Immunochromatography test strip marked by time-resolved fluorescent microspheres

Abstract

The invention discloses a time-resolved fluorescent microsphere-labeled immunochromatography test strip, which comprises a bottom plate with an NC film attached to the surface, and a sample pad, a time-resolved fluorescent microsphere combination pad, a chromatography pad and a water absorption pad which are sequentially lapped are laid on the bottom plate; a time-resolved fluorescent microsphere conjugate is coated on the time-resolved fluorescent microsphere conjugate pad; the chromatography pad is divided into three parts by test lines, the test line close to one side of the latex labeling combination pad is a detection line, the test line close to one side of the water absorption pad is a control line, the detection line is coated with an antibody LEC, and the control line is coated with a goat-anti-mouse second antibody; the coating amount of the time-resolved fluorescent microsphere conjugate on the time-resolved fluorescent microsphere conjugate pad is 9.9 L / cm; the detection line is coated with a phosphate buffer solution with the antibody LEC concentration of 1.5 mg / mL, and the coating amount is 1.5 L / cm; the contrast control line is coated with a phosphate buffer solution with the goat-anti-mouse second antibody concentration of 350 g / mL, and the coating amount is 1.5 L / cm.

Description

Technical Field

[0001] The invention belongs to the technical field of protein detection, and in particular relates to an immunochromatographic test strip marked with time-resolved fluorescent microspheres. Background Art

[0002] Increased expression and excessive deposition of amyloid-β in cerebral blood vessels is one of the pathological mechanisms of Alzheimer's disease (AD). As an early marker, the concentration of amyloid-β will increase significantly during the cognitive dysfunction period and the early stages of Alzheimer's disease. In the middle and late stages, as the deposition increases, the concentration will decrease. Therefore, the detection of amyloid-β is of great value in the early screening of Alzheimer's disease, the dynamic observation of progressive disease changes, and the dynamic evaluation of treatment effects.

[0003] Currently, the detection kits for Aβ on the market mainly use blood samples, and most of the invention forms are laboratory test kits rather than rapid in vitro diagnostic reagents. There are few in vitro diagnostic reagents for rapid detection of urinary Aβ, and the chromatographic test strips based on time-resolved fluorescent microspheres are blank for detecting β-amyloid products in urine.

[0004] The chromatographic test strips of the traditional colloidal gold immunochromatography can also be used for rapid screening and on-site testing. The test results are clear in color and can be judged visually by the naked eye, but the readers are subjective and there is a certain reading deviation. Colloidal gold chromatography uses physical adsorption to bind, which has low binding efficiency. The antigen may be detached from the surface of the antibody-gold particle marker, and the marker binding is unstable. Summary of the invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provide an immunochromatographic test strip labeled with time-resolved fluorescent microspheres.

[0006] In order to achieve the above object, the technical solution provided by the present invention is: The time-resolved fluorescent microsphere-labeled immunochromatographic test strip comprises a bottom plate (7) with an NC film attached to the surface, and the bottom plate (7) is provided with a sample pad (1), a time-resolved fluorescent microsphere binding pad (2), a chromatography pad (3) and a water absorption pad (4) which are overlapped in sequence; the time-resolved fluorescent microsphere binding pad (2) is coated with a time-resolved fluorescent microsphere conjugate; the chromatography pad (3) is divided into three parts by a test line, the test line close to the latex-labeled binding pad (2) is a test line (5), and the test line close to the water absorption pad (4) is a control line (6), the test line (5) is coated with an antibody LEC, and the control line (6) is coated with a goat anti-mouse secondary antibody; the coating amount of the time-resolved fluorescent microsphere conjugate on the time-resolved fluorescent microsphere binding pad (2) is 9.9 μL. / cm; the test line (5) is coated with a phosphate buffer solution with an antibody LEC concentration of 1.5 mg / mL, and the coating amount is 1.5 µL / cm; the control line (6) is coated with a phosphate buffer solution with a goat anti-mouse secondary antibody concentration of 350 µg / mL, and the coating amount is 1.5 µL / cm.

[0007] Preferably, the preparation method of each mL of the time-resolved fluorescent microsphere conjugate comprises the following steps: (1) Prepare coupling solution, blocking solution and storage solution; the coupling solution is a MES solution with a concentration of 300-500 mM and a pH value of 7, the blocking solution is 0.2% ethanolamine, the storage solution is 25 mM Tris solution + 1% BSA + 0.1% Triton or S9 + 2% sucrose + 0.05% Proclin300, and the pH value of the storage solution is 8.5; the % is the mass percentage unit of the component in the liquid system; (2) Activation: Take 900 μL of coupling solution and put it into a 1.5 mL centrifuge tube, add 100-200 μg of time-resolved fluorescent microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to another 0.01 mL of coupling solution to dissolve, then add the coupling solution to the centrifuge tube containing the coupling solution and time-resolved fluorescent microspheres, react at room temperature for 30 minutes, centrifuge at 10000-12000 rpm for 15 minutes, and resuspend with coupling solution to obtain an activated time-resolved fluorescent microsphere suspension; (3) Coupling: Add 100-200 μg of Aβ 42 N-terminal antibody (e.g., monoclonal antibody W0-2) to the activated time-resolved fluorescent microsphere suspension and react at room temperature for 120-150 min to obtain a time-resolved fluorescent microsphere conjugate suspension; (4) Blocking: Add blocking solution to the time-resolved fluorescent microsphere conjugate suspension and react at room temperature for 30-60 minutes; (5) Storage: Centrifuge the time-resolved fluorescent microsphere conjugate suspension treated in step (4), remove the supernatant, and then add 1 mL of storage solution for storage.

[0008] Preferably, the sample pad (1) needs to be immersed in a treatment solution for hydration and sealing before being prepared into a test strip. The immersion time is 37 minutes and then dried at 37°C overnight. The treatment solution is BSA, PVP, S9, S12, sucrose, casein, ProClin300 or PBS buffer.

[0009] In order to improve the performance of Aβ early screening rapid detection products, the present invention provides a chromatographic test strip based on time-resolved fluorescent microsphere labeling technology, which can quickly and conveniently detect amyloid beta in urine. The excitation wave and emission wave span of the time-resolved fluorescent microspheres used in the present invention are higher than those of general fluorescent microspheres, which are suitable for the reaction between the Aβ 42 N-terminal antibody used in the present invention and the sample. Aβ 42 N-terminal antibody and Lec antibody (lecanemab, an Aβ monoclonal antibody drug jointly developed by Biogen and Eisai, with a trade name of Leqembi) are used for sandwiching to achieve the ectopic binding technology of Lec antibody connection on time-resolved fluorescent microspheres, protect the long fluorescence lifetime of the antibody and sample reaction under fluorescence excitation in this technology, enhance stability and repeatability, reduce interference effects, and improve sensitivity.

[0010] In short, the present invention is simple and convenient to test with good stability, good repeatability and high sensitivity, and provides more reliable technical support for the detection of related β-amyloid protein in the early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a schematic diagram of the immunochromatographic test strip labeled with time-resolved fluorescent microspheres of the present invention; Figure 2 This is a test result diagram of the present invention; from left to right: negative result, positive result, invalid result; Figure 3 This is a sensitivity experiment diagram of the present invention; Figure 4 This is a luminescence diagram of the pure water non-interference negative control of the present invention.

[0012] In the figure: 1. sample pad; 2. time-resolved fluorescent microsphere binding pad; 3. chromatography pad; 4. absorbent pad; 5. detection line; 6. control line; 7. bottom plate. DETAILED DESCRIPTION

[0013] Coupling solution: 300-500 mM MES solution, pH 7.0; Blocking solution: 0.2% ethanolamine; Storage solution: 25 mM PBS solution, pH 8.5, 1% BSA, 0.1% Triton or S9, 2% sucrose, 0.05% Proclin300 Example

[0014] according to Figure 1 The preparation of a time-resolved fluorescent microsphere-labeled immunochromatographic test strip for qualitative detection of urine beta-amyloid protein comprises the following steps: (1) Preparation of immunotime-resolved fluorescent microspheres: Activation: Take 900 μL of coupling solution and put it in a 1.5 mL centrifuge tube, add 100 μg of time-resolved fluorescent microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to another 0.01 mL of coupling solution to dissolve, then add the coupling solution to the centrifuge tube containing coupling solution and latex microspheres, react at room temperature for 30 minutes, centrifuge at 12000 rpm for 15 minutes, and resuspend with coupling solution to obtain activated time-resolved fluorescent microspheres; Coupling: Add 100 μg of Aβ 42 N-terminal antibody to the activated time-resolved fluorescent microspheres and react at room temperature for 120 min; Blocking: Add 0.2% ethanolamine to the coupled time-resolved fluorescent microspheres and react at room temperature for 30 minutes; Storage: Centrifuge the blocked time-resolved fluorescent microspheres to remove the supernatant and add 1 mL of storage solution for storage; (2) Fixation of time-resolved fluorescent microsphere conjugates: Ultrasound: Treat the high-concentration time-resolved fluorescent microsphere conjugate with ultrasound for 6 minutes to disperse the fluorescent microspheres; Dilution: Dilute the ultrasonically treated time-resolved fluorescent microsphere conjugate 1:2 to the working concentration with time-resolved fluorescent microsphere diluent and mix well; Spraying: The diluted time-resolved fluorescent microsphere conjugate was sprayed at 9.9 μL / cm sprayed on the treated conjugate pad On the bonding pad, make the bonding pad absorb water evenly; Drying: Place the sprayed conjugate pad in a drying room at 37°C and dry for 60 minutes, then take it out and store it away from moisture to obtain a conjugate pad combined with immuno-time-resolved fluorescent microspheres; (3) Preparation of base plate and NC membrane: Dilution: Dilute LEC and IgG goat anti-mouse to the working concentration according to the required antibody concentration, T line: 1.5mg / mL; C line: 0.35mg / mL; Film pasting: Take PVC board and NC film, paste NC film on the PVC board, and balance the pasted NC film in the film cutting room for 30 minutes; Membrane marking: Use a membrane marking machine to evenly mark the prepared IgG goat anti-mouse and antibody on the T and C lines of the NC membrane; Drying: Transfer the scribed NC membrane to a drying room at 37°C and dry overnight for 10 hours to obtain the NC membrane; (4) Assemble the test strip components together and cut them into 4 mm thin strips. Figure 1 As shown, put it into the shell to obtain a time-resolved fluorescent microsphere-labeled immunochromatographic test strip for detecting urine beta-amyloid protein.

[0015] In this embodiment, the sample pad, as the site for adding the sample to be tested, is connected to the binding pad and the NC membrane; the binding pad is combined with immune time-resolved fluorescent microspheres; the NC membrane is connected to IgG goat anti-mouse antibodies, and the absorbent paper contains substances in the liquid that are not combined with the binding pad or the NC membrane. Example 2 according to Figure 1 The preparation of a colored latex microsphere labeled test strip for qualitative detection of urine beta-amyloid protein comprises the following steps: (1) Preparation of immunofluorescence microspheres: Activation: Take 900 μL of coupling solution and put it in a 1.5 mL centrifuge tube, add 200 μg of time-resolved fluorescent microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to another 0.01 mL of coupling solution to dissolve, then add the coupling solution to the centrifuge tube containing coupling solution and latex microspheres, react at room temperature for 30 minutes, centrifuge at 10000 rpm for 15 minutes, and resuspend with 0.5 M pH 7 MES solution to obtain activated time-resolved fluorescent microspheres; Coupling: Add 200 μg of Aβ 42 N-terminal antibody to the activated time-resolved fluorescent microspheres and react at room temperature for 150 min; Blocking: Add 0.2% ethanolamine to the coupled time-resolved fluorescent microspheres and react at room temperature for 60 minutes; Storage: Centrifuge the blocked time-resolved fluorescent microspheres to remove the supernatant and add 1 mL of storage solution for storage; (2) Fixation of time-resolved fluorescent microsphere conjugates: Ultrasound: Treat the high-concentration time-resolved fluorescent microsphere conjugate with ultrasound for 6 min to disperse the fluorescent microspheres; Dilution: dilute the ultrasonically treated time-resolved fluorescent microsphere conjugate 1:4 to the working concentration with time-resolved fluorescent microsphere diluent and mix well; Spraying: The diluted time-resolved fluorescent microsphere conjugate was sprayed at 9.9 μL / cm sprayed on the treated conjugate pad On the bonding pad, make the bonding pad absorb water evenly; Drying: Place the sprayed conjugate pad in a drying room at 37°C and dry for 60 minutes, then take it out and store it away from moisture to obtain a conjugate pad combined with immuno-time-resolved fluorescent microspheres; (3) Preparation of base plate and membrane: Dilution: Dilute LEC and IgG goat anti-mouse to the working concentration according to the required antibody concentration, T line: 1.8mg / mL; C line: 0.35mg / mL; Film pasting: Take the PVC board and NC film, paste the NC film on the bottom plate first, and balance the pasted NC film in the film cutting room for 30 minutes; Membrane marking: Use a membrane marking machine to evenly mark the prepared IgG goat anti-mouse and antibody on the T and C lines of the NC membrane; Drying: Transfer the scribed NC membrane to a drying room at 37°C and dry overnight for 10 hours to obtain the NC membrane; (4) Assemble the test strip components together and cut them into 4 mm thin strips. Figure 1 As shown, put it into the shell to obtain a time-resolved fluorescent microsphere-labeled immunochromatographic test strip for detecting urine beta-amyloid protein.

Claims

1. An immunochromatographic test strip labeled with time-resolved fluorescent microspheres, the test strip comprising a bottom plate (7) with an NC film attached to the surface, characterized in that: The bottom plate (7) is provided with a sample pad (1), a time-resolved fluorescent microsphere binding pad (2), a chromatography pad (3) and a water absorption pad (4) which are overlapped in sequence; the time-resolved fluorescent microsphere binding pad (2) is coated with a time-resolved fluorescent microsphere conjugate; the chromatography pad (3) is divided into three parts by a test line, the test line close to the latex labeling binding pad (2) is a test line (5), and the test line close to the water absorption pad (4) is a control line (6), the test line (5) is coated with an antibody LEC, and the control line (6) is coated with a goat anti-mouse secondary antibody; the coating amount of the time-resolved fluorescent microsphere conjugate on the time-resolved fluorescent microsphere binding pad (2) is 9.9 µL / cm; the test line (5) is coated with a phosphate buffer solution with an antibody LEC concentration of 1.5 mg / mL, and the coating amount is 1.5 µL / cm; the control line (6) is coated with a goat anti-mouse secondary antibody with a concentration of 350 µg / mL. / mL phosphate buffer, the coating volume is 1.5µL / cm.

2. The immunochromatographic test strip labeled with time-resolved fluorescent microspheres as claimed in claim 1, characterized in that: The preparation method of each mL of the time-resolved fluorescent microsphere conjugate comprises the following steps: (1) Prepare coupling solution, blocking solution and storage solution; the coupling solution is a MES solution with a concentration of 300-500 mM and a pH value of 7, the blocking solution is 0.2% ethanolamine, the storage solution is 25 mM Tris solution + 1% BSA + 0.1% Triton or S9 + 2% sucrose + 0.05% Proclin300, and the pH value of the storage solution is 8.5; the % is the mass percentage unit of the component in the liquid system; (2) Activation: Take 900 μL of coupling solution and put it into a 1.5 mL centrifuge tube, add 100-200 μg of time-resolved fluorescent microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to another 0.01 mL of coupling solution to dissolve, then add the coupling solution to the centrifuge tube containing the coupling solution and time-resolved fluorescent microspheres, react at room temperature for 30 minutes, centrifuge at 10000-12000 rpm for 15 minutes, and resuspend with coupling solution to obtain an activated time-resolved fluorescent microsphere suspension; (3) Coupling: Add 100-200 μg of Aβ 42 N-terminal antibody (e.g., monoclonal antibody W0-2) to the activated time-resolved fluorescent microsphere suspension and react at room temperature for 120 min to obtain a time-resolved fluorescent microsphere conjugate suspension; (4) Blocking: Add blocking solution to the time-resolved fluorescent microsphere conjugate suspension and react at room temperature for 30 min; (5) Storage: Centrifuge the time-resolved fluorescent microsphere conjugate suspension treated in step (4), remove the supernatant, and then add 1 mL of storage solution for storage.

3. The immunochromatographic test strip labeled with time-resolved fluorescent microspheres as claimed in claim 1, characterized in that: The sample pad (1) needs to be immersed in a treatment solution for hydration and sealing before being prepared into a test strip. The immersion time is 37 minutes and then dried at 37°C overnight. The treatment solution is BSA, PVP, S9, S12, sucrose, casein, ProClin300 or PBS buffer.

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