CN112147332B - Colloidal gold immunochromatography test strip for rapidly detecting endosulfan and preparation and detection methods thereof - Google Patents
Colloidal gold immunochromatography test strip for rapidly detecting endosulfan and preparation and detection methods thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—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
- G01N33/587—Nanoparticles
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D327/00—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms
- C07D327/10—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms two oxygen atoms and one sulfur atom, e.g. cyclic sulfates
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Abstract
The invention provides a colloidal gold immunochromatography test strip for rapidly detecting endosulfan, and a preparation and detection method thereof, comprising the preparation of hapten: the method comprises the steps of derivatizing endosulfan alcohol with a similar structure to endosulfan by a succinic anhydride method, and introducing carboxyl to prepare endosulfan hapten; preparation of complete antigen: respectively preparing immunogen and coating antigen from the endosulfan hapten coupled with protein by an activated ester method; preparation of monoclonal antibodies: immunizing a mouse with the immunogen to prepare a thiodan monoclonal antibody; preparing colloidal gold immunochromatography test paper: and coating the secondary antibody on a nitrocellulose membrane to serve as a quality control line, and spraying the coating antigen on the nitrocellulose membrane to serve as a detection line. The method is suitable for rapid screening of a large number of samples on site, has high sensitivity, high detection speed and low cost, does not need instrument assistance, can realize the on-site detection of the endosulfan, and is suitable for the field of food safety detection.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a colloidal gold immunochromatography test strip for rapidly detecting endosulfan, and a preparation method and a detection method thereof.
Background
The chemical names of the endosulfan, shuodan and Anshadan are 1,2,3,4,7,7-hexachlorobicyclo (2, 1) -heptene- (2) dihydroxymethyl-5, 6-sulfite, and the molecular formula is C 9 H 6 Cl 6 O 3 S exists mainly in the form of a mixture of two isomers (alpha-endosulfan and beta-endosulfan). The endosulfan is a cyclopentanediyl organic chlorine pesticide developed and synthesized in 1945, and has good insecticidal effectThe pesticide composition has strong lasting effect and is widely applied to pest control of crops such as fruits, vegetables, tea and the like, and the pesticide composition can block the inflow of chloride ions by combining with a chloride ion channel to cause neurotoxic effect so as to kill insects. Endosulfan is a typical Persistent Organic Pollutant (POPs), which has been proven by the national cancer agency to be one of the important causes of cancer induction by animal experiments. In addition, the endosulfan has the characteristics of high biotoxicity, durability, long-distance migration and the like in the environment, can be accumulated in water sources, food chains and human tissues for a long time, and has potential carcinogenicity to cause sustainable harm to human bodies, so that the endosulfan has attracted wide attention at home and abroad. In 2011, the endosulfan is listed in a forbidden substance list in the 'Stockholm convention', and in the 3 rd month and the 26 th year of 2019, the application of the endosulfan in agriculture is completely forbidden in China, and the maximum residual limit of pesticides in foods is regulated to be 0.05mg/kg in vegetable and fruit residual limits.
Currently, the detection method for endosulfan mainly depends on the traditional instrument analysis method, and mainly comprises High Performance Liquid Chromatography (HPLC), gas chromatography-tandem mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS). The analysis method has low detection speed, requires expensive instruments and professional operators, is greatly influenced by sample pretreatment, has a certain limit on application range, and cannot meet the rapid detection of a large number of samples on site. The immunological detection method is a method based on a specific reaction of an antigen-antibody and detecting by color development. There are only two immunoassay detection methods for endosulfan which are authorized or announced in China at present. The minimum detection limit of the colloidal gold test paper for rapidly detecting the endosulfan and the DDT residues in the colloidal gold test paper (authorized bulletin number: CN 2844924Y) is 0.5-1 mg/kg, and the national limit standard can not be met. The organic chlorine pesticide endosulfan artificial antigen and antibody, and the preparation method and the ELISA method established in the application (publication No. CN 1700001A) thereof have the disadvantages of complicated experimental operation steps and long time consumption, and the detection result is not suitable for on-site rapid detection due to the assistance of instruments.
Disclosure of Invention
Aiming at the problems, the invention provides the colloidal gold immunochromatography test strip for rapidly detecting the endosulfan, and the preparation and detection method thereof, wherein the colloidal gold immunochromatography test strip with rapid detection speed, low cost and high sensitivity is established on the basis of the monoclonal antibody by preparing the high-quality endosulfan monoclonal antibody, and the test strip is only used for specifically detecting the endosulfan, has rapid detection speed and low cost, does not need instrument assistance and is suitable for rapidly detecting and screening a large number of samples on site.
A preparation method of a colloidal gold immunochromatographic test strip for rapidly detecting endosulfan comprises the following steps:
preparation of hapten: the method comprises the steps of derivatizing endosulfan alcohol with a similar structure to endosulfan by a succinic anhydride method, and introducing carboxyl to prepare endosulfan hapten;
preparation of complete antigen: respectively preparing immunogen and coating antigen from the endosulfan hapten coupled with protein by an activated ester method;
preparation of monoclonal antibodies: preparing a monoclonal antibody by using the immunogen, detecting and screening by using the coating antigen through ic-ELISA, and performing cell fusion to obtain a endosulfan monoclonal antibody;
preparing colloidal gold immunochromatography test paper: and coating the secondary antibody on a nitrocellulose membrane to serve as a quality control line, spraying the coating antigen on the nitrocellulose membrane to serve as a detection line, and then assembling according to the structure of the test strip.
In the above scheme, the preparation method of the hapten specifically comprises the following steps:
dissolving endosulfan alcohol in pyridine, adding succinic anhydride and 4-dimethylaminopyridine, rotary evaporating, adding pure water to dissolve precipitate, adding NaOH solution to regulate pH to 9-10, extracting with n-hexane, collecting all water phase, regulating pH of water phase solution to 3-4 with HCl solution, separating out solid, filtering and stoving to obtain endosulfan hapten.
Further, the concentration of the HCl solution and the NaOH solution is 1M.
Further, the dosage ratio of the thiodanol, the pyridine, the succinic anhydride and the 4-dimethylaminopyridine is 20-50 mg:1-3 mL: 8-20 mg: 1-3 mg.
In the above scheme, the preparation of the complete antigen is to couple the hapten with bovine serum albumin to prepare immunogen through an activated ester method, and couple the hapten with chicken ovalbumin to prepare coating antigen through an activated ester method, specifically comprising the following steps:
dissolving the endosulfan hapten in dimethylformamide, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC, and performing room-temperature reaction for activation to obtain the activated endosulfan hapten;
respectively dissolving bovine serum albumin BSA and chicken egg albumin OVA in a carbonate buffer solution CBS, dropwise adding the activated endosulfan hapten into the bovine serum albumin BSA and chicken egg albumin OVA solution, reacting at room temperature, and dialyzing with phosphate buffer PBS to respectively obtain immunogen and coating antigen.
Further, in the activation process of the endosulfan hapten, the dosage mole ratio of the endosulfan hapten, N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC is 1: 2-3: 2 to 3.
Further, in the preparation process of the immunogen and the coating antigen, the mol ratio of the bovine serum albumin BSA, the chicken ovalbumin OVA and the endosulfan hapten is 30-50: 30-50: 1.
in the above scheme, the preparation of the colloidal gold immunochromatographic test strip specifically comprises the following steps:
the colloidal gold immunochromatographic test strip comprises a sample pad, a Nitrocellulose (NC) membrane, a water absorption pad and a bottom plate, wherein the nitrocellulose membrane is stuck in the middle of the bottom plate, the water absorption pad and the sample pad are respectively stuck on the upper side and the lower side of the bottom plate, the secondary antibody is coated on the nitrocellulose membrane to serve as a quality control line, and the coating raw material is sprayed on the nitrocellulose membrane to serve as a detection line, so that the colloidal gold immunochromatographic test strip is prepared.
A colloidal gold immunochromatographic test strip for rapidly detecting endosulfan is prepared according to a preparation method of the colloidal gold immunochromatographic test strip for detecting endosulfan.
According to the detection method of the colloidal gold immunochromatographic test strip for rapidly detecting the endosulfan, the monoclonal antibody is marked with a colloid Jin Dedao gold-labeled antibody, a endosulfan sample extracting solution to be detected and the gold-labeled antibody are incubated for 5min, the colloidal gold immunochromatographic test strip is inserted, and the detection result is interpreted after 3 min.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the thiodanol which has a core structure of the thiodan and is similar to the structure of the thiodan is selected as a hapten, and is derivatized and coupled with carrier protein to prepare immunogen and coating antigen; the prepared immunogen is immunized with mice to prepare monoclonal antibodies with high sensitivity and strong specificity. And then a colloidal gold immunochromatographic test strip which has high detection speed, low detection cost and high sensitivity and only specifically recognizes the endosulfan is established on the basis of the monoclonal antibody. The colloidal gold immunochromatographic test strip prepared by the invention has strong specificity, high detection speed, no need of instrument assistance, low cost and suitability for rapid detection of endosulfan on site.
2. The colloidal gold immunochromatographic test paper prepared by the invention only specifically recognizes the endosulfan, and the endosulfan has high biotoxicity and accumulation, so that the colloidal gold immunochromatographic test paper has great value and significance for detecting the endosulfan.
3. The test strip prepared by the colloidal gold labeled monoclonal antibody has high sensitivity, the detection result can be directly and visually interpreted, the detection cost is low, the detection speed is high, the sample extracting solution and the gold labeled antibody are directly incubated for 5min, the test strip is inserted, the detection result can be interpreted after 3min, and the method is suitable for rapid detection of a large number of samples.
4. The colloidal gold immunochromatographic test strip prepared by the invention has simple detection steps and does not need professional operators; the detection can be carried out at any time and any place without the assistance of instruments, and the requirements of crowds such as law enforcement departments, farmers, consumers and the like can be met.
Drawings
FIG. 1 is a schematic structural diagram of a colloidal gold immunochromatographic test strip according to an embodiment of the present invention.
Fig. 2 is a diagram of a gel Jin Biaozheng according to an embodiment of the present invention: (A) is an ultraviolet diagram; and (B) is a Transmission Electron Microscope (TEM) of colloidal gold.
Fig. 3 is a diagram showing the practical effect of detecting the endosulfan residue by the colloidal gold immunochromatographic test strip according to an embodiment of the present invention.
Detailed Description
Embodiments of the present invention will now be described with reference to the accompanying drawings, which are given by way of illustration only and are not to be construed as limiting the invention.
The invention relates to a colloidal gold immunochromatographic test strip for rapidly detecting endosulfan, and a preparation and detection method thereof, comprising the preparation of hapten, complete antigen, monoclonal antibody, gold-labeled antibody and colloidal gold immunochromatographic test strip; the detection method of the colloidal gold immunochromatography test strip suitable for rapidly screening a large number of samples on site is prepared based on the monoclonal antibody. The detection method has the advantages of high sensitivity, high detection speed, low cost and no need of instrument assistance, and can realize the on-site detection of the endosulfan.
A method for preparing colloidal gold immunochromatography test strip for rapidly detecting endosulfan includes preparing colloidal gold immunochromatography test strip of endosulfan, preparing immunogen and monoclonal antibody of anti endosulfan, and assembling and preparing test strip. The method specifically comprises the following steps:
preparation of hapten: the method comprises the steps of derivatizing endosulfan alcohol with a similar structure to endosulfan by a succinic anhydride method, and introducing carboxyl to prepare hapten, wherein the hapten is specifically as follows: sulfosanol is dissolved in pyridine, succinic anhydride and 4-dimethylaminopyridine are added for reaction at 50 ℃ for 6 hours, rotary evaporation is carried out, 0.5mL of pure water is added for dissolving precipitate, the pH of the solution is adjusted to 9-10 by NaOH solution, and 5mL of n-hexane is used for extraction. All water phases are collected, the pH value of the water phase solution is adjusted to 3-4 by using HCl solution, solid is separated out, and the product obtained after filtration and drying is the endosulfan hapten.
Preferably, the concentration of the HCl solution and the NaOH solution is 1M; the dosage ratio of the endosulfan alcohol, the pyridine, the succinic anhydride and the 4-dimethylaminopyridine is 20-50 mg: 1-3 mL: 8-20 mg: 1-3 mg.
Preparation of complete antigen: the hapten is respectively coupled with bovine serum albumin and chicken ovalbumin by an activated ester method to prepare immunogen and coating antigen, and the method specifically comprises the following steps:
1) Dissolving the endosulfan hapten in dimethylformamide DMF, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC, and reacting for 6 hours at room temperature for activation to obtain the activated endosulfan hapten.
2) Respectively dissolving bovine serum albumin BSA and chicken egg albumin OVA in a carbonate buffer solution CBS, dropwise adding the activated endosulfan hapten into the bovine serum albumin BSA and chicken egg albumin OVA solution, reacting for 5 hours at room temperature, and dialyzing for 3 days by using phosphate buffer PBS to obtain the immunogen and the coating antigen.
Preferably, in the activation process of the endosulfan hapten, the molar ratio of the endosulfan hapten to the NHS to the EDC is 1: 2-3: 2 to 3.
Preferably, in the preparation process of the immunogen and the coating antigen, the mol ratio of the bovine serum albumin BSA, the chicken ovalbumin OVA and the endosulfan hapten is 30-50: 30-50: 1.
the bovine serum albumin BSA and chicken ovalbumin OVA were purchased from Shanghai Sigma-Aldrich chemical reagent company and endosulfan alcohol were purchased from Shanghai carbofuran biological reagent Co.
Preparation of monoclonal antibodies: the immunogen is used for immunizing mice, the serum of the mice is detected by ic-ELISA, and the mice with the best quality are screened for cell fusion to prepare the endosulfan monoclonal antibody, which comprises the following steps:
the prepared immunogen was diluted to 2mg/mL with physiological saline, and then added with an equal volume of Freund's complete adjuvant, and emulsified sufficiently with an emulsifier. Mice were immunized by subcutaneous multipoint injection on the back, with 100 μg per mouse, the first immunization of the mice. The mice were then boosted every three weeks, fully emulsified with immunogen diluted to 2mg/mL and an equal volume of Freund's incomplete adjuvant, and immunized in the same manner as the first immunization, with an immunization dose of 50 μg per mouse. Starting from the third immunization, mice with high titers and high inhibition were screened by detecting mouse tail serum by ic-ELISA and were intraperitoneally injected with 20. Mu.g of immunogen. Three days later, mice were sacrificed and spleen cells were taken and ground to prepare spleen cell suspensions. The spleen cells and myeloma cells of the mice are fully and evenly mixed according to the ratio of 10:1, the mixture is centrifuged for 10min under the condition of 1200r/min, and the supernatant is discarded. Lightly knocking the bottom of the centrifuge tube with a finger to uniformly disperse cells; PEG1500 was slowly added over the first minute, 10mL of RMI-1640 medium was added after 1min of rest to terminate the PEG effect, and the fused cells were left in the cell incubator for 10min and then centrifuged at 3500r/min for 10min. Cells were resuspended in HAT medium containing 12% fetal bovine serum, mixed and plated in 96-well plates at 150. Mu.L/well and cultured in a cell incubator. And screening cell supernatant by adopting an indirect competition ic-ELISA method, selecting a cell hole with high inhibition rate and strong positive to subclone, and carrying out expansion culture after 3 times of subcloning to obtain the target hybridoma cell strain.
Preparation and purification of ascites: the hybridoma cells obtained above were diluted to 1X 10 per ml with PRMI-1640 medium 6 ～1×10 8 And (3) injecting hybridoma cells of cells into each mouse, performing intraperitoneal injection according to 300-500 uL, swelling the abdomen of the mouse, collecting ascites, centrifuging at 3500r/min for 10min, collecting supernatant, and purifying by using n-octanoic acid-ammonium sulfate to obtain the monoclonal antibody.
The purification method of the n-octanoic acid-ammonium sulfate comprises the following specific steps: 3mL of ascites is taken in a 50mL centrifuge tube, 6mL of acetic acid buffer is added, and after uniform mixing, the pH of the solution is adjusted to 5 by using 1M HCl. Along the tube wall, 99. Mu.L of n-octanoic acid was slowly added dropwise with stirring. Standing at 4deg.C for 2 hr, centrifuging at 10000r/min for 20min, removing precipitate, collecting supernatant, and filtering. 1mL of 0.01M PBS (pH 7.4) was added and the pH of the solution was adjusted to 7.4 with 1M NaOH. Slowly adding 2.43g of ammonium sulfate into the solution, standing at 4 ℃ for 3h, centrifuging for 20min and discarding the supernatant. The precipitate was dissolved in an appropriate amount of 0.01M PBS (pH 7.4) solution, transferred to a dialysis bag, dialyzed at 4 ℃ for three days under magnetic stirring, and the dialysate was changed twice daily.
Preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold, freeze-drying and preserving for later use, wherein the method specifically comprises the following steps: an appropriate amount of ultrapure water was taken in the flask, and heated and boiled for 30min. Adding 10% chloroauric acid solution under magnetic stirring, adding 10% trisodium citrate solution after 5min, and continuing stirring until the color of the solution does not change to obtain colloidal gold solution. 1-5 mL of the colloidal gold solution is placed on a magnetic stirrer, boric acid buffer solution with pH of 8.5 is added dropwise, the prepared endosulfan monoclonal antibody is slowly added for reaction for 35min, and then 10% BSA is added dropwise for blocking. The solution was centrifuged at 4℃for 15min at 10000r/min for 20min, the supernatant was discarded, and the pellet was dissolved in 300. Mu.L of a heavy suspension containing 2% BSA, 2% sucrose, 3% trehalose and 0.02% sodium azide in PBS. The gold-labeled antibody prepared above was diluted 10 to 30 times, and 50. Mu.L was lyophilized in a 96-well plate.
The dosage ratio of the pure water, the chloroauric acid solution and the trisodium citrate solution is 100-200 mL: 1-2 mL: 1.2-2.4 mL; the dosage ratio of the colloidal gold solution, the monoclonal antibody, the boric acid buffer solution and the 10% BSA is 1-3 mL: 5-25 mug: 100-200 mu L: 100-300 mu L.
Preparing colloidal gold immunochromatography test paper: and (3) sticking NC films in the middle of the bottom plate, and respectively sticking a sample pad and a water absorption pad on the left side and the right side of the bottom plate to overlap the NC films by 2-3 mm. Spraying the secondary antibody of 0.5-1 mg/mL and the endosulfan coating antigen of 0.6-1.2 mg/mL on NC film by using a film spraying instrument to serve as C line and T line, and standing overnight in a 37 ℃ oven. And (3) assembling according to the structure of the test strip, cutting the test strip into test strips with the length of 3.8-4 mm by using a slitter, placing the test strips into a self-sealing bag, and storing the test strips in a drying oven for standby.
Detection of endosulfan: and incubating the sample extracting solution with the gold-labeled antibody for 5min, inserting the colloidal gold immunochromatographic test strip, and judging the detection result after 3 min.
The detection principle of the method is as follows: according to capillary action, the object to be detected and the gold-labeled antibody can be combined with the coating antigen of the T line and the secondary antibody of the C line in specificity to develop color when climbing upwards. If the color of the T line is deeper than or the same as that of the C line, the T line is a negative sample; if the color of the T line is lighter than that of the C line or the T line is not color, the sample is positive.
The secondary anti-goat anti-mouse IgG was purchased from Shanghai Biotechnology Inc.
Example 1
As shown in figure 1, the colloidal gold immunochromatographic test strip for rapidly detecting endosulfan is supported by a bottom Plate (PVC), a nitrocellulose membrane is arranged in the middle, and a sample pad on the left side and a water absorption pad on the right side are respectively connected with the nitrocellulose membrane. The nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is sprayed with a secondary antibody, and the detection line is sprayed with a synthetic endosulfan coating source. In the 96-well plate are conjugates of colloidal gold-labeled endosulfan monoclonal antibodies.
The main component of the sample pad material in the test strip is a glass fiber film, and the bottom plate is a polyvinyl chloride material.
The sample pad and the water absorbing pad in the test strip are overlapped with the nitrocellulose membrane by 2-3 mm.
Example 2
The preparation method of the colloidal gold immunochromatographic test strip for rapidly detecting the endosulfan comprises the following steps of:
preparation of hapten: 35.7mg of endosulfan alcohol was dissolved in 1mL of pyridine, 12mg of succinic anhydride and 2mg of 4-dimethylaminopyridine were added, reacted at 50℃for 6 hours, rotary evaporated, 0.5mL of pure water was added to dissolve the precipitate, the pH of the solution was adjusted to 10 with 1M NaOH solution, and extraction was performed with 5mL of n-hexane. All aqueous phases are collected, the pH of the aqueous phase solution is adjusted to 4 by using 1M HCl solution, solid is separated out, and the product obtained after filtration and drying is the endosulfan hapten.
Preparation of complete antigen:
4mg of endosulfan hapten is taken and dissolved in 0.5mL of DMF, 2.2mg of NHS and 4mg of EDC are added and stirred at room temperature for 6h for activation. 15mg BSA and 10mg OVA were dissolved in CBS buffer, respectively. Under magnetic stirring at 4 ℃, dropwise adding the activated endosulfan hapten solution into the BSA solution and the OVA solution, reacting for 5 hours at room temperature to obtain immunogen and coating antigen, dialyzing for 72 hours by using PBS buffer solution, and sub-packaging for later use.
Preparation of monoclonal antibodies: the prepared immunogen was diluted to 2mg/mL with physiological saline, and then added with an equal volume of Freund's complete adjuvant, and emulsified sufficiently with an emulsifier. Mice were immunized by subcutaneous multipoint injection on the back, with 100 μg per mouse, the first immunization of the mice. The mice were then boosted every three weeks, fully emulsified with immunogen diluted to 2mg/mL and an equal volume of Freund's incomplete adjuvant, and immunized in the same manner as the first immunization, with an immunization dose of 50 μg per mouse. Starting from the third immunization, mice with high titers and high inhibition were screened by detecting mouse tail serum by ic-ELISA and were intraperitoneally injected with 20. Mu.g of immunogen. Three days later, mice were sacrificed and spleen cells were taken and ground to prepare spleen cell suspensions. The spleen cells and myeloma cells of the mice are fully and evenly mixed according to the ratio of 10:1, the mixture is centrifuged for 10min under the condition of 1200r/min, and the supernatant is discarded. Lightly knocking the bottom of the centrifuge tube with a finger to uniformly disperse cells; PEG1500 was slowly added over the first minute, 10mL of RMI-1640 medium was added after 1min of rest to terminate the PEG effect, and the fused cells were left in the cell incubator for 10min and then centrifuged at 3500r/min for 10min. Cells were resuspended in HAT medium containing 12% fetal bovine serum, mixed and plated in 96-well plates at 150. Mu.L/well and cultured in a cell incubator. And screening cell supernatant by adopting an indirect competition ic-ELISA method, selecting a cell hole with high inhibition rate and strong positive to subclone, and carrying out expansion culture after 3 times of subcloning to obtain the target hybridoma cell strain.
Preparation and purification of ascites: the hybridoma cells obtained above were diluted to 1X 10 per ml with PRMI-1640 medium 6 ～1×10 8 And (3) injecting hybridoma cells of cells into each mouse, performing intraperitoneal injection according to 300uL, swelling the abdomen of the mouse, collecting ascites, centrifuging at 3500r/min for 10min, collecting supernatant, and purifying n-octanoic acid-ammonium sulfate to obtain the monoclonal antibody.
The purification method of the n-octanoic acid-ammonium sulfate comprises the following specific steps: 3mL of ascites is taken in a 50mL centrifuge tube, 6mL of acetic acid buffer is added, and after uniform mixing, the pH of the solution is adjusted to 5 by using 1M HCl. Along the tube wall, 99. Mu.L of n-octanoic acid was slowly added dropwise with stirring. Standing at 4deg.C for 2 hr, centrifuging at 10000r/min for 20min, removing precipitate, collecting supernatant, and filtering. 1mL of 0.01M PBS (pH 7.4) was added and the pH of the solution was adjusted to 7.4 with 1M NaOH. Slowly adding 2.43g of ammonium sulfate into the solution, standing at 4 ℃ for 3h, centrifuging for 20min and discarding the supernatant. The precipitate was dissolved in an appropriate amount of 0.01M PBS (pH 7.4) solution, transferred to a dialysis bag, dialyzed at 4 ℃ for three days under magnetic stirring, and the dialysate was changed twice daily.
Determination of antibody sensitivity and crossover rate: seven chlorine, toxafen and chlordane with similar structures to endosulfan are selected and their half Inhibition Concentration (IC) is measured by IC-ELISA method 50 ) And then measuring the antibody crossing rate according to a calculating formula of the crossing rate. The calculation formula of the crossing rate is as follows: crossover rate (%) = (endosulfan IC) 50 Analog IC 50 ) X 100%. The results are shown in Table 1, which shows the IC of the antibody against endosulfan, alpha-endosulfan and beta-endosulfan 50 5.16, 4.95 and 6.65ng/mL, respectively, and no crossover to heptachlor, toxafen and chlordane, indicating that the antibody only specifically recognizes the endosulfan. Development of two enzyme-linked immunosorbent assays for detection of endosulfan residues in agricultural products only the inhibition of endosulfan was analyzed, but not both isomers of endosulfan, and the antibody sensitivity herein was improved compared to that. In conclusion, the antibody prepared by the invention only specifically recognizes the endosulfan, has higher sensitivity and provides strong support for the preparation of endosulfan colloidal gold immunochromatography test strip.
TABLE 1 determination of antibody Cross Rate
Preparation of gold-labeled antibody: 100mL of ultrapure water was taken in the flask, and boiled under heating for 30min. 1mL of 10% chloroauric acid solution was added under magnetic stirring, after 5min, 1.2mL of 10% trisodium citrate solution was added, and stirring was continued until the color of the solution did not change, to obtain a colloidal gold solution. 1mL of the colloidal gold solution is placed on a magnetic stirrer, 100 mu L of boric acid buffer with pH of 8.5 is added dropwise, 8 mu g of the prepared endosulfan monoclonal antibody is slowly added for reaction for 35min, and then 100 mu L of 10% BSA is added dropwise for blocking. The solution was centrifuged at 4℃for 15min at 10000r/min for 20min, the supernatant was discarded, and the pellet was dissolved in 300. Mu.L of a heavy suspension containing 2% BSA, 2% sucrose, 3% trehalose and 0.02% sodium azide in PBS. The gold-labeled antibody prepared above was diluted 15-fold, and 50. Mu.L was lyophilized in a 96-well plate.
Preparing a colloidal gold immunochromatography test strip: coating antigen (0.8 mg/mL) and secondary antibody (0.6 mg/mL) were sprayed on NC film as T line and C line, respectively, using a film spray instrument, and oven at 37℃overnight. And (3) assembling according to the structure of the test strip, cutting the test strip into test strips with the length of 3.8mm by a slitter, putting the test strips into a self-sealing bag, and storing the test strips in a drying oven for standby. The detection method comprises the following steps: and (3) adding 150 mu L of sample extracting solution into the freeze-dried gold-labeled antibody, incubating for 5min at room temperature, inserting the prepared test strip into the hole, taking out the test strip after 3min, and directly judging the detection result.
Colloidal gold immunochromatography test strip detection limit determination: weighing 5g of pears, placing the pears into a 50mL centrifuge tube, adding the standard substances of the endosulfan, the alpha-endosulfan and the beta-endosulfan with different concentrations, adding 50mL of methanol into the centrifuge tube, vigorously shaking for 5min at 5000r/min, filtering, diluting the filtrate by using PBST, and detecting. The limit of detection is the lowest concentration where the T line color is lighter than the C line compared to the negative control. The detection results are shown in fig. 3: the detection limits of the endosulfan, the alpha-endosulfan and the beta-endosulfan in the pears are all 40ng/g.
Fig. 2 is a representation of colloidal gold: wherein (A) is an ultraviolet graph of the colloidal gold, the ultraviolet absorption peak is 522nm, and the peak is high and narrow, which indicates that the colloidal gold is successfully prepared; (B) Is a Transmission Electron Microscope (TEM) of the colloidal gold, has good dispersibility when observed under the TEM, the aggregation phenomenon is avoided, the particle size of the colloidal gold is uniform, and the average diameter is about 14 nm; the prepared colloidal gold can be used for preparing a test strip.
FIG. 3 is a graph showing the effect of detecting the endosulfan residue by using the colloidal gold immunochromatographic test strip. Wherein A, B, C is endosulfan, alpha-endosulfan and beta-endosulfan respectively, and the concentration gradients are 0, 20, 40 and 80ng/g; the test result of the test strip in the graph D shows that the detection limits of the endosulfan, the alpha-endosulfan and the beta-endosulfan are 40ng/g, and the national limit standard is reached.
It should be understood that although the present disclosure has been described in terms of various embodiments, not every embodiment is provided with a separate technical solution, and this description is for clarity only, and those skilled in the art should consider the disclosure as a whole, and the technical solutions in the various embodiments may be combined appropriately to form other embodiments that will be understood by those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (4)
1. The preparation method of the colloidal gold immunochromatographic test strip for rapidly detecting the endosulfan is characterized by comprising the following steps of:
preparation of hapten: the method comprises the steps of derivatizing endosulfan alcohol with a similar structure to endosulfan by a succinic anhydride method, and introducing carboxyl to prepare endosulfan hapten; the preparation method of the hapten specifically comprises the following steps: fully dissolving endosulfan alcohol in pyridine, adding succinic anhydride and 4-dimethylaminopyridine, performing rotary evaporation, adding pure water to dissolve precipitate, adding NaOH solution to adjust the pH to 9-10, extracting with n-hexane, collecting all water phases, adjusting the pH of the water phase solution to 3-4 with HCl solution, and separating out solids, wherein the product obtained after filtering and drying is endosulfan hapten; the concentration of the HCl solution and the NaOH solution is 1M; the dosage ratio of the endosulfan alcohol, the pyridine, the succinic anhydride and the 4-dimethylaminopyridine is 20-50 mg: 1-3 mL: 8-20 mg: 1-3 mg;
preparation of complete antigen: respectively preparing immunogen and coating antigen from the endosulfan hapten coupled with protein by an activated ester method; the complete antigen is prepared by coupling hapten with bovine serum albumin through an activated ester method to prepare immunogen, coupling hapten with chicken ovalbumin through an activated ester method to prepare coating antigen, and the preparation method specifically comprises the following steps: dissolving the endosulfan hapten in dimethylformamide, adding N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC, and performing room-temperature reaction for activation to obtain the activated endosulfan hapten; respectively dissolving bovine serum albumin BSA and chicken egg albumin OVA in a carbonate buffer solution CBS, dropwise adding the activated endosulfan hapten into the bovine serum albumin BSA and chicken egg albumin OVA solution, reacting at room temperature, and dialyzing with phosphate buffer PBS to respectively obtain an immunogen and a coating antigen; in the activation process of the endosulfan hapten, the dosage mole ratio of the endosulfan hapten, N-hydroxysuccinimide NHS and 1-ethylcarbodiimide hydrochloride EDC is 1: 2-3: 2-3; in the preparation process of the immunogen and the coating antigen, the using molar ratio of the bovine serum albumin BSA, the chicken ovalbumin OVA and the endosulfan hapten is 30-50: 30-50: 1, a step of;
preparation of monoclonal antibodies: immunizing a mouse by using the immunogen, detecting and screening by an ic-ELISA method, and performing cell fusion to obtain a endosulfan monoclonal antibody;
preparing colloidal gold immunochromatography test paper: and coating a secondary antibody on the nitrocellulose membrane to serve as a quality control line, spraying the coating antigen on the nitrocellulose membrane to serve as a detection line, and assembling according to the structure of the test strip.
2. The method for preparing the colloidal gold immunochromatographic test strip for rapidly detecting endosulfan according to claim 1, which is characterized by comprising the following steps:
the colloidal gold immunochromatographic test strip comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the nitrocellulose membrane is stuck in the middle of the bottom plate, the water absorption pad and the sample pad are respectively stuck on the upper side and the lower side of the bottom plate, secondary antibodies are coated on the nitrocellulose membrane to serve as quality control lines, and the coating raw materials are sprayed on the nitrocellulose membrane to serve as detection lines, so that the colloidal gold immunochromatographic test strip is prepared.
3. The colloidal gold immunochromatographic test strip for rapidly detecting endosulfan is characterized in that the colloidal gold immunochromatographic test strip for detecting endosulfan is prepared by the preparation method of the colloidal gold immunochromatographic test strip for detecting endosulfan according to any one of claims 1-2.
4. A method for detecting a colloidal gold immunochromatographic test strip for rapidly detecting endosulfan according to claim 3, which is characterized in that the monoclonal antibody is marked with a colloidal Jin Dedao gold-labeled antibody, a endosulfan sample extracting solution to be detected is incubated with the gold-labeled antibody for 5min, the colloidal gold immunochromatographic test strip is inserted for 3min, and then the detection result is interpreted.
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| CN111187346A (en) * | 2020-01-15 | 2020-05-22 | 江苏大学 | Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof |
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| CN1700001A (en) * | 2005-02-24 | 2005-11-23 | 黄永 | Organic chlorine pesticide benzoepin artificial antigen and antibody, their preparation and use thereof |
| WO2018121677A1 (en) * | 2016-12-31 | 2018-07-05 | 中国农业科学院油料作物研究所 | Time-resolved fluorescence immunochromatography kit for simultaneous detection of mixed pollutant of aflatoxin and carbaryl, preparation method therefor, and application thereof |
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