CN113640522A - Colloidal gold test strip for detecting T-2 toxin, kit and detection method - Google Patents
Colloidal gold test strip for detecting T-2 toxin, kit and detection method Download PDFInfo
- Publication number
- CN113640522A CN113640522A CN202111028602.4A CN202111028602A CN113640522A CN 113640522 A CN113640522 A CN 113640522A CN 202111028602 A CN202111028602 A CN 202111028602A CN 113640522 A CN113640522 A CN 113640522A
- Authority
- CN
- China
- Prior art keywords
- solution
- toxin
- colloidal gold
- line
- antibody
- 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.)
- Withdrawn
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
- G01N33/58—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
-
- 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
-
- 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
-
- 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
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to the field of detection preparations, in particular to a colloidal gold test strip for detecting T-2 toxin, a kit and a detection method. The colloidal gold test strip comprises a sample pad, an NC membrane and an absorption pad, wherein the NC membrane is provided with a T line and a C line; the sample pad is coated with T-2 toxin monoclonal antibody labeled colloidal gold, the T line is coated with T-2 toxin complete antigen for competitive binding with the T-2 toxin monoclonal antibody labeled colloidal gold, and the C line is coated with goat anti-mouse antibody for capturing unbound T-2 toxin monoclonal antibody labeled colloidal gold. The colloidal gold test strip provided by the invention can be used for detecting T-2 toxin with a concentration range within 15min, has the technical advantages of wide linear range, low detection limit and high sensitivity, and is matched with a handheld card reader to realize quantitative detection.
Description
Technical Field
The invention relates to the field of detection preparations, in particular to a colloidal gold test strip for detecting T-2 toxin, a kit and a detection method.
Background
The T-2 toxin is a kind of trichothecene toxin A with strong toxicity, can be produced by various fungi, and is widely present in food crops and feeds. T-2 toxin has wide toxic effect, can cause toxic effect on organs or tissues such as bone marrow, thymus, lymph and the like, and can cause damage to the content of white blood cells and platelets in blood, thereby reducing the blood coagulation capability; meanwhile, research shows that the compound has carcinogenicity and teratogenicity. T-2 toxin enters human body mainly through polluted grains or by stockbreeding containing T-2 toxin, and causes great harm to human health.
Methods for detecting T-2 toxin are based mainly on chromatography (high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry, gas chromatography-tandem mass spectrometry) and immunological methods. The method based on chromatography has the advantages of high sensitivity and the like, but the required instrument is expensive, needs professional personnel and cannot meet the requirement of on-site rapid detection. Immunoassay has the characteristics of simple and convenient operation, analysis time period and easy on-site detection, and is widely applied. The lateral flow immunochromatographic test paper immunoassay method has the advantages of simple operation, rapid reaction, easy reading, low cost and the like in rapid on-site detection.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a colloidal gold test strip for detecting T-2 toxin.
The second purpose of the invention is to provide a kit for detecting T-2 toxin.
The third object of the present invention is to provide a method for detecting T-2 toxin.
In order to achieve the purpose of the invention, the technical scheme is as follows:
the invention provides a1, a colloidal gold test strip for detecting T-2 toxin, which comprises a sample pad, an NC membrane and an absorption pad, wherein the NC membrane is provided with a T line and a C line;
the sample pad is coated with colloidal gold labeled by a T-2 toxin monoclonal antibody; 4-6 mu L of colloidal gold solution marked by the T-2 toxin monoclonal antibody with the coating amount of 2-5 mu g/mL, preferably 5 mu L of colloidal gold solution marked by the T-2 toxin monoclonal antibody with the coating amount of 4 mu g/mL;
the T line is coated with a T-2 toxin complete antigen for competitive binding with the colloidal gold labeled by the T-2 toxin monoclonal antibody; 4-6 mu L of T-2 toxin complete antigen solution with the coating amount of 1-1.5 mg/mL; preferably 5 mu L of T-2 toxin complete antigen solution with the concentration of 1 mg/mL;
the C line is coated with a goat anti-mouse antibody for capturing the colloidal gold marked by the unbound T-2 toxin monoclonal antibody; the coating amount is 4-6 muL of 0.1-0.3 mg/mL sheep anti-mouse antibody solution, and preferably 5 muL of 0.3mg/mL sheep anti-mouse antibody solution.
Optionally, the distance between the T line and the C line is 4-7 mm, preferably 4.5-5.5 mm.
Optionally, the preparation method of the colloidal gold labeled by the T-2 toxin monoclonal antibody comprises the following steps:
s1, preparing a colloidal gold solution;
s2, taking 1mL of colloidal gold solution with OD value of 1, adjusting pH to 8.5, adding 4 mu g T-2 toxin monoclonal antibody, mixing uniformly, sealing, centrifuging, adding 1mL of antibody stabilizing solution, redissolving and precipitating, centrifuging, and finally adding 100 mu L of antibody stabilizing solution for resuspension; obtaining colloidal gold marked by the T-2 toxin monoclonal antibody;
the antibody stabilizing solution comprises the following components: contains 0.05% PEG6000, 10% sucrose and 0.02M PB buffer solution, and has a pH of 6.2.
Optionally, in S1, 1.5mL of 1% sodium citrate solution and 1mL of 1% chloroauric acid solution are used to prepare a colloidal gold solution;
preferably: 1.5mL of 1% sodium citrate solution is added into a container in boiling water bath, 1mL of 1% chloroauric acid solution is rapidly added under the stirring state, after the solution turns to wine red, a heat source is removed, and the stirring is continued; and (3) cooling the temperature to 45-55 ℃, stopping stirring, and taking the colloidal gold solution to be constant volume to 100mL by using a volumetric flask.
Optionally, the T line is prepared from a T line antigen streaking solution, and the concentration of a T-2 toxin complete antigen in the T line antigen streaking solution is 1-1.5 mg/mL;
preferably, the pH of the T-line antigen streaking solution is 8.0-8.5, preferably 8.5;
more preferably, the T-line antigen streaking solution is a solution containing 1-1.5 mg/mL of T-2 toxin complete antigen, 2.5% of sucrose by mass and 50mM of PB.
Optionally, the C line is prepared from a C line antibody marking solution, and the concentration of goat anti-mouse antibodies in the C line antibody marking solution is 0.1-0.3 mg/mL, preferably 0.3 mg/mL;
more preferably, the C-line antibody streaking solution is a PB solution containing 0.1-0.3 mg/mL goat anti-mouse antibody, 2.5% sucrose by mass and 50 mM;
more preferably, the pH of the C-line antibody streaking solution is 8.5.
Optionally, the flow rate of the scribing liquid is 1 muL/cm,
preferably, the marked NC film is placed into an oven to be dried for 4-8 hours at the temperature of 36-37 ℃.
The invention also relates to a kit for detecting the T-2 toxin, which contains the colloidal gold test strip, and also contains a sample treatment solution, a T-2 toxin labeling solution and a sample diluent;
the sample treatment solution is a methanol water solution with the volume percentage concentration of 70%;
the concentration range of the T-2 toxin in the T-2 toxin labeling solution is 0-200 ng;
the sample diluent is: contains 0.05% of PEG6000, 5% of sucrose and 20mM of PB solution, and has a pH of 6.2.
The invention also relates to a detection method of T-2 toxin, which adopts the kit for detection and at least comprises the following steps:
s1, adding the sample treatment fluid into a sample to be detected to obtain a mixture;
s2, adding the T-2 toxin labeling solution into the mixture to enable the concentration of the T-2 toxin labeling sample to be 0-200 ng/g, mixing, centrifuging, taking 100 mu L of supernatant, diluting with 0.6mL of the sample diluent, and mixing uniformly to obtain a solution to be detected;
s3, adding 200 mu L of the liquid to be detected into a sample hole of the test paper detection card, timing for 6min, and then determining the color rendering value through a handheld card reader.
Optionally: the sample to be detected is selected from edible oil or powder prepared from grain crops.
The invention has at least the following beneficial effects:
the colloidal gold test strip provided by the invention can be used for detecting T-2 toxin with a concentration range within 15min, has the technical advantages of wide linear range, low detection limit and high sensitivity, and is matched with a handheld card reader to realize quantitative detection. Specifically, the linear range of the sample for detecting the T-2 toxin is as follows: 3.13 to 50 μ g/kg (1.78 to 28.57 ppb); the detection limit is: 1.52. mu.g/kg (0.869 ppb); the linear range for detecting T-2 toxin in the labeled recovery detection of corn flour is as follows: the detection limit of 6.25 to 100 [ mu ] g/kg (0.44 to 7.14ppb) is: 6.68 mu g/kg (0.47ppb) can meet daily detection requirements.
Drawings
FIG. 1 shows the results of an experiment for optimizing the synthesis conditions of colloidal gold in Experimental example 1;
FIG. 2 shows the results of the test for stability of the label in Experimental example 1;
FIG. 3 shows the color development effect of T-line antigen streaking liquid chromatography of different pH values in Experimental example 2;
FIG. 4 is a graph showing the color development effect after streaking concentration chromatography of different antigens in Experimental example 2;
FIG. 5 shows the color development effect of the test paper in Experimental example 2 with optimized distance between the line T and the line C;
FIG. 6 shows the results of the test of the edible oil with different concentrations in Experimental example 3;
FIG. 7 is a linear curve of T-2 toxin in edible oil detected in Experimental example 3;
FIG. 8 is a standard curve of T-2 toxin in edible oil tested in Experimental example 3;
FIG. 9 shows the effect of labeling corn meal with different concentrations in Experimental example 4;
FIG. 10 is a standard curve of T-2 toxin in corn meal detected in Experimental example 4;
FIG. 11 is a linear plot of T-2 toxin in corn meal detected in Experimental example 4.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a colloidal gold test strip for detecting T-2 toxin, which can qualitatively detect the T-2 toxin in a sample by matching with a handheld card reader. According to the embodiment of the invention, the condition of the test strip is deeply researched, so that the lowest detection limit of the T-2 toxin is greatly reduced, and the T-2 toxin quantitative detection method with wide linear range, low detection limit and high sensitivity is obtained. The colloidal gold test strip comprises a sample pad, an NC membrane and an absorption pad, wherein a T line and a C line are arranged on the NC membrane.
Wherein, the sample pad is coated with colloidal gold marked by the T-2 toxin monoclonal antibody; the coating amount is 4-6 mu L of T-2 toxin monoclonal antibody labeled colloidal gold of 2-5 mu g/mL, and preferably 5 mu L of T-2 toxin monoclonal antibody labeled colloidal gold of 4 mu g/mL. The colloidal gold labeled by the T-2 toxin monoclonal antibody of 3 mu g/mL is most stable as determined by a sodium chloride coagulation method, and in order to ensure that the coupling of the colloidal gold and the monoclonal antibody is more sufficient, 20 percent of monoclonal antibody is added, namely the colloidal gold with the final concentration of the monoclonal antibody labeling of 4 mu g/mL is used for preparing the test strip.
T-2 toxin complete antigen is coated on the T line and is used for competitively binding with the colloidal gold marked by the T-2 toxin monoclonal antibody; 4-6 mu L of T-2 toxin complete antigen with the coating amount of 1-1.5 mg/mL; preferably 5. mu.L of T-2 toxin complete antigen at 1 mg/mL. According to screening tests, the T-2 toxin with the coating amount of 1-1.5 mg/mL has good complete antigen color development effect and small difference, and the antigen concentration of 1mg/mL can be adopted from the cost perspective. If the concentration of the complete antigen of the T-2 toxin is lower than the range, the maximum color rendering value is reduced due to the small amount of the combined colloidal gold, so that the detection range is narrowed; if the concentration of the complete antigen of the T-2 toxin is higher than this concentration, the detection limit may be increased due to the excessive amount of the bound colloidal gold.
The C line is coated with a goat anti-mouse antibody for capturing the colloidal gold marked by the unbound T-2 toxin monoclonal antibody; the coating amount is 4-6 muL of goat anti-mouse antibody with 0.1-0.3 mg/mL, preferably 5 muL of goat anti-mouse antibody with 0.3 mg/mL. The line C antibody marking solution is prepared and contains 0.1-0.3 mg/mL goat anti-mouse antibody, 2.5% sucrose by mass and 50mM PB solution; preferably, the concentration of the goat anti-mouse antibody is 0.3 mg/mL; more preferably, the pH of the C-line antibody streaking solution is 8.5.
Experiments show that the sensitivity of the kit can be improved by matching the concentration and the coating amount with the T line. If the concentration of the goat anti-mouse antibody is lower than the range, the color value may be reduced because of the colloidal gold which cannot be completely combined, so that the judgment on the performance of the colloidal gold marked by the T-2 toxin monoclonal antibody is influenced; if the concentration of goat anti-mouse antibody is higher than this concentration, it may cause waste of materials and increase the production cost.
In the embodiment of the invention, the distance between the T line and the C line is 4-7 mm, preferably 4.5-5.5 mm, most preferably 5mm, and the T line with the distance of 5mm from the C line has the best negative color development effect. If the distance is too wide, the gold-labeled antibody is excessively adsorbed on an NC membrane in the chromatography process, so that the background value is high, and the negative and positive T-line color development inhibition signals are not obvious.
In the embodiment of the invention, the preparation method of the colloidal gold labeled by the T-2 toxin monoclonal antibody specifically comprises the following steps:
s1, preparing a colloidal gold solution; wherein, 1.5mL of 1% sodium citrate solution and 1mL of 1% chloroauric acid solution are adopted to prepare colloidal gold solution; experiments prove that the colloidal gold solution prepared under the condition has good stability.
S2, taking 1mL of colloidal gold solution with OD value of 1, adjusting pH to 8.5, adding 4 mu g T-2 toxin monoclonal antibody, mixing uniformly, sealing, centrifuging, adding 1mL of antibody stabilizing solution, redissolving and precipitating, centrifuging, and finally adding 100 mu L of antibody stabilizing solution for resuspension; obtaining the colloidal gold marked by the T-2 toxin monoclonal antibody. The colloidal gold labeled by the T-2 toxin monoclonal antibody with the concentration has the best stability.
The composition of the antibody stabilizing solution is as follows: contains 0.05% PEG6000, 10% sucrose and 0.02M PB buffer solution, and has a pH of 6.2.
Further preferably, the preparation method of the colloidal gold comprises the following steps: in S1, 1.5mL of 1% sodium citrate is added into a container in boiling water bath, 1mL of 1% chloroauric acid is rapidly added under the stirring state, after the solution turns to wine red, the heat source is removed, and the stirring is continued; and (3) cooling the temperature to 45-55 ℃, preferably 50 ℃, stopping stirring, and taking the colloidal gold to be constant volume to 100mL by using a volumetric flask.
In the embodiment of the invention, the T line is prepared from a T line antigen streaking solution, and the pH of the T line antigen streaking solution is 7.5-8.5, preferably 8.0-8.5, and more preferably 8.5; the concentration of the complete antigen of the T-2 toxin in the T line antigen streaking solution is 1-1.5 mg/mL. Preferably, the T-line antigen streaking solution is a solution containing 1-1.5 mg/mL of T-2 toxin complete antigen, 2.5% by mass of sucrose and 50mM of PB.
In the embodiment of the invention, the C line is prepared from a C line antibody marking solution, and the C line antibody marking solution is a PB solution containing 0.1-0.3 mg/mL goat anti-mouse antibody, 2.5% of sucrose by mass and 50 mM. The concentration of goat anti-mouse antibody is preferably 0.3 mg/mL.
In the embodiment of the invention, the flow rate of the scribing liquid is 1 muL/cm, preferably, the scribed NC film is placed into an oven to be dried for 4-8 h at the temperature of 36-37 ℃.
The embodiment of the invention also relates to a kit for detecting T-2 toxin, which contains the colloidal gold test strip, a sample treatment solution, a T-2 toxin labeling solution and a sample diluent;
wherein: the sample treatment solution is a methanol water solution with the volume percentage concentration of 70%;
the concentration range of the T-2 toxin in the T-2 toxin labeling solution is 0-200 ng;
the sample diluent was: contains 0.05 percent of PEG6000, 5 percent of cane sugar and 20mM of PB solution by mass percent, and the pH value is 6.2.
The embodiment of the invention also relates to a detection method of T-2 toxin, which adopts the kit for detection and at least comprises the following steps:
s1, adding a sample treatment fluid into the sample to be detected to obtain a mixture;
s2, adding the T-2 toxin labeling solution into the mixture to enable the concentration of the T-2 toxin labeling sample to be 0-200 ng/g, mixing, centrifuging, taking 100 mu L of supernatant, diluting with 0.6mL of sample diluent, and mixing uniformly to obtain a solution to be detected;
s3, adding 200 mu L of liquid to be detected into a sample hole of the test paper detection card, timing for 6min, and then determining the color rendering value through a handheld card reader.
Optionally: the sample to be tested is selected from edible oil or powder prepared from grain crops.
The following examples and experimental examples further illustrate the embodiments of the present invention:
examples and experimental examples the main experimental reagents used were: t-2 standard substance, chloroauric acid, sodium citrate and sodium carbonate are purchased from Tianjin Bohai reagent, bovine serum albumin and SDS are purchased from Solaibao (Beijing) biotechnology limited, and Sartorius-95 Nitrocotton (NC) membrane, sample pad, absorbent paper, glass fiber and polyvinyl chloride substrate are purchased from Shanghai Jie biological technology limited. The T-2 toxin monoclonal antibody is purchased from Shanghai Biotechnology Inc., the T-2 toxin complete antigen (T2-BSA) is purchased from Tianjin Haotai technology Inc., and other chemicals are analytical grade and are from Shanghai national medicine chemical reagent Inc., China. The relevant solutions were prepared using Mill-Q purified ultrapure water (18.2M. omega. cm-1).
The handheld card reader is an FD-200 handheld food safety rapid detector purchased from Shanghai-Fei-Measure biotechnology Limited.
Example 1
Step one preparation of colloidal gold nanoparticles
Soaking the stirring paddle and the glassware in an acid jar overnight, and cleaning; assembling a stirring paddle on the stirrer, and opening an electric heating sleeve to preheat for 5 min; after the water in the flask boiled, 1.5mL of 1% sodium citrate was added.
Then, the stirrer was turned on to mix the mixture uniformly, the rotation speed was adjusted to 500rpm, and 1mL of 1% chloroauric acid was added rapidly. After the solution turns into wine red, removing the heat source and continuing stirring; the temperature is reduced to about 50 ℃, the stirring is stopped, and the colloidal gold is taken out and is added into a volumetric flask to be 100 mL.
Step two preparation of T-2 toxin monoclonal antibody-colloidal gold marker
Adding 20 mu L of 0.1M sodium carbonate into 1mL of colloidal gold, adjusting the pH value to about 8.5, adding 4 mu g/mL of T-2 toxin monoclonal antibody, uniformly mixing for 30min, adding 120 mu L of 10% BSA, sealing for 15min, centrifuging at 10000rpm for 15min, discarding the supernatant, adding 1mL of antibody stabilizing solution, redissolving and precipitating, continuing to centrifuge at 8000rpm for 15min, repeating for 2 times, and finally adding 100 mu L of antibody stabilizing solution for re-suspension.
The composition of the antibody stabilizing solution is as follows: 0.02M PB buffer containing 0.05% PEG6000 and 10% sucrose, pH 6.2.
Example 2 preparation of test strips
Preparing a T-line antigen streaking solution: a solution of sucrose 2.5 wt%, 50mM PB containing 1.5mg/mL of the complete antigen of T-2 toxin (T2-BSA), pH 8.5;
preparing a C-line antibody scribing solution: contains 0.3mg/mL goat anti-mouse antibody, and is diluted with 2.5 wt% sucrose and 50mM PB solution, and has a pH value of 8.5.
And respectively scribing and coating the T-line antigen scribing liquid and the C-line antibody scribing liquid on an NC membrane, wherein the scribing flow is 1 mu L/cm, and the distance between the T line and the C line is 5 mm. And (4) putting the marked NC film into an oven for drying for 4h at 37 ℃, and taking out.
Spraying the colloidal gold labeled by the T-2 toxin monoclonal antibody prepared in the example 1 on the bonding pads at the speed of 5 mu L/cm, repeatedly spraying each bonding pad for 3 times, drying in an oven at 37 ℃ for 6h after finishing the spraying, and taking out; pasting a sample pad, a combination pad, an NC film and a water absorption pad.
The specific pasting method comprises the following steps: firstly pasting an NC film, then pasting a combination pad at the lower end of the NC film, overlapping the combination pad with the NC film for 2mm, and then pasting a sample pad at the lower end of the combination pad, and overlapping the sample pad with the NC film for 2 mm; and (3) sticking the water absorption pad on the NC membrane, overlapping the water absorption pad with the NC membrane by 2mm, pressing the material, cutting the material into test strips with the width of 5mm, and placing the test strips into a detection clamping groove for later use.
The use method of the test strip comprises the following steps:
s1, adding a sample treatment fluid into the sample to be detected to obtain a mixture; the sample treatment solution is a methanol water solution with the volume percentage concentration of 70%;
s2, adding a T-2 toxin labeling solution into the mixture to enable the concentration of a T-2 toxin labeling sample to be 0-200 ng/g, mixing, centrifuging, taking 100 mu L of supernatant, diluting with 0.6mL of sample diluent, and mixing uniformly to obtain a solution to be detected; the concentration range of the T-2 toxin in the T-2 toxin labeling solution is 0-200 ng; the product diluent is: a 20mM PB solution containing 0.05% PEG6000 and 5% sucrose by weight, and having a pH of 6.2;
s3, adding 200 mu L of liquid to be detected into a sample hole of the test paper detection card, timing for 6min, and then determining the color rendering value through a handheld card reader.
Experimental example 1
1. Optimization of colloidal gold nanoparticles
Soaking the stirring paddle and the glassware in an acid jar overnight, and cleaning; assembling a stirring paddle on the stirrer, and opening an electric heating sleeve to preheat for 5 min; after the water in the flask is boiled, the stability of the synthesized gold colloid is optimized by adding 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL of 1% sodium citrate.
Then, the stirrer was turned on to mix the mixture uniformly, the rotation speed was adjusted to 500rpm, and 1mL of 1% chloroauric acid was added rapidly. After the solution turns into wine red, removing the heat source and continuing stirring; and (3) cooling the temperature to about 50 ℃, stopping stirring, taking the colloidal gold, fixing the volume to 100mL by using a volumetric flask, and measuring an ultraviolet absorption peak. The results are shown in FIG. 1.
As is clear from FIG. 1, when 1.5mL of 1% sodium citrate was added, the maximum absorption peak of the synthesized gold colloid was 524nm, and the gold colloid was not violet or coagulated in the destruction test by adding acetic acid, indicating that the gold colloid had good stability.
2. Optimization of T-2 toxin monoclonal antibody labeling quantity
Taking 1mL of colloidal gold with OD value of 1, placing in an EP tube, and adding 20 μ L of 0.1M Na into each tube2CO3Mixing, adding 0, 1 μ g, 2 μ g and 3 μ g of T-2 toxin monoclonal antibody, shaking at 37 deg.C for 30min, adding 10% NaCl 100 μ L, mixing, standing for 30min, and analyzing the result.
An Mey-th stability experiment, namely a sodium chloride coagulation method is adopted to estimate the optimal monoclonal antibody labeling amount, as shown in figure 2, the monoclonal antibody labeling final concentration is 3 mug/mL colloidal gold, and almost no color change is caused after the sodium chloride is added, so that the monoclonal antibody labeling final concentration can be selected to be 3 mug/mL colloidal gold, and 20% monoclonal antibody labeling amount, namely the colloidal gold with the monoclonal antibody labeling final concentration of 4 mug/mL is added for preparing the test paper.
Experimental example 2 preparation of reaction Membrane and concentration of coating antigen
Coating the T-2 toxin-bovine serum albumin conjugate, namely the T-2 toxin complete antigen, on a T line to form a detection line, and coating the goat anti-mouse antibody on a C line to form a quality control line. In order to improve the sensitivity and stability of the test strip, the pH value and the antigen concentration of the T-line antigen streaking solution and the distance between the T line and the C line are optimized.
The specific experimental method comprises the following steps: (1) the pH values of the T-line antigen streaking solutions were adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, and the rest of the procedure was the same as in example 2. Test paper strips A1-A6 (not loaded in the test card slot) are prepared. The experimental results of pH optimization obtained by adding 4 of each test strip, 2 of each test strip to a negative test solution (0.02M PB buffer), and 2 of each test strip to a positive test solution (20ng/mL T2 toxin) are shown in FIG. 3.
As can be seen from fig. 3, although the change in pH has a certain effect on the color development effect of T-line, the color development effect can be improved by alkaline coating, because the adsorption efficiency of protein on the membrane can be improved by alkaline coating. Subsequent experiments all used a streaking buffer system at pH 8.5.
(2) The concentrations of the streaked antigen in the adjusted T-line antigen streaks were 1mg/mL, 1.25mg/mL and 1.5mg/mL, respectively, and the rest of the procedure was the same as in example 2. Test strips B1-B3 (not loaded in the test card slot) are prepared. The results of experiments in which 4 of each test strip was added with positive test solution (20ng/mL of T2 toxin) to optimize antigen concentration are shown in FIG. 4.
As can be seen from FIG. 4, the difference of the concentration of the antigen streaked is not large between 1 and 1.5mg/mL, and the obvious color development effect can be achieved within 2 min.
(3) Adjusting the distance between a T line and a C line of the scribing instrument, wherein the distance is respectively adjusted to be 5-8 mm, and the rest steps are the same as those of the embodiment 2; and preparing to obtain test paper strips C1-C4 (not arranged in the detection card slot). For each test strip, 4 samples were taken, 2 samples were added with negative test solutions (distilled water), and 2 samples were added with positive test solutions (20ng/mL of T2 toxin), and the distance between the T line and the C line was optimized as shown in FIG. 5.
As can be seen from fig. 5, the test strip has the best negative color development effect of the T line with a distance of 5mm from the C line, and the T line color development inhibition signals of the negative sample and the positive sample of the relatively wide experimental group (8 mm apart) are not obvious.
Experimental example 3
And (4) taking the edible oil as a sample, and carrying out actual sample detection.
Pretreatment of edible oil: weighing 1g of edible oil, adding a sample treatment solution, namely 0.5mL of 70% methanol aqueous solution, adding a T-2 toxin labeling solution to make the labeling concentration 200ng/g, shaking for 2min, centrifuging at 5000rpm for 5min, taking 100 mu L of supernatant, diluting with 0.6mL of sample diluent, and mixing uniformly to obtain a solution to be detected.
And (3) adding 200 mu L of the liquid to be detected into the sample hole of the colloidal gold test paper detection card prepared in the embodiment 2, standing for 2min, inserting a paper strip, timing for 6min, and then measuring the color rendering value through a handheld card reader. The results of the edible oil detection are shown in FIGS. 6 to 8.
As can be seen from FIGS. 6 to 8, the linear range of the samples for detecting T-2 toxin is: 3.13 to 50 μ g/kg (1.78 to 28.57 ppb); the detection limit is: 1.52. mu.g/kg (0.869 ppb).
Experimental example 4
And (4) taking corn flour as a sample, and carrying out actual sample detection.
Pretreatment aiming at a corn sample: weighing 1g of corn flour, adding a sample treatment solution, namely 2mL of 70% methanol aqueous solution, adding a T-2 toxin labeling solution to make the labeling concentration 200ng/g, shaking for 2min, centrifuging at 5000rpm for 5min, taking 100 mu L of supernatant, diluting with 0.6mL of sample diluent, and mixing uniformly to obtain a solution to be detected.
And (3) adding 200 mu L of the liquid to be detected into the sample hole of the colloidal gold test paper detection card prepared in the embodiment 2, standing for 2min, inserting a paper strip, and timing for 6min to measure the color rendering value through a handheld card reader. The results of detecting corn meal are shown in fig. 9-11.
As can be seen from FIGS. 9 to 11, the linear range for detecting T-2 toxin is: 6.25 to 100. mu.g/kg (0.44 to 7.14 ppb); the detection limit is: 6.68. mu.g/kg (0.47 ppb).
Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.
Claims (10)
1. A colloidal gold test strip for detecting T-2 toxin comprises a sample pad, an NC membrane and an absorption pad, wherein a T line and a C line are arranged on the NC membrane; the method is characterized in that:
the sample pad is coated with colloidal gold labeled by a T-2 toxin monoclonal antibody; 4-6 mu L of colloidal gold solution marked by the T-2 toxin monoclonal antibody with the coating amount of 2-5 mu g/mL, preferably 5 mu L of colloidal gold solution marked by the T-2 toxin monoclonal antibody with the coating amount of 4 mu g/mL;
the T line is coated with a T-2 toxin complete antigen for competitive binding with the colloidal gold labeled by the T-2 toxin monoclonal antibody; 4-6 mu L of T-2 toxin complete antigen solution with the coating amount of 1-1.5 mg/mL; preferably 5 mu L of T-2 toxin complete antigen solution with the concentration of 1 mg/mL;
the C line is coated with a goat anti-mouse antibody for capturing the colloidal gold marked by the unbound T-2 toxin monoclonal antibody; the coating amount is 4-6 muL of sheep anti-mouse antibody solution with 0.1-0.3 mg/mL, and preferably 5 muL of sheep anti-mouse antibody solution with 0.3 mg/mL.
2. The colloidal gold test strip of claim 1, wherein: the distance between the T line and the C line is 4-7 mm, and preferably 4.5-5.5 mm.
3. The colloidal gold test strip of claim 1, wherein: the preparation method of the colloidal gold solution labeled by the T-2 toxin monoclonal antibody comprises the following steps:
s1, preparing a colloidal gold solution;
s2, taking 1mL of colloidal gold solution with OD value of 1, adjusting pH to 8.5, adding 4 mu g T-2 toxin monoclonal antibody, mixing uniformly, sealing, centrifuging, adding 1mL of antibody stabilizing solution, redissolving and precipitating, centrifuging, and finally adding 100 mu L of antibody stabilizing solution for resuspension; obtaining colloidal gold solution marked by the T-2 toxin monoclonal antibody;
the antibody stabilizing solution comprises the following components: contains 0.05% PEG6000, 10% sucrose and 0.02M PB buffer solution, and has a pH of 6.2.
4. The colloidal gold test strip of claim 1, wherein: in S1, preparing a colloidal gold solution by using 1.5mL of 1% sodium citrate solution and 1mL of 1% chloroauric acid solution;
preferably: 1.5mL of 1% sodium citrate solution is added into a container in boiling water bath, 1mL of 1% chloroauric acid solution is rapidly added under the stirring state, after the solution turns to wine red, a heat source is removed, and the stirring is continued; and (3) cooling the temperature to 45-55 ℃, stopping stirring, and taking the colloidal gold solution to be constant volume to 100mL by using a volumetric flask.
5. The colloidal gold test strip of claim 1, wherein: the T line is prepared from T line antigen streaking liquid, and the concentration of a T-2 toxin complete antigen in the T line antigen streaking liquid is 1-1.5 mg/mL;
preferably, the pH of the T-line antigen streaking solution is 8.0-8.5, preferably 8.5;
more preferably, the T-line antigen streaking solution is a solution containing 1-1.5 mg/mL of T-2 toxin complete antigen, 2.5% of sucrose by mass and 50mM of PB.
6. The colloidal gold test strip of claim 1, wherein: the C line is prepared from a C line antibody marking solution, and the concentration of goat anti-mouse antibodies in the C line antibody marking solution is 0.1-0.3 mg/mL, preferably 0.3 mg/mL;
more preferably, the C-line antibody streaking solution is a PB solution containing 0.1-0.3 mg/mL goat anti-mouse antibody, 2.5% sucrose by mass and 50 mM;
further preferably, the pH of the C-line antibody streaking solution is 8.5.
7. The colloidal gold test strip of claim 5 or 6, wherein: the flow rate of the scribing liquid is 1 mu L/cm,
preferably, the marked NC film is placed into an oven to be dried for 4-8 hours at the temperature of 36-37 ℃.
8. A kit for detecting T-2 toxin, which comprises the colloidal gold test strip of any one of claims 1 to 7, and is characterized in that the kit further comprises a sample treatment solution, a T-2 toxin labeling solution and a sample diluent;
the sample treatment solution is a methanol water solution with the volume percentage concentration of 70%;
the concentration range of the T-2 toxin in the T-2 toxin labeling solution is 0-200 ng;
the sample diluent is: contains 0.05% of PEG6000, 5% of sucrose and 20mM of PB solution, and has a pH of 6.2.
9. A method for detecting T-2 toxin, which is characterized in that the kit of claim 8 is used for detection, and the method at least comprises the following steps:
s1, adding the sample treatment fluid into a sample to be detected to obtain a mixture;
s2, adding the T-2 toxin labeling solution into the mixture to enable the concentration of the T-2 toxin labeling sample to be 0-200 ng/g, mixing, centrifuging, taking 100 mu L of supernatant, diluting with 0.6mL of the sample diluent, and mixing uniformly to obtain a solution to be detected;
s3, adding 200 mu L of the liquid to be detected into a sample hole of the test paper detection card, timing for 6min, and then determining the color rendering value through a handheld card reader.
10. The method of claim 9, wherein the sample is selected from the group consisting of edible oil and flour made from food crops.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111028602.4A CN113640522A (en) | 2021-09-02 | 2021-09-02 | Colloidal gold test strip for detecting T-2 toxin, kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111028602.4A CN113640522A (en) | 2021-09-02 | 2021-09-02 | Colloidal gold test strip for detecting T-2 toxin, kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113640522A true CN113640522A (en) | 2021-11-12 |
Family
ID=78425003
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111028602.4A Withdrawn CN113640522A (en) | 2021-09-02 | 2021-09-02 | Colloidal gold test strip for detecting T-2 toxin, kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113640522A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203465275U (en) * | 2013-06-04 | 2014-03-05 | 北京勤邦生物技术有限公司 | Rapid detection test strip for T-2 toxin |
| CN104215763A (en) * | 2013-06-04 | 2014-12-17 | 北京勤邦生物技术有限公司 | Test strip for detecting T-2 toxin and application thereof |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN112816701A (en) * | 2020-12-28 | 2021-05-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit |
-
2021
- 2021-09-02 CN CN202111028602.4A patent/CN113640522A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203465275U (en) * | 2013-06-04 | 2014-03-05 | 北京勤邦生物技术有限公司 | Rapid detection test strip for T-2 toxin |
| CN104215763A (en) * | 2013-06-04 | 2014-12-17 | 北京勤邦生物技术有限公司 | Test strip for detecting T-2 toxin and application thereof |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN112816701A (en) * | 2020-12-28 | 2021-05-18 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit |
Non-Patent Citations (1)
| Title |
|---|
| 朱亮亮 等: "胶体金免疫层析法快速检测T-2毒素的研究" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114137206A (en) * | 2021-12-02 | 2022-03-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip, kit and detection method for detecting fumonisins |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2020199501A1 (en) | Quick detection method for food allergens based on quantum dot fluorescence | |
| CN109884306B (en) | Small molecule detection test strip, kit and detection method thereof | |
| CN107132359B (en) | Pepsinogen Cgene and Pepsinogen II detection method and its kit | |
| WO2023025074A1 (en) | Immunoassay kit having multiple test scales, and application thereof | |
| CN107449898B (en) | Kanamycin residue fluorescence immunochromatographic test paper and preparation method thereof | |
| CN111751527A (en) | Novel coronavirus IgG and IgM antibody detection kit and preparation method and application thereof | |
| CN107271669A (en) | Propepsin, helicobacter pylori antibody and G17 detection method and its kit | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| KR101451733B1 (en) | Labeling agent for aflatoxin B1 detection and the kit for detecting aflatoxin B1 comprising thereof | |
| CN115639366A (en) | Beta 2-microglobulin fluorescence immunochromatography assay kit and detection method thereof | |
| CN113640522A (en) | Colloidal gold test strip for detecting T-2 toxin, kit and detection method | |
| CN108761075A (en) | A kind of deoxynivalenol quantifies rapid detection card and its detection method | |
| CN110988365A (en) | Quantitative immune colloidal gold detection card and kit for cystatin C, microalbuminuria and urinary creatinine | |
| CN107192819A (en) | One kind centrifuges detection method | |
| CN111896749A (en) | Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method | |
| CN116773807A (en) | Method for detecting African swine fever virus by using time-resolved fluorescence immunoassay probe | |
| WO2005078446A1 (en) | Method for a rapid test | |
| CN114675028A (en) | Preparation method and application of fenitrothion nano antibody-colloidal gold marker | |
| CN114113597A (en) | Novel coronavirus antigen detection kit and preparation method thereof | |
| CN208140718U (en) | A kind of deoxynivalenol quantifies rapid detection card | |
| CN114371298A (en) | SERS immunochromatographic test strip for detecting cow milk allergen alpha-lactalbumin and application thereof | |
| CN113834934A (en) | Immunochromatography test strip and preparation method thereof | |
| CN113406330A (en) | Kit for detecting norfloxacin and detection method | |
| CN106248976A (en) | Four kinds of metabolites of nitrofuran colloidal gold strips of a kind of detection and preparation method thereof | |
| CN113820492A (en) | Detection preparation for detecting atrazine, kit and detection method 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 | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20211112 |