Test strip and kit for detecting Alzheimer-related neurofilament protein AD7c-NTP

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a test strip and a kit for detecting Alzheimer-related neurofilament protein AD7c-NTP. The detection test strip for the Alzheimer-related neurofilament protein AD7c-NTP provided by the invention comprises a PVC (Polyvinyl Chloride) bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC bottom plate; a mouse anti-AD7c-NTP monoclonal antibody I marked by latex is contained on the combination pad; a detection line T and a quality control line C are arranged on the nitrocellulose membrane; the detection line T is coated with a mouse anti-AD7c-NTP monoclonal antibody II, and the quality control line C is coated with a goat anti-mouse IgG polyclonal antibody. The detection test strip for Alzheimer-related neurofilament protein AD7c-NTP provided by the invention has the advantages of high sensitivity, strong specificity, good stability and the like, can realize auxiliary diagnosis of Alzheimer's disease, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to a test strip and kit for detecting Alzheimer's-related neurofilament protein AD7c-NTP. Background Technology

[0002] Alzheimer's-associated neurofilament protein AD7c-NTP is a key biomarker for AD. Its concentration in urine is positively correlated with the degree of neuronal damage and the pathological progression of AD. Therefore, the detection of AD7c-NTP in urine is of great significance for the early diagnosis and monitoring of AD.

[0003] Colloidal gold immunochromatography is a novel immunolabeling technique that uses colloidal gold as a tracer marker for antigen-antibody interactions. Due to its ease of operation, rapid detection, and lack of specialized equipment, it has been widely used for the rapid detection of Alzheimer's-related neurofilament protein AD7c-NTP. The core of this technology is the test strip. However, existing test strips for AD7c-NTP still have significant shortcomings in practical applications: First, the detection sensitivity is relatively low, making it difficult to identify low concentrations of AD7c-NTP in the urine of early-stage AD patients, leading to missed diagnoses and missed opportunities for optimal treatment. Second, the complex matrix of the urine sample, containing proteases, lipids, and salts, easily generates non-specific interference, leading to false positives and affecting the accuracy of the test. Third, the reagent kit has poor stability, with antibodies easily degrading, affecting the repeatability of the test and resulting in inconsistent results between different batches.

[0004] Therefore, there is an urgent need to optimize the preparation method of Alzheimer's-related neurofilament protein AD7c-NTP test strips to improve detection sensitivity, accuracy and stability, thereby providing a more reliable tool for the diagnosis and screening of Alzheimer's disease. Summary of the Invention

[0005] To improve the detection sensitivity, accuracy, and stability of Alzheimer's-related neurofilament protein AD7c-NTP test strips, this application provides a test strip and kit for Alzheimer's-related neurofilament protein AD7c-NTP.

[0006] Firstly, this application provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP, which adopts the following technical solution: A test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP includes a PVC base plate; the PVC base plate is provided with a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the conjugate pad contains mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres; the nitrocellulose membrane has a detection T line and a control C line; the detection T line is coated with mouse anti-AD7c-NTP monoclonal antibody II, and the control C line is coated with goat anti-mouse IgG polyclonal antibody; The preparation of the nitrocellulose membrane includes the following steps: (1) Preparation of T-line coating solution and C-line coating solution: The mouse anti-AD7c-NTP monoclonal antibody II was diluted with coating diluent to a concentration of 1.0-1.5 mg / ml to obtain the T-line coating solution; the goat anti-mouse IgG polyclonal antibody was diluted with coating diluent to a concentration of 0.5-1.0 mg / ml to obtain the C-line coating solution; The coating diluent comprises: 0.5-1.5% trehalose, 0.5-1.5% BSA, 0.3-1% pullulan, 0.1-0.3% Tween-80, with the balance being 10mM PBS buffer at pH 7.2-7.4; (2) Modification of nitrocellulose membrane: Soak the nitrocellulose membrane in anhydrous ethanol for 10-15 min, then rinse with water 2-3 times and drain to obtain the pretreated nitrocellulose membrane; mix epichlorohydrin and 5-10 wt% sodium hydroxide solution at a volume ratio of 1:(4-6), stir evenly, and adjust the pH to 10.0±0.2 to obtain the modification solution; immerse the pretreated nitrocellulose membrane in the modification solution and shake at 25-35℃ for 2-3 h, then remove the nitrocellulose membrane, wash with water, and dry at 37℃; (3) Draw the detection T line and the quality control C line on the surface of the modified nitrocellulose membrane; use a gold spraying membrane drawing instrument to spray the T line coating solution onto the detection T line and the C line coating solution onto the quality control C line, and dry to obtain the coated nitrocellulose membrane.

[0007] This application provides a test strip for the Alzheimer's-related neurofilament protein AD7c-NTP. Through improvements in the preparation method of the nitrocellulose membrane, the test strip exhibits significant enhancements in sensitivity, accuracy, and stability, providing a more reliable tool for the diagnosis and screening of Alzheimer's disease. Specifically, the addition of pullulan to the coating solution enhances the stability of mouse anti-AD7c-NTP monoclonal antibody II and goat anti-mouse IgG polyclonal antibody, effectively preventing antibody degradation and improving the detection stability and repeatability of the kit. Trehalose and BSA help protect antibody activity and reduce the impact of external factors on the antibodies. Tween-80 reduces surface tension, allowing the antibody to be better dispersed in the diluent, ensuring effective coating. Furthermore, by using epichlorohydrin to perform a ring-opening reaction under alkaline conditions to hydrophilically modify the nitrocellulose membrane, more hydroxyl groups are introduced onto the membrane surface. These hydroxyl groups can form hydrogen bonds with the amino groups of antibodies, allowing the antibodies to be fully and stably immobilized on the nitrocellulose membrane surface. This increases the number of antigen-antibody complexes captured by the T-line and enhances the fluorescence signal intensity, thereby significantly improving detection sensitivity and accuracy. In summary, the optimization of the nitrocellulose membrane in this application enables the test strip to more effectively identify low concentrations of the target protein AD7c-NTP in urine, reducing non-specific interference and the probability of false positives. This provides a more reliable screening tool for early AD patients and better meets the needs of rapid testing in primary healthcare settings.

[0008] Optionally, the spraying amount of the T-line coating liquid and the C-line coating liquid is 0.8-1 μl / cm.

[0009] Optionally, the preparation method of the conjugate pad includes the following steps: activating latex microspheres, then adding mouse anti-AD7c-NTP monoclonal antibody I for labeling, blocking, reconstituted, and diluted, then spraying onto the pretreated conjugate pad surface, drying, and obtaining a conjugate pad containing mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres.

[0010] Optionally, the pretreatment method for the conjunctival pad is as follows: immerse the conjunctival pad in a conjunctival pad pretreatment solution for 30-60 minutes, and then dry it to obtain the pretreated conjunctival pad; The conjugation pad pretreatment solution comprises the following components in the following amounts: 1-3% sucrose, 0.1-0.4% sodium caseinate, 0.5-1.5% BSA, 0.3-0.7% trehalose, 0.1-0.5% BIO-TERGE AS-40, 0.1-0.5‰ Proclin 300, with the balance being 8-12 mM PBS buffer at pH 7.2-7.4.

[0011] In this application, the binding sites are pretreated using a conjugate pad pretreatment solution. Sucrose in the solution acts as a protectant, maintaining the structural stability of the membrane. Sodium caseinate acts as an anti-interference component, inhibiting antibody degradation by proteases in urine. BSA reduces non-specific adsorption. Trehalose provides protection and stability. The molecular structure of BIO-TERGE AS-40 contains hydrophilic sulfonic acid groups and hydrophobic alkyl chains; its surface activity reduces the surface tension of the conjugate pad material, changing the surface from hydrophobic to hydrophilic, accelerating sample diffusion and penetration within the conjugate pad, and improving the reaction efficiency between labeled antibodies and antigens. Proclin 300 inhibits microbial growth and prevents membrane contamination. This pretreatment significantly improves the anti-interference ability, stability, and detection accuracy of the test strip.

[0012] Optionally, the diluent used for the dilution comprises the following components: 0.5-1.5% BSA, 3-7% trehalose, 0.5-2% PVP-40, 0.1-0.3% PEG-20000, with the balance being a 50mM Tris-HCl buffer at pH 8.0.

[0013] In the diluent provided in this application, the pyrrolidone group (-NCO-) on the PVP-40 molecular chain is polar and hydrophilic, and the hydroxyl groups (-OH) at both ends of the PEG-20000 molecule are strongly hydrophilic. Both of these components can bind to water through hydrogen bonds to form a "hydration film" on the surface of the latex microspheres, reducing the surface tension of the microspheres, reducing van der Waals forces between particles, and preventing the latex microspheres labeled with antibodies from agglomerating due to charge attraction or hydrophobic effects. This ensures uniform distribution when subsequently sprayed onto the conjugate pad and improves the stability of the detection signal.

[0014] Optionally, the distance between the detection T-line and the quality control C-line is 4±0.5mm.

[0015] Optionally, the preparation of the sample pad includes the following steps: placing the sample pad in a tray, using a pipette to draw sample pad treatment solution to wet the sample pad, soaking for 20-30 minutes and then drying to obtain the sample pad; The sample pad treatment solution comprises the following components in the following amounts: 0.3-0.7% Triton-100, 0.5-1.5% casein, 0.2-0.6‰ Proclin 300, 0.05-0.2% EDTA-2Na, with the balance being 8-12mM PBS buffer at pH 7.2-7.4.

[0016] Secondly, this application provides an Alzheimer's-related neurofilament protein AD7c-NTP detection kit, including a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0017] In summary, this application has the following beneficial effects: This application provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP. By optimizing the nitrocellulose membrane and conjugate pad, the test strip can effectively identify low concentrations of the target protein in urine when detecting Alzheimer's-related neurofilament protein AD7c-NTP, reducing non-specific interference and lowering the probability of false positives. This provides a reliable screening tool for early AD patients and better meets the needs of rapid testing in primary healthcare. Detailed Implementation

[0018] This application provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP, comprising a PVC base plate; the PVC base plate is provided with a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the conjugate pad contains mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres; the nitrocellulose membrane is provided with a detection T line and a control C line; the detection T line is coated with mouse anti-AD7c-NTP monoclonal antibody II, and the control C line is coated with goat anti-mouse IgG polyclonal antibody.

[0019] In the embodiments of this application, the latex microspheres were purchased from Shenzhen Maifu New Material Technology Co., Ltd., product number DPR300C; the nitrocellulose membrane product number was Equ LFN. 120, 20mm × 100m, purchased from Tianren Membrane Technology (Shaoxing) Co., Ltd.; conjugate pad, catalog number IVDT8975, purchased from Guangzhou Yuwei Biotechnology Instruments Co., Ltd.; sample pad, catalog number NJ-Y8, purchased from Shanghai Jieyi Biotechnology Co., Ltd.; mouse anti-AD7c-NTP monoclonal antibody I, catalog number V3003, purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; mouse anti-AD7c-NTP monoclonal antibody II, catalog number V3005, purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; goat anti-mouse IgG polyclonal antibody purchased from Feipeng Biotechnology; pullulan polysaccharide, CAS number 9057-02-7; sodium caseinate, CAS number 9005-46-3; BSA, bovine serum albumin, purchased from SIGMA, catalog number V900933; BIO-TERGE AS-40 is an S6 surfactant, purchased from Jinan Yili; the CAS number of PEG-20000 is 25322-68-3; the reagents, solvents, materials, etc. used in this application are all commercially available.

[0020] The present application will be further described in detail below with reference to embodiments and performance testing.

[0021] Example 1 Example 1 provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0022] The preparation method of the above test strip includes the following steps: (1) Preparation of the binding pad: (1-1) Antibody labeling: Microsphere activation: Dissolve 25 μL of latex microspheres (10 mg / mL) in 375 μL of activation solution (50 mM, pH 6.0 MES buffer containing 0.05% Tween-20); add 8 μL each of EDC (15 mg / mL) and NHS (15 mg / mL), and incubate at 37 °C with shaking for 40 min; centrifuge at 12000g for 15 min and discard the supernatant; Labeling: Add 400 μL of 10 mM borax-boric acid buffer (pH 7.5-7.7) to the activated microspheres, and add 50 μg of mouse anti-AD7c-NTP monoclonal antibody I. Shake for 60 min. Sealing: Add 40 μL of 10% BSA solution, shake for 30 min, centrifuge for 10 min, and discard the supernatant; Reconstitution: The blocked antibody was reconstituted using 200 μL of 10 mM borax-borate buffer at pH 7.6 to obtain mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres; The method for preparing the borax-boric acid buffer solution is as follows: First, weigh 0.9535g of borax (Na2B4O7·10H2O), dissolve it in 50mL of pure water to obtain a 0.05M borax solution; weigh 0.6185g of boric acid (H3BO3), dissolve it in 50mL of pure water to obtain a 0.2M boric acid solution; mix 1.5mL of the 0.05M borax solution with 8.5mL of the 0.2M boric acid solution, adjust the pH, and dilute it 20 times to obtain a 10mM borax-boric acid buffer solution with a pH of 7.6.

[0023] (1-2) Pretreatment of the conjunctival pad: The conjunctival pad was immersed in the conjunctival pad pretreatment solution for 30 min, and then dried at 37±3℃ to obtain the pretreated conjunctival pad. The conjugation pad pretreatment solution comprises the following components: 2% sucrose, 0.2% sodium caseinate, 1% BSA, 0.5% trehalose, 0.3% BIO-TERGE AS-40, 0.3‰ Proclin 300, with the remainder being 10mM PBS buffer at pH 7.4. (1-3) Dilution and spraying: The mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres was diluted with diluent at a volume ratio of 1:2 and then sprayed onto the pretreated conjugate pad at a rate of 4 μl / cm. The conjugate pad was then dried at 37±3℃. The diluent comprises the following components: 1% BSA, 5% trehalose, 1% PVP-40, 0.2% PEG-20000, with the balance being a 50mM Tris-HCl buffer at pH 8.0.

[0024] (2) Preparation of nitrocellulose membrane: (1) Preparation of T-line coating solution and C-line coating solution: The mouse anti-AD7c-NTP monoclonal antibody II was diluted with coating diluent to a concentration of 1.2 mg / ml to obtain T-line coating solution; the goat anti-mouse IgG polyclonal antibody was diluted with coating diluent to a concentration of 0.8 mg / ml to obtain C-line coating solution. The coating diluent contains: 1% trehalose, 1% BSA, 0.5% pullulan, 0.2% Tween-80, and the balance is 10mM PBS buffer at pH 7.4. (2) Modification of nitrocellulose membrane: The nitrocellulose membrane was soaked in anhydrous ethanol for 12 min, then rinsed with water 3 times and drained to obtain the pretreated nitrocellulose membrane; epichlorohydrin and 5 wt% sodium hydroxide solution were mixed at a volume ratio of 1:5, stirred evenly, and adjusted to pH 10.0±0.2 to obtain the modification solution; the pretreated nitrocellulose membrane was soaked in the modification solution and shaken at 30℃ for 2 h, the nitrocellulose membrane was taken out, washed with water, and dried at 37℃; (3) Draw the detection T line and the quality control C line on the surface of the modified nitrocellulose membrane; use a gold spraying membrane drawing instrument to spray the T line coating liquid onto the detection T line and the C line coating liquid onto the quality control C line. The spraying amount of the T line coating liquid and the C line coating liquid is 1 μl / cm. Dry to obtain the coated nitrocellulose membrane.

[0025] (3) Preparation of sample pad: Place the sample pad in the tray, use a pipette to draw sample pad treatment solution to wet the sample pad, soak for 20-30 minutes and then dry at 37±3℃ to obtain the sample pad. The sample pad treatment solution comprises the following components in the following amounts: 0.5% Triton-100, 1% casein, 0.4‰ Proclin 300, 0.1% EDTA-2Na, with the balance being 10mM PBS buffer at pH 7.4.

[0026] (4) Assembly of test strips for Alzheimer's-related neurofilament protein AD7c-NTP: At 20-30% humidity and 20-25℃, the prepared sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are sequentially overlapped and glued onto the PVC base plate with a thickness of 2±1mm. Then, the strips are placed in a cutting machine to obtain test strips for Alzheimer's-related neurofilament protein AD7c-NTP with a width of 4±0.1mm.

[0027] Examples 2-6 Examples 2-6 provide a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0028] The difference between the above embodiments and Embodiment 1 is that the content of each component of the coating diluent used in the preparation of the T-line coating solution and the C-line coating solution is shown in Table 1 below.

[0029] Comparative Examples 1-3 Examples 1-3 provide a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0030] The difference between the above comparative examples and Example 1 is that the content of each component of the coating diluent used in the preparation of the T-line coating solution and the C-line coating solution is shown in Table 1 below.

[0031] Table 1. Components and contents of the coating solutions used in Examples 1-6 and Comparative Examples 1-3 Examples 7-11 Examples 7-11 provide a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0032] The difference between the above embodiments and Embodiment 1 is that the content of each component in the pretreatment solution of the binding pad is shown in Table 2 below.

[0033] Comparative Example 4-5 Comparative Examples 4 and 5 each provide a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0034] The difference between the above comparative example and Example 1 is that the content of each component in the pretreatment solution of the binding pad is as shown in Table 2 below.

[0035] Table 2. Contents of each component in the conjugate pad pretreatment solution in Examples 1, 7-11, and Comparative Examples 4-5. Comparative Example 6 Comparative Example 6 provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0036] The difference between the above comparative example and Example 1 is that: (1-3) In the dilution and spraying steps, the diluent used for dilution includes the following components: 1% BSA, 5% trehalose, and the balance is pH 8.0, 50mM Tris-HCl buffer.

[0037] Comparative Example 7 Comparative Example 7 provides a test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP.

[0038] The difference between the above comparative example and Example 1 is that the nitrocellulose membrane was not modified.

[0039] Performance testing (a) Accuracy The test strips for detecting Alzheimer's-related neurofilament protein AD7c-NTP prepared in Examples 1-11 and Comparative Examples 1-7 were used to detect the test samples. The specific detection process is as follows, and the results are shown in Table 3 below.

[0040] 1. Detection method: Use a pipette to draw up urine and add 3-4 drops into the sample well of the test card. Let it stand for 10-15 minutes. Then, visually observe whether bands appear on the test strip's T line and control C line to determine the positive or negative result of the sample. The determination rules are as follows: A band appeared on the quality control line C, but no band appeared on the test line T, so the result was negative. A band appears on the quality control line C, and a band appears on the test line T, indicating a positive result. No bands appeared on the quality control line C, so the result is invalid.

[0041] 2. The urine samples (sample ① and sample ②) were tested according to the above testing method. Each sample was tested in parallel for 30 groups to obtain the positive and negative results of the samples. The results are shown in Table 3.

[0042] Note: Sample ① is a urine sample from an Alzheimer's disease patient, with a high level of Alzheimer's-related neurofilament protein AD7c-NTP; Sample ② is a urine sample from a healthy person, with a low level of Alzheimer's-related neurofilament protein AD7c-NTP.

[0043] Table 3. Accuracy test results of various Alzheimer's-related neurofilament protein AD7c-NTPs. According to the test results in Table 3, the accuracy of the test strips for Alzheimer's-related neurofilament protein AD7c-NTP provided in Examples 1-11 for detecting positive and negative urine samples was 93.33-100%. However, the accuracy of the test strips for Alzheimer's-related neurofilament protein AD7c-NTP provided in Comparative Examples 1-5 for detecting positive and negative urine samples was 66.67-96.67%. Therefore, this application demonstrates that the test strips for Alzheimer's-related neurofilament protein AD7c-NTP prepared by using a specific coating diluent to dilute the antibodies on the T and C lines, a specific conjugate pad pretreatment solution to soak the conjugate pad, and modifying the nitrocellulose membrane can stably and accurately detect AD7c-NTP in samples, thus achieving Alzheimer's disease screening.

[0044] (II) Sensitivity The sensitivity of the test strips for detecting Alzheimer's-related neurofilament protein AD7c-NTP provided in Examples 1-11 and Comparative Examples 1-7 was investigated, and the specific process is as follows: The AD7c-NTP recombinant antigen was serially diluted to obtain linear dilutions with concentrations of 50 ng / ml, 10 ng / ml, and 4 ng / ml, respectively. The test strips for Alzheimer's-related neurofilament protein AD7c-NTP in Examples 1-11 and Comparative Examples 1-7 were used to detect the above linear dilutions. Each linear dilution was tested in parallel for 30 groups to obtain the positive and negative results of the test samples. The results are shown in Table 4.

[0045] Table 4. Sensitivity test results of various Alzheimer's-related neurofilament protein AD7c-NTPs. Table 4 shows that the test strips for Alzheimer's-related neurofilament protein AD7c-NTP provided in Examples 1-11 have an accuracy of 93.33-100.00% (≥90%) for detecting AD7c-NTP reference dilutions with concentrations as low as 4 ng / ml; while the test strips for Alzheimer's-related neurofilament protein AD7c-NTP provided in Comparative Examples 1-7 have an accuracy of only 0-6.67% for detecting AD7c-NTP reference dilutions with concentrations as low as 4 ng / ml. Therefore, this demonstrates that the test strips for Alzheimer's-related neurofilament protein AD7c-NTP provided in the embodiments of this application have excellent detection sensitivity, and can achieve high detection accuracy even for samples containing Alzheimer's-related neurofilament protein AD7c-NTP with a concentration as low as 4 ng / ml.

[0046] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP, characterized in that, The device includes a PVC base plate; the PVC base plate is provided with a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the conjugate pad contains mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres; the nitrocellulose membrane is provided with a detection T line and a quality control C line; the detection T line is coated with mouse anti-AD7c-NTP monoclonal antibody II, and the quality control C line is coated with goat anti-mouse IgG polyclonal antibody; The preparation of the nitrocellulose membrane includes the following steps: (1) Preparation of T-line coating solution and C-line coating solution: The mouse anti-AD7c-NTP monoclonal antibody II was diluted with coating solution diluent to a concentration of 1.0-1.5 mg / ml to obtain T-line coating solution; the goat anti-mouse IgG polyclonal antibody was diluted with coating solution diluent to a concentration of 0.5-1.0 mg / ml to obtain C-line coating solution; The coating diluent comprises: 0.5-1.5% trehalose, 0.5-1.5% BSA, 0.3-1% pullulan, 0.1-0.3% Tween-80, with the balance being 10mM PBS buffer at pH 7.2-7.4; (2) Modification of nitrocellulose membrane: Soak the nitrocellulose membrane in anhydrous ethanol for 10-15 min, then rinse with water 2-3 times, drain, and obtain the pretreated nitrocellulose membrane; mix epichlorohydrin and 5-10 wt% sodium hydroxide solution at a volume ratio of 1:(4-6), stir evenly, adjust the pH to 10.0±0.2, and obtain the modified solution; immerse the pretreated nitrocellulose membrane in the modified solution, shake and react at 25-35℃ for 2-3 h, take out the nitrocellulose membrane, wash with water, and dry at 37℃; (3) Draw the detection T line and the quality control C line on the surface of the modified nitrocellulose membrane; use a gold spraying membrane drawing instrument to spray the T line coating solution onto the detection T line and the C line coating solution onto the quality control C line, and dry to obtain the coated nitrocellulose membrane.

2. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 1, characterized in that, The spraying amount of the coating liquid for both the T-line and C-line is 0.8-1 μl / cm.

3. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 1, characterized in that, The method for preparing the conjugate pad includes the following steps: activating latex microspheres, then adding mouse anti-AD7c-NTP monoclonal antibody I for labeling, blocking, reconstitution, dilution, then spraying onto the pretreated conjugate pad surface, drying, and obtaining a conjugate pad containing mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres.

4. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 3, characterized in that, The pretreatment method for the conjunctival pad is as follows: immerse the conjunctival pad in a conjunctival pad pretreatment solution for 30-60 minutes, then dry it to obtain the pretreated conjunctival pad. The conjugation pad pretreatment solution comprises the following components: 1-3% sucrose, 0.1-0.4% sodium caseinate, 0.5-1.5% BSA, 0.3-0.7% trehalose, 0.1-0.5% BIO-TERGE AS-40, 0.1-0.5‰ Proclin 300, with the balance being 8-12 mM PBS buffer at pH 7.2-7.

4.

5. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 3, characterized in that, The diluent used for the dilution comprises the following components: 0.5-1.5% BSA, 3-7% trehalose, 0.5-2% PVP-40, 0.1-0.3% PEG-20000, with the balance being 50mM Tris-HCl buffer at pH 8.

0.

6. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 1, characterized in that, The distance between the detection T-line and the quality control C-line is 4 ± 0.5 mm.

7. The test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP according to claim 1, characterized in that, The preparation of the sample pad includes the following steps: placing the sample pad in a tray, using a pipette to draw sample pad treatment solution to wet the sample pad, soaking for 20-30 minutes and then drying to obtain the sample pad; The sample pad treatment solution comprises the following components in the following amounts: 0.3-0.7% Triton-100, 0.5-1.5% casein, 0.2-0.6‰ Proclin 300, 0.05-0.2% EDTA-2Na, with the balance being 8-12mM PBS buffer at pH 7.2-7.

4.

8. A detection kit for Alzheimer's-related neurofilament protein AD7c-NTP, characterized in that, Test strips comprising the Alzheimer's-associated neurofilament protein AD7c-NTP as described in any one of claims 1-7.

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