CN116903524B - Chlorfluazuron hapten, antigen, antibody, detection device and preparation and application thereof - Google Patents
Chlorfluazuron hapten, antigen, antibody, detection device and preparation and application thereof Download PDFInfo
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- CN116903524B CN116903524B CN202311184280.1A CN202311184280A CN116903524B CN 116903524 B CN116903524 B CN 116903524B CN 202311184280 A CN202311184280 A CN 202311184280A CN 116903524 B CN116903524 B CN 116903524B
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- chlorfluazuron
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- hapten
- antigen
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- UISUNVFOGSJSKD-UHFFFAOYSA-N chlorfluazuron Chemical compound FC1=CC=CC(F)=C1C(=O)NC(=O)NC(C=C1Cl)=CC(Cl)=C1OC1=NC=C(C(F)(F)F)C=C1Cl UISUNVFOGSJSKD-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 239000000427 antigen Substances 0.000 title claims abstract description 25
- 102000036639 antigens Human genes 0.000 title claims abstract description 25
- 108091007433 antigens Proteins 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims description 45
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 33
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 19
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- 238000003756 stirring Methods 0.000 claims description 15
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- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 4
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- KGEXISHTCZHGFT-UHFFFAOYSA-N 4-azaniumyl-2,6-dichlorophenolate Chemical compound NC1=CC(Cl)=C(O)C(Cl)=C1 KGEXISHTCZHGFT-UHFFFAOYSA-N 0.000 claims description 3
- DMAYBPBPEUFIHJ-UHFFFAOYSA-N 4-bromobut-1-ene Chemical compound BrCCC=C DMAYBPBPEUFIHJ-UHFFFAOYSA-N 0.000 claims description 3
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- WYSPMXSNCAFCFV-UHFFFAOYSA-N methyl 2-fluoro-4-hydroxybenzoate Chemical compound COC(=O)C1=CC=C(O)C=C1F WYSPMXSNCAFCFV-UHFFFAOYSA-N 0.000 claims description 3
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- YPSCQJTUAKNUNF-UHFFFAOYSA-N 2-chloro-n-[(4-chlorophenyl)carbamoyl]benzamide Chemical compound C1=CC(Cl)=CC=C1NC(=O)NC(=O)C1=CC=CC=C1Cl YPSCQJTUAKNUNF-UHFFFAOYSA-N 0.000 description 1
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 239000005944 Chlorpyrifos Substances 0.000 description 1
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- HRYILSDLIGTCOP-UHFFFAOYSA-N N-benzoylurea Chemical compound NC(=O)NC(=O)C1=CC=CC=C1 HRYILSDLIGTCOP-UHFFFAOYSA-N 0.000 description 1
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- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
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- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/63—One oxygen atom
- C07D213/64—One oxygen atom attached in position 2 or 6
- C07D213/643—2-Phenoxypyridines; Derivatives thereof
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/58—Preparation of carboxylic acid halides
- C07C51/60—Preparation of carboxylic acid halides by conversion of carboxylic acids or their anhydrides or esters, lactones, salts into halides with the same carboxylic acid part
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
- C07C67/31—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of functional groups containing oxygen only in singly bound form
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- 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
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- Engineering & Computer Science (AREA)
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- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application discloses a chlorfluazuron hapten, an antigen, an antibody, a detection device and preparation and application thereof, and relates to a chlorfluazuron hapten, an antigen, an antibody, a colloidal gold chromatography detection device and preparation and application thereof in detecting chlorfluazuron residues in foods. The application provides an artificial antigen prepared from a chlorfluazuron hapten coupled carrier protein, and an antibody which is specifically directed against chlorfluazuron and is produced by a body of the animal after the animal is immunized, wherein the titer is high, the IC50 is 10.85ng/mL, and the detection range is 2.963-39.742 ng/mL. The application utilizes the specific immunological reaction of antigen and antibody and the easily identified immune marking amplification technology, can qualitatively and semi-quantitatively detect the residual chlorfluazuron in food, has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, on-site rapid detection and the like, and can better meet the on-site supervision requirements of supervision departments and detection institutions.
Description
Technical Field
The application relates to the technical field of detection, in particular to a chlorfluazuron hapten, an antigen, an antibody, a detection device and preparation and application thereof.
Background
The chlorfluazuron (also known as chlorfluazuron, imazalil, chlorpyrifos, chlorbenzuron) is a novel benzoyl urea pesticide, and is mainly used for preventing and controlling cotton bollworms, beetles, noctuid and the like in solanaceous vegetables. The highest residual limit (MRL) of chlorfluazuron in vegetables formulated in China is 0.2-2 mg/kg. The current detection of the residual of the chlorfluazuron is based on SN/T-2095-2008 high performance liquid chromatography of detection method of residual quantity of chlorfluazuron in import and export vegetables and GB/T19648-2006 gas chromatography-mass spectrometry of determination of residual of 500 pesticides and related chemicals in fruits and vegetables.
Common detection methods of chlorfluazuron comprise gas chromatography, and the like, and the detection methods have higher accuracy, but rely on large-scale detection equipment, have complex operation and high detection cost, and are not suitable for large-scale popularization and application; and the result cannot be immediately displayed. The immunological detection analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has a plurality of advantages compared with methods such as instrument detection and the like.
When an immunological detection method is established and the detection method is applied to detect the residual quantity of the chlorfluazuron, the key technology is that the antibody with strong specificity and high sensitivity can be obtained, and the aim is to be fulfilled if a proper chlorfluazuron hapten is required to be synthesized and prepared. However, no related report on chlorfluazuron hapten is available in China at present.
Disclosure of Invention
The application aims to provide a chlorfluazuron hapten, an antigen, an antibody, a detection device and a preparation and detection method thereof, aiming at detecting residual chlorfluazuron in food.
According to one aspect of the present application, there is provided a chlorfluazuron hapten having a structure as shown in formula (I):
(Ⅰ)。
according to another aspect of the present application, there is provided a method for preparing a chlorfluazuron hapten, comprising the steps of:
s1, adding 2-fluoro-4-hydroxybenzoic acid methyl ester, 4-bromo-1-butene and potassium carbonate, dissolving by using DMF, heating and stirring for reaction, extracting, combining organic phases, evaporating to dryness and distilling under reduced pressure to obtain an intermediate 1, wherein the reaction in the step is shown as the following formula (II):
formula (II);
s2, heating and stirring the intermediate 1, a sodium hydroxide solution and ethanol for reaction, performing rotary evaporation under reduced pressure, adding dilute hydrochloric acid, extracting, merging organic phases, and evaporating to dryness to obtain an intermediate 2, wherein the reaction in the step is shown in the following formula (III):
formula (III);
s3, taking the intermediate 2 and thionyl chloride, heating, stirring, reacting, decompressing and steaming to obtain the intermediate 3, wherein the reaction formula in the step is shown as a formula (IV):
formula (IV);
s4, reacting the intermediate 3 with ammonia water to separate out white solid to obtain an intermediate 4, wherein the reaction formula of the step is shown as a formula (V):
(V)
S5, dissolving the intermediate 4 by using dichloromethane, adding oxalyl chloride, heating and refluxing for reaction, and evaporating the solvent after the reaction is finished to obtain an intermediate 5, wherein the reaction formula in the step is shown as a formula (VI):
formula (VI);
s6, dissolving 2, 6-dichloro-4-aminophenol, 2, 3-dichloro-5-trifluoromethylpyridine and potassium carbonate in DMF, stirring overnight at 80 ℃, distilling under reduced pressure to remove DMF, extracting, washing the combined organic phases with water, evaporating to dryness, and purifying by a column to obtain an intermediate 6, wherein the reaction formula of the step is shown as a formula (VII):
formula (VII);
s7, taking the intermediate 6 and the intermediate 5 to react in 1, 2-dichloroethane, precipitating a large amount of solids, filtering to obtain an intermediate 7, wherein the reaction formula in the step is shown as a formula (VIII):
formula (VIII);
s8, putting the intermediate 7 into a single-neck flask, adding tert-butyl acrylate and GuRBB' S second generation catalyst, adding dichloromethane for dissolution, carrying out reflux reaction, evaporating to dryness after the reaction is completed, and directly passing through a column to obtain an intermediate 8, wherein the reaction formula in the step is shown as a formula (IX):
formula (IX);
s9, dissolving the intermediate 8 in dichloromethane, adding trifluoroacetic acid, reacting at room temperature, evaporating the solvent, and purifying by a column to obtain a product 9, namely the chlorfluazuron hapten, wherein the reaction formula in the step is shown as a formula (X):
formula (X).
In some embodiments, the mass volume fraction of sodium hydroxide solution in step S2 is 10%.
In some embodiments, the molar ratio of intermediate 4 to oxalyl chloride in step S5 is 1:1.1.
according to a further aspect of the application, the fluazinam antigen is a conjugate of a fluazinam hapten and a carrier protein, the carrier protein being one of bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
According to the fourth aspect of the application, the fluazinam antibody is prepared by animal immunization with a fluazinam antigen, and the fluazinam antibody is a fluazinam monoclonal antibody.
According to a fifth aspect of the application, there is provided the use of a chlorfluazuron hapten and a chlorfluazuron antigen in the immunological detection of chlorfluazuron.
According to a sixth aspect of the present application there is provided the use of a chlorfluazuron antibody in the immunological detection of chlorfluazuron.
According to a seventh aspect of the application, there is provided a rapid detection method of residual chlorfluazuron in food, which is to detect residual chlorfluazuron in food by using chlorfluazuron antibody.
The application has the beneficial effects that: the application provides an artificial antigen prepared from a chlorfluazuron hapten coupled carrier protein, and an antibody specific to chlorfluazuron is produced by a body of the animal after the animal is immunized, so that the antibody has high titer, and the IC50 value of the antibody is 10.85 ng/mL. The application utilizes the specific immunological reaction of antigen and antibody and the easily identified immune labeling amplification technology, can qualitatively and semi-quantitatively detect the residual chlorfluazuron in food, and has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, on-site rapid detection and the like.
Drawings
FIG. 1 is a mass spectrum of a chlorfluazuron hapten according to one embodiment of the present application.
FIG. 2 is a standard curve of an indirect competition ELISA established based on a chlorfluazuron monoclonal antibody according to an embodiment of the present application.
Detailed Description
The application will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the application, as various equivalent modifications of the application will occur to those skilled in the art upon reading the application, as defined in the appended claims. Unless otherwise specified, all materials and reagents of the application are those commercially available in the conventional market.
Example 1 a method for preparing a chlorfluazuron hapten comprising the steps of:
s1, sequentially adding 5.73g of 2-fluoro-4-hydroxybenzoic acid methyl ester, 9.1g of 4-bromo-1-butene and 7g of potassium carbonate into a clean 250mL single-mouth bottle, dissolving with DMF, stirring overnight at 80 ℃, after TLC detection reaction is complete, distilling the reaction liquid under reduced pressure by using an oil pump, removing DMF, adding water and ethyl acetate into the residues for extraction twice, and combining organic layers and passing through a column to obtain 5.8g of intermediate 1, wherein the intermediate 1 is colorless oily liquid;
s2, sequentially adding 5.8g of intermediate 1 and 20mL of 10% sodium hydroxide solution into another clean 250mL single-port bottle, adding 50mL of ethanol, refluxing at 80 ℃ for overnight reaction, adjusting the pH of the reaction solution to be acidic by using 6M hydrochloric acid solution after TLC detection reaction is complete, extracting the reaction solution with ethyl acetate for two times, merging organic phases, evaporating the solvent, and drying to obtain 5.1g of intermediate 2, wherein the intermediate 2 is a white solid;
s3, sequentially adding 5.1g of intermediate 2 and 30mL thionyl chloride into a clean 100mL single-port bottle, carrying out reflux reaction for 3-4 h at 80 ℃, carrying out reduced pressure distillation on the reaction solution at 45 ℃ until no obvious liquid flows out, and adding dichloromethane into the residue for twice to obtain 5.2g of intermediate 3;
s4, sequentially adding 20. 20mL ammonia water into a clean 100mL single-mouth bottle, slowly adding 5.2g intermediate 3 into the ammonia water, precipitating a large amount of solids, reacting for 1h at room temperature after the addition, filtering, washing a filter cake twice, and drying to obtain 4.8 g intermediate 4, wherein the intermediate 4 is a white solid;
s5, sequentially adding 2.4g of intermediate 4 into a clean 100mL single-mouth bottle, dissolving with dichloromethane, adding 1.5mL of oxalyl chloride, carrying out reflux reaction for 3-4 h at 80 ℃, carrying out reduced pressure distillation on the reaction solution at 45 ℃ until no obvious liquid flows out, and adding dichloromethane into residues for twice to obtain 2.5 g intermediate 5;
s6, sequentially adding 8g of 2, 6-dichloro-4-aminophenol, 10.68g of 2, 3-dichloro-5-trifluoromethylpyridine and 9.32g of potassium carbonate into a clean 250mL single-mouth bottle, adding DMF for dissolution, stirring overnight at 80 ℃, after TLC detection is completed, distilling the reaction liquid under reduced pressure by an oil pump to remove DMF, adding water and ethyl acetate into residues for extraction twice, combining organic layers, passing through a column, and recrystallizing to obtain 15.6g of intermediate 6, wherein the intermediate 6 is orange crystals;
s7, sequentially adding 3.276g of the intermediate 6 into a clean 100mL single-port bottle, dissolving the intermediate 6 by using 1, 2-dichloroethane, slowly adding the intermediate 5 of 2.5 g into the solution of the intermediate 6, separating out a large amount of solids, reacting for 1h at room temperature after the addition, filtering the reaction solution, washing a filter cake twice, and drying to obtain the intermediate 7 of 4.5 g;
s8, sequentially adding 3.7g of intermediate 7, 4g of tert-butyl acrylate and 0.18g of GuRBB' S second generation catalyst into a clean 100mL single-mouth bottle, adding dichloromethane for dissolution, carrying out reflux reaction at 45 ℃ for 3-4 h, and detecting the reaction completely by TLC. Directly passing the reaction solution through a column to obtain 4.2 g intermediate 8;
s9, sequentially adding 4.2 g intermediate 8, 20mL dichloromethane 20 and 10mL trifluoroacetic acid into a clean 100mL single-port bottle, reacting for 2-3 hours at room temperature, carrying out reduced pressure distillation on the reaction liquid after TLC detection reaction is finished, carrying out twice with trifluoroacetic acid by using petroleum ether, and passing through a column to obtain 3.4g of product 9, namely chlorfluazuron hapten, wherein the structure of the chlorfluazuron hapten is shown as a formula (I).
And identifying the chlorfluazuron hapten by adopting a mass spectrometry method, wherein the obtained mass spectrum is shown in a figure 1 of the specification. As can be seen from the mass spectrum, the molecular ion peak of the chlorfluazuron hapten is M/z 636.11 [ M-H ]] - And the peak is the highest peak, and meanwhile, the isotope peak 634.11 is consistent with the molecular weight 636.76 of the chlorfluazuron hapten, which shows that the chlorfluazuron hapten shown in the formula (I) is successfully synthesized.
EXAMPLE 2 preparation of antigen for fluazinam immunization and antigen for coating
Preparation of antigen for fluazinam immunization: weighing 25.5mg of fluazinam hapten, dissolving in 2mL of N, N-Dimethylformamide (DMF), adding 20mg of N-hydroxysuccinimide (NHS) and 30mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), and reacting for 6h at room temperature to obtain an activated liquid; dissolving 65mg Bovine Serum Albumin (BSA) in 3mL of L0.05M boric acid buffer solution with pH of 9.0, adding 1mL of DMF and 0.5mL of the activating solution, reacting for 4 hours at room temperature, dialyzing with PBS (0.02 mol/L phosphate buffer solution with pH of 7.4), changing the solution for 1 time every 4h, changing the solution for 7-8 times, centrifuging for 5 minutes at 4000 revolutions/min after dialyzing, and taking supernatant to obtain a chlorfluazuron hapten-BSA conjugate, namely an antigen for chlorfluazuron immunization, split charging and storing at-20 ℃.
Preparation of antigen for coating chlorfluazuron: 13 mg chlorfluazuron hapten is weighed and dissolved in 1mL anhydrous N, N-Dimethylformamide (DMF), then 4.3 mu L diisopropylethylamine and 6 mu L isobutyl chloroformate are sequentially added for reaction for 30min at 0 ℃ to prepare solution A; dissolving 70 mg chicken Ovalbumin (OVA) with 0.1M boric acid buffer solution 7mL to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, dialyzing with PBS (0.02 mol/L, phosphate buffer solution with pH of 7.4), changing the solution for 1 time every 4h times, changing the solution for 7-8 times, centrifuging for 5 minutes at 4000 revolutions/min after dialyzing, taking the supernatant, and obtaining the chlorfluazuron hapten-OVA conjugate, namely the chlorfluazuron antigen for coating, subpackaging and storing at-20 ℃.
Example 3 preparation, purification of Fluorofluazuron monoclonal antibody and establishment of an Indirect competitive ELISA Standard Curve
The preparation method of the chlorfluazuron monoclonal antibody comprises the following steps:
after antigen identification for the chlorfluazuron immunization obtained in example 2, 10 8-week-old BALB/C mice were immunized, three times of booster immunization were performed, the titers were measured by blood sampling, and after the serum titers were no longer increased, the mice were immunized with twice the dose of chlorfluazuron immunization antigen without adjuvant, and after three days, the mice were cervical-removed and killed. Spleen cells were prepared under aseptic conditions, mixed with growing mouse myeloma cells in a 50mL centrifuge tube at a ratio of 8:1, added with 30mL serum-free IPMI1640 medium, centrifuged at 1100r/min for 5min to discard supernatant, and the cell mass was gently shaken loose and placed in a 37 ℃ water bath. Slowly adding 1mL 50% PEG-4000 into cells, dripping over 1min while gently stirring the bottom sediment, standing for 1min, slowly adding 1mL of serum-free medium along the tube wall at constant speed for the first 30s, adding 2mL for the last 30s, rapidly adding 27mL, stopping fusion process, centrifuging at 1100r/min for 5min, discarding supernatant, re-suspending with HAT selective medium, adding into 96-well cell culture plate with feeder cells spread thereon, adding CO with volume fraction of 5% at 37deg.C, and concentrating under reduced pressure 2 Culturing under the condition.
And after 7 days, changing into HT culture solution, screening by using an indirect ELISA method when the number of hybrid cells in the holes reaches more than 300, selecting holes with strong positivity, good inhibition effect and vigorous cell growth for limited dilution cloning, carrying out cloning culture and detection for more than 3 times, wherein the cells in the holes which are positive are hybridoma cells secreting monoclonal antibodies, and carrying out expanded culture on the hybridoma cells for preparing the monoclonal antibodies.
The monoclonal antibody of the fluazinam is produced by adopting an in-vivo induced ascites method. Selecting 4 produced Kunming mice, injecting liquid paraffin oil into abdominal cavity 0.5 mL/mouse, and injecting hybridoma cells into abdominal cavity 3-5×10 after 7 days 6 After 10 days, ascites was collected when the abdomen of the mice had significantly distended. Purifying ascites by precipitation with n-octanoic acid-ammonium sulfate, and ultraviolet measurementContent of chlorfluazuron monoclonal antibody. A standard curve is established according to indirect ELISA, see figure 2 of the specification. The IC50 value of the curve is 10.85ng/mL, the detection range (mass concentration of chlorfluazuron at the binding rate of 20% -80%) is 2.963-39.742 ng/mL, and the detection requirement of chlorfluazuron residues in agricultural products such as fruits and vegetables in GB2763-2021 can be met.
Example 4 preparation of fluazinam colloidal gold chromatography detection device
4.1 Preparation of colloidal gold solution
Diluting 1% chloroauric acid solution into 0.01% (mass fraction) by using double distilled deionized water, placing 100mL of 0.01% chloroauric acid solution into a conical flask, heating to boiling by using a constant-temperature electromagnetic stirrer, rapidly adding 1.6 mL of 1% trisodium citrate solution at one time, continuously stirring at a constant speed, heating until the solution is transparent red, stopping stirring, continuously heating for 5min after color stabilization, cooling to room temperature, recovering to original volume by using deionized water, obtaining colloidal gold solution, and preserving at 4 ℃;
4.2 Preparation of fluazinam monoclonal antibody-colloidal gold marker
Taking 100mL of prepared 0.01% colloidal gold solution, adjusting the pH value of the colloidal gold solution to about 8.5 by using 0.1mol/L potassium carbonate under magnetic stirring, adding 30-40 mug of fluazinam monoclonal antibody per milliliter of colloidal gold solution, adding the fluazinam monoclonal antibody prepared in the example 3 into the colloidal gold solution, stirring uniformly, and standing for 20min at room temperature of 25 ℃. Adding 1mL of 10% Bovine Serum Albumin (BSA) solution, stirring by using a magnetic stirrer (rotating speed is 150r/min,30 min), standing for 20min, centrifuging at 12000rpm for 40min at 4 ℃, discarding supernatant, re-suspending the rest precipitate by using 20mL of colloid Jin Chong suspension (2% sucrose, 1% BSA and 0.3% Tween 20 and prepared by 0.01mol/L phosphate buffer solution with pH of 7.4), and standing at 4 ℃ for later use to prepare a fluazuron monoclonal antibody-colloidal gold marker;
4.3 preparation of fluazinam colloidal gold detection device
Uniformly spraying a chlorfluazuron monoclonal antibody-colloidal gold marker on a gold-labeled pad according to the amount of 5 mu L/cm by a film spraying instrument, drying for 2 hours at 37 ℃, coating a detection area (T line) and a quality control area (C line) on an NC film, coating the antigen for chlorfluazuron coating prepared in the embodiment 2 on the detection area (T line), sequentially adhering the NC film, the gold-labeled pad, a sample pad and a water-absorbing pad on a PVC bottom plate, cutting into test strips with the width of 4mm after the test strips are adhered, placing the test strips in a special plastic card, placing in a sealing bag, adding a drying agent, sealing, and obtaining the chlorfluazuron colloidal gold chromatography detection card, and preserving at room temperature.
Example 5 method for rapidly detecting residual fluazinam in food
5.1 sample pretreatment: 1.0 g+/-0.01 g of crushed food sample to be detected is weighed into a 50mL polystyrene centrifuge tube, tris-PBS buffer solution with pH of 7.2 is added for dilution by 10 times, a cover is covered, a vortex mixer is evenly mixed or manually oscillated up and down for evenly mixing 30s, and the mixture is kept stand for 5min, and supernatant fluid is sucked to be detected.
5.2, detection: 3-5 drops are added into the sample adding hole of the chlorfluazuron colloidal gold chromatography detection card prepared in the example 4, and after 3-5min, the result is judged.
Result determination
And judging the result by comparing the color shades of the quality control area (C line) and the detection area (T line).
Positive: when the quality control area (C line) shows a strip, the detection area (T line) does not develop color, and the food is judged to be positive, namely the food has chlorfluazuron residue, which is indicated by "+";
negative: when the quality control area (C line) and the detection area (T line) both show strips, judging as negative, namely that no residual chlorfluazuron exists in the food or the residual chlorfluazuron in the food is lower than the detection limit, and using the formula of "-;
invalidation: when the quality control area (C line) does not show the strip, the test paper fails.
The foregoing is merely illustrative of some embodiments of the application, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.
Claims (6)
1. The chlorfluazuron hapten is characterized in that the structure is shown as a formula (I):
(Ⅰ)。
2. a method for preparing the chlorfluazuron hapten according to claim 1, which comprises the following steps:
s1, adding 2-fluoro-4-hydroxybenzoic acid methyl ester, 4-bromo-1-butene and potassium carbonate, dissolving by using DMF, heating and stirring for reaction, extracting, combining organic phases, evaporating to dryness and distilling under reduced pressure to obtain an intermediate 1, wherein the reaction in the step is shown as the following formula (II):
formula (II);
s2, heating and stirring the intermediate 1, a sodium hydroxide solution and ethanol for reaction, performing rotary evaporation under reduced pressure, adding dilute hydrochloric acid, extracting, merging organic phases, and evaporating to dryness to obtain an intermediate 2, wherein the reaction in the step is shown in the following formula (III):
formula (III);
s3, taking the intermediate 2 and thionyl chloride, heating, stirring, reacting, decompressing and steaming to obtain an intermediate 3, wherein the reaction formula of the step is shown as a formula (IV):
formula (IV);
s4, reacting the intermediate 3 with ammonia water to separate out white solid to obtain an intermediate 4, wherein the reaction formula of the step is shown as a formula (V):
formula (V);
s5, dissolving the intermediate 4 by using dichloromethane, adding oxalyl chloride, heating and refluxing for reaction, and evaporating the solvent after the reaction is finished to obtain an intermediate 5, wherein the reaction formula in the step is shown as a formula (VI):
formula (VI);
s6, dissolving 2, 6-dichloro-4-aminophenol, 2, 3-dichloro-5-trifluoromethylpyridine and potassium carbonate in DMF, stirring overnight at 80 ℃, distilling under reduced pressure to remove DMF, extracting, washing the combined organic phases with water, evaporating to dryness, and purifying by a column to obtain an intermediate 6, wherein the reaction formula of the step is shown as a formula (VII):
formula (VII);
s7, taking the intermediate 6 and the intermediate 5 to react in 1, 2-dichloroethane, precipitating a large amount of solids, filtering to obtain the intermediate 7, wherein the reaction formula of the step is shown as a formula (VIII):
formula (VIII);
s8, putting the intermediate 7 into a single-neck flask, adding tert-butyl acrylate and GuRBB' S second generation catalyst, adding dichloromethane for dissolution, carrying out reflux reaction, evaporating to dryness after the reaction is completed, and directly passing through a column to obtain an intermediate 8, wherein the reaction formula in the step is shown as a formula (IX):
formula (IX);
s9, dissolving the intermediate 8 in dichloromethane, adding trifluoroacetic acid, reacting at room temperature, evaporating the solvent, and purifying by a column to obtain a product 9, namely the chlorfluazuron hapten, wherein the reaction formula in the step is shown as a formula (X):
formula (X).
3. The method according to claim 2, characterized in that the concentration of sodium hydroxide solution in step S2 is 10%.
4. The method according to claim 2, wherein the molar ratio of the intermediate to oxalyl chloride in step S5 is 1:1.1.
5. The chlorfluazuron antigen is characterized in that the chlorfluazuron antigen is a conjugate of the chlorfluazuron hapten and a carrier protein, wherein the carrier protein is one of bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
6. The application of the chlorfluazuron hapten as claimed in claim 1 and the chlorfluazuron antigen as claimed in claim 5 in immunological detection of chlorfluazuron.
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JP2001261631A (en) * | 2000-03-16 | 2001-09-26 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of flufenoxuron, antibody and method of measuring the same |
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Non-Patent Citations (2)
Title |
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Development of a Class-Specific Competitive ELISA for the Benzoylphenylurea Insecticides;Wang Shuo等;J. Agric. Food Chem.;第46卷(第8期);3330−3338 * |
Development of a Compound-Specific ELISA for Flufenoxuron and an Improved Class-Specific Assay for Benzoylphenylurea Insect Growth Regulators;Wang Shuo 等;J. Agric. Food Chem.;第47卷(第8期);3416−3424 * |
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