Colored latex microsphere labeled test strip

Abstract

The invention discloses a colored latex microsphere labeled test strip which comprises a bottom plate with an NC film attached to the surface, and a sample pad, a latex labeled combination pad, a chromatography pad and a water absorption pad which are sequentially lapped are laid on the bottom plate; the latex marking combination pad is coated with a latex microsphere antibody conjugate; 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 Adu, and the control line is coated with a goat-anti-mouse second antibody; the coating amount of the latex microsphere antibody conjugate on the latex labeled conjugate pad is 8.5 L / cm; the detection line is coated with a phosphate buffer solution with the antibody Adu concentration of 0.2 mg / mL, and the coating amount is 1.5 L / cm; the contrast control line (6) is coated with a phosphate buffer solution with the goat-anti-mouse second antibody concentration of 400 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 a colored latex microsphere-marked test strip. Background Art

[0002] With the increase of age, the incidence of Alzheimer's disease (AD) is getting higher and higher. At the same time, Alzheimer's disease (AD) is not only prevalent in the elderly, but also tends to be younger. The increased expression and excessive deposition of β-amyloid protein in cerebral blood vessels is one of the pathological mechanisms of Alzheimer's disease (AD). In the early stages of Alzheimer's disease, the concentration of β-amyloid protein will increase significantly. In the middle and late stages, as the precipitation increases, the concentration will decrease. Therefore, β-amyloid protein detection 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] Chemiluminescent immunoassay (CLIA) has high sensitivity and can detect analytes at low concentrations, but it is costly and time-consuming, and is not suitable for rapid detection of early-stage amyloid-β.

[0004] Although traditional colloidal gold testing is simple, the results of repeatability and stability vary greatly.

[0005] There is still a gap in the market for the application of colored latex microsphere labeling in early detection of amyloid-β. Summary of the invention

[0006] The present invention aims to overcome the deficiencies of the prior art and provides a colored latex microsphere-marked test strip.

[0007] In order to achieve the above object, the technical solution provided by the present invention is: The colored latex microsphere-labeled 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 latex-labeled binding pad (2), a chromatography pad (3) and a water-absorbing pad (4) which are overlapped in sequence; the latex-labeled binding pad (2) is coated with a latex microsphere antibody 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-absorbing pad (4) is a control line (6), the test line (5) is coated with antibody Adu, and the control line (6) is coated with goat anti-mouse secondary antibody; the amount of the latex microsphere antibody conjugate coated on the latex-labeled binding pad (2) is 8.5 µL. / cm; the test line (5) is coated with a phosphate buffer solution with an antibody Adu concentration of 0.2 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 400 µg / mL, and the coating amount is 1.5 µL / cm.

[0008] Preferably, the preparation method of each mL of the latex microsphere antibody 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 50-100 mM and a pH value of 6-6.5; the blocking solution is 0.1% ethanolamine alone or a combination of 0.1% ethanolamine and 1% BSA; the storage solution is 25 mMTris solution + 1% BSA + 0.1% Triton + 2% trehalose + 0.05% sodium azide; the pH value of the storage solution is 8.5; the % is the mass percentage 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 50-100 μg of latex microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to another 0.05-0.1 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 10 minutes, and resuspend with coupling solution to obtain activated latex microsphere suspension. After ultrasonication for 3 minutes, the microspheres are fully dispersed to improve the coupling efficiency; (3) Coupling: Add 50 μg of Lecanemab antibody to the activated latex microsphere suspension and react at room temperature for 120 min to obtain a latex microsphere antibody conjugate suspension; (4) Blocking: Add blocking solution to the latex microsphere antibody conjugate suspension and react at room temperature for 30 minutes; (5) Storage: Centrifuge the latex microsphere antibody conjugate suspension treated in step (4), remove the supernatant, and then add 1 mL of storage solution for storage.

[0009] Preferably, if the blocking solution added in step (4) is 0.1% ethanolamine and 1% BSA, 0.1% ethanolamine is first added and reacted at room temperature for 30 minutes, and then 1% BSA is added and reacted at room temperature for 30 minutes.

[0010] More 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 2% sodium chloride + 0.5% Tween 20.

[0011] The present invention uses lec antibody and Adu antibody to provide a simplified test strip preparation and detection method based on colored latex microsphere labeling and PVC plate streaking coating. The test strip prepared by the method can be used to detect urine beta amyloid protein, with simple detection, high sensitivity and good stability, providing more reliable technical support for the detection of beta amyloid protein in the early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of a colored latex microsphere-labeled test strip 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 the sensitivity and pure water experimental diagram of the present invention; Figure 4 This is a graph of fresh urine test results for children under 10 years old collected by the present invention.

[0013] In the figure: 1. sample pad; 2. latex labelled binding pad; 3. chromatography pad; 4. absorbent pad; 5. test line; 6. control line; 7. bottom plate. DETAILED DESCRIPTION Example 1

[0014] 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: Step 1: Pre-treatment of the conjugate pad and the sample pad. Cut the conjugate pad into 9 mm strips. After cutting, the sample pad is immersed in the treatment solution for hydration and sealing. After immersion for 37 minutes, it is dried at 37°C overnight. The sample pad treatment solution includes 2% sodium chloride and 0.5% Tween 20. The latex microsphere antibody conjugate pad is treated with the improved conjugate pad to produce an ideal conjugate pad release effect. Step 2: Activation of latex microspheres: by connecting the activated carboxyl groups on the surface of latex microspheres through chemical bonds, and then combining with antibody proteins to form latex microsphere-antibody conjugates, which can be directly read after chromatography; during detection, the operation is simple, time-saving and sensitive; Step 3: When labeling the Lecanemab monoclonal antibody with latex microspheres, a method of simultaneously adding a protective agent and an antibody is used to prepare the latex microsphere antibody, so that the addition of the protective agent is more conducive to the labeling of the antibody; Step 4: Spraying the conjugate pad: Use the GoldBiao Bio-Gold Sprayer to evenly spray the latex microsphere antibody conjugate on the conjugate pad. The spraying volume of the conjugate pad is 8.5 μl / cm and dried at 37°C. Step 5. PVC plate streaking (coating of detection line antibody and control line secondary antibody): Dilute another coated antibody Adu (an antibody with a different site from that attached to the latex microsphere marker) jointly developed by Biogen and Eisai to 0.2 mg / ml and coat 1.5 μL / cm The secondary antibody was a commercial goat anti-mouse secondary antibody diluted to 0.4 mg / ml and coated at 1.5 μL / cm, The T / C line spacing was 5 mm and dried at 37°C overnight; Step 6: Assemble the test strips: Figure 1 Assemble the test strip components together and cut into 4 mm thin strips; Step 7, qualitative detection method: 1. Use a pipette to draw the sample and add 3 drops of sample in sequence, 2. Use a pipette to draw the sample and add 80 μL of sample in sequence; Step 8. Dilute the Aβ42 (Qiangyao Biotechnology) quality control product into 12ng, 8ng, 4ng, 2ng, 1ng, 0.5ng, and 0.1ng, and drip them onto the β-amyloid protein latex microsphere test strip in sequence. Use water as the control group to observe the experimental results. Figure 2 The actual sample test results are shown in Figure 4 .

[0015] The preparation method of the latex microsphere antibody conjugate mentioned above comprises the following steps: Coupling solution: 50 mM MES solution, pH 6.5; Blocking solution: 0.1% ethanolamine and 1% BSA; Storage solution: 25 mM Tris solution, pH 8.5, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% sodium azide; a. Activation: Take 900 μL of coupling solution and put it in a 1.5 mL centrifuge tube, add 50 μg of latex microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to another 0.05 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 10 minutes, resuspend with coupling solution to obtain activated latex microspheres, and ultrasonicate for 3 minutes to fully disperse the microspheres to improve coupling efficiency; b. Coupling: Add 50 μg of Lecanemab antibody (Eisai and Biogen) to the activated latex microspheres and react at room temperature for 120 minutes; c. Blocking: Add 0.1% ethanolamine to the coupled latex microspheres and react at room temperature for 30 minutes; Then, 1% BSA was added and reacted at room temperature for 30 min; d. Storage: Centrifuge the blocked latex microspheres, remove the supernatant, and add 1 mL of storage solution for storage.

[0016] 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: Step 1: Pre-treatment of the conjugate pad and the sample pad. After cutting, the conjugate pad is passed through the conjugate pad treatment solution, soaked for 37 minutes, and then dried at 37°C overnight. After cutting, the sample pad is immersed in the treatment solution for hydration and sealing. After soaking for 37 minutes, it is dried at 37°C overnight. The sample pad treatment solution: 2% sodium chloride, 0.5% Tween 20. The latex microsphere antibody conjugate pad uses the improved conjugate pad treatment to produce an ideal conjugate pad release effect. Step 2: Activation of latex microspheres: by connecting the activated carboxyl groups on the surface of latex microspheres through chemical bonds, and then combining with antibody proteins to form latex microsphere-antibody conjugates, which can be directly read after chromatography; during detection, the operation is simple, time-saving and sensitive; Step 3: When labeling the Lecanemab monoclonal antibody with latex microspheres, a method of simultaneously adding a protective agent and an antibody is used to prepare the latex microsphere antibody, so that the addition of the protective agent is more conducive to the labeling of the antibody; Step 4: Spraying the conjugate pad: Use the GoldBiao Bio-Gold Sprayer to evenly spray the latex microsphere antibody conjugate on the conjugate pad. The spraying volume of the conjugate pad is 8.5 μl / cm and dried at 37°C. Step 5, PVC plate streaking (coating of detection line antibody and control line secondary antibody): Dilute another coated antibody Adu (an antibody with a different site from that attached to the latex microsphere marker) jointly developed by Biogen and Eisai to 0.2 mg / ml and coat 1.5 μL / cm The secondary antibody was a commercial goat anti-mouse secondary antibody diluted to 0.4 mg / ml and coated at 1.5 μL / cm, The T / C line spacing was 5 mm and dried at 37°C overnight; Step 6: Assemble the test strips: Figure 1 Assemble the test strip components together and cut into 4 mm thin strips; Step 7, qualitative detection method: 1. Use a pipette to draw the sample and add 4 drops of sample in sequence, 2. Use a pipette to draw the sample and add 100 μL of sample in sequence; Step 8. Dilute the Aβ42 (Qiangyao Biotechnology) quality control product into 12ng, 8ng, 4ng, 2ng, 1ng, 0.5ng, and 0.1ng, and drip them onto the β-amyloid protein latex microsphere test strip in sequence. Use water as the control group to observe the experimental results. Figure 3 The actual sample test results are shown in Figure 4 .

[0017] The preparation method of the latex microsphere antibody conjugate mentioned above comprises the following steps: Coupling solution: 100 mM MES solution, pH 6; Blocking solution: 0.1% ethanolamine; Storage solution: 25 mM Tris solution, pH 8.5, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% sodium azide a. Activation: Take 900 μL of coupling solution and put it in a 1.5 mL centrifuge tube, add 100 μg of latex microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to another 0.1 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 10 minutes, and resuspend with coupling solution to obtain activated latex microspheres. After ultrasonication for 3 minutes, the microspheres are fully dispersed to improve the coupling efficiency; b. Coupling: Add 100 μg of Lecanemab antibody (Eisai and Biogen) to the activated latex microspheres and react at room temperature for 150 minutes; c. Blocking: Add 0.1% ethanolamine to the coupled latex microspheres and react at room temperature for 30 minutes; d. Storage: Centrifuge the blocked latex microspheres, remove the supernatant, and add 1 mL of storage solution for storage.

Claims

1. A colored latex microsphere-labeled test strip, 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 latex marker binding pad (2), a chromatography pad (3) and a water-absorbing pad (4) which are overlapped in sequence; the latex marker binding pad (2) is coated with a latex microsphere antibody conjugate; the chromatography pad (3) is divided into three parts by a test line, the test line close to the latex marker binding pad (2) is a test line (5), and the test line close to the water-absorbing pad (4) is a control line (6), the test line (5) is coated with antibody Adu, and the control line (6) is coated with goat anti-mouse secondary antibody; the coating amount of the latex microsphere antibody conjugate on the latex marker binding pad (2) is 8.5 µL / cm; the test line (5) is coated with a phosphate buffer solution with an antibody Adu concentration of 0.2 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 400 µg / mL. / mL phosphate buffer, the coating volume is 1.5µL / cm.

2. The colored latex microsphere-labeled test strip according to claim 1, characterized in that: The preparation method of each mL of the latex microsphere antibody 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 50-100 mM and a pH value of 6-6.5; the blocking solution is 0.1% ethanolamine alone or a combination of 0.1% ethanolamine and 1% BSA; the storage solution is 25 mMTris solution + 1% BSA + 0.1% Triton + 2% trehalose + 0.05% sodium azide; the pH value of the storage solution is 8.5; the % is the mass percentage 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 50-100 μg of latex microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to another 0.05-0.1 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 10 minutes, and resuspend with coupling solution to obtain activated latex microsphere suspension. After ultrasonication for 3 minutes, the microspheres are fully dispersed to improve the coupling efficiency; (3) Coupling: Add 50 μg of Lecanemab antibody to the activated latex microsphere suspension and react at room temperature for 120 min to obtain a latex microsphere antibody conjugate suspension; (4) Blocking: Add blocking solution to the latex microsphere antibody conjugate suspension and react at room temperature for 30 minutes; (5) Storage: Centrifuge the latex microsphere antibody conjugate suspension treated in step (4), remove the supernatant, and then add 1 mL of storage solution for storage.

3. The colored latex microsphere-labeled test strip according to claim 2, characterized in that: In step (4), if the blocking solution added is 0.1% ethanolamine and 1% BSA, first add 0.1% ethanolamine and react at room temperature for 30 minutes, and then add 1% BSA and react at room temperature for 30 minutes.

4. The colored latex microsphere-labeled test strip according to 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 2% sodium chloride + 0.5% Tween 20.

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