CN111273018B - ELISA kit for detecting bromadiolone and preparation and application thereof - Google Patents

ELISA kit for detecting bromadiolone and preparation and application thereof Download PDF

Info

Publication number
CN111273018B
CN111273018B CN202010285672.7A CN202010285672A CN111273018B CN 111273018 B CN111273018 B CN 111273018B CN 202010285672 A CN202010285672 A CN 202010285672A CN 111273018 B CN111273018 B CN 111273018B
Authority
CN
China
Prior art keywords
bromadiolone
solution
antibody
pbs buffer
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010285672.7A
Other languages
Chinese (zh)
Other versions
CN111273018A (en
Inventor
温凯
马立才
刘河冰
聂靖东
邢维维
刘薇
杨柳
丁亚芳
崔乃元
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Wdwk Biotechnology Co ltd
Original Assignee
Beijing Wdwk Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Wdwk Biotechnology Co ltd filed Critical Beijing Wdwk Biotechnology Co ltd
Priority to CN202010285672.7A priority Critical patent/CN111273018B/en
Publication of CN111273018A publication Critical patent/CN111273018A/en
Application granted granted Critical
Publication of CN111273018B publication Critical patent/CN111273018B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses an ELISA method for detecting bromadiolone and a kit thereof, and the ELISA kit for detecting bromadiolone provided by the invention comprises an ELISA plate (a conjugate of a bromadiolone drug hapten and a carrier protein of a coating formula I), an antibody working solution (a bromadiolone drug monoclonal antibody), an enzyme marker working solution (an anti-antibody of an anti-bromadiolone drug monoclonal antibody marked by horseradish peroxidase), a washing solution, a sample diluent, a sample extracting solution, a bromadiolone standard substance solution containing different gradient concentrations, a substrate color development solution and a termination solution. The bromadiolone detection kit provided by the invention can be used for detecting poison in food, can also be used for detecting human serum, has the detection limit of bromadiolone in pork, milk and serum of 5 mug/kg, has the inter-batch variation coefficient of less than 10% and the intra-batch variation coefficient of less than 15%, has the advantages of simplicity and rapidness in operation, high sensitivity, strong specificity and strong accuracy, and has great value for rapid detection of food poisoning.

Description

ELISA 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, a preparation method and application thereof.
Background
The chemical name of the bromadiolone is 3- (4-hydroxy-3-coumarin) -3-phenyl-1- (p-bromobiphenyl) -propanol-1, and belongs to 4-hydroxy biscoumarin anticoagulation raticide. It can inhibit the production of prothrombin and coagulation factor by the liver, and raise capillary permeability and brittleness, resulting in hemorrhage death in mouse. With the massive administration of rodenticides, the rodenticides inevitably remain in the food, affecting human health. In recent years, the occurrence of poisoning caused by the rodenticide is serious harm to the health and life safety of people.
The existing detection methods of bromadiolone mainly comprise a high-performance liquid phase method and a liquid chromatography-mass spectrometry method, and the methods are high in specificity and sensitivity, complex in operation, expensive in instrument and not suitable for screening and detecting a large number of samples. The rapid detection method is mainly a colorimetric detection method by a chemical method, has poor specificity and sensitivity, is easy to misjudge, and cannot meet the field detection requirement.
Disclosure of Invention
The invention aims to provide a combined immunity detection kit of bromadiol Long Mei, and provides a detection method which has high sensitivity, strong specificity and low detection cost and is suitable for screening a large number of samples.
In order to achieve the above object, the technical scheme of the present invention is as follows:
an ELISA kit for detecting bromadiolone comprises a bromadiolone detection ELISA plate, bromadiolone series standard substance working solution, bromadiolone antibody working solution, enzyme marker working solution, sample dilution solution, sample extraction solution, washing solution, substrate color development solution and termination solution.
The bromadiolone Long Mei target is prepared by coating a conjugate of a bromadiolone drug hapten and a carrier protein, wherein the bromadiolone hapten is a compound shown in a formula I:
i is a kind of
The bromadiolone hapten is obtained by reacting bromadiolone with phthalic anhydride, and the specific preparation method is as follows: 500mg bromadiolone is weighed and dissolved by 6mL pyridine, 154.45mg of phthalic anhydride is added, the mixture is heated to 60 ℃ and stirred, purified and dried by spin, and the reaction is terminated, thus obtaining the compound shown in the formula I.
The carrier protein is Bovine Serum Albumin (BSA), human Serum Albumin (HSA), murine serum protein (MSA), thyroxine (BCG), rabbit serum protein (RSA), hemocyanin (KLH) or Ovalbumin (OVA).
The conjugate of the bromadiolone hapten and the carrier protein refers to a product obtained by the bromadiolone hapten and the carrier protein through an active ester method, and specifically comprises the following steps of:
1) Dissolving the bromadiolone hapten (formula I) in Dimethylformamide (DMF), and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and magnetically stirring at 20-25 ℃ to react for 2-3 h 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) Placing the carrier protein in 0.1M sodium bicarbonate buffer solution, stirring at 200 rpm for 10min, and fully dissolving to obtain solution II; the ratio of the carrier protein to the 0.1M sodium bicarbonate buffer 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 is 50 mg:3.5 mL; if the carrier protein is Ovalbumin (OVA), the ratio of Ovalbumin (OVA) to the 0.1M carbonic acid buffer 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 stirring at 1000 rpm at the temperature of 0-4 ℃, and stirring at 500 rpm for reaction 24 h to obtain a solution III;
4) The solution III was dialyzed with PBS buffer (0.01M PBS, pH 7.2) at 4℃for 3 days with stirring to give the bronopol Long Bao antigen.
The bromadiolone antibody is a specific antibody of a bromadiolone drug.
The bromadiolone antibody working solution is obtained by diluting a monoclonal antibody of bromadiolone by 1000 times with an antibody diluent.
The antibody diluent is PBS buffer solution containing Proclin300 and Triton X-100; 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, proclin300 and Triton X-100, and the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively.
The enzyme marker working solution is obtained by diluting an anti-antibody of a horseradish peroxidase-marked anti-bromadiolone drug monoclonal antibody by 500 times with an anti-antibody diluent.
The anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and the solute is anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentration of the solute in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively. In the kit, the conjugate of the bromadiolone drug hapten and the carrier protein is coated on an ELISA plate.
In the kit, the specific antibody of the bromadiolone drug is the bromadiolone Long Shan clone antibody.
In the kit, the clone antibody of bromadiol Long Shan consists of a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region 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 of 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 concentration of the solute in the 6 standard working solutions is 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 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.
In the kit, the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 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.
In the kit, 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 solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 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.
In the kit, the washing solution is PBS buffer solution containing Tween 20 and Proclin 300; the solvent of the washing liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 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 a sulfuric acid aqueous solution of 0.05 mol/L.
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 of the above kits;
3) And analyzing the detection result.
In the above method, the detection with the kit comprises the steps of: adding standard working solution or 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 bromadiolone medicine; after incubation, washing and beating to dry, adding the enzyme-labeled anti-antibody, developing with a substrate, stopping and measuring the absorbance value with an enzyme-labeled instrument.
In the above method, the method for pre-treating the sample specifically comprises the following steps:
accurately weighing 1+/-0.01 g (or mL) sample, adding the sample into 5mL sample extracting solution, carrying out high-speed whirling for 1 min at room temperature (25+/-2 ℃), and centrifuging for 5 min at 4000 g to obtain a sample solution. 200 mu L of sample solution is taken, 200 mu L of sample diluent is added, high-speed vortex is carried out for 1 min, and 50 mu L of supernatant is taken for analysis.
The detection principle of the kit provided by the invention is as follows: an indirect competitive ELISA method for detecting bromadiolone features that the conjugate of bromadiolone hapten and carrier protein is used to coat ELISA plate, the monoclonal antibody of bromadiolone is used as the first antibody, the antibody of monoclonal antibody of Horse Radish Peroxidase (HRP) resisting bromadiolone is used as the second antibody, the substrate liquid is added for developing reaction, and the concentration of said antibody is 0.05mol/L H 2 SO 4 The 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 value (B) of the standard working solution at 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) ×100%
And drawing a standard curve graph by taking the half-logarithmic value of the concentration (mu g/L) of the bromadiolone standard working solution as an X axis and the percentage absorbance value as a Y axis. The percentage absorbance value 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 in the invention can also utilize the 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 only in a short time.
Experiments prove that the kit can detect bromadiolone in pork, milk and serum; the detection limit of detecting bromadiolone in pork, milk and serum is 5 mug/kg, the inter-batch variation coefficient is less than 10%, the intra-batch variation coefficient is less than 15%, and the stability is good.
The bromadiolone detection kit provided by the invention can be used for detecting poison 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 rapid detection of food poisoning.
Drawings
FIG. 1 identification mass spectrum of bromadiolone hapten.
Fig. 2 standard graph of bromadiolone.
FIG. 3 is a graph comparing the results of the present kit with LC-MS/MS detection results.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of ELISA kit for detecting bromadiolone
The kit comprises the following components: the kit comprises an ELISA plate (conjugate of coated bromadiolone drug and carrier protein), an antibody working solution (monoclonal antibody containing bromadiolone drug), an enzyme marker working solution (monoclonal antibody containing horseradish peroxidase-labeled bromadiolone drug), a washing solution, a sample diluent, a sample extracting solution, a standard working solution containing different gradient concentrations of bromadiolone, a substrate color development solution and a termination solution.
The preparation method comprises the following steps:
1. preparation of ELISA plate
1. Preparation of bromadiol Long Bao quilt
(1) Preparation of bromadiolone hapten
500 The mg of bromadiolone was completely dissolved with 6mL pyridine, phthalic anhydride 154.45mg was weighed, added thereto, stirred, and heated to 60 ℃. When the reaction is not changed by TLC, treating, spin-drying pyridine, dissolving with methanol, adding silica gel for sample stirring, spin-drying the sample for column chromatography, collecting the required solution, spin-drying, and collecting to obtain the bromadiolone hapten. The structure of the mass spectrum is confirmed, and the structural formula is shown as formula I.
I is a kind of
(2) Preparation of bromadiol Long Bao quilt
30.22. 30.22 mg bromadiolone hapten is dissolved by 1.5 mL of DMF, stirred at 200 rpm for 10min, 25.75 mg of EDC and 15.46 mg of NHS are added for dissolution, and the mixture is stirred at room temperature (500 rpm) for activation of 2-3 h; weighing OVA 33.6 and mg, dissolving in 3.5 mL of 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10min to enable the solution to be fully dissolved, cooling the solution by using an ice bath to 0-4 ℃, dropwise adding the reaction solution obtained in the step 1 (1 mL/min) under stirring at 1000 rpm, and stirring at 500 rpm for reaction 24 h; the reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d with 1L of 0.01M PBS (1X, pH 7.2) stirred (100 rpm) at 4℃and 3 times daily (each time in the morning, in the evening) and 9 times total, and the dialyzed product was centrifuged at 5000 rpm for 6 min to obtain bromadiolone Long Bao as the starting material.
2. Preparation of ELISA plate
Diluting the obtained bromadiolone Long Bao with coating buffer solution to 10.0 mu g/mL, adding 100 mu L of coating source into each hole, incubating at 37 ℃ for 2 h, pouring off the coating solution, washing with 20 times of diluted washing solution for 2 times for 30 seconds each time, beating to dry, adding 150 mu L of sealing solution into each hole, incubating at 37 ℃ for 1 h, pouring off the liquid in the holes, drying to obtain the ELISA plate coated with the coating source (conjugate of bromadiolone drug hapten and carrier protein), and vacuum sealing and storing with an aluminum film.
Coating buffer solution: sodium carbonate buffer solution with pH of 9.6 and 0.03 mol/L;
sealing liquid: a phosphate buffer solution of 0.2 mol/L pH7.7 containing 50 g/L sucrose, 2.5 g/L casein, 0.5% calf serum, 3% sodium azide.
2. Preparation of antibody working solution
1. Preparation of clone antibody of bromadiol Long Shan
(1) Preparation of bromadiolone immunogen
30.22. 30.22 mg bromadiolone hapten is dissolved by 1.5 mL of DMF, stirred at 200 rpm for 10min, 25.75 mg of EDC and 15.46 mg of NHS are added for dissolution, and the mixture is stirred at room temperature (500 rpm) for activation of 2-3 h; weighing BSA 50 mg, dissolving in 3.5 mL 0.1M sodium bicarbonate solution, stirring at 200 rpm for 10min to fully dissolve, cooling with ice bath to 0-4 ℃, dropwise adding (1 mL/min) the reaction solution obtained in the step 1 under stirring at 1000 rpm, and stirring at 500 rpm to react for 24 h; the reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d with 1L of 0.01M PBS (1X, pH 7.2) stirred (100 rpm) at 4℃and 3 times daily (each time in the morning, in the evening) and 9 times total, and the dialyzed product was centrifuged at 5000 rpm for 6 min to obtain bromadiolone immunogen.
(2) Immunization of animals
The prepared bromadiolone immunogen is dissolved by normal saline according to the volume of 100 mug/mouse, the immunogen is uniformly mixed with Freund's complete adjuvant, 6-8 week old Balb/c female mice are subcutaneously injected into the back of the neck, the immunogen and Freund's incomplete adjuvant are uniformly mixed in the volume of 7, 14 and 28 days after primary immunization, the immunization is carried out once each time, 100 mug/mouse immune complex is added 3 days before fusion, and the Freund's adjuvant is not added for one time.
(3) Cell fusion and cloning
The spleen cells of immunized mice were mixed with mouse myeloma cells (SP 2/0) in logarithmic growth phase and then in conventional mannerSlowly adding preheated fusion agent (PEG 4000) for fusion within 45s, suspending with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate, and culturing at 37deg.C and 5% CO 2 Culturing in an incubator, half-changing liquid with HT culture medium after 5 days, and full-changing liquid at 9 days.
After the cells are fused, when the cells grow to 1/4 of the area of the culture hole, the step-by-step screening method is adopted to screen the hybridoma cells. The primary selection adopts an indirect ELISA method, an ELISA plate is coated with coating antigen (the optimal coating concentration and positive serum dilution are conventionally titrated in advance by a square method), the culture supernatant of a tested hole is added, the culture supernatant is incubated, and after washing, goat anti-mouse IgG-HRP and IgM-HRP and OPD are added for color reaction. The positive holes are screened by an indirect competition ELISA method, the cell supernatant is firstly mixed with 100 mug/mL bromodiuron in equal volume, the mixture is subjected to water bath at 37 ℃ for 30 min, and then the mixture is added into the coated ELISA plate. Meanwhile, PBS is used for replacing bromothalonil Long Zuodui, and the rest steps are the same as the above. OD after blocking with bromadiolone 450 If the nm value is reduced to less than 50% of the control hole, the hole is judged to be positive, and the hole which is positive after 2-3 times of detection is immediately subcloned by a limiting dilution method.
(4) Preparation and purification of monoclonal antibodies
Amplifying and culturing hybridoma cells after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing; and taking 0.5 mL/mouse of 8-10 week old Balb/c mice to be injected with liquid paraffin in the intraperitoneal injection, and injecting hybridoma cells in the intraperitoneal injection for 7-10 days for 1-2X 10 5 And (3) extracting ascites of the mice after 7-10 days. Collecting cell supernatant or ascites, and measuring its titer by indirect ELISA (P/N for measuring titer)>2.1 Expressed as the maximum dilution of cell supernatants or ascites), the results indicated that the titer of cell supernatants was 1:10000, ascites titer is 1:60000. then purifying the monoclonal antibody by an octanoic acid-saturated ammonium sulfate method, and taking the supernatant to obtain the monoclonal antibody of the purified bromadiolone drug.
The determination of the monoclonal antibody titer is carried out by using a chessboard method, and the result shows that: the titer of the bromadiolone drug monoclonal antibody is 1:128000, half-maximal inhibitory amount (IC 50 ) 0.35. Mu.g/L.
Through detection, the amino acid sequence of the variable region of the heavy chain of the clone antibody of the bromadiol Long Shan is shown as a sequence 1 of a sequence table, and the amino acid sequence of the variable region of the light chain of the clone antibody of the bromadiol Long Shan is shown as a sequence 2 of the sequence table.
2. Preparation of antibody working solution
And diluting the obtained monoclonal antibody of the bromadiolone drug by 1000 times by using an antibody diluent to obtain an antibody working solution containing the monoclonal antibody of the bromadiolone drug.
The antibody diluent is PBS buffer solution containing Proclin300 and Triton X-100; 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, proclin300 and Triton X-100, and the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively.
3. Preparation of enzyme-labeled working fluid
1. Preparation of anti-antibodies
The monoclonal antibody of the bromadiolone medicine is used as an immunogen, and the goat is used as an immune animal, so that the goat anti-mouse antibody of the monoclonal antibody of the bromadiolone medicine is obtained.
2. Preparation of horseradish peroxidase-labeled anti-antibodies
Coupling the anti-antibody of the monoclonal antibody of the anti-bromadiolone drug obtained in the step 1 with horseradish peroxidase (HRP) by adopting an improved sodium periodate method, and the steps are as follows:
dissolving 8 mg horseradish peroxidase in 2 mL distilled water; adding the prepared 100 mmol/L NaIO 4 Solution 0.4 and mL, stirring and reacting for 20 min at room temperature; dialyzing overnight at 4deg.C with 1 mmol/L acetate buffer; removing excess NaIO 4 Simultaneously reducing the self-coupled enzyme; 40 μLPBS buffer (pH 8.6,0.5 mol/L) and 2.0mL of anti-antibody PBS buffer (pH 8.6,5 mol/L) containing 16mg of anti-bromadiolone drug monoclonal antibody were added, and the reaction was stirred at room temperature for 4 hours; adding 0.1 mol/L NaBH mL which is prepared at present 4 The aqueous solution was reacted at 4℃for 4 hours, and the mixture was purified and stored.
3. Preparation of enzyme-labeled working fluid
Diluting the anti-antibody of the monoclonal antibody of the horseradish peroxidase-marked anti-bromadiolone drug obtained in the step 2 by 500 times by using an anti-antibody diluent to obtain an enzyme marker working solution of the anti-antibody of the monoclonal antibody of the horseradish peroxidase-marked anti-bromadiolone drug.
The anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and the solute is anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, and the concentration of the solute in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively.
4. Preparation of standard working solution
The kit also comprises 6 standard working fluids, wherein the concentration of the solute in the 6 standard working fluids is 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 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 comprise a sample diluent and/or a sample extract and/or a washing solution and/or a substrate color developing solution and/or a stop solution.
The sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 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 light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, light stabilizer, bovine serum albumin and surfactant, the concentration of the solute in the PBS buffer solution is 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 liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively.
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.
The stop solution is 0.05mol/L sulfuric acid aqueous solution.
Example 2, method of Using the kit of example 1
1. Pretreatment of samples
1. Pretreatment of meat-like, dairy, serum samples
Accurately weighing 1+/-0.01 g (or mL) sample, adding the sample into 5mL sample extracting solution, carrying out high-speed whirling for 1 min at room temperature (25+/-2 ℃), and centrifuging for 5 min at 4000 g to obtain a sample solution. 200 mu L of sample solution is taken, 200 mu L of sample diluent is added, high-speed vortex is carried out for 1 min, and 50 mu L of supernatant is taken for analysis.
2. Detection Using the example 1 kit
1. The ELISA plate is inserted into the ELISA plate frame, the positions of each standard substance and each sample are recorded, each sample is parallel to 3, and the unused ELISA plate strips are immediately stored in an environment of 2-8 ℃ after being sealed by a self-sealing bag;
2. adding 50 mu L of each standard working solution or sample solution into the corresponding standard or sample hole respectively;
3. add 50. Mu.L of antibody working fluid to each plate well;
4. covering the cover plate film, lightly oscillating the ELISA plate 10 s, fully mixing, and performing light-shielding reaction at room temperature (25+/-2 ℃) for 30 minutes;
5. uncovering the cover plate film, pouring out the 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, fully washing for 3-4 times, and soaking for 15-30 s each time; the method comprises the steps of carrying out a first treatment on the surface of the
6. Pouring out the liquid in the plate hole, inverting the ELISA plate on the absorbent paper, and drying;
7. adding 100 mu L of enzyme marker working solution into each plate hole; covering the cover plate film, lightly oscillating the ELISA plate 10 s, fully mixing, and performing light-shielding reaction for 30 min at room temperature (25+/-2 ℃);
8. repeating the steps 5-6;
9. immediately adding 100 mu L of 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 film, and carrying out light-proof reaction for 15 min;
10. uncovering the cover plate film, adding 50 mu L of stop solution into each plate hole, gently oscillating the ELISA plate 10 s, and fully and uniformly mixing;
11. the absorbance values of the elisa plates were read with an microplate reader at two wavelengths 405 nm, 630 nm within 5 minutes after termination.
3. Analyzing the detection result
1. Calculation of the percent absorbance values
The average absorbance value of each standard (or sample to be measured) is divided by the absorbance value of the zero standard (standard with the concentration of 0 mug/L), and the absorbance value is multiplied by 100%, so that the percentage of the absorbance corresponding to each standard (or sample to be measured), namely the percentage absorbance value, can be obtained.
Absorbance percentage = B/B 0 ×100%
Wherein: average absorbance value of B-standard (or sample); b (B) 0 -average absorbance value of a standard at a concentration of 0 ppb.
2. Making a standard curve
Drawing a standard curve graph by taking the percentage absorbance value of each standard substance as an ordinate and the bromadiolone concentration (mug/L) in each standard substance working solution as an abscissa, and carrying out nonlinear fitting analysis by using origin8.0 (Originlab Corp., northampton, mass., 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 four parameters of the standard curve.
Fitting of the test data shows that the standard curve of bromadiolone is: y= -0.141+ (2.304+0141) (1+ (x 1.007)/(0.822)) linear correlation R 2 0.997.
The standard graph is shown in fig. 2.
3. Calculating the content of bromadiolone in the sample
Substituting the percentage absorbance value of the sample to be measured into a standard curve to obtain the corresponding residual concentration of the sample to be measured, and multiplying the residual concentration by the dilution multiple of the corresponding sample to obtain the actual content of bromadiolone in the original sample to be measured.
Example 3 specificity, accuracy, precision detection of the kits of example 1
1. Specificity test of the kit:
the specificity of the bromadiol Long Mei combined immunoassay kit was determined by cross-reacting with the corresponding substances.
The serial dilutions of bromadiolone and the common raticides (Dalong, de-Kazak, warfarin, and De-Kazak) were performed respectively, the operations were performed according to example 2, serial dilutions of bromadiolone and analogues thereof were used to replace the "bromadiolone standard working solution" therein, a standard curve was prepared, and 50% Inhibition Concentrations (IC) were found on the curve 50 ) The specific method comprises the following steps: the corresponding bromadiolone concentration (mug/L) with the ordinate value equal to 50 percent is obtained, namely IC 50 Values. The cross-reactivity of the kit to bromadiolone and each of the commonly used rodenticides was calculated using the following formula:
cross-reactivity (%) = (concentration of bromadiolone causing 50% inhibition/concentration of bromadiolone analog 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 on bromadiolone, and the kit can detect the raticide bromadiolone.
2. Minimum detection limit measurement
Pork, milk and serum blank samples (negative in LC-MS/MS detection) were taken, detected according to the method of example 2, measured values were obtained according to a standard curve, the average value was calculated, and the minimum detection Limit (LOD) was obtained by adding 3 times of standard deviation. The results are shown in Table 2.
TABLE 2 statistical table of blank sample measurement results (μg/L)
The results show that the minimum detection limit of the bromadiolone in the pork is 4.87 mug/L, the minimum detection limit of the bromadiolone in the milk is 4.86 mug/L, and the minimum detection limit of the bromadiolone in the serum is 4.09 mug/L, and in order to ensure the stability of the kit, the minimum detection limit of the bromadiolone detection kit in the pork, the milk and the serum is considered to be 5.0 mug/L.
3. Accuracy and precision test of kit
Pork, milk and serum blank samples (LC-MS/MS detection negative) are respectively pretreated according to the method in the step one of the example 2, and then bromadiolone is added to obtain detection sample solutions, wherein the final concentration of the bromadiolone in the samples is 5.0 mug/L and 10.0 mug/L respectively.
3 different batches of kits were used for detection, each experiment was repeated 5 times, and the coefficient of variation was calculated separately. The results are shown in Table 3.
The calculation method of the intra-batch variation coefficient comprises the following steps: intra-lot coefficient of variation = coefficient of variation for each parallel sample in the same assay.
The calculation method of the variation coefficient between batches comprises the following steps: inter-lot coefficient of variation = coefficient of variation of the same sample measured in different lots, and the average value is taken.
TABLE 3 accuracy and precision
The results show that the recovery rate of each addition concentration of all samples is between 80 and 120 percent. The intra-batch variation coefficient of each additive concentration is lower than 10% and the inter-batch variation coefficient is lower than 15%.
4. The kit is compared with LC-MS/MS detection results
The samples of pork, milk and serum were tested as in example 2, and the results of the tests were confirmed and compared with the results of the LC-MS/MS tests, respectively.
And (3) taking the concentration of the bromadiolone detected by the kit as an X axis, taking the concentration of the bromadiolone detected by the LC-MS/MS as a Y axis, and drawing a scatter diagram. The measurement results of the two methods are subjected to linear analysis, the result is shown in fig. 3, and the regression equation is as follows: y=0.916x+0.056, which indicates that the method established by the invention has good consistency with LC-MS/MS detection results.
5. Shelf life test of kit
The storage conditions of the kit of example 1 were 2-8deg.C, and the maximum absorbance (zero standard), 50% inhibition concentration and actual measurement of bromadiolone addition of the kit were all within the normal range after 12 months of measurement. Considering that abnormal preservation conditions appear in the transportation and use processes, the kit is placed for 8 days at 37 ℃ for accelerated aging test, and the result shows that the indexes of the first to fourth steps of the kit completely meet the requirements. Considering the occurrence of the freezing condition of the kit, the kit is placed for 8 days at the temperature of-20 ℃, and all indexes of the steps one to four completely meet the requirements. From the above results, the kit of example 1 can be stored at 2-8℃for at least 12 months.
Sequence listing
<110> Beijing Verdyvelkang Biotechnology Co., ltd
<120> an ELISA kit for detecting bromadiolone, its preparation and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Phe Ser Pro Ser Lys Leu Leu Gln His Ser Leu Gln Cys Leu His Gly
1 5 10 15
Arg Arg Ser Pro Ala Ala Ala Pro Val Pro Gln Val Ser Val Thr Cys
20 25 30
Thr Thr Gly Ser Arg Ser Gln Asp Pro Pro Pro Asn Pro Leu Phe His
35 40 45
Ile Ser Pro Thr Trp Leu Leu Glu Ser Leu Leu Ala Ser Val Ala Val
50 55 60
Gly Leu Gly Pro Ser Trp Gln Leu Ile Leu Ser Thr Ala Arg Leu Lys
65 70 75 80
Met Leu Ile Thr Ala Leu Pro Ser Ser Gly Val Val Thr Arg Ser Arg
85 90 95
Ser Val Leu Gly Pro Ser Trp Ser Ala Leu Leu Lys Thr Gly Ser Leu
100 105 110
<210> 2
<211> 110
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Glu Leu Glu Glu Ser Glu Val Gly Gly Gly Ser Met Leu Val Gln Pro
1 5 10 15
Gly Gly Lys Leu Cys Val Ala Ser Ser Gly Phe Thr Phe Ser Asn Trp
20 25 30
Trp Met Asn Arg Tyr Gln Val Ser Pro Glu Glu Val Lys Gly Leu Trp
35 40 45
Ala Glu Ile Arg Leu Ile Ser Asn Asn Tyr Val Pro Tyr Ala His Glu
50 55 60
Ser Val Lys Arg Asp Gly Arg Ile Ser Phe Thr Asp Ser Lys Ser Val
65 70 75 80
Gly Ile Leu Arg Gln Glu Asp Thr Tyr Tyr Cys Ser Phe Gly Asn Ser
85 90 95
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Ala Thr Val Ser
100 105 110

Claims (4)

1. An enzyme-linked immunosorbent assay kit for detecting bromadiolone, comprising: the detection kit comprises a bromadiolone detection ELISA plate, a bromadiolone series standard substance working solution, a bromadiolone antibody working solution, an enzyme marker working solution, a sample dilution solution, a sample extraction solution, a washing solution, a substrate color development solution and a termination solution, and is characterized in that:
the bromadiolone detection ELISA plate is prepared by coating a conjugate of a bromadiolone drug hapten and a carrier protein, wherein the bromadiolone drug hapten is a compound shown in a formula I:
the preparation method of the bromadiolone drug hapten comprises the following steps: weighing 500mg of bromadiolone, dissolving with 6mL of pyridine, adding 154.45mg of phthalic anhydride, heating to 60 ℃, stirring, purifying, spin-drying, and terminating the reaction to obtain the compound shown in the formula I;
the bromadiolone antibody is a specific antibody of a bromadiolone drug;
the specific antibody of the bromadiolone drug is the bromadiolone Long Shan clone antibody, the bromadiolone Long Shan clone antibody consists of a heavy chain and a light chain, the amino acid sequence of a variable region of the heavy chain is shown as a sequence 1 of a sequence table, and the amino acid sequence of a variable region of the light chain is shown as a sequence 2 of the sequence table;
the bromadiolone antibody working solution is obtained by diluting a monoclonal antibody of bromadiolone by 1000 times with an antibody diluent to obtain an antibody working solution of the monoclonal antibody containing bromadiolone medicine;
the antibody diluent is PBS buffer containing Proclin300 and Triton X-100; the solvent of the antibody diluent is deionized water, and solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, proclin300 and Triton X-100, wherein the concentration of the solutes in the PBS buffer is 1.072g/L, 0.59g/L, 8.5g/L, 0.4g/L, 200 mu L/L and 500 mu L/L respectively;
the enzyme marker working solution is obtained by diluting an anti-antibody of a horseradish peroxidase-marked anti-bromadiolone drug monoclonal antibody by 500 times with an anti-antibody diluent;
the anti-antibody diluent is PBS buffer solution containing calf serum and Proclin 300; the solvent of the antibody diluent is deionized water, and solutes are anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium chloride, potassium chloride, calf serum and Proclin300, wherein the concentration of the solutes in the PBS buffer is 1.072g/L, 0.6g/L, 16g/L, 0.4g/L, 50mL/L and 200 mu L/L respectively;
the bromadiolone series standard working solution is 6 bromadiolone standard working solutions, a solvent of the solution is PBS buffer solution containing light stabilizer and bovine serum albumin, and a solute is bromadiolone; the concentration of the solute in the working solution of the 6 bromadiolone standard substances is 0 mug/L, 0.15 mug/L, 0.45 mug/L, 1.35 mug/L, 4.05 mug/L and 12.15 mug/L respectively; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer and bovine serum albumin, the concentration of the solute in the PBS buffer solution is 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;
the sample diluent is PBS buffer solution containing light stabilizer, bovine serum albumin and surfactant; the solvent of the PBS buffer solution is deionized water, the solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentration of the solute in the PBS buffer solution is 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 solute is disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, a light stabilizer, bovine serum albumin and a surfactant, the concentration of the solute in the PBS buffer solution is 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 liquid is deionized water, and solutes are disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, tween-20 and Proclin300, and the concentration of the solutes in the PBS buffer is 23.2g/L, 2.0g/L, 64g/L, 0.036g/L, 20mL/L and 300 mu L/L respectively;
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;
the stop solution is 0.05mol/L sulfuric acid aqueous solution.
2. A method for detecting bromadiolone residue in a sample, comprising the steps of:
1) Pretreating a sample;
2) Performing a test with the kit of claim 1;
3) And analyzing the detection result.
3. The method for detecting bromadiolone residue in a sample according to claim 2, wherein: the detection by the kit comprises the following steps: adding standard working solution or solution of the sample into a bromadiolone detection ELISA plate coated with the conjugate of the bromadiolone drug hapten and the carrier protein; adding bromadiolone antibody working solution; washing and beating after incubation, adding enzyme label working solution, developing color by a substrate, stopping and measuring the absorbance value by an enzyme label instrument.
4. The method for detecting bromadiolone residue in a sample according to claim 2, wherein: the kit can detect the bromadiolone in pork, milk or serum; the detection limit of detecting bromadiolone in pork, milk or serum is 5 mug/kg, the inter-batch variation coefficient is less than 10%, and the intra-batch variation coefficient is less than 15%.
CN202010285672.7A 2020-04-13 2020-04-13 ELISA kit for detecting bromadiolone and preparation and application thereof Active CN111273018B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010285672.7A CN111273018B (en) 2020-04-13 2020-04-13 ELISA kit for detecting bromadiolone and preparation and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010285672.7A CN111273018B (en) 2020-04-13 2020-04-13 ELISA kit for detecting bromadiolone and preparation and application thereof

Publications (2)

Publication Number Publication Date
CN111273018A CN111273018A (en) 2020-06-12
CN111273018B true CN111273018B (en) 2024-02-20

Family

ID=70999546

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010285672.7A Active CN111273018B (en) 2020-04-13 2020-04-13 ELISA kit for detecting bromadiolone and preparation and application thereof

Country Status (1)

Country Link
CN (1) CN111273018B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102079788A (en) * 2009-11-27 2011-06-01 北京维德维康生物技术有限公司 Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof
CN103575885A (en) * 2012-07-19 2014-02-12 北京勤邦生物技术有限公司 Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof
CN103630689A (en) * 2013-12-03 2014-03-12 河北省科学院生物研究所 ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit
CN109897025A (en) * 2019-02-28 2019-06-18 中国农业大学 Anticoagulation raticide haptens and artificial antigen and the preparation method and application thereof
CN110950961A (en) * 2019-12-11 2020-04-03 中国农业大学 Bromadiolone nano antibody and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102079788A (en) * 2009-11-27 2011-06-01 北京维德维康生物技术有限公司 Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof
CN103575885A (en) * 2012-07-19 2014-02-12 北京勤邦生物技术有限公司 Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof
CN103630689A (en) * 2013-12-03 2014-03-12 河北省科学院生物研究所 ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit
CN109897025A (en) * 2019-02-28 2019-06-18 中国农业大学 Anticoagulation raticide haptens and artificial antigen and the preparation method and application thereof
CN110950961A (en) * 2019-12-11 2020-04-03 中国农业大学 Bromadiolone nano antibody and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Hongfang Li et.al.Novel inner filter effect-based fluorescence immunoassay with gold nanoclusters for bromadiolone detection in human serum.Sensors and Actuators B: Chemical.2019,第297卷第126787(1-9)页. *

Also Published As

Publication number Publication date
CN111273018A (en) 2020-06-12

Similar Documents

Publication Publication Date Title
AU2007257105B2 (en) Elisa kit for detecting Sudan red and method thereof
CN106324240B (en) Detect enzyme linked immunological kit and its application of chlopyrifos
CN110441512B (en) Colloidal gold immunochromatography detection device for ethyl maltol hapten and ethyl maltol
WO2016011852A1 (en) Bladder tumor-associated antigen detection kit
CN109307761B (en) Indirect competitive ELISA method for detecting furaldehyde
CN109061169B (en) Enzyme linked immunosorbent assay kit for detecting acetamiprid and application thereof
CN101307303B (en) Kit for detecting clenobuterol hydrochloride residue and method for preparing same
CN105807055A (en) Test strip for detecting quinclorac and preparation method and application of test strip
CN105785011A (en) Test strip for detecting maleic hydrazide, as well as preparation method and application thereof
CN111273015B (en) Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof
CN109180519B (en) Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method
CN111443202B (en) ELISA kit for detecting anticoagulant rodenticide, preparation and application thereof
CN111273041B (en) ELISA kit for detecting phalloidin and preparation and application thereof
CN111289753B (en) Enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticide and preparation and application thereof
CN105675858A (en) Enzyme-linked immunosorbent assay kit for detecting quinclorac and application of enzyme-linked immunosorbent assay kit
CN111273018B (en) ELISA kit for detecting bromadiolone and preparation and application thereof
CN101561439A (en) Nitrofurantoin residue enzyme-linked immunoassay kit
CN111308100B (en) ELISA kit for detecting beta-amatoxin and preparation and application thereof
CN110684188A (en) Nonylphenol polyoxyethylene ether hapten and holoantigen as well as preparation method and application thereof
CN111812316B (en) Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit
CN109942624B (en) Glufosinate hapten, artificial antigen, antibody, preparation method and detection device thereof
CN110746286B (en) Eugenol hapten, artificial antigen, preparation method and application thereof
CN112904008A (en) Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof
CN109265395B (en) Preparation method and application of quinclorac hapten and antigen
CN113125729A (en) Enzyme linked immunosorbent assay kit for detecting citrinin and detection method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant