CN109061157B - Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof - Google Patents
Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof Download PDFInfo
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- 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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- 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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- 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
A time-resolved fluorescence immunochromatographic test strip for detecting flumetralin, a preparation method and application thereof. The test strip comprises a bottom plate and a sample absorption pad, the conjugate release pad is embedded with a Flumetralin monoclonal antibody marked by fluorescent microspheres, a detection area and a quality control area are fixed on the nitrocellulose membrane, a Flumetralin hapten-carrier protein conjugate is sprayed on the detection area, a goat anti-mouse antibody is sprayed on the quality control area, and the Flumetralin hapten is obtained by reacting 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid to generate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid and then reacting with iodoethane. The test strip and the detection method have the advantages of simple operation, high sensitivity, high detection speed and low cost, and can realize the rapid detection of the flumetralin 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 flumetralin in tobacco and tobacco products, and a preparation method and application thereof.
Background
Flumetralin is a dinitroaniline plant growth regulator which is contact and local systemic and is used for inhibiting lateral buds of tobacco, and is an excellent tobacco bud inhibitor. Was successfully developed in 1977 by Ciba-Geigy, Switzerland. In 1990, Prime (Prime) is officially registered in China as a trade name with a registration number of PDll 6-90. It is a new type of highly effective sprout inhibitor which is popular internationally, and is suitable for flue-cured tobacco, open fire flue-cured tobacco, Maryland tobacco, sun-cured tobacco and cigar. The national Zhejiang chemical research institute is researched and developed at the earliest time and is successfully developed in 1998, and temporary registration of pesticides is obtained in 1999. The pesticide is applied once within 24 hours after the tobacco is artificially topped, and bud picking is not needed in the whole growing season. 60-70 mL of 25% flumetralin emulsifiable concentrate is used per mu, the effect is quick, the absorption is quick, the effect can be achieved as long as no rain exists after the pesticide is applied for 2 hours, and the pesticide application is convenient in rainy seasons. The contact of the medicament with the fully extended leaves does not cause phytotoxicity and does not contain harmful residues. The use of the flumetralin can save a great amount of artificial bud picking, lead the natural maturity to be consistent, increase the yield and improve the upper-middle proportion of the tobacco leaves and the internal quality of the tobacco leaves. In addition, the use of flumetralin can also reduce contact infection of mosaic disease in the field. Compared with other bud inhibitors, the flumetralin has high drug effect. The international cooperation center for tobacco science research (CORESTA) stipulates that the directive residual limit of the flunomide in the tobacco is 5 mg/kg, and the maximum residual limit of the flunomide in the food is not established in China. Currently, methods for detecting flumetralin are commonly used, such as gas chromatography, gas chromatography-mass spectrometry, gas chromatography tandem 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 urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples.
The fluorescent microsphere 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. Until now, no time-resolved fluoroimmunoassay test strip has appeared on the market.
Disclosure of Invention
The invention aims to provide a time-resolved fluorescence immunochromatographic test strip for detecting flumetralin, which has the advantages of high sensitivity, simple and convenient operation, rapid 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 the flumetralin.
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 a fluorescein microsphere labeled fluoroacetylamine monoclonal antibody is embedded on the conjugate release pad, a detection area and a quality control area are fixed on the nitrocellulose membrane, a fluoroacetylamine hapten-carrier protein conjugate is sprayed on the detection area, and a goat anti-mouse anti-antibody is sprayed on the quality control area.
The flumetralin monoclonal antibody is prepared by taking a flumetralin hapten-carrier protein conjugate as an immunogen; the flumetralin hapten-carrier protein conjugate is obtained by coupling flumetralin hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin, the flumetralin hapten is obtained by reacting 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid to generate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, and then reacting the 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid with iodoethane, and the molecular structural formula is as follows:
the preparation method of the fluvalinate hapten specifically comprises the following steps:
1) taking 1.00 g of 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene, and adding 20 mL of absolute ethyl alcohol to dissolve the 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene to obtain solution A; dissolving 0.88 g of 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid in 10 mL of absolute ethanol, adding 1 mL of aqueous solution containing 0.37 g of sodium bicarbonate to obtain solution B, dropwise adding the solution A into the solution B, and reacting at room temperature for 3 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, carrying out rotary evaporation, removing ethanol, adding 80 mL of water for dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for shaking and layering, washing an organic phase by using water, carrying out rotary evaporation, applying to a silica gel column, and carrying out elution separation on n-hexane and ethyl acetate according to the volume ratio of 10:1 to obtain 1.53 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid;
2) taking 1.50 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, dissolving the intermediate in 50 mL of acetonitrile, adding 0.20 g of potassium hydroxide, adding 0.57 g of iodoethane, reacting for 4 h at 50 ℃, detecting, stopping the reaction when the raw materials completely react, removing the acetonitrile by rotary evaporation, adding 100 mL of water, dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate, extracting, washing and drying an organic phase, evaporating to dryness to obtain a yellow oily substance, and recrystallizing with a dichloromethane solution by using n-hexane and a dichloromethane solution in a volume ratio of 1:1 to obtain 1.51 g of a flumetralin hapten product.
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 the flumetralin is provided, which comprises the following steps:
1) preparation of conjugate release pad: marking the flumetralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the flumetralin 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 fluetralin monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the fluvalinate 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: and sequentially bonding a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled flumetralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate in a lap joint manner, and shearing the nitrocellulose membrane and the water absorption pad into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
Specifically, the steps include:
1) reacting 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene with 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid to generate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, and reacting with iodoethane to prepare the flumetralin hapten;
2) coupling the flumetralin hapten with carrier protein to prepare a flumetralin hapten-carrier protein conjugate;
3) immunizing a mouse by using the flunomide hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the flunomide monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) respectively spraying the fluvalinate 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;
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 flumetralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the flumetralin monoclonal antibody by using a specific buffer system, soaking a conjugate release pad in a dilution buffer solution, and performing vacuum freeze drying for later use;
8) and sequentially overlapping and adhering a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled flumetralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate, and shearing the nitrocellulose membrane and the water absorption pad 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 the flumetralin in the detection of the flumetralin is provided, which comprises the following steps:
1) pretreating a sample;
2) detecting by using the time-resolved fluorescence immunochromatographic test strip for detecting the flumetralin;
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 Flumetralin 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, rapid rewetting, sufficient release of the antibody conjugate, good performance, rapid 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 flumetralin 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 the flumetralin by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and 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 shows a scheme for synthesis of flumetralin hapten (the figure is taken as an abstract figure).
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 Flumetralin
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, a flumetralin hapten-carrier protein conjugate (flumetralin 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 flumetralin
The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting the flumetralin mainly comprises the following steps:
1) preparation of conjugate release pad: marking the flumetralin monoclonal antibody by using a commercially available fluorescent microsphere, diluting the flumetralin 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 fluetralin monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the fluvalinate 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: and sequentially bonding a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled flumetralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate in a lap joint manner, and shearing the nitrocellulose membrane and the water absorption pad 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 fluvalinate hapten-carrier protein conjugate
Flumetralin is a small molecular substance, has immunoreactivity and 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 flumetralin hapten
Taking 1.00 g of 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene, and adding 20 mL of absolute ethyl alcohol to dissolve the 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene to obtain solution A; dissolving 0.88 g of 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid in 10 mL of absolute ethanol, adding 1 mL of aqueous solution containing 0.37 g of sodium bicarbonate to obtain solution B, dropwise adding the solution A into the solution B, and reacting at room temperature for 3 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, carrying out rotary evaporation, removing ethanol, adding 80 mL of water for dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for shaking and layering, washing an organic phase by using water, carrying out rotary evaporation, applying to a silica gel column, eluting and separating normal hexane and ethyl acetate according to the volume ratio of 10:1 to obtain 1.53 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid with the yield of 92.17%;
taking 1.50 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, dissolving the intermediate in 50 mL of acetonitrile, adding 0.20 g of potassium hydroxide, adding 0.57 g of iodoethane, reacting for 4 hours at 50 ℃, detecting, stopping the reaction when the raw materials completely react, removing the acetonitrile by rotary evaporation, adding 100 mL of water, dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate, extracting, washing and drying an organic phase, evaporating to dryness to obtain a yellow oily substance, and recrystallizing a dichloromethane solution by using n-hexane and a dichloromethane solution in a volume ratio of 1:1 to obtain 1.51 g of a flumetralin hapten product with the yield of 94.97%.
Nuclear magnetic identification of 1H NMR (CDCl)3300 MHZ) δ:11.00 (1H, s), 7.141 (1H, dd, J =8.271, J =1.347), 7.373 (2H, dd, J =8.373, J =8.271), 4.23 (2H, dd, J =8.373, J =1.347), 2.880 (2H, d, J =6.843), 3.631 (2H, q, J =7.108), 1.238 (3H, t, J =7.108), 8.75 (2H, d, J =0.000), chemical shift δ =11.0 is the carboxyhydrogenresonance absorption peak on the spacer arm, δ =2.88 is the resonance absorption peak of methylenehydrogenon the spacer arm, the presence of these peaks, proving successful spacer arm coupling.
(2) Preparation of immunogens
The immunogen is obtained by coupling the fluvalin hapten and Bovine Serum Albumin (BSA).
Dissolving 18 mg of flumetralin hapten in 0.3 mL of Dimethylformamide (DMF), clarifying, adding 8.6 mg of carbodiimide (EDC), stirring, clarifying, adding 5.2 mg of N-hydroxysuccinimide (NHS), stirring at room temperature, and activating for 3h to obtain solution A; 50 mg of BSA was dissolved in 8mL of 0.05 mol/L Phosphate Buffer (PB) having a pH of 7.2 to obtain solution B, and solution A was slowly added dropwise to solution B, followed by reaction with stirring at room temperature for 5 hours. Stopping reaction, dialyzing with 0.02M Phosphate Buffer Solution (PBS) for 3 days, changing the solution three times per day to obtain flumetralin-BSA immunogen, subpackaging, and storing at-20 ℃ for later use.
(3) Preparation of coating antigen
Coupling the fluvalin hapten and Ovalbumin (OVA) to obtain the coating antigen.
Dissolving 8 mg of flumetralin hapten in 0.2 mL of DMF, clarifying, adding 4.13 mg of Dicyclohexylcarbodiimide (DCC) and 2.3 mg of NHS, stirring at room temperature for 2h, filtering, and removing precipitates to obtain a hapten activation solution A; adding OVA 50 mg into 8mL of 0.05 mol/L PB buffer solution with pH value of 7.2 to dissolve to obtain solution B, slowly dripping the solution A into the solution B, and stirring at room temperature for reaction for 5 h. Stopping reaction, dialyzing with 0.02M PBS buffer solution for 3 days, changing the solution three times per day to obtain flumetralin-OVA coating antigen, subpackaging, and storing at-20 ℃ for later use.
2. Preparation of Flumetralin monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting an indirect competitive ELISA method, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Making hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during resuscitation, immediately putting into 37 deg.C water bath for rapid melting, and separatingRemoving the frozen stock solution from the heart, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
(5) Determination of the potency of monoclonal antibodies
The titer of the antibody is 1 (100000-400000) by using an indirect competition ELISA method.
Indirect competitive ELISA method: coating an enzyme label plate with a flumetralin hapten-OVA conjugate, adding a flumetralin standard solution, a flumetralin monoclonal antibody solution and a horse radish peroxidase labeled goat anti-mouse anti-antibody solution, reacting at 25 ℃ for 30 min, 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.
(6) 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, dinitroaniline herbicides (flumetralin, butralin, pendimethalin and trifluralin) are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained through analysis50Then, the cross-reactivity was calculated as follows:
the results show that the cross-reactivity rate of each analog is: 100 percent of flumetralin, less than 1 percent of butralin, less than 1 percent of pendimethalin and less than 1 percent of trifluralin. The antibody of the invention has no cross reaction to other dinitroaniline herbicides such as butralin, pendimethalin, trifluralin and the like, and only has specific binding to flumetralin.
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 flumetralin 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 flumetralin monoclonal antibody solution (with the protein concentration of 1 mg/mL), uniformly mixing, and performing oscillation 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 Flumetralin monoclonal antibody marked by the fluorescent microspheres 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 flumetralin 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 flumetralin 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 Flumetralin 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 flumetralin
1. Tobacco sample pretreatment
Weighing 1.0 +/-0.05 g of crushed tobacco sample into a polystyrene centrifuge tube; adding 10 mL 50% methanol water solution, vortexing with a vortex instrument for 3 min, and centrifuging at 6000 rpm at room temperature for 5 min; and transferring 100 mu L of supernatant and 400 mu L of deionized water, and uniformly mixing the supernatant and the 400 mu L of deionized water for detection.
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) Semi-quantitative detection
After the test is finished, the instrument automatically calculates the concentration value of the flumetralin 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 (-): and if the result on the display screen of the fluorescence detector is negative, the result indicates that the sample does not contain the flumetralin or the concentration of the flumetralin is lower than the detection limit.
Positive (+): and if the result on the display screen of the fluorescence detector is positive, the concentration of the flumetralin 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.
(2) 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 the flumetralin in the extracting solution 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 the flumetralin, and finally obtains the content of the flumetralin in the sample to be tested through conversion.
Example 4 sample testing example
1. Limit of detection test
Respectively adding the flumetralin standard substances into blank tobacco samples until the final concentration is 2.5, 5 and 10 mg/kg, and detecting by using a time-resolved fluorescence immunochromatography test strip, wherein the result is as follows: when the concentration of the flumetralin is 2.5 mg/kg, the detection of the fluorescence detector is negative; when the concentration of the flumetralin is 5 and 10 mg/kg, the detection of the fluorescence detector is positive, which shows that the detection limit of the test strip on the flumetralin in the tobacco is 5 mg/kg.
2. Test for false positive and false negative rates
And (3) taking 20 parts of positive tobacco samples with the known flumetralin content being more than 5 mg/kg and 20 parts of negative tobacco samples without flumetralin, respectively detecting by using 3 time-resolved fluorescence immunochromatographic test strips produced in batches, and calculating the negative and positive rates of the positive and negative samples. The results are shown in Table 2.
TABLE 2 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 the flumetralin can be used for quickly detecting the flumetralin in the tobacco.
3. Specificity test
The test paper of flumetralin is used for detecting 500 mu g/L dinitroaniline herbicides such as pendimethalin, butralin, trifluralin and the like. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to dinitroaniline herbicides such as pendimethalin, butralin, trifluralin and the like with the concentration of 500 mu g/L, and has good specificity.
Claims (7)
1. The utility model provides a detect time-resolved fluorescence immunochromatographic test paper strip of flumetralin, includes the bottom plate and on the bottom plate in proper order the sample absorption pad, conjugate release pad, nitrocellulose membrane and the pad that absorbs water of overlap joint pasting, its characterized in that: the conjugate release pad is embedded with a Flumetralin monoclonal antibody marked by fluorescent microspheres, a detection area and a quality control area are fixed on the nitrocellulose membrane, the detection area is sprayed with a Flumetralin hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody; the flumetralin monoclonal antibody is prepared by taking a flumetralin hapten-carrier protein conjugate as an immunogen; the flumetralin hapten-carrier protein conjugate is obtained by coupling flumetralin hapten and carrier protein, wherein the flumetralin hapten is obtained by reacting 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene with 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid to generate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid and reacting with iodoethane, and the molecular structural formula of the conjugate is as follows:
2. the time-resolved fluorescence immunochromatographic test strip for detecting flumetralin of claim 1, which comprises: the preparation reaction process of the fluvalinate hapten is as follows:
3. the time-resolved fluorescence immunochromatographic test strip for detecting flumetralin 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.
4. The time-resolved fluorescence immunochromatographic test strip for detecting flumetralin according to claim 3, characterized in that: the fluorescent substance is a lanthanide series.
5. The time-resolved fluorescence immunochromatographic test strip for detecting flumetralin according to claim 1, characterized in that: the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin.
6. A method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting flumetralin according to any one of claims 1 to 5, which is characterized by comprising the following steps:
1) preparation of conjugate release pad: labeling a flumetralin monoclonal antibody by using fluorescent microspheres, diluting the flumetralin monoclonal antibody by using a storage buffer solution, soaking a conjugate release pad in the dilution buffer solution, and performing vacuum freeze drying to prepare the flunomide monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the fluvalinate 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: and sequentially bonding a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled flumetralin monoclonal antibody, a nitrocellulose membrane fixed with a detection area and a quality control area and a water absorption pad on the bottom plate in a lap joint manner, and shearing the nitrocellulose membrane and the water absorption pad into required widths, namely the time-resolved fluorescence immunochromatographic test strip.
7. The use of the time-resolved fluorescence immunochromatographic strip for detecting flumetralin according to any one of claims 1 to 5, wherein: the method comprises the following steps:
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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