CN112180080B - Small molecule triple immunochromatography detection method, test strip and kit - Google Patents
Small molecule triple immunochromatography detection method, test strip and kit Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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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
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
The invention belongs to the field of small molecule detection, and relates to a small molecule triple immunochromatography detection method, a test strip and a kit. The detection method comprises the following steps: (1) Incubating the to-be-detected object, the labeled antibody and the labeled antigen to obtain a mixture; the labeled antibody is an antibody against a target small molecule and is labeled with a first signal substance; the labeled antigen is a derivative of a target small molecule with a modified protein and is labeled with a second signal substance; (2) Sequentially passing the mixture through a detection area and a quality control area; the detection area is fixedly provided with an antibody for resisting the modified protein in the labeled antigen; the quality control area is fixedly provided with a secondary antibody corresponding to the labeled antibody; (3) And obtaining the content of the target small molecules in the object to be detected according to the triple signal intensity of the detection area and the quality control area. The detection method is sensitive, can realize triple detection fusion, provides a wider detection range for small molecule detection, and has wide market prospect.
Description
Technical Field
The invention belongs to the field of small molecule detection, and particularly relates to a small molecule triple immunochromatography detection method, a small molecule triple immunochromatography detection test strip and a small molecule triple immunochromatography detection kit.
Background
There are various existing detection methods for small molecules, wherein the detection principle of immunochromatographic test paper based on the chromatographic separation principle is to fix the specific antigen of the to-be-detected object to the detection area of the test paper in advance, and the small molecules to be detected in the sample and the labeled antibodies flow to the position of the detection area under the action of capillary force, so that the immobilized antigen and the to-be-detected object in the solution compete for binding with the labeled antibodies in the solution. The signal intensity of the measuring area is inversely proportional to the content of the small molecules to be measured.
Dual immunochromatographic assays based on different signal responses of the labeling materials under different excitation light sources have been reported, such as: in the Raman detection based on the gold core silver shell as the surface enhanced substrate, the combination of visual detection and Raman detection can be realized.
Because of rapid measurement, low cost, visual signal response and easy field use, immunochromatography detection methods based on traditional structures have been increasingly widely applied in various fields such as food safety, clinical medicine and the like. However, the test paper technology has certain defects: 1. in the double detection, the detection ranges are difficult to link, which is not beneficial to data analysis; triple detection techniques have not been reported; 2. the immobilization of the antigen of the object to be detected is unfavorable for the full exposure of the binding site, the full progress of competitive binding reaction and the limitation of the detection sensitivity; 3. the competitive binding reaction in the detection area of the chromatographic test paper has a plurality of influencing conditions, is difficult to control accurately and is unfavorable for the exertion of the detection sensitivity.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a small molecule triple immunochromatography detection method, and a test strip and a kit based on the method. The detection method is sensitive, can realize triple detection fusion, has wide application range, provides a wider detection range for small molecule detection, and has wide market prospect.
In order to achieve the above object, a first aspect of the present invention provides an immunochromatographic method for triple detection of small molecules, comprising the steps of:
(1) Incubating the object to be detected, the labeled antibody and the labeled antigen to obtain a mixture; wherein the labeled antibody is an antibody against a small molecule of interest and is labeled with a first signal substance; the labeled antigen is a derivative of a target small molecule with a modified protein and is labeled with a second signal substance; the first signal species or the second signal species has two signals;
(2) Sequentially passing the mixture obtained in the step (1) through a detection area and a quality control area; wherein the detection zone is fixedly provided with an antibody against a modified protein in the labeled antigen; the quality control area is fixedly provided with a secondary antibody corresponding to the labeled antibody;
(3) And obtaining the content of the target small molecules in the object to be detected according to the triple signal intensity of the detection area and the quality control area.
Compared with the existing detection method, the method provided by the invention has the advantages that the antibody of the small molecule to be detected is labeled, and the small molecule to be detected, the labeled antibody and the labeled antigen are subjected to 'competitive' binding reaction in an incubation mode before chromatographic detection. In the invention, antigens and antibodies are marked by using different materials, and the triple detection of the small molecules to be detected can be realized according to the signal intensity of the different materials in the detection area and the quality control area, so that the detection application range is wider. In addition, the detection area is immobilized with a specific antibody of the modified protein in the antigen, so that any antigen obtained by a small molecule which can be modified by the protein can be identified, and the detection has the characteristic of universal type. In addition, the "competing" binding reaction in the incubation can be performed under the most favorable conditions, and the detection sensitivity can be sufficiently improved.
In the invention, if the sample to be detected contains small molecules, the obtained mixture is a mixture of a labeled antibody, the small molecules to be detected and a labeled antibody, and a labeled antigen; if the sample to be tested does not contain small molecules, the mixture formed is only a labeled antibody-labeled antigen. The content of small molecular substances in the object to be detected is judged through the signal intensity of different materials of the detection area and the quality control area, so that the method is more accurate and more sensitive. When the content is lower than the detectable value, the object to be detected can be determined to be free of the target small molecule. Therefore, the method of the present invention can be either a qualitative or a quantitative detection method.
According to the present invention, preferably, one of the two signals that the first signal substance or the second signal substance has is a visual signal; the two marking materials can realize double detection, and one of the two marking materials can also realize visual detection, so that triple detection of small molecules is finally realized.
Specifically, the first signal substance and the second signal substance may be selected from any two of fluorescent compounds, fluorescent quantum dots, biotin, radioisotopes, electron dense substances, colloidal gold, materials based on colloidal gold or gold core silver shell materials as surface enhanced raman detection, or enzymes; preferably, the first signal substance and the second signal substance are respectively fluorescent quantum dots (QB) and gold core silver shell materials (au@ag-R) modified with raman reporter molecules. The raman reporter R may be selected from CBT (4-chlorophenylthiol), ABT (4-aminophenylthiophenol), HBT (4-hydroxyphenyl thiophenol), DTNB (5, 5' -dithiobis (2-nitrobenzoic acid)). The above materials are all commercially available or prepared according to conventional methods.
According to the detection method of the invention, the modified protein is preferably universal recognition protein, and the universal recognition protein is used as a binding substance of a small molecule to be detected, so that the application range is wide. Specifically, the modified protein may be Ovalbumin (OVA), hemocyanin (KLH), bovine Serum Albumin (BSA), human serum albumin (HAS) or chicken ovalbumin (THY).
The method of the present invention can be used to detect a variety of small molecules of interest that meet the principles of the present invention. For example, sedatives such as Diazepam (DAP); chemical dyes such as malachite green; pesticides, such as organophosphorus pesticides; veterinary drugs such as chloramphenicol and tetracycline; additives such as melamine; mycotoxins, such as aflatoxins and trichothecenes, can be used in the fields of food safety, clinical diagnosis, and the like.
According to one embodiment of the invention, the small molecule of interest is Diazepam (DAP); the labeling material of the antibody is quantum dot microsphere (QB); the labeling material of the antigen is gold core silver shell material (Au@Ag-R) modified with Raman reporter molecules.
The specific incubation method is not particularly limited in the present invention, and preferably, in the step (1), the temperature of the incubation is 25 to 40 ℃, preferably 35 to 37 ℃; the incubation time is 5-20min; the incubation is performed in a buffer, the composition of which comprises: based on 0.04-0.06mol/L phosphate buffer, the following substances in percentage by weight are added: 1.5 to 2.5 percent of sucrose, 4 to 6 percent of fructose, 0.8 to 1.2 percent of PEG, 2.5 to 3.5 percent of Tween-20.
According to the method of the present invention, the concentration and the amount of each substance in the incubation system may be adjusted as required, and the present invention is not particularly limited. For example, the concentration of the labeled antibody is 0.2-0.8mg/mL and the concentration of the labeled antigen is 0.01-0.04mg/mL.
According to the detection method provided by the invention, standard graphs under signal response of different materials can be prepared according to test conditions, and finally the content of small molecules is judged according to signal response intensity of different materials.
The second aspect of the invention provides a small molecule triple immunochromatography detection test strip, which comprises a sample pad, a water absorption pad and a bottom plate fixed with a nitrocellulose membrane, wherein the sample pad and the water absorption pad are arranged on the bottom plate and are respectively positioned at two sides of the nitrocellulose membrane; wherein,
the sample pad is used for bearing an incubation of a to-be-detected object, a labeled antibody and a labeled antigen;
the labeled antibody is an antibody against a target small molecule and is labeled with a first signal substance; the labeled antigen is a derivative of a target small molecule with a modified protein and is labeled with a second signal substance; the first signal species or the second signal species has two signals;
the nitrocellulose membrane is provided with a detection area and a quality control area; the detection area is close to the sample pad, and an antibody for resisting the modified protein in the labeled antigen is fixedly arranged on the detection area; the quality control area is close to the water absorption pad, and a secondary antibody corresponding to the labeled antibody is fixedly arranged on the quality control area.
In the test strip of the present invention, the first signal substance, the second signal substance, the modified protein, and the target small molecule are as described above, and are not described in detail herein. The amounts of the components on the test strip are not particularly limited in the present invention, and can be determined by one skilled in the art based on the principle of the present invention and according to actual needs.
The small molecule triple immunochromatography detection test paper provided by the invention realizes triple detection of signal response of an object to be detected through the respective labeling of the antigen and the antibody; the 'competition' combination reaction is realized through an incubation step, a detection area and a quality control area are positioned on a nitrocellulose membrane pad and are distributed vertically to the long axis of test paper, and the detection area is coated with an antibody which can identify the modified protein of immune complex formed in the pre-incubation step, such as an anti-BSA antibody (anti-BSA-anti); the quality control region is coated with a secondary antibody corresponding to the labeled antibody, such as goat anti-mouse antibody (goat anti-mouse antibody).
When detecting a to-be-detected object, incubating the to-be-detected object containing the to-be-detected small molecules, a labeled antibody and a labeled antigen, adding the obtained mixture into chromatographic test paper, enabling the mixture to enter a reaction zone through the water absorption force of a water absorption pad, and judging the content of the to-be-detected small molecules in the to-be-detected object through the triple signal intensity of different labeling materials of the detection zone and the quality control zone if the to-be-detected object contains the to-be-detected small molecules and the labeled antibody-labeled antigen, wherein the labeled antibody-labeled antigen is combined with an antibody of an anti-modification protein of the detection zone, and the labeled antibody-to-be-detected small molecules are combined with a second antibody of the corresponding labeled antibody of the quality control zone; if the test substance does not contain the small molecule to be tested, only the labeled antibody-labeled antigen is formed. The small molecule detection test strip provided by the invention provides convenience for detection of small molecules.
The small molecule detection test strip provided by the invention has the advantages that the signal response of immune response is subjected to triple analysis, and simultaneously, the sensitivity, the specificity and the universality of immunochromatography detection are organically fused.
In the invention, the bottom plate can be made of non-water-absorbing materials, PVC or other hard materials; the sample pad can be a suction filter paper or a glass fiber membrane; the absorbent pad may be absorbent paper.
In addition, in the present invention, the terms "detection zone" and "quality control zone" are used only for functional differentiation, and the "zone" may be in the form of a "line" or a "region" having a certain geometry. According to one embodiment of the invention, the detection zone and the quality control zone are present in the form of detection lines and quality control lines.
In a third aspect, the invention provides a small molecule triple immunochromatographic assay kit comprising:
the small molecule triple immunochromatography detection test strip;
an incubation liquid, wherein the composition of the incubation liquid is as follows: based on 0.04-0.06mol/L phosphate buffer, the following substances in percentage by weight are added: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG, 2.5-3.5% of Tween-20; the method comprises the steps of,
and (3) optionally selecting a standard curve chart card of the target small molecules, and conveniently obtaining the content of the small molecules by referring to the standard curve chart.
In the present invention, the terms "target small molecule" and "small molecule to be detected" have the same meaning and refer to the small molecule to be detected. The terms "sample to be measured", "analyte" refer to the object to be measured.
According to a specific embodiment of the invention, the small molecule to be detected is taken as DAP, the modified protein is taken as BSA, the first signal molecule is QB, and the second signal molecule is taken as Au@Ag-R as an example:
the labeled antigen signal substance is prepared from a Raman signal surface enhanced material Au@Ag-R by the following method: the chloroauric acid is reduced into gold seeds by sodium citrate, and the silver nitrate is further reduced by a sodium citrate reduction method and coated on the gold seeds, so that the preparation of Au@Ag is realized. After the Au@Ag material is prepared, adding a Raman reporter dissolved in a methanol aqueous solution; the raman reporter may be: CBT: 4-chlorophenylthiol; ABT: 4-amino thiophenol; HBT-4-hydroxybenzene thiophenol; DTNB:5,5' -dithiobis (2-nitrobenzoic acid), and the like.
The antibody to diazepam (anti-DAP-mAb) was prepared as follows: the complete antigen DAP-BSA is obtained by carboxylation modification of DAP and further coupling with BSA by an active ester method. The antibody-secreting cells are obtained by immunization of animals, and the cell line is further preferred after fusion with tumor cells. Injecting the optimized cell strain into the abdominal cavity of the mouse to obtain the ascites containing the antibody, and purifying to obtain the high specificity antibody of the DAP.
The method comprises the steps of pre-incubating a small molecule DAP, a small molecule QB labeled antibody and an Au@Ag-R labeled antigen to be detected to obtain a mixture, wherein the antibody is an anti-DAP antibody, and the antigen is a derivative (DAP-BSA) formed by DAP and a modified protein BSA.
The mixture passes through a detection area and a quality control area in sequence, an anti-BSA antibody is fixedly arranged in the detection area, and a goat anti-mouse secondary antibody (goat anti-michantibody) corresponding to the labeled antibody is fixedly arranged in the quality control area.
And judging the content of small molecules DAP in the object to be detected according to the signal intensity of the detection area and the quality control area.
A pre-incubation step, wherein a certain content of small molecules DAP to be detected in the solution and a marked BSA derivative Au@Ag-R-DAP-BSA of the small molecules to be detected "compete" to bind with a marked antibody QB-DAP-mAb, and the more the content of the small molecules DAP to be detected in the solution; the less the immune complex formed by Au@Ag-R-DAP-BSA and the labeled antibody QB-DAP-mAb, the weaker the visual signal and fluorescent signal of the detection area and the Raman signal based on the surface enhanced material; namely, the content of the object to be detected is inversely proportional to the signal intensity of the detection area; the goat anti-mouse secondary antibody of the quality control region is combined with immune complexes of the small molecules to be detected and the labeled antibodies; because the quantity of the content of the labeled antibody determines the intensity of the fluorescent signal response and the quantity of the labeled antigen determines the intensity of the visual signal and the Raman signal, the intensity of the visual signal, the fluorescence signal and the Raman signal in the detection area is inversely proportional to the content of the small molecule to be detected, the intensity of the visual signal and the fluorescence signal in the quality control area is directly proportional to the content of the small molecule to be detected, and the content of the small molecule to be detected can be judged by comparing the relative intensities of the signals in the detection area and the quality control area.
Compared with the existing immunochromatography detection technology based on the traditional structure, the invention has the beneficial effects that:
(1) In the invention, the use of different labeling materials can realize triple signal analysis on the small molecules to be detected, obviously widen the detection range of the small molecules, and facilitate the further application of immunochromatography detection technology in practice.
(2) In the invention, the 'competing' reaction is carried out in the pre-incubation step, the time controllability is strong, the antigen epitope is more fully exposed, and the reaction can be more fully carried out due to the more sufficient reaction time and the more sufficient epitope exposure, so that the 'competing' reaction has higher sensitivity and the detection method has higher sensitivity.
(3) The detection area is fixed with the antibody for universally recognizing the protein, which is not changed by the change of the object to be detected, thus realizing the organic fusion of the sensitivity, the specificity and the universality of the immune reaction and realizing the high-sensitivity high-flux high-universality quantitative detection of the object to be detected. Therefore, the detection method and the test strip thereof provided by the invention have wider application range.
(4) The invention can be used in various fields of food safety, clinical diagnosis, inspection and quarantine, and the like, and has wide market prospect.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a schematic diagram of a small molecule three-fold test strip according to example 1 of the present invention.
Fig. 2 is a standard graph based on different marking materials in example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
The specific conditions not specified in the examples were either conventional or manufacturer-recommended. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
This example illustrates the preparation of a test strip according to the invention for detection of diazepam. The modified protein is BSA, and the labeled antibody signal material is carboxylated modified fluorescent quantum dot microsphere (QB, available from Beijing Najingjingsu Biotechnology Co., ltd., product model: QBB12117, H07187M).
The labeled antigen signal substance is prepared from a Raman signal surface enhanced material Au@Ag-R by the following method: the chloroauric acid is reduced into gold seeds by sodium citrate, and the silver nitrate is further reduced by a sodium citrate reduction method and coated on the gold seeds, so that the preparation of Au@Ag is realized. After the material preparation is completed, adding the Raman reporter dissolved in 5% methanol aqueous solution, wherein the concentration is 10mg/mL; the raman reporter is DTNB:5,5' -dithiobis (2-nitrobenzoic acid).
The antibody to diazepam (anti-DAP-mAb) was prepared as follows: the complete antigen DAP-BSA is obtained by carboxylation modification of DAP and further coupling with BSA by an active ester method. The antibody-secreting cells are obtained by immunization of animals, and the cell line is further preferred after fusion with tumor cells. Injecting the optimized cell strain into the abdominal cavity of the mouse to obtain the ascites containing the antibody, and purifying to obtain the high specificity antibody of the DAP.
The preparation method of the test strip comprises the following steps:
1) NC membrane detection area coated anti-BSA antibody (anti-BSA-mAb), the coating process is as follows: diluting the BSA antibody to 0.5mg/ml with a mixture of 0.01mol/L phosphate buffer solution with pH7.4 and methanol (methanol content 15%), and coating the BSA antibody on a detection area on a nitrocellulose membrane by using an isolow metal spraying membrane-drawing instrument with the coating amount of 1.0 mu L/cm; and (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
2) NC membrane quality control area coats sheep anti-mouse anti-body (coat anti-mouse anti-body), the coating process is as follows: diluting the goat anti-mouse secondary antibody to the concentration of 0.5mg/mL by using a mixed solution (methanol content of 15%) of phosphate buffer solution with the pH of 0.01mol/L and methanol, and coating the goat anti-mouse secondary antibody on a quality control area on a nitrocellulose membrane by using an isolow metal spraying membrane drawing instrument, wherein the coating amount is 1.0 mu L/cm; and (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
3) Preparing a labeled antigen, a labeled antibody: the antibody (anti-DAP-mAb) of the diazepam to be detected is modified by carboxylated modified quantum dot microspheres to prepare the QB marked antibody (QB-DAP-mAb). The antigen marker Au@Ag-R-DAP-BSA was prepared using Au@Ag-R, DAP-BSA.
4) Sequentially assembling an NC film, a glass fiber pad and a water absorbing paper pad on a bottom plate to prepare a test strip; storing in 2-8deg.C environment, and has a shelf life of 12 months.
Example 2
In this example, the detection method of the present invention is illustrated by the detection of diazepam, and the test strip prepared in example 1 is used for the test:
preparation of a Standard Curve
Diazepam (DAP) standards were prepared as DAP solutions of different concentrations using phosphate buffer: 1.28ng/mL, 0.64ng/mL, 0.32ng/mL, 0.16ng/mL, 0.08ng/mL, 0.04ng/mL, 0.02ng/mL, 0.01ng/mL. The composition of the phosphate buffer was as follows: based on 0.05mol/L phosphate buffer, the following substances in percentage by weight are added: sucrose 2%, fructose 5%, PEG 1%, tween-20%.
Au@Ag-R-DAP-BSA 0.1. Mu.g/100. Mu.L solution, QB-DAP-mAb at 1: the dilution ratio of 3000 was mixed with DAP standard of different concentrations, respectively, the final concentration ranges of QB-DAP-mAb and Au@Ag-R-DAP-BSA were 0.2-0.8mg/mL and 0.01-0.04mg/mL, respectively, DAP was incubated with the labeled antibodies QB-DAP-mAb and Au@Ag-R-DAP-BSA at 37℃for 10min, forming a mixture of immune complexes QB-DAP-mAb-Au@Ag-R-DAP-BSA and QB-DAP-mAb-DAP.
After incubation, adding a glass fiber pad into the solution, and sequentially passing through a detection area and a quality control area of a reaction membrane (NC membrane) under the suction action of a water absorption pad, wherein the QB-DAP-mAb-Au@Ag-R-DAP-BSA immune complex is combined with an anti-BSA-mAb in the detection area; through the quality control zone, QB-DAP-mAb-DAP is combined with goat anti-mouse secondary antibody.
After incubation, adding immunochromatographic test paper into the solution for chromatographic reaction for 10min, reading visual, fluorescent and Raman data by using different readers, quantitatively judging the result by detecting the relative signal intensity of the color development strips of the measuring area and the quality control area, and analyzing and recording the result.
The standard curve obtained from the data obtained is shown in figure 2.
As can be seen from the standard curve calculation in FIG. 2, the DAP visualized minimum detection concentration is 0.08ng mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The minimum fluorescence detection concentration is 0.01ng mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The lowest detection concentration of Raman is 0.006ng mL -1 。
Example 3
In this embodiment, DAP in an aquatic product is taken as an example, which illustrates that the method of the present invention is used to detect a sample to be detected, and the steps are as follows:
sample to be detected: adding DAP into fish meat; the labeling concentrations are shown in table 1.
The test strip prepared in example 1 was equilibrated to room temperature.
DAP-added fish was diluted in equal volume with a mixture of phosphate buffer and methanol (10%) and the resulting dilution was subjected to immunochromatography.
The fish meat standard sample is diluted, and the obtained diluted solution is incubated with a labeled antibody and a labeled antigen for 11min at 37 ℃ to form a mixture of immune complexes QB-DAP-mAb-DAP and QB-DAP-mAb-Au@Ag-R-DAP-BSA; after incubation, adding the solution containing the mixture into a glass fiber pad, and sequentially passing through a detection area and a quality control area of a reaction membrane (NC membrane) under the suction action of a water absorption pad, wherein QB-DAP-mAb-Au@Ag-R-DAP-BSA is combined with an anti-BSA antibody after passing through the detection area; through the quality control zone, QB-DAP-mAb-DAP is combined with goat anti-mouse secondary antibody.
And detecting the visualization of the color development strips of the measuring area and the quality control area by a detector, and carrying out quantitative interpretation on the result by fluorescence and Raman relative signal intensity. The detection results were calculated from the standard curve obtained in example 2, as shown in table 1.
TABLE 1 detection results of DAP sample under different Signal measurement modes
a Mean ± standard deviation.
From the detection results in table 1, the accuracy of the three signal measurement is higher, which indicates that the detection method provided by the invention can realize triple detection of the DAP.
Compared with the existing dual signal detection based on the traditional structural immunochromatography detection technology, the method provided by the invention is used for marking both the antigen and the antibody, and can finally realize triple detection of the object to be detected. The "competition" reaction is already fully performed in the pre-incubation step and the antigen DAP-BSA is in solution rather than immobilized, and the epitope can be more fully exposed and the competition reaction can be more fully performed, making the "competition" reaction more sensitive. In addition, the detection region of the present invention is immobilized with a specific antibody (anti-BSA-mAb) of a modified protein (BSA) and is not changed by the change of the analyte, so that the detection region has high versatility.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.
Claims (5)
1. A small molecule triple immunochromatography detection method comprises the following steps:
(1) Incubating the object to be detected, the labeled antibody and the labeled antigen to obtain a mixture; wherein the labeled antibody is an antibody against a small molecule of interest and is labeled with a first signal substance; the labeled antigen is a derivative of a target small molecule with a modified protein and is labeled with a second signal substance; the first signal substance and the second signal substance are respectively fluorescent quantum dots and gold core silver shell materials modified with Raman reporter molecules;
(2) Sequentially passing the mixture obtained in the step (1) through a detection area and a quality control area; wherein the detection zone is fixedly provided with an antibody against a modified protein in the labeled antigen; the quality control area is fixedly provided with a secondary antibody corresponding to the labeled antibody;
(3) Obtaining the content of target small molecules in the object to be detected according to the triple signal intensity of the detection area and the quality control area;
the target small molecule is diazepam;
wherein the modified protein is ovalbumin, hemocyanin, bovine serum albumin, human serum albumin or chicken ovalbumin.
2. The small molecule triple immunochromatographic assay of claim 1, wherein in step (1), the temperature of the incubation is 25-40 ℃; the incubation time is 5-20min; the incubation is performed in a buffer, the composition of which comprises: based on 0.04-0.06mol/L phosphate buffer, the following substances in percentage by weight are added: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG, 2.5-3.5% of Tween-20;
in the incubation system, the concentration of the labeled antibody is 0.2-0.8mg/mL, and the concentration of the labeled antigen is 0.01-0.04mg/mL.
3. The small molecule triple immunochromatographic assay of claim 2, wherein in step (1), the incubation temperature is 35-37 ℃.
4. The test strip is characterized by comprising a sample pad, a water absorption pad and a bottom plate fixed with a nitrocellulose membrane, wherein the sample pad and the water absorption pad are arranged on the bottom plate and are respectively positioned at two sides of the nitrocellulose membrane; wherein,
the sample pad is used for bearing an incubation of a to-be-detected object, a labeled antibody and a labeled antigen;
the labeled antibody is an antibody against a target small molecule and is labeled with a first signal substance; the labeled antigen is a derivative of a target small molecule with a modified protein and is labeled with a second signal substance; the first signal substance and the second signal substance are respectively fluorescent quantum dots and gold core silver shell materials modified with Raman reporter molecules;
the nitrocellulose membrane is provided with a detection area and a quality control area; the detection area is fixedly provided with an antibody for resisting the modified protein in the labeled antigen; the quality control area is fixedly provided with a secondary antibody corresponding to the labeled antibody;
the target small molecule is diazepam;
wherein the modified protein is ovalbumin, hemocyanin, bovine serum albumin, human serum albumin or chicken ovalbumin.
5. A small molecule triple immunochromatography detection kit is characterized in that the kit comprises:
the small molecule triple immunochromatographic test strip of claim 4;
an incubation liquid, wherein the composition of the incubation liquid is as follows: based on 0.04-0.06mol/L phosphate buffer, the following substances in percentage by weight are added: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG, 2.5-3.5% of Tween-20; the method comprises the steps of,
standard graph card for small molecules of interest.
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| CN109884306A (en) * | 2019-03-14 | 2019-06-14 | 中国人民解放军军事科学院军事医学研究院 | A kind of small molecule test strip, kit and its detection method |
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