muscat antibody working solution is prepared by diluting a Muscat monoclonal antibody by 1000 times with an antibody diluent to obtain the antibody working solution containing the monoclonal antibody of the Muscat 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 1.072g/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-mouse killer 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, and the solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 muL/L respectively. In the kit, the conjugate of the rat decok drug hapten and the carrier protein is coated on an enzyme label plate.

In the kit, the specific antibody of the murder g drug is the murder g monoclonal antibody.

In the kit, the murek 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 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 solvents of the 6 standard substance working solutions are PBS buffer solutions containing light stabilizers and bovine serum albumin, and the solute is Pedick; 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, and the solutes of disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/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, and the solutes of the PBS buffer solution are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizers, bovine serum albumin and surfactants, wherein the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.1g/L respectively, 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, light stabilizers, bovine serum albumin and surfactants, and the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.2g/L respectively, 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 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively.

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

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

Another object of the present invention is to provide a method for detecting rhodak 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 a standard substance working solution or a sample solution into an enzyme label plate coated with the conjugate of the mouse killer drug hapten and the carrier protein; adding a specific antibody solution containing a murder drug; after incubation, the swatter is washed dry, the enzyme-labeled anti-antibody is added, the substrate is used for color development, the reaction is stopped, and the light absorption value is measured by an enzyme-labeled instrument.

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

accurately weighing 1+ -0.01 g (or mL) sample, adding 5mL sample extract, whirling at high speed for 1 min at room temperature (25 + -2 ℃), and centrifuging at 4000 g for 5min 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 1 min, 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 mouse gram features that the conjugate of mouse gram's hapten and carrier protein is used to coat enzyme label plate, the monoclonal antibody of mouse gram's drug is used as the primary antibody, the anti-antibody of horseradish peroxidase (HRP) is used as the secondary antibody, and the substrate liquid is added for developing reaction, and the reaction is performed at 0.05mol/L H ₂ SO ₄ The reaction was stopped and absorbance was measured at 450 nm for the detection of murek's drug.

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

the absorbance average (B) of the obtained standard working solution of each concentration 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 (microgram/L) of the Mudeke standard substance working solution as an X axis and the percent absorbance value as a Y axis. The percent absorbance of the sample solution was calculated in the same manner and the amount of murek in the sample was 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 the Mudecog, the clomazone and the diphacinone in pork, milk and serum with the detection limit of 5.0 mug/kg, the chlorophacinone with the detection limit of 2.5 mug/kg, the bromadiolone, the dazomet and the warfarin with the detection limit of 10.0 mug/kg, the fluoromurin with the detection limit of 15.0 mug/kg, the inter-batch variation coefficient of less than 10 percent, the intra-batch variation coefficient of less than 15 percent and good stability. The coumarin and indandione rodenticide detection kit provided by the invention can be used for detecting toxicants in food, can also be used for detecting human serum, has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapidly detecting food poisoning.

Drawings

FIG. 1 is a graph of the Murdack standard.

FIG. 2 is a graph comparing the results of the detection by the kit and 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 rodenticide

The kit comprises the following components: the kit comprises an enzyme label plate (a conjugate of an encapsulated mouse killer and a carrier protein), an antibody working solution (containing a mouse killer monoclonal antibody), an enzyme marker working solution (containing an anti-mouse killer monoclonal antibody marked by horseradish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a working solution containing mouse killer standard products with different gradient concentrations, a substrate developing solution and a stop solution.

The preparation method comprises the following steps:

1. preparation of ELISA plates

1. Preparation of mouse dexrazoxane coating antigen

(1) Preparation of mouse-tek hapten

Weighing 100mg of murraya paniculata raw material, dissolving the raw material by using 10ml of methanol, adding 30mg of sodium hydride and 36mg of tetrabromobutyric acid to react at room temperature, adding 50ml of saturated salt solution into a system after TLC detection reaction is completed, and filtering, washing and drying the mixture to obtain the hapten shown in the formula I.

Formula I

(2) Preparation of mouse dexrazoxane coating antigen

Dissolving 36.4 mg Mudecoy hapten in 1.5 mL DMF, stirring at 200 rpm for 10 min, adding 38.3 mg EDC and 23mg NHS to dissolve, stirring at room temperature (500 rpm) to activate 2-3h to obtain solution I; weighing OVA 50 mg, dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve, cooling at 0-4 ℃ in an ice bath, dropwise adding the solution I (1 mL/min) under stirring at 1000 rpm, and stirring at 500 rpm to react with 24 h; the reaction product is filled into a distilled water to wash clean dialysis bag (10 cm), 1L of 0.01M PBS (1 x, pH7.2) is stirred at 4 ℃ (100 rpm) and dialyzed for 3 d, the liquid is changed 3 times (once in the morning, in the evening) every day, the total time is 9 times, and the dialysis product is centrifuged for 6 min at 5000 rpm, thus obtaining the rat decok coating antigen.

2. Preparation of ELISA plate

Diluting the obtained Mudeke coating antigen to 10.0 mu g/mL by using a coating buffer solution, adding 100 mu L of the coating antigen into each hole, incubating at 37 ℃ for 2 h, pouring out the coating solution, washing for 2 times by using a washing solution diluted by 20 times, each time for 30 seconds, beating to dry, then adding 150 mu L of a sealing solution into each hole, incubating at 37 ℃ for 1 h, pouring out the liquid in the holes, drying to obtain an ELISA plate coated with the coating antigen (a conjugate of a Mudeke drug hapten and a carrier protein), and performing vacuum sealing and preservation 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 0.2 mol/L pH7.7 phosphate buffer solution of 3 ‰ sodium azide.

2. Preparation of antibody working solution

1. Preparation of mouse monoclonal antibody

(1) Preparation of mouse Dekker immunogen

Dissolving 24.4 mg Mudeke hapten in 1.5 mL DMF, stirring at 200 rpm for 10 min, adding 25.7 mg EDC and 15.4 mg NHS to dissolve, stirring at room temperature (500 rpm) to activate for 2-3h to obtain solution I; weighing 50 mg of BSA and dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10 min to fully dissolve the BSA, cooling at 0-4 ℃ in an ice bath, dropwise adding the solution I (1 mL/min) under stirring at 1000 rpm, and stirring at 500 rpm to react with 24 h; the reaction product is filled into a dialysis bag (10 cm) which is washed clean by distilled water, 1L of 0.01M PBS (1 x, pH7.2) is stirred at 4 ℃ (100 rpm) and dialyzed for 3 d, the solution is changed 3 times (once in the morning, in the evening) every day, the total time is 9 times, and the dialysis product is centrifuged for 6 min at 5000 rpm, so that the mouse dexgen immunogen is obtained.

(2) Animal immunization

Dissolving the prepared mouse dexraz immunogen by using normal saline according to 100 mu g/mouse, uniformly mixing the dissolved immunogen with Freund complete adjuvant in equal volume, injecting 6~8 week-old Balb/c female mice subcutaneously in the neck and back, uniformly mixing the immunogen and Freund incomplete adjuvant in equal volume in 7, 14 and 28 days after primary immunization, performing additional immunization once respectively, and performing additional immunization once by using 100 mu g/mouse of immune complex in3 days before fusion without adding Freund adjuvant.

(3) Cell fusion and cloning

Mixing splenocytes of immunized mice with myeloma cells (SP 2/0) of mice 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, and culturing at 37 deg.C and 5% CO ₂ Culturing 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 cell grows to 1/4 of the culture hole area, adopting step-by-step sieveSelecting and screening hybridoma cells. 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 Kong Zaiyong indirect competition ELISA method is screened, firstly, cell supernatant is mixed with 100 mu g/mL difenax in equal volume, and the mixture is subjected to water bath at 37 ℃ for 30 min and then added into a coated ELISA plate. The control was also performed by replacing mouse control with PBS and the procedure was the same as above. OD after Cork blocking ₄₅₀ When the nm value was decreased to 50% or less of the control well, the wells were judged to be positive, and the wells were all positive in 2~3 determinations and immediately subcloned by the limiting dilution method.

(4) Preparation and purification of monoclonal antibodies

Carrying out amplification culture on the hybridoma after 2~3 times of subclone strain establishment, collecting supernate, measuring the titer by using indirect ELISA, and freezing and storing; injecting 0.5 mL/mouse into the abdominal cavity of a Balb/c mouse aged 8 to 10 weeks, and injecting 1~2 x 10 hybridoma cells into the abdominal cavity after 7 to 10 days ⁵ And extracting the ascites of the mice 7 to 10 days later. Collecting cell supernatant or ascites, and measuring titer by indirect ELISA (P/N for measuring titer)>2.1 Expressed as the maximal dilution of the cell supernatant or ascites), the results showed that the titer of the cell supernatant was 1:10000, ascites titer 1:60000. then, the mixture was purified by an octanoic acid-saturated ammonium sulfate method, and the supernatant was collected to obtain a purified monoclonal antibody against mouse killer.

The chessboard method is utilized to measure the titer of the monoclonal antibody, and the result shows that: the titer of the murder 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 Mudecoy 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 Mudecoy 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 mouse killer by 1000 times by using an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the mouse killer.

3. Preparation of enzyme-labeled working solution

1. Preparation of anti-antibodies

The obtained monoclonal antibody of the mouse killer is used as immunogen, and the goat is used as immune animal to obtain the goat anti-mouse anti-antibody of the monoclonal antibody of the mouse killer.

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

Coupling the anti-antibody of the monoclonal antibody of the anti-mouse killer obtained in the step 1 with Horse Radish Peroxidase (HRP) by adopting a modified sodium periodate method, and comprising the following steps:

dissolving 8 mg horse radish peroxidase in 2 mL distilled water; adding 100 mmol/L NaIO prepared now ₄ The solution is 0.4 mL, and the reaction is stirred at room temperature for 20 min; dialyzing with 1 mmol/L acetate buffer solution at 4 deg.C overnight; removing excess NaIO ₄ Simultaneously reducing the enzyme coupled with the enzyme; 40 mu LPBS buffer (pH 8.6,0.5 mol/L) and 2.0 mL anti-antibody PBS buffer (pH 8.6,5 mol/L) containing 16 mg anti-mouse killer monoclonal antibody were added and the reaction was stirred at room temperature for 4 hours; adding 0.1 mL of 1 mol/L NaBH prepared now ₄ Reacting 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 monoclonal antibody of the horseradish peroxidase-labeled anti-mouse killer drug obtained in the step (2) by 500 times with an anti-antibody diluent to obtain an enzyme-labeled substance working solution of the anti-antibody of the monoclonal antibody of the horseradish peroxidase-labeled anti-mouse killer drug.

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, and the solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentrations of the solutes in the PBS buffer are 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 muL/L respectively.

4. Preparation of standard working solution

The kit also comprises 6 standard substance working solutions, wherein the solvents of the 6 standard substance working solutions are PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is Pedick; the concentrations of the solute in the 6 standard substance working solutions are respectively 0 mug/L, 0.1 mug/L, 0.3 mug/L, 0.9 mug/L, 2.7 mug/L and 8.1 mug/L; the solvent of the PBS buffer solution is deionized water, and the solutes of the PBS buffer solution are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizers and bovine serum albumin, wherein the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L and 0.1g/L respectively, and the pH value is 7.2.

5. Preparation of 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, light stabilizers, bovine serum albumin and surfactants, and the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.1g/L respectively, 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, light stabilizers, bovine serum albumin and surfactants, the concentrations of the solutes in the PBS buffer solution are 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L, 0.1g/L and 0.2g/L respectively, 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 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively.

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

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

Example 2 and example 1 methods of Using the kits

1. Pretreatment of samples

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

2. 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 plate strips 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, lightly vibrating the ELISA plate 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 hole, adding 260 mu L of washing working solution (the washing solution is diluted by deionized water by 20 times) into each hole, and fully washing for 3-4 times, wherein the soaking time is 15-30 s each time; (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 membrane, lightly shaking the ELISA plate 10 s, fully and uniformly mixing, and reacting for 30 min in a dark place at room temperature (25 +/-2 ℃);

8. repeating the step 5-6;

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

10. uncovering the cover plate film, adding 50 mu L of stop solution into each plate hole, lightly vibrating the ELISA plate 10 s, and fully and uniformly mixing;

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

3. Analyzing the results of the detection

1. Calculating percent absorbance value

The average absorbance value of each standard (or sample to be detected) is divided by the absorbance value of zero standard (standard with the 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 detected), namely the percentage absorbance value.

Percent absorbance = B/B ₀ ×100%

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

2. Making a standard curve

The percent absorbance values of the standards are used as ordinate, the concentration of the Murdack in the working solution of the standards (mu g/L) is used as abscissa to draw a standard curve graph, origin8.0 (originLab Corp., northamapton, MA, USA) is used for nonlinear fitting analysis, and a four-parameter fitting curve is formed:

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.

Through the test data, the standard curve equation of murek is: y = -0.143+ (2.368 + 0.143) (1 + (x 0.948) ^ 0.820), linear correlation R ² Is 0.998.

The standard graph is shown in figure 1.

3. Calculating the content of mouse 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 murek in the original sample to be detected.

Example 3 and example 1 detection of specificity, detection Limit, accuracy, precision of the kits

1. Specific test of the kit:

the specificity of the enzyme-linked immunosorbent assay kit of the rat-duke is determined by carrying out cross reaction tests with corresponding substances.

Respectively diluting rodenticide and conventional rodenticides (bromadiolone, dazomet, warfarin, clomazone, diphacinone, chlorophacinone, flumiclorac, rodenticide ether and rodenticide) in series, respectively operating according to example 2, replacing the 'rodenticide standard working solution' with the dilution series of rodenticide and its analogues, making standard curve, and finding out respective 50% Inhibition Concentrations (IC) on the curve ₅₀ ) The specific method comprises the following steps: the concentration of Mucor (μ g/L) corresponding to a value of 50% on the ordinate, i.e. IC, is obtained ₅₀ The value is obtained. The cross-reactivity of the kit to difenax and each of the commonly used muricides was calculated using the following formula:

cross-reactivity (%) = (concentration of rodenticide causing 50% inhibition/concentration of rodenticide analogue causing 50% inhibition) × 100%.

The results are shown in Table 1.

TABLE 1 specificity of the kit

Name (R)	Cross reaction Rate (%)
		Mouse killer	100.0
Chlorfenapyr	126.3
		Diphacinone	105.2
Chlorophacinone	230.3
		Bromadiolone	56.8
Root of Dalong-noded Fangqi	46.6
		Warfarin	42.3
Flumazole	38.2
		Deratization control	<1
Rodenticide	<1
		Ratcidal ketones	<1

Experiments show that the kit has good cross reaction rate on difenac, bromadiolone, dazomet, warfarin, clomazone, diphacinone, chlorophacinone and flocoumazone, namely the kit can detect the raticide, difenoconazole, dazomet, warfarin, clomazone, diphacinone, chlorophacinone and flocoumazone.

2. Detection Limit determination of the kit

A blank sample of pork, milk and serum (negative in LC-MS/MS) is taken, detection is carried out according to the method of the embodiment 2, the measured values are obtained according to a standard curve, the average value of the measured values is calculated, and 3 times of standard deviation is added, so that the lowest detection Limit (LOD) is obtained. The results are shown in tables 2 to 9.

TABLE 2 rat Degram blank sample determination results (μ g/kg)

Table 3 clomazone blank sample determination results (μ g/kg)

TABLE 4 Dipper blank sample determination results (mug/kg)

TABLE 5 chlorine diphacinone blank sample determination results (mug/kg)

TABLE 6 Bromodiolone blank sample assay results (μ g/kg)

TABLE 7 bloom blank sample determination results (μ g/kg)

TABLE 8 warfarin blank sample determination results (μ g/kg)

TABLE 9 measurement results of the Flumazone blank samples (μ g/kg)

The result shows that in order to prevent the occurrence of false positive, the kit can be used for determining the detection limit of 5.0 mug/kg for murrex, clomazone and diphacinone in pork, milk and serum, the detection limit of 2.5 mug/kg for chlorophacinone, bromadiolone, dazomet and warfarin as 10.0 mug/kg and the detection limit of 15.0 mug/kg for flocoumazone.

3. Accuracy and precision testing of the kit

Accuracy refers to the degree of coincidence between a measured value and a true value, and is often expressed in terms of recovery and precision in terms of coefficient of variation. Pork, milk and serum blank samples (LC-MS/MS detection negative) are respectively pretreated according to the method in the first step of the embodiment 2, and then, rodenticide, clomiphene, diphacinone, chlordiazepoxide, brodifuron, warfarin and flocoumafen standard substances are added to the required concentrations (table 10) to obtain detection sample solutions.

TABLE 10 blank sample addition concentration

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 tables 11 to 18, 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 11 accuracy and precision of Muteck

TABLE 12 accuracy and precision of clomiprinol

TABLE 13 accuracy and precision of diphacinone

TABLE 14 accuracy and precision of chlorophacinone

TABLE 15 accuracy and precision of bromadiolone

TABLE 16 high bump accuracy and precision

TABLE 17 warfarin accuracy and precision

TABLE 18 Flumazone accuracy and precision

The results show that the recovery rate of each added concentration of all samples is between 80 and 120 percent. The intra-batch coefficient of variation of each addition concentration is lower than 10%, and the inter-batch coefficient of variation is lower than 15%.

4. The detection result of 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 mouse-loving gram measured by the kit as an X axis and the concentration of the mouse-loving gram 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. 2, and the regression equation is: y =0.941x +0.067, which shows that the method established by the invention has good consistency with the detection result of LC-MS/MS.

5. 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 the mouse killer 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 for 8 days at the temperature of-20 ℃, 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 Vierweikang Biotech. Ltd

<120> enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticide, and preparation and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Lys Asp Gly Ser Leu Leu Glu Ser Val Gln Gly Gly Gly Leu Pro

1 5 10 15

Gly Arg Lys Leu Ser Cys Ala Ala Gly Ser Thr Phe Phe Ser Ser Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Trp Val Glu

35 40 45

Tyr Ile Ala Ser Gly Gly Thr Ile Ser Asn Tyr Tyr Ala Asp Thr Val

50 55 60

Arg Pro Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asn Thr Leu Phe

65 70 75 80

Leu Met Gln Thr Ser Ala Tyr Thr Val Arg Met Leu Asp Thr Phe Cys

85 90 95

Ala Arg Trp Gly Thr Gly Thr Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Val Ser Ser Thr

115

<210> 2

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Glu Leu Val Met Thr Gln Met Tyr Ser Ser Thr Pro Asp Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Ala Asn Ser Gln Lys Asp Ile Ser Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Val

35 40 45

Asp Val Tyr Arg Asp Asn Arg Leu Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Thr Ser Leu Gln Asp Gly Tyr Ser Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Phe Pro Cys Leu Gln Tyr Asp Glu Phe

85 90 95

Thr Phe Gly Ser Gly Lys Leu Glu Gln Asn Val Ser

100 105

Claims

1. An enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticides comprises: the kit comprises an enzyme label plate for Mudeke detection, a Mudeke series standard substance working solution, a Mudeke antibody working solution, an enzyme marker working solution, a sample diluent, a sample extracting solution, a washing solution, a substrate developing solution and a stop solution, and is characterized in that: the rat-gram detection enzyme label plate is prepared by coating a conjugate of a rat-gram drug hapten and a carrier protein in a formula I, wherein the rat-gram antibody is a specific antibody of the rat-gram drug.

2. The enzyme linked immunosorbent assay kit for detecting the coumarins and the indandione rodenticides according to claim 1, wherein the enzyme linked immunosorbent assay kit comprises: the preparation method of the mouse killer hapten comprises the following steps: weighing 100mg of murrax, dissolving the murrax in 10ml of methanol, adding 30mg of sodium hydride and 36mg of tetrabromobutyric acid, reacting at room temperature, detecting by TLC (thin layer chromatography), adding 50ml of saturated saline solution into a system after the reaction is completed, and filtering, washing and drying to obtain the compound shown in the formula I.

3. The ELISA kit for detecting coumarin and indandione rodenticide according to claim 1, wherein the kit comprises: the specific antibody of the mouse killer drug is a mouse killer monoclonal antibody, the mouse killer monoclonal antibody is composed of a heavy chain and a light chain, the amino acid sequence of the variable region of the heavy chain is shown as the sequence 1 in a sequence table, and the amino acid sequence of the variable region of the light chain is shown as the sequence 2 in the sequence table.

4. The ELISA kit for detecting coumarin and indandione rodenticide according to claim 1 or 3, wherein the kit comprises: diluting the monoclonal antibody of the mouse killer by 1000 times by using an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the mouse killer;

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 solution are 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 muL/L and 500 muL/L respectively.

5. The ELISA kit for detecting coumarin and indandione rodenticide according to claim 1, wherein the kit comprises: the enzyme marker working solution is obtained by diluting an anti-antibody of an anti-mouse gram 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 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 muL/L respectively.

6. The ELISA kit for detecting coumarin and indandione rodenticide according to claim 1, wherein the kit comprises: the mouse dexrazoxane series standard working solution is 6 mouse dexrazoxane standard working solutions, the solvent of the standard working solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and the solute is mouse dexrazoxane; the concentrations of the solutes in the 6 murder 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, and the solutes of the PBS buffer solution are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, wherein the concentrations of the solutes in the PBS buffer solution are respectively 2.68g/L, 0.1g/L, 4g/L, 0.1g/L, 0.2g/L and 0.1g/L, and the pH value is 7.2.

7. The ELISA kit for detecting coumarin and indandione rodenticide according to claim 1, wherein the kit comprises: 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, 0.2g/L, 0.1g/L and 0.1g/L, and the pH value is 7.2;

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, 0.2g/L, 0.1g/L and 0.2g/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 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 muL/L respectively;

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

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

8. A method of detecting murek's residue in a sample, comprising the steps of:

1) Pretreating a sample;

2) Detecting with the kit of claim 1;

3) And analyzing the detection result.

9. The method of claim 8, wherein the step of detecting murek's residue in the sample comprises: the detection by using the kit comprises the following steps: adding standard substance working solution or the solution of the sample into an enzyme label plate coated with the conjugate of the mouse killer drug hapten and the carrier protein and the conjugate of the quinolone drug hapten and the carrier protein; adding a specific antibody of the murder drug; washing and drying after incubation, adding the enzyme-labeled anti-antibody composition, developing by using a substrate of alkaline phosphatase, stopping, and measuring a light absorption value by using an enzyme-labeled instrument; then, the substrate of horseradish peroxidase is used for developing, stopping and measuring the light absorption value by an enzyme-linked immunosorbent assay instrument.

10. The method of claim 8, wherein the step of detecting murek's residue in the sample comprises: the kit is used for detecting coumarin and indandione rodenticide in pork, milk and serum, the detection limits of rodenticide, clomiprinine and diphacinone in the pork, milk and serum are 5.0 mu g/kg, the detection limit of chlorophacinone is 2.5 mu g/kg, the detection limits of bromadiolone, dazomet and warfarin are 10.0 mu g/kg, the detection limit of flocoumafen is 15.0 mu g/kg, the inter-batch variation coefficient is less than 10 percent, and the intra-batch variation coefficient is less than 15 percent.