CN109061158B - Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof - Google Patents
Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof Download PDFInfo
- Publication number
- CN109061158B CN109061158B CN201811104821.4A CN201811104821A CN109061158B CN 109061158 B CN109061158 B CN 109061158B CN 201811104821 A CN201811104821 A CN 201811104821A CN 109061158 B CN109061158 B CN 109061158B
- Authority
- CN
- China
- Prior art keywords
- acetamiprid
- test strip
- detection
- nitrocellulose membrane
- hapten
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—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 acetamiprid 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 acetamiprid 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 acetamiprid hapten-carrier protein conjugate, the quality control area is sprayed with a goat anti-mouse anti-antibody, and the acetamiprid hapten is formed by sequentially overlapping and adhering a sample absorption pad, a conjugate release pad, a nitrocellulose membrane and a water absorption pad on the base plateE‑N- (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide with aminopropionic acid. The invention also provides a preparation method of the test strip and a method for detecting acetamiprid in a sample by applying the test strip. 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 and on-site monitoring of acetamiprid 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 acetamiprid in tobacco and tobacco products, and a preparation method and application thereof.
Background
Acetamiprid belongs to a neonicotinoid insecticide, which is an important insecticide following organophosphorus, carbamate and pyrethroid insecticides, and is mainly used for blocking normal conduction of a central nervous system of an insect by selectively controlling nicotinic acetylcholinesterase receptors (nAChRs) of the central nervous system of the insect so as to cause the insect to be paralyzed systemically and die. Acetamiprid is a representative agent of a neonicotinoid insecticide, is widely used for preventing and controlling various pests in seeds, leaf surfaces and soil in agricultural production, has ideal effect on preventing and controlling pests such as aphids, leafhoppers, bemisia tabaci, leaf miners and the like, and is mainly used for preventing and controlling myzus persicae in tobacco. Pesticide residue control is an important content of product quality safety control and is a key point of common attention of government agencies, manufacturing enterprises and consumers. Acetamiprid is widely used in recent years, has high detection rate and is frequently over-limited. In order to strictly control the quality, GB 2763-2016 (national Standard for food safety food) specifies that the Maximum Residual Limit (MRL) of acetamiprid in vegetables is 0.2-1 mg/kg, the maximum residual limit in fruits is 0.5-2 mg/kg, the maximum residual limits in brown rice, wheat, cottonseed and tea are 0.5, 0.1 and 10 mg/kg respectively, China does not specify the maximum residual limit of acetamiprid in tobacco, and International Cooperation center for tobacco science research (CORESTA) specifies that the guiding residual limit of acetamiprid in tobacco is 3 mg/kg. Therefore, in order to avoid the harm of the residual acetamiprid to human bodies and break through foreign trade barriers, it is necessary to establish a simple, rapid, accurate and reliable method for detecting the residual acetamiprid.
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.
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.
Disclosure of Invention
The invention aims to provide a time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid, which has the advantages of 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 acetamiprid.
In order to achieve the purpose, the invention adopts a technical scheme that:
the time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid 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 acetamiprid 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 acetamiprid hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody.
The acetamiprid monoclonal antibody is prepared by taking an acetamiprid hapten-carrier protein conjugate as an immunogen; the acetamiprid hapten-carrier protein conjugate is obtained by coupling acetamiprid hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroxine or human serum albumin, and the acetamiprid hapten is prepared by mixing acetamiprid hapten and carrier proteinE-N- (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide and aminopropionic acid, the molecular structural formula of which is:
the preparation method of the acetamiprid hapten specifically comprises the following steps:
getE-N0.50 g of- (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide is dissolved by adding 50 mL of methanol, 0.30 g of aminopropionic acid is added, the mixture is stirred, 0.37 mL of 37 percent formaldehyde aqueous solution is added, the mixture is stirred and mixed evenly, and the mixture is reacted for 4 hours at 80 ℃; stopping reaction, removing methanol by rotary evaporation, adding 50 mL of water, extracting with 100 mL of ethyl acetate for three times, combining organic phases, drying by rotary evaporation, loading on a silica gel column, and adding dichloromethane/methanol (C/E)V/V10/1) to obtain 0.69 g of carboxyl acetamiprid as 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 goat anti-mouse anti-antibody is obtained by immunizing a goat by taking a murine antibody as an immunogen.
The invention adopts another technical scheme that a method for preparing the time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid is provided, which comprises the following steps:
1) preparation of conjugate release pad: marking the acetamiprid monoclonal antibody by using a commercially available fluorescent microsphere, diluting the 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 acetamiprid monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the acetamiprid 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 acetamiprid 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) by passingE-NReacting (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide with aminopropionic acid to prepare acetamiprid hapten;
2) the acetamiprid hapten and carrier protein are coupled to prepare an acetamiprid hapten-carrier protein conjugate;
3) immunizing a mouse by using an acetamiprid hapten-carrier protein conjugate, and fusing and screening spleen cells of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting an acetamiprid monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) respectively spraying the acetamiprid 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 2 h, and drying at 37 deg.C for 2 h;
7) marking the acetamiprid monoclonal antibody by using a commercially available fluorescent microsphere, diluting the 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 a sample absorption pad, a conjugate release pad embedded with a fluorescent microsphere labeled acetamiprid 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 acetamiprid in detecting acetamiprid 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 acetamiprid;
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 acetamiprid 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 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 acetamiprid in tobacco and tobacco products exists, and the invention fills the gap. 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 acetamiprid 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 shows a scheme for synthesis of acetamiprid 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 composition of time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid
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 acetamiprid hapten-carrier protein conjugate (acetamiprid 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 fluorescence immunochromatographic test strip for detecting acetamiprid
The preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid mainly comprises the following steps:
1) preparation of conjugate release pad: marking the acetamiprid monoclonal antibody by using a commercially available fluorescent microsphere, diluting the 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 acetamiprid monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the acetamiprid 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 acetamiprid 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 acetamiprid hapten-carrier protein conjugate
Acetamiprid 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 acetamiprid hapten
GetE-N0.50 g of- (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide is dissolved by adding 50 mL of methanol, 0.30 g of aminopropionic acid is added, the mixture is stirred, 0.37 mL of 37 percent formaldehyde aqueous solution is added, the mixture is stirred and mixed evenly, and the mixture is reacted for 4 hours at 80 ℃; stopping reaction, removing methanol by rotary evaporation, adding 50 mL of water, extracting with 100 mL of ethyl acetate for three times, combining organic phases, drying by rotary evaporation, loading on a silica gel column, and adding dichloromethane/methanol (C/E)V/V10/1) to obtain 0.69 g of carboxyl acetamiprid as hapten product with yield of 92.81%.
Taking the above hapten, and performing nuclear magnetic resonanceHydrogen resonance spectroscopy identification, 1H NMR (CDCl)3, 300MHZ）δ:11.0 (1H, -COOH), 8.53 (1H, s, ArH), 7.86 (1H, d, ArH), 7.21 (1H, d, ArH), 4.16 (1H, dd, =CH-), 3.91 (1H, s, -CH2-), 3.21 (2H, d, -CH2-), 2.82 (2H, t, -CH2-), 2.49 (2H, t, -CH2-), 2.26 (3H, s, -CH3) 2.0 (1H, s, -NH-). The chemical shift δ =11 is the resonance absorption peak of carboxyl hydrogen on the spacer arm, δ =2.82, 2.49 is the resonance absorption peak of methylene hydrogen on the spacer arm, and the existence of these peaks proves the success of spacer arm coupling.
(2) Preparation of immunogens
The acetamiprid hapten and Bovine Serum Albumin (BSA) are coupled to obtain the immunogen.
Dissolving 11 mg of hapten carboxyl acetamiprid in 1 mL of dimethyl sulfoxide, adding 0.18 mL of isobutyl chloroformate and 0.1 mL of triethylamine, and reacting at 0-4 ℃ for 1h to obtain a hapten activating solution A; taking 50 mg of BSA, and adding 3 mL of 0.8% saline to dissolve the BSA to obtain solution B; dripping the A solution into the B solution, continuously stirring for reaction for 5h, dialyzing and purifying 0.02 mol/L PB for 3 days, changing the solution 3 times per day to obtain immunogen, subpackaging, and storing at-20 ℃.
(3) Preparation of coating antigen
The acetamiprid hapten is coupled with Ovalbumin (OVA) to obtain the coating antigen.
Dissolving hapten carboxyl acetamiprid 5 mg in dimethyl sulfoxide 1 mL, adding Dicyclohexylcarbodiimide (DCC) 9 mg and N-hydroxysuccinimide (NHS) 6 mg, and reacting at room temperature for 1h to obtain hapten activation solution A; dissolving OVA 50 mg in 0.8% saline 3 mL to obtain solution B; dripping the A solution into the B solution, continuously stirring for reaction for 5h, dialyzing and purifying 0.02 mol/L PB for 3 days, changing the solution 3 times per day to obtain coating source, subpackaging, and storing at-20 deg.C.
(4) Identification
And (2) carrying out ultraviolet (200 nm-400 nm) scanning measurement according to the proportion of the hapten, the carrier protein and the coupling product used in the reaction for synthesizing the acetamiprid coupling antigen, and calculating the binding ratio of the three by comparing the light absorption values of the three at 260 nm and 280 nm respectively. Compared with the maximum absorption peaks of the acetamiprid hapten and carrier protein, the maximum absorption peak of the conjugate acetamiprid hapten-carrier protein is obviously changed, which indicates that the synthesis of the acetamiprid hapten-carrier protein is successful. The binding ratio of hapten to BSA was calculated to be 14:1 and OVA was calculated to be 9: 1.
2. Preparation of acetamiprid monoclonal antibody
(1) Obtaining hybridoma cells
1) First immunization: fully emulsifying an acetamiprid hapten-BSA conjugate (immunogen) with an equal amount of Freund complete adjuvant, and injecting 0.2 mL of 6-week-old Balb/c mice each subcutaneously;
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 holes by using a limiting dilution method to obtain and establish a hybridoma cell strain which stably secretes the acetamiprid monoclonal antibody, preparing the hybridoma cells in the logarithmic growth phase into cell suspension by using a freezing medium, subpackaging in a freezing tube, and storing in liquid nitrogen for a long time.
(2) Preparation of monoclonal antibodies
1) Cell recovery: taking out the freeze storage tube of the acetamiprid monoclonal antibody hybridoma cell strain, immediately putting the freeze storage tube into a water bath at 37 ℃ for quick melting, centrifugally removing a frozen stock solution, and transferring the frozen stock 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 acetamiprid 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 acetamiprid hapten-OVA conjugate, adding an acetamiprid standard solution, an acetamiprid monoclonal antibody solution and a horseradish 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.
(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, nicotine insecticides (acetamiprid, imidacloprid, nitenpyram, thiacloprid, thiamethoxam, clothianidin and dinotefuran) are diluted in series, are 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% of acetamiprid, less than 1% of imidacloprid, less than 1% of nitenpyram, less than 1% of thiacloprid, less than 1% of thiamethoxam, less than 1% of clothianidin and less than 1% of dinotefuran. The antibody of the invention has no cross reaction to other nicotine insecticides such as imidacloprid, nitenpyram, thiacloprid, thiamethoxam, clothianidin and dinotefuran, and only has specific binding to acetamiprid.
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 acetamiprid 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 acetamiprid 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 acetamiprid monoclonal antibody marked by the fluorescent microspheres with a storage buffer solution, soaking the conjugate release pad in the dilution buffer solution, and performing vacuum freeze drying for later use.
6. Preparation of cellulose Nitrate (NC) membranes
Diluting the acetamiprid 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 acetamiprid 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 2 h 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 acetamiprid 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 acetamiprid
1. Tobacco sample pretreatment
Weighing 1.0 +/-0.05 g of crushed tobacco leaf sample into a polystyrene centrifuge tube; adding 5 mL of 50% methanol aqueous solution, and sufficiently crushing the mixture by using a homogenizer to obtain a sample solution; and transferring 75 mu L of sample liquid, mixing with 425 mu L of deionized water uniformly, and detecting.
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 the acetamiprid 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 acetamiprid concentration, and finally obtains the content of the acetamiprid 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 the acetamiprid 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, indicating that the sample does not contain the acetamiprid or the concentration of the acetamiprid is lower than the detection limit.
Positive (+): and if the result on the display screen of the fluorescence detector is positive, indicating that the concentration of the acetamiprid in the sample is equal to or higher than the detection limit.
No effect: 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. Sensitivity test
The acetamiprid standard substance is respectively diluted to 1, 2 and 4 mu g/L, and the used dilution is phosphate buffer solution with the pH value of 7.2 and 0.2 mol/L.
The detection is carried out by using a time-resolved fluorescence immunochromatographic test strip, and the result is as follows: when the concentration of the acetamiprid standard substance is 1 mug/L, the detection of the fluorescence detector is negative; when the concentration of the acetamiprid standard substance is 2 mug/L and 4 mug/L, the detection of the fluorescence detector is positive, which shows that the sensitivity of the test strip for detecting the acetamiprid is 2 mug/L.
2. Limit of detection test
Respectively adding acetamiprid standard substances into blank tobacco samples until the final concentrations are 0.05, 0.1 and 0.2 mg/kg, and detecting by using a time-resolved fluorescence immunochromatographic test strip, wherein the results are as follows: when the concentration of the acetamiprid is 0.05 mg/kg, the detection of the fluorescence detector is negative; when the concentrations of the acetamiprid are 0.1 and 0.2 mg/kg, the detection of the fluorescence detector is positive, which shows that the detection limit of the test strip on the acetamiprid in the tobacco is 0.1 mg/kg.
3. Test for false positive and false negative rates
20 parts of positive tobacco leaf samples with known acetamiprid content of more than 0.1 mg/kg and 20 parts of negative tobacco leaf samples without acetamiprid are respectively detected by 3 time-resolved fluorescence immunochromatographic test strips produced in batches, and the negative and positive rates of the positive tobacco leaf samples and the negative tobacco leaf samples are calculated. The results are shown in the following table.
Results of positive and negative samples of the tabular detection
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 acetamiprid can be used for quickly detecting acetamiprid in tobacco leaves.
4. Specificity test
And detecting imidacloprid, nitenpyram, thiacloprid, thiamethoxam, clothianidin and dinotefuran at the concentration of 10 mg/L by using an acetamiprid test strip. The result shows that the detection of the fluorescence detector is negative. The test paper strip has no cross reaction to imidacloprid, nitenpyram, thiacloprid, thiamethoxam, clothianidin and dinotefuran.
Claims (6)
1. A time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid 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 acetamiprid 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 acetamiprid hapten-carrier protein conjugate, and the quality control area is sprayed with a goat anti-mouse anti-antibody; the acetamiprid monoclonal antibody is prepared by taking an acetamiprid hapten-carrier protein conjugate as an immunogen; the acetamiprid hapten-carrier protein conjugate is obtained by conjugating acetamiprid hapten and carrier protein, wherein the acetamiprid hapten is prepared byE-N- (4- (6-chloropyridin-3-yl) 2-buten-2-yl) cyanamide and aminopropionic acid, the molecular structural formula of which is:
the reaction process is as follows:
2. the time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid according to claim 1, which is 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 acetamiprid according to claim 2, characterized in that: the fluorescent substance is a lanthanide.
4. The time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid according to claim 1, which is characterized in that: the carrier protein is bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein or human serum albumin.
5. The preparation method of the acetamiprid detection time-resolved fluorescence immunochromatographic test strip 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 acetamiprid monoclonal antibody by using fluorescent microspheres, diluting the 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 acetamiprid monoclonal antibody;
2) preparation of nitrocellulose membrane: spraying the acetamiprid 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 acetamiprid 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 acetamiprid detection time-resolved fluorescence immunochromatographic strip according to any one of claims 1 to 4, characterized by comprising the following steps:
1) pretreating a sample;
2) detecting with the test strip;
3) and analyzing the detection result by using a fluorescence detector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104821.4A CN109061158B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811104821.4A CN109061158B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109061158A CN109061158A (en) | 2018-12-21 |
| CN109061158B true CN109061158B (en) | 2021-09-03 |
Family
ID=64763364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811104821.4A Active CN109061158B (en) | 2018-09-21 | 2018-09-21 | Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061158B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111308069B (en) * | 2020-03-09 | 2023-06-06 | 中国农业科学院烟草研究所 | Time-resolved fluorescence immunochromatography test paper card for testing clothianidin |
| CN112698026B (en) * | 2020-11-18 | 2023-12-12 | 山东勤邦生物技术有限公司 | Test strip for detecting clothianidin and application thereof |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010041724A1 (en) * | 1998-08-04 | 2001-11-15 | Carla Jean Klittich | Method for the control of flea beetles in canola |
| AU2184099A (en) * | 1999-01-27 | 2000-08-18 | Sumitomo Chemical Company, Limited | Insecticidal compositions and insecticidal methods |
| CZ303846B6 (en) * | 1999-03-12 | 2013-05-29 | Basf Aktiengesellschaft | Synergistic insecticidal composition |
| UA76708C2 (en) * | 1999-12-08 | 2006-09-15 | Сінгента Патисипейшонс Аг | Antibody used in immunological analysis of sample for determining neonicotinoid insecticide, protein conjugate for obtaining antibody, method for determination of neonicotinoid insecticide concentration in sample and set for determination of amount of neonicotinoid insecticide |
| CN102174013A (en) * | 2011-03-10 | 2011-09-07 | 江苏长青农化股份有限公司 | New synthesis technology of acetamiprid |
| 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 |
| CN104897863B (en) * | 2014-03-06 | 2016-08-17 | 北京勤邦生物技术有限公司 | A kind of fluorescent micro-ball immune chromatography test paper strip detecting Aflatoxins M1 and method |
| CN105158461A (en) * | 2015-09-22 | 2015-12-16 | 浙江大学 | Rapid detecting method and corresponding test strip for acetamiprid residues in tea |
| CN106771238B (en) * | 2016-12-14 | 2018-08-10 | 北京勤邦生物技术有限公司 | Detect the fluorescent micro-ball immune chromatography test paper strip and its preparation method and application of melamine residual |
| CN108051489B (en) * | 2017-09-15 | 2020-06-02 | 浙江省农业科学院 | Electrochemical sensor for detecting acetamiprid and preparation method and application thereof |
| CN107602678B (en) * | 2017-09-21 | 2021-03-05 | 福建省农业科学院农业生物资源研究所 | Acetamiprid-coupled Bt insecticidal toxin and preparation method and application thereof |
| CN108508215A (en) * | 2018-06-01 | 2018-09-07 | 北京勤邦生物技术有限公司 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application of detection tetracycline medication |
-
2018
- 2018-09-21 CN CN201811104821.4A patent/CN109061158B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109061158A (en) | 2018-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109111394B (en) | Preparation method and application of acetamiprid hapten and antigen | |
| JP4980536B2 (en) | Neonicotine pesticide immunoassay | |
| CN109324182B (en) | Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof | |
| CN109061146B (en) | Test strip for detecting acetamiprid and preparation method and application thereof | |
| CN109061169B (en) | Enzyme linked immunosorbent assay kit for detecting acetamiprid and application thereof | |
| US10234470B2 (en) | Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof | |
| CN109061152A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting carbendazim | |
| CN109061158B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting acetamiprid and preparation method and application thereof | |
| CN108912090B (en) | Test strip for rapidly detecting total amount of alternariol and alternariol monomethyl ether | |
| CN110850090A (en) | Test strip for detecting bifenthrin and application thereof | |
| CN109307761B (en) | Indirect competitive ELISA method for detecting furaldehyde | |
| CN109053477B (en) | Preparation method and application of butralin hapten and butralin antigen | |
| CN109061149B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting butralin and preparation method and application thereof | |
| CN109061157B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting flumetralin and preparation method and application thereof | |
| CN109061156B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting pendimethalin and preparation method and application thereof | |
| US20050043515A1 (en) | Tobacco-specific nitrosamine detection assays and reagents | |
| CN109061150B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting metalaxyl and preparation method and application thereof | |
| CN109061153B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting iprodione and preparation method and application thereof | |
| CN109061154B (en) | Fluorescent microsphere immunochromatography test strip for detecting metalaxyl and preparation method and application thereof | |
| CN109061148B (en) | Test strip for detecting butralin and preparation method and application thereof | |
| CN108844932B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting safrole as well as preparation method and application thereof | |
| CN109061151B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting quinclorac and preparation method and application thereof | |
| CN109265395B (en) | Preparation method and application of quinclorac hapten and antigen | |
| CN108680738B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting coumarin and preparation method and application thereof | |
| CN111751535A (en) | Test strip for detecting endosulfan and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |