CN109061153B - Time-resolved fluorescence immunochromatographic test strip for detecting iprodione and preparation method and application thereof - Google Patents
Time-resolved fluorescence immunochromatographic test strip for detecting iprodione and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
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- 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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Abstract
The invention discloses a time-resolved fluorescence immunochromatographic test strip for detecting iprodione and a preparation method and application thereof. The test strip comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, wherein the conjugate release pad is embedded with an iprodione monoclonal antibody marked by fluorescent microspheres, the nitrocellulose membrane is fixed with a detection area and a quality control area, the detection area is sprayed with an iprodione hapten-carrier protein conjugate, the quality control area is sprayed with a goat anti-mouse anti-antibody, and the iprodione hapten is obtained by a series of reactions of 3, 5-dichlorophenylisocyanate, glycine ethyl ester hydrochloride, 6-methyl aminocaproate hydrochloride and the like. The test strip and the detection method provided by the invention have the advantages of simple operation, high sensitivity, high detection speed and low cost, and can realize rapid detection of iprodione in a large batch of samples.
Description
Technical Field
The invention belongs to the field of pesticide residue detection, and particularly relates to a time-resolved fluorescence immunochromatographic test strip for detecting iprodione in tobacco and tobacco products, and a preparation method and application thereof.
Background
Iprodione belongs to dicarboximide, is a broad-spectrum contact-killing protective bactericide, and is widely applied to disease control of tobacco, fruit trees and vegetables and storage and preservation of fruits. The iprodione can absorb and absorb systemic action through roots, and can effectively prevent and control fungi with resistance to benzimidazole systemic bactericides. The main prevention and treatment objects are diseases caused by botrytis, alternaria, sclerotinia and the like, such as gray mold, early blight, black spot, sclerotinia and the like. China sets the maximum residual limit standard of iprodione aiming at different crops, wherein the maximum residual limit in rapeseeds and cucumbers is 2 mg/kg, the maximum residual limit in tomatoes, apples and pears is 5 mg/kg, and the maximum residual limit in grapes and bananas is 10 mg/kg. The international cooperation center for tobacco science research (CORESTA) stipulates that the guiding residual limit of iprodione in tobacco is 0.5 mg/kg, the maximum residual limit of iprodione in China is not established, and 0.5 mg/kg is used as the maximum residual quantity judgment standard of tobacco in actual production.
At present, the common detection methods include high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry and the like. The methods all need advanced detection instruments, are expensive in detection cost, complex in steps and time-consuming, have high requirements on the professional performance of operators, and are not suitable for high-throughput rapid screening and detection of the primary enterprises and public institutions. Therefore, it is an object of the present invention to develop a product and a method for rapidly detecting a large number of samples without being limited by the detection equipment.
The fluorescence immunochromatography technology is developed on the fluorescent dye labeling technology, is a combination of an immunoaffinity technology, an immunolabeling technology and an immunochromatography technology as an immunological detection method, and has the advantages of rapidness, simple and convenient operation and the like. Compared with the traditional marker, the luminous intensity of the fluorescent microsphere can be enhanced along with the enhancement of the intensity of exciting light, so that the fluorescent microsphere marker is expected to improve the detection limit of the immunochromatography technology; under the action of the microsphere shell structure, the fluorescent microsphere has a relatively stable morphological structure, uniform granularity, good monodispersity, good stability, high luminous efficiency, good repeatability and better biocompatibility; after the microsphere is formed, the fluorescence quenching of the dye is greatly reduced, the emission is strong and stable, and the influence of the change of an external environment medium is basically avoided. Therefore, compared with the detection method, the fluorescent microsphere immunochromatography technology has the advantages of high detection sensitivity, simple and convenient operation and good stability. At present, no time-resolved fluorescence immunochromatographic test strip for detecting iprodione in tobacco and tobacco products exists.
Disclosure of Invention
The invention aims to provide a time-resolved fluorescence immunochromatographic test strip for detecting iprodione, which has high sensitivity, simple and convenient operation, quick detection and low cost aiming at the defects of the prior art; the invention also aims to provide a preparation method of the test strip; the invention further aims to provide the application of the test strip in detecting iprodione.
In order to achieve the purpose, the invention adopts a technical scheme that:
the time-resolved fluorescence immunochromatographic test strip comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, wherein the conjugate release pad is embedded with a fluorescent microsphere-labeled iprodione monoclonal antibody, the nitrocellulose membrane is fixedly provided with a detection area and a quality control area, the detection area is sprayed with an iprodione hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody.
The iprodione monoclonal antibody is prepared by taking a iprodione hapten-carrier protein conjugate as an immunogen; the iprodione hapten-carrier protein conjugate is obtained by coupling iprodione hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein, rabbit serum protein or human serum albumin, the iprodione hapten is obtained by reacting 3, 5-dichlorophenylisocyanate with glycine ethyl ester hydrochloride to generate 2- (3- (1, 5-dichlorophenylureido)) ethyl acetate, hydrolyzing to obtain 2- (3- (1, 5-dichlorophenylureido)) acetic acid, performing cyclization reaction to obtain 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinyl diketone, reacting with 6-methyl aminocaproate hydrochloride and triphosgene to obtain 6- (3- (3), 5-dichlorophenyl) -2, 4-dioxy imidazole alkyl-1-formamido) methyl caproate, and finally hydrolyzing under acidic condition to obtain the compound with the molecular structural formula:
the preparation method of the iprodione hapten specifically comprises the following steps:
1) 8.37 g (60 mmol) of glycine ethyl ester hydrochloride and 16.5 mL (120 mmol) of triethylamine were added to 100 mL of dichloromethane, a 10 g (53 mmol) dichloromethane solution of 3, 5-dichlorophenylisocyanate was added dropwise at 0 ℃, the mixture was stirred overnight at room temperature, insoluble solids were removed by filtration, the filtrate was washed with 2N hydrochloric acid, a saturated sodium bicarbonate solution and a saturated brine, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated to give 2- (3- (1, 5-dichlorophenylureido)) ethyl acetate as a white solid.
2) Adding the white solid into 150 mL of 6% sodium hydroxide aqueous solution, heating to 90 ℃ under stirring for reaction for 3 h, cooling to room temperature, extracting a small amount of unreacted raw materials and byproducts from 150 mL of ethyl acetate, adjusting the pH value of the aqueous phase to 2 at 0 ℃ by using 4N hydrochloric acid, and filtering to obtain the white solid 2- (3- (1, 5-dichlorophenylureido)) acetic acid.
3) The above white solid was added to 100 mL of 20% hydrochloric acid, and the mixture was refluxed (105 ℃ C. and 115 ℃ C.) with stirring for 4 hours, cooled to room temperature, filtered to give a white solid, and dried with water to give 9.50 g of 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinedione as a white solid.
4) In a reaction flask, 2.20 g (7.36 mmol) of triphosgene was dissolved in 30 mL of dichloromethane. A solution of 3.34 g (18.4 mmol) of methyl 6-aminocaproate hydrochloride and 7.13 g (55.2 mmol) of Diisopropylethylamine (DIEPA) in methylene chloride was slowly dropped into the above reaction solution at 0 ℃ to react at room temperature for 1 hour with stirring, methylene chloride was distilled off, 50 mL of anhydrous ether was added to the residual solid to stir, insoluble salts were filtered off, and the filtrate was concentrated to obtain 2.30 g of methyl 6-isocyanatohexanoate.
5) 3.00 g (12.2 mmol) of 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinedione and 2.67 g (18.4 mmol) of DBU (1, 8-diazabicyclo [5.4.0] -7-undecene) were added to 50 mL of dichloromethane, a dichloromethane solution of 2.30 g (13.5 mmol) of methyl 6-isocyanatohexanoate was slowly added dropwise at 0 ℃ and the reaction was stirred at room temperature for 4 hours, the solvent was distilled off from the reaction mixture, and the residue was purified by silica gel column chromatography and eluted with ethyl acetate/petroleum ether at a volume ratio of 1:5 to obtain 1.83 g of methyl 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoate.
6) 1.83 g (4.4 mmol) of methyl 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoate was dissolved in 50 mL of tetrahydrofuran, and 1 mL of 20% hydrochloric acid was added thereto to carry out a reaction at 50 ℃ for 4 hours and at room temperature overnight. The solvent was evaporated from the reaction solution, and the residue was purified by silica gel column chromatography and eluted with methylene chloride/methanol at a volume ratio of 30:1 to give 1.12 g of iprodione hapten 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoic acid.
The fluorescent microspheres are microspheres with the diameter of 100-300 nm and are prepared by wrapping fluorescent materials with polystyrene, the surfaces of the microspheres are connected with-COOH groups, and the fluorescent materials are lanthanide series.
The invention adopts another technical scheme that a method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting iprodione is provided, which comprises the following steps:
1) preparation of conjugate release pad: marking the iprodione monoclonal antibody by using a commercially available fluorescent microsphere, diluting the iprodione monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the iprodione monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the iprodione hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;
3) assembling and shearing: a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled iprodione monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad are sequentially overlapped and adhered on a bottom plate, and the nitrocellulose membrane and the water absorption pad are cut into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
Specifically, the steps include:
1) reacting 3, 5-dichlorophenylisocyanate with glycine ethyl ester hydrochloride to generate 2- (3- (1, 5-dichlorophenylureido)) ethyl acetate, hydrolyzing to obtain 2- (3- (1, 5-dichlorophenylureido)) acetic acid, performing cyclization reaction to obtain 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinyl diketone, reacting with 6-isocyanatomethyl hexanoate obtained by reacting 6-aminocaproate hydrochloride with triphosgene to generate 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-formamido) methyl hexanoate, and finally hydrolyzing under an acidic condition to obtain an iprodione hapten;
2) coupling the iprodione hapten with carrier protein to prepare an iprodione hapten-carrier protein conjugate;
3) immunizing a mouse by using the iprodione hapten-carrier protein conjugate, and fusing and screening splenocytes of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the iprodione monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) spraying the iprodione hapten-carrier protein conjugate and the goat anti-mouse anti-antibody to a detection area range (T) and a quality control area range (C) of a nitrocellulose membrane respectively;
6) soaking the sample absorption pad in 0.5% bovine serum albumin (volume fraction), pH 7.2, 0.1 mol/L phosphate buffer solution for 2h, and drying at 37 deg.C for 2 h;
7) marking the iprodione monoclonal antibody by using a commercially available fluorescent microsphere, diluting the iprodione monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and freezing and drying in vacuum for later use;
8) and a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled iprodione monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad are sequentially overlapped and adhered on the bottom plate, and the nitrocellulose membrane and the water absorption pad are cut into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
The invention adopts another technical scheme that an application of the time-resolved fluorescence immunochromatographic test strip for detecting iprodione in the detection of iprodione is provided, which comprises the following steps:
1) pretreating a sample;
2) detecting by using the time-resolved fluorescence immunochromatographic test strip for detecting iprodione;
3) and analyzing the detection result by using a fluorescence detector.
Compared with the prior art, the invention has the following beneficial effects:
(1) strong specificity and high sensitivity: the test strip embeds the iprodione monoclonal antibody marked by the fluorescent microspheres on the conjugate release pad, and has the advantages of good hydrophilicity, capability of adsorbing the antibody conjugate in a large capacity, quick re-wetting, full release of the antibody conjugate, good performance, quick release, good shape and the like, thereby reducing errors, reducing the cost and increasing the reaction sensitivity of the whole system.
(2) The time-resolved fluorescence has larger stock displacement, so that the interference of specific stray light caused by exciting light on detection is reduced, and the fluorescence detection stability is improved; the service life is long, and the interference of fluorescent substances in the environment to an object to be detected is eliminated; the excitation wavelength is wide, the emission spectrum range is narrow, the background fluorescence intensity is reduced, and the resolution ratio is improved.
(3) Polystyrene is wrapped on the surface of the fluorescent microsphere, so that the lanthanide series of the fluorescent substance is protected, the interference of the external environment is reduced, and the stability and the fluorescent life of the fluorescent microsphere are improved.
(4) The surface of the fluorescent microsphere is modified with active groups-COOH, and the antibody is marked by adopting a chemical coupling method to form stable combination of the antibody and the microsphere.
At present, no time-resolved fluorescence immunochromatographic test strip for detecting iprodione in tobacco and tobacco products exists, and the invention fills the blank. The test strip has the advantages of low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period, and the method for detecting iprodione by using the test strip is simple, convenient, rapid, visual and accurate, does not need large-scale instruments, has low cost and is easy to popularize and use.
Drawings
FIG. 1 is a schematic diagram of a cross-sectional structure of a time-resolved fluorescence immunochromatographic test strip;
FIG. 2 is a scheme showing the synthesis of iprodione hapten.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 constitution of time-resolved fluoroimmunoassay test strip for detecting iprodione
Test paper strip
Referring to fig. 1: the test strip consists of a bottom plate, a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad;
the sample absorption pad 1, the conjugate release pad 2, the nitrocellulose membrane 3 and the water absorption pad 4 are sequentially overlapped and adhered to the bottom plate 7, the conjugate release pad is covered by the sample absorption pad from an area 1/3 at the starting end, the tail end of the conjugate release pad is connected with the starting end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate;
a detection area 5 and a quality control area 6 are fixed on the nitrocellulose membrane, an iprodione hapten-carrier protein conjugate (iprodione hapten-ovalbumin conjugate) is sprayed on the detection area, and a goat anti-mouse anti-antibody is sprayed on the quality control area;
the bottom plate is a PVC bottom plate; the conjugate release pad is glass wool; the absorbent pad is absorbent paper.
Example 2 preparation of time-resolved fluoroimmunoassay test strip for detecting iprodione
The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting iprodione mainly comprises the following steps:
1) preparation of conjugate release pad: marking the iprodione monoclonal antibody by using a commercially available fluorescent microsphere, diluting the iprodione monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the iprodione monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the iprodione hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;
3) assembling and shearing: a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled iprodione monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad are sequentially overlapped and adhered on a bottom plate, and the nitrocellulose membrane and the water absorption pad are cut into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
The following steps are detailed:
preparation of the Components
1. Synthesis and identification of iprodione hapten-carrier protein conjugate
The iprodione is a small molecular substance, has immunoreactivity, has no immunogenicity, cannot induce an organism to generate immune response, and has immunogenicity only after being coupled with a macromolecular carrier protein.
(1) Preparation of iprodione hapten
1) 8.37 g (60 mmol) of glycine ethyl ester hydrochloride and 16.5 mL (120 mmol) of triethylamine were added to 100 mL of dichloromethane, a 10 g (53 mmol) dichloromethane solution of 3, 5-dichlorophenylisocyanate was added dropwise at 0 ℃, the mixture was stirred overnight at room temperature, insoluble solids were removed by filtration, the filtrate was washed with 2N hydrochloric acid, a saturated sodium bicarbonate solution and a saturated brine, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated to give 2- (3- (1, 5-dichlorophenylureido)) ethyl acetate as a white solid.
2) Adding the white solid into 150 mL of 6% sodium hydroxide aqueous solution, heating to 90 ℃ under stirring for reaction for 3 h, cooling to room temperature, extracting a small amount of unreacted raw materials and byproducts from ethyl acetate, adjusting the pH value of the water phase to 2 at 0 ℃ by using 4N hydrochloric acid, and filtering to obtain the white solid 2- (3- (1, 5-dichlorophenylureido)) acetic acid.
3) The white solid is added into 100 mL of 20% hydrochloric acid, stirred, heated and refluxed for 4h, cooled to room temperature, filtered to obtain a white solid, and washed and dried to obtain 9.50 g of white solid 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinedione, wherein the total yield of the three steps is 73.2%.
4) In a reaction flask, 2.20 g (7.36 mmol) of triphosgene was dissolved in 30 mL of dichloromethane. A solution of 3.34 g (18.4 mmol) of methyl 6-aminocaproate hydrochloride and 7.13 g (55.2 mmol) of Diisopropylethylamine (DIEPA) in methylene chloride was slowly dropped into the above reaction solution at 0 ℃ to react at room temperature for 1 hour with stirring, methylene chloride was distilled off, 50 mL of anhydrous ether was added to the residual solid to stir, insoluble salts were filtered off, and the filtrate was concentrated to obtain 2.30 g of methyl 6-isocyanatohexanoate.
5) 3.00 g (12.2 mmol) of 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinedione and 2.67 g (18.4 mmol) of DBU (1, 8-diazabicyclo [5.4.0] -7-undecene) were added to 50 mL of dichloromethane, a dichloromethane solution of 2.30 g (13.5 mmol) of methyl 6-isocyanatohexanoate was slowly added dropwise at 0 ℃ and the reaction was stirred at room temperature for 4 hours, the reaction mixture was evaporated to remove the solvent, and the residue was purified by silica gel column chromatography and eluted with ethyl acetate/petroleum ether at a volume ratio of 1:5 to give 1.83 g of methyl 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoate in a yield of 36.1%.
6) 1.83 g (4.4 mmol) of methyl 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoate was dissolved in 50 mL of tetrahydrofuran, and 1 mL of 20% hydrochloric acid was added thereto to carry out a reaction at 50 ℃ for 4 hours and at room temperature overnight. The solvent was evaporated from the reaction solution, and the residue was purified by silica gel column chromatography and eluted with methylene chloride/methanol at a volume ratio of 30:1 to give 1.12 g of iprodione hapten 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxoimidazolidinyl-1-carboxamido) hexanoic acid in a yield of 63.3%.
Nuclear magnetic identification1H NMR (300 MHz, DMSO-d6): δ 12.04 (1H, s), 10.07 (1H, s, NH), 7.73 (2H, d, J = 1.80 Hz), 7.34 (1H, t, J = 1.80 Hz), 4.32 (2H, s), 3.44 (2H, t, J = 6.81 Hz), 2.20 (2H, t, J= 6.25 Hz), 1.62-1.44 (4H, m), 1.36-1.22 (2H, m). In the chromatogram, the chemical shift delta =12.04 is the resonance absorption peak of carboxyl hydrogen on the spacer arm, the delta =3.44, 2.20, 1.62-1.44 and 1.36-1.22 are the resonance absorption peaks of methylene hydrogen on the spacer arm, and the existence of the peaks is matched with the existence of the intrinsic absorption peaks of other iprodione hydrogen to prove the success of hapten synthesis.
(2) Preparation of immunogens
The iprodione hapten is coupled with Bovine Serum Albumin (BSA) to obtain the immunogen.
Dissolving 9.0 mg of iprodione hapten in 1.0 mL of Dimethylformamide (DMF), adding 0.18 mL of isobutyl chloroformate and 0.3 mL of pyridine, and stirring at room temperature for 5 hours to obtain hapten activating solution A; taking 50 mg of Bovine Serum Albumin (BSA), fully dissolving in 3.8 mL of phosphate buffer PBS to obtain solution B, dripping the solution A into the solution B, stirring for 5 hours at room temperature, dialyzing for 3 days at 4 ℃ by using 0.01 mol/L PBS to remove unreacted small molecular substances to obtain iprodione-BSA immunogen; storing at-20 deg.C for use.
(3) Preparation of coating antigen
Coupling the iprodione hapten with Ovalbumin (OVA) to obtain the coating antigen.
Dissolving 7.0 mg of iprodione hapten in 1.0 mL of DMF, adding 0.27 mL of thionyl chloride, reacting at 60 ℃ for 6 h, stopping the reaction, and cooling to room temperature to obtain hapten activating solution A; dissolving 50 mg of Ovalbumin (OVA) fully in 3.8 mL of PBS to obtain solution B, dripping the solution A into the solution B, reacting for 8 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.01 mol/L PBS buffer solution, changing the solution 3 times every day, and subpackaging to obtain iprodione-OVA coating antigen, and storing at-20 ℃ for later use.
(4) Identification
Determining whether the coupling was successful: whether the coupling of the hapten and the carrier protein is effective coupling is generally identified by an ultraviolet scanning method, because the hapten and the protein have different characteristic absorptions under ultraviolet, when the coupling is successful, the ultraviolet absorption of the conjugate has the additive effect of the two, so that certain deviation occurs compared with the characteristic absorption of the protein alone, and the method can be used for detecting whether the coupling is successful.
Determination of coupling ratio: diluting iprodione hapten, bovine serum albumin, ovalbumin and a combination of two proteins and iprodione hapten by pure water to prepare a solution with a certain concentration, and then carrying out full-wavelength scanning by an ultraviolet spectrophotometer to obtain an ultraviolet absorption spectrogram of the solution.
And respectively calculating the molar extinction coefficients of the iprodione hapten, the bovine serum albumin and the ovalbumin according to a formula K = A/CL. Detecting the light absorption value of the conjugate at the maximum wavelength of the carrier protein and the iprodione hapten, and calculating the molar concentration ratio of the two substances in the conjugate according to a formula, namely the coupling ratio:
Ca/Cb=（Adoll 260×KBSA280-ACouple 280 ×KBSA260）/（ACouple 280×KIprodione 260-ADoll 260×KIprodione 280）
Determination of protein content: after the conjugate is diluted to an appropriate multiple, the spectrophotometric values at 280 nm and 260 nm are determined, and the concentration of the protein, i.e., the concentration of the conjugate, is calculated according to the formula:
protein (mg/mL) =1.45 × OD280－0.74×OD260
Identification of immunogen and coatingen: the effective coupling of the hapten and the carrier protein is identified by an ultraviolet scanning method, the coupling ratio of the hapten to the carrier protein is estimated to be 15:1 and 11:1 respectively according to the molar absorption coefficients of the iprodione hapten, the carrier protein and the conjugate at specific wavelengths, the coupling effect is good, the protein content of the immunogen is 15.1 mg/mL, and the protein content of the coating antigen is 6.9 mg/mL.
2. Preparation of iprodione monoclonal antibody
(1) Obtaining hybridoma cells
1) First immunization: fully emulsifying the iprodione hapten-BSA conjugate (immunogen) with an equal amount of Freund complete adjuvant, and injecting 6-week-old Balb/c mice subcutaneously, wherein the immunization dose is 150 mu g/mouse;
2) two booster immunizations: from the first immunization, boosting once every two weeks, and replacing Freund's complete adjuvant with Freund's incomplete adjuvant in the same method and dosage as the first immunization;
3) after one week of last boosting immunization, measuring the titer and inhibition in fundus venous blood sampling, and performing the following last immunization when the titer reaches more than 1: 10000: injecting 0.1 mL of immunogen solution without any adjuvant into the abdominal cavity, killing the mouse after three days, and fusing the spleen with myeloma cells;
4) and (3) measuring cell supernatant by adopting an indirect competitive enzyme-linked immunoassay method, and screening positive holes. Cloning the positive hole by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the iprodione monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging the cell suspension in a freezing tube, and storing the cell suspension in liquid nitrogen for a long time.
(2) Preparation of monoclonal antibodies
1) Cell recovery: taking out the cryopreservation tube of the hybridoma cell strain of the iprodione monoclonal antibody, immediately putting the cryopreservation tube into a water bath at 37 ℃ for medium-speed thawing, centrifuging to remove a cryopreservation solution, and transferring the frozen cryopreservation solution into a culture bottle for culture;
2) preparing ascites and purifying antibodies: injecting sterilized paraffin oil 0.5 mL/mouse in Balb/c mouse (8 weeks old) into abdominal cavity by in vivo induction method, injecting hybridoma cells 5 × 10 into abdominal cavity 7 days later5Ascites were collected 7 days later. Purifying by octanoic acid-saturated ammonium sulfate method to obtain iprodione monoclonal antibody solution (preservation at-20 deg.C).
(3) Determination of the potency of monoclonal antibodies
The titer of the antibody is 1 (100000-300000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with an iprodione hapten-OVA conjugate, adding an iprodione standard solution, an iprodione monoclonal antibody solution and a horseradish peroxidase-labeled goat anti-mouse anti-antibody solution, reacting for 30 min at 25 ℃, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.
(4) Determination of monoclonal antibody specificity
Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.
In the experiment, iprodione and other dicarboximide bactericides (procymidone, dimethachlon and ethenobiosporin) are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained by analysis50Then, the cross-reactivity was calculated as follows:
the results show that the cross-reactivity of iprodione and its structural analogues is: 100% of iprodione, less than 1% of procymidone, less than 1% of dimethachlon and less than 1% of vinclozolin. The antibody of the invention has no cross reaction to other dicarboximide bactericides such as procymidone, dimethachlon, vinclozolin and the like, and only has specific combination aiming at iprodione.
3. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
4. Preparation of fluorescent microsphere labeled iprodione monoclonal antibody
(1) And (3) activation: suspending 100 mu L of microsphere suspension which is embedded with fluorescent dye and modified with carboxyl functional groups on the surface and is sold in market in 900 mu L of activation buffer solution, centrifuging for 10min at 4 ℃ at 10000 r/min, then discarding supernatant, resuspending microspheres in 1 mL of activation buffer solution, washing the microspheres for 2 times by the method, adding a proper amount of activating agent, uniformly mixing, and then oscillating and activating for 10min at room temperature;
(2) coupling: centrifuging the suspension of the step (1) at 4 ℃ and 10000 r/min for 10min, then discarding the supernatant, suspending the suspension in a coupling buffer solution, washing the microspheres for 2 times by the method, adding 10-20 mu L of iprodione monoclonal antibody solution (with the protein concentration of 1 mg/mL), uniformly mixing, and oscillating and coupling at room temperature for 120 min;
(3) and (3) sealing: centrifuging the suspension of (2) at 4 ℃ and 10000 r/min for 10min, then discarding the supernatant, suspending in a closed buffer solution, washing the microspheres for 1 time by the method, uniformly mixing, and oscillating at room temperature and sealing for 30 min;
(4) and (3) storage: centrifuging the suspension of (3) at 4 ℃ 10000 r/min for 10min, then discarding the supernatant, suspending in a storage buffer solution, washing the microspheres for 1 time by the method, mixing uniformly, and storing at 4 ℃ in a dark place.
The activating buffer solution is a 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution with the pH value of 5.5-6.5 and the mol/L of 0.05.
The activating agent is water-soluble carbodiimide, wherein the molar mass ratio of EDC to NHS to COOH = (1.5-3) to (8-20) to 1, and the activating agent is diluted to a required concentration by using an activating buffer solution before use.
The coupling buffer is borate buffer with the pH value of 7.5-8.50.05 mol/L (solvent with free amine is avoided).
The blocking buffer solution is a PB buffer solution which contains 0.1-0.4 mol/L primary amine (hydroxylamine hydrochloride, ethanolamine or aminoethanol) and 1% -10% BSA and has a pH value of 7.4.
The storage buffer solution contains 0.01 percent of NaN30.1% BSA at pH 7.4.
5. Preparation of conjugate Release pad
Diluting the stored fluorescent microsphere labeled iprodione monoclonal antibody with a storage buffer solution, soaking the conjugate release pad in the dilution buffer solution, and freeze-drying in vacuum for later use.
6. Preparation of cellulose Nitrate (NC) membranes
Diluting the iprodione hapten-ovalbumin conjugate to 100 mu g/mL by using 0.05 mol/L, pH PBS buffer solution with the value of 7.2, and spraying the iprodione hapten-ovalbumin conjugate to a detection area (T) on an NC membrane by using an Isoflow point membrane instrument, wherein the spraying amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01 mol/L, pH value of 7.4 PBS buffer, and sprayed on the quality control area (C) on the NC membrane in an Isoflow point membrane machine in an amount of 1.0. mu.L/cm. And (3) drying the prepared NC membrane for 2h at 37 ℃ for later use.
7. Preparation of sample absorbent pad
The sample absorption pad is soaked in 0.5 percent bovine serum albumin (volume fraction), pH value of 7.2 and 0.1 mol/L phosphate buffer solution for 2 hours and dried for 2 hours at 37 ℃.
(II) Assembly of test strip
A sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad are sequentially overlapped and stuck and fixed on a bottom plate from left to right, the conjugate release pad is covered by the sample absorption pad from the area 1/3 at the starting end, the tail end of the conjugate release pad is connected with the starting end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the bottom plate, the tail end of the water absorption pad is aligned with the tail end of the bottom plate, and then the sample absorption pad is cut into small strips with the width of 3.96 mm by a machine and is arranged in a special plastic card to form a test paper card. The iprodione fluorescent microsphere immunochromatographic test paper card is stored in a shady, cool and dark dry mode at the temperature of 2-8 ℃, and the effective period is 12 months.
Example 3 application of time-resolved fluorescence immunochromatographic test strip for detecting iprodione
1. Tobacco sample pretreatment
Weighing 1.0 +/-0.05 g of crushed sample to be detected into a 50 mL centrifuge tube, adding 10 mL of 50% methanol aqueous solution, carrying out vortex for 1 min, and centrifuging for 5 min at more than 3000 rpm; and adding 400 mu L of sample complex solution into 100 mu L of supernatant, and uniformly mixing to be tested.
2. Detection with test strips
Sucking 100 mu L of sample solution to be detected, vertically dropping the sample solution into a sample adding hole of a test paper card, starting timing when the liquid flows, and reacting for 10 min; inserting the test paper card into a carrier of a KFT-100A type fluorescence detector, selecting an item to be detected by touching a display screen, pressing a 'start detection' button, automatically carrying out scanning test on the test paper card by the fluorescence detector, and reading or printing a detection result on a display screen of the fluorescence detector.
3. Analysis of detection results
(1) Quantitative detection
After the test is finished, the instrument obtains the ratio of the time-resolved fluorescence intensity of the detection area on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control area, obtains the content of iprodione in the extract of the sample to be tested based on the relation curve between the preset ratio of the time-resolved fluorescence intensity of the detection area on the fluorescent test strip to the time-resolved fluorescence intensity of the quality control area and the concentration of iprodione, and finally obtains the content of iprodione in the sample to be tested through conversion.
(2) Semi-quantitative detection
After the test is finished, the instrument automatically calculates the concentration value of iprodione in the extracting solution according to the ratio of the time-resolved fluorescence intensity of the detection area to the time-resolved fluorescence intensity of the quality control area, and gives out positive and negative judgment according to a preset threshold value.
Negative (-): if the result on the display screen of the fluorescence detector is negative, the result indicates that the specimen does not contain iprodione or the concentration of the iprodione is lower than the detection limit.
Positive (+): if the result on the display screen of the fluorescence detector is positive, the concentration of the iprodione in the sample is equal to or higher than the detection limit.
And (4) invalidation: if the quality control area does not detect the intensity of the fluorescence signal, the incorrect operation process or the failure of the test paper card is indicated.
Example 4 sample testing example
1. Limit of detection test
Adding iprodione standard substances into blank tobacco samples respectively until the final concentration is 0.2, 0.4 and 0.8 mg/kg, and detecting by using a time-resolved fluorescence immunochromatographic test strip, wherein the result is as follows: when the concentration of the iprodione is 0.2 mg/kg, the detection of the fluorescence detector is negative; when the concentration of the iprodione is 0.4 mg/kg and 0.8 mg/kg, the detection of the fluorescence detector is positive, which shows that the detection limit of the test strip on the iprodione in the tobacco is 0.4 mg/kg.
2. Test for false positive and false negative rates
And (3) taking 20 parts of positive tobacco leaf samples with known iprodione content being more than 0.4 mg/kg and 20 parts of negative tobacco leaf samples without known iprodione, respectively detecting by using 3 time-resolved fluorescence immunochromatographic test strips produced in batches, and calculating the negative and positive rates of the positive tobacco leaf samples and the negative tobacco leaf samples. The results are shown in Table 1.
TABLE 1 test results for positive and negative samples
The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The time-resolved fluorescence immunochromatographic test strip for detecting iprodione can be used for quickly detecting iprodione in tobacco leaves.
3. Specificity test
The iprodione test strip is used for detecting other dicarboximide bactericides such as 1 mg/L procymidone, dimethachlon, vinclozolin and the like, and the result shows that the detection of the fluorescence detector is negative. The test paper strip has no cross reaction to the compounds with similar structures with iprodione, such as procymidone, dimethachlon, etc., and has good specificity.
Claims (6)
1. A time-resolved fluorescence immunochromatographic test strip for detecting iprodione comprises a base plate, and a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and adhered on the base plate, and is characterized in that the conjugate release pad is embedded with an iprodione monoclonal antibody marked by fluorescent microspheres, the nitrocellulose membrane is fixed with a detection area and a quality control area, the detection area is sprayed with an iprodione hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody; the iprodione monoclonal antibody is prepared by taking a iprodione hapten-carrier protein conjugate as an immunogen; the iprodione hapten-carrier protein conjugate is obtained by coupling iprodione hapten and carrier protein, the iprodione hapten is obtained by reacting 3, 5-dichlorophenylisocyanate with glycine ethyl ester hydrochloride to generate 2- (3- (1, 5-dichlorophenylureido)) ethyl acetate, hydrolyzing to obtain 2- (3- (1, 5-dichlorophenylureido)) acetic acid, performing cyclization reaction to obtain 3- (3, 5-dichlorophenyl) -2, 4-imidazolidinyl diketone, and reacting with 6-isocyanatomethyl hexanoate obtained by reacting 6-aminocaproic acid methyl ester hydrochloride with triphosgene to generate 6- (3- (3, 5-dichlorophenyl) -2, 4-dioxy imidazolidinyl-1-formamido) methyl hexanoate, finally hydrolyzing under acidic condition to obtain the product with molecular structural formula:
the preparation reaction process of the iprodione hapten is as follows:
2. the time-resolved fluorescence immunochromatographic test strip for detecting iprodione according to claim 1, characterized in that: the fluorescent microspheres are microspheres with the diameter of 100-300 nm and fluorescent materials wrapped by polystyrene, and the surfaces of the microspheres are connected with-COOH groups.
3. The time-resolved fluorescence immunochromatographic test strip for detecting iprodione according to claim 2, characterized in that: the fluorescent substance is a lanthanide.
4. The time-resolved fluorescence immunochromatographic test strip for detecting iprodione according to claim 1, characterized in that: the carrier protein is thyroid protein, bovine serum albumin, rabbit serum protein, human serum protein, ovalbumin or hemocyanin.
5. A method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting iprodione according to any one of claims 1 to 4, which is characterized by comprising the following steps:
1) preparation of conjugate release pad: labeling an iprodione monoclonal antibody by using fluorescent microspheres, diluting the iprodione monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying to prepare the iprodione monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the iprodione hapten-carrier protein conjugate to a detection area range on a nitrocellulose membrane to prepare a detection area; spraying goat anti-mouse anti-antibody to the range of the quality control area on the nitrocellulose membrane to prepare the quality control area;
3) assembling and shearing: a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled iprodione monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad are sequentially overlapped and adhered on a bottom plate, and the nitrocellulose membrane and the water absorption pad are cut into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
6. The use of the time-resolved fluoroimmunoassay test strip for the detection of iprodione of claims 1 to 4, comprising the steps of:
1) pretreating a sample;
2) detecting with the test strip;
3) and analyzing the detection result by using a fluorescence detector.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5342771A (en) * | 1990-12-03 | 1994-08-30 | American Cyanamid Company | Functionalized imidazolinone haptens and protein conjugates thereof useful in assays for the detection of imidazolinone herbicides |
| CN105424939A (en) * | 2015-12-23 | 2016-03-23 | 中国烟草总公司郑州烟草研究院 | Test paper strip for detecting carbendazim, preparation method and application thereof |
| CN105675874A (en) * | 2016-01-11 | 2016-06-15 | 北京勤邦生物技术有限公司 | Colloidal gold test strip for detection of imidacloprid and application thereof |
| CN105807055A (en) * | 2016-03-15 | 2016-07-27 | 中国烟草总公司郑州烟草研究院 | Test strip for detecting quinclorac and preparation method and application of test strip |
| CN106771238A (en) * | 2016-12-14 | 2017-05-31 | 北京勤邦生物技术有限公司 | Detect fluorescent micro-ball immune chromatography test paper strip of melamine residual and its preparation method and application |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3947283B2 (en) * | 1997-11-19 | 2007-07-18 | 株式会社堀場製作所 | Hapten compound, antibody and measuring method of iprodione and its metabolite |
| JP2001281250A (en) * | 2000-03-30 | 2001-10-10 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Method for detecting low-molecule compound using antigen/antibody reaction |
| CN104849467B (en) * | 2014-02-15 | 2016-08-17 | 北京勤邦生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip of detection clenbuterol hydrochloride residual and its preparation method and application |
| CN105388281B (en) * | 2015-12-23 | 2017-09-12 | 中国烟草总公司郑州烟草研究院 | Detect enzyme linked immunological kit and its application of iprodione |
| CN107022024A (en) * | 2017-04-25 | 2017-08-08 | 江南大学 | A kind of synthetic method of iprodione artificial antigen |
-
2018
- 2018-09-21 CN CN201811104585.6A patent/CN109061153B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5342771A (en) * | 1990-12-03 | 1994-08-30 | American Cyanamid Company | Functionalized imidazolinone haptens and protein conjugates thereof useful in assays for the detection of imidazolinone herbicides |
| CN105424939A (en) * | 2015-12-23 | 2016-03-23 | 中国烟草总公司郑州烟草研究院 | Test paper strip for detecting carbendazim, preparation method and application thereof |
| CN105675874A (en) * | 2016-01-11 | 2016-06-15 | 北京勤邦生物技术有限公司 | Colloidal gold test strip for detection of imidacloprid and application thereof |
| CN105807055A (en) * | 2016-03-15 | 2016-07-27 | 中国烟草总公司郑州烟草研究院 | Test strip for detecting quinclorac and preparation method and application of test strip |
| CN106771238A (en) * | 2016-12-14 | 2017-05-31 | 北京勤邦生物技术有限公司 | Detect fluorescent micro-ball immune chromatography test paper strip of melamine residual and its preparation method and application |
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