A test strip and kit for Alzheimer-related neurofilament protein AD7c-NTP

Abstract

The application relates to the technical field of biological detection, and particularly discloses a test strip and a kit for Alzheimer-related neurofilament protein AD7c-NTP. The test strip for Alzheimer-related neurofilament protein AD7c-NTP provided by the application comprises a PVC bottom plate; a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC bottom plate; the combination pad contains latex-labeled mouse anti-AD7c-NTP monoclonal antibody I; 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 test strip for Alzheimer-related neurofilament protein AD7c-NTP provided by the application 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] The application relates to the technical field of biological detection, in particular to an Alzheimer-related neurofilament protein AD7c-NTP detection test strip and kit. BACKGROUND

[0002] The Alzheimer-related neurofilament protein AD7c-NTP is a key biomarker related to AD, and its concentration in urine is positively correlated with the degree of neuronal damage and the AD pathological process, so the detection of AD7c-NTP in urine is of great significance for the early diagnosis and disease monitoring of AD.

[0003] The colloidal gold immunochromatography technology is a novel immunolabeling technology for antigen-antibody using colloidal gold as a tracer marker. The technology has been widely used in the rapid detection of Alzheimer-related neurofilament protein AD7c-NTP due to its simple operation, rapid detection and the absence of special equipment. The core of the technology is a detection test strip. However, the existing Alzheimer-related neurofilament protein AD7c-NTP detection test strip still has obvious deficiencies in practical application. First, the detection sensitivity is low, and it is difficult to identify low-concentration AD7c-NTP in the urine of early AD patients, so that early AD patients are easily missed diagnosis and miss the best treatment opportunity. Second, due to the complex matrix of the urine sample to be tested, it contains proteases, lipids, salts and other substances, which can easily produce non-specific interference and easily lead to false positives, thereby affecting the accuracy of the detection. Third, the stability of the kit is poor, the antibody is easily degraded, which affects the repeatability of the detection, and the detection results of different batches lack consistency.

[0004] Therefore, it is urgent to optimize the preparation method of the Alzheimer-related neurofilament protein AD7c-NTP detection test strip to improve the detection sensitivity, accuracy and stability, and thus provide a more reliable tool for the diagnosis and screening of Alzheimer's disease. SUMMARY

[0005] In order to improve the detection sensitivity, accuracy and stability of the Alzheimer-related neurofilament protein AD7c-NTP detection test strip, the application provides an Alzheimer-related neurofilament protein AD7c-NTP detection test strip and kit.

[0006] In a first aspect, the application provides an Alzheimer-related neurofilament protein AD7c-NTP detection test strip, which adopts the following technical solution:

[0007] An Alzheimer-related neurofilament protein AD7c-NTP detection test strip, comprising a PVC base plate; a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC base plate; the binding pad contains latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I; 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;

[0008] The preparation of the nitrocellulose membrane comprises the following steps:

[0009] (1) Preparation of T line coating solution and C line coating solution: dilute mouse anti-AD7c-NTP monoclonal antibody II with coating diluent to obtain a T line coating solution with a dilution concentration of 1.0-1.5 mg / ml; dilute goat anti-mouse IgG polyclonal antibody with coating diluent to obtain a C line coating solution with a dilution concentration of 0.5-1.0 mg / ml;

[0010] The coating diluent comprises 0.5-1.5% trehalose, 0.5-1.5% BSA, 0.3-1% pullulan, 0.1-0.3% Tween-80, and the rest is PBS buffer with a pH of 7.2-7.4 and a concentration of 10 mM;

[0011] (2) Modification of the nitrocellulose membrane: soak the nitrocellulose membrane in anhydrous ethanol for 10-15 min, then rinse with water for 2-3 times, drain, and obtain a pretreated nitrocellulose membrane; mix epichlorohydrin and 5-10 wt% sodium hydroxide solution at a volume ratio of 1:(4-6), stir uniformly, adjust to pH 10.0±0.2, and obtain a modification solution; soak the pretreated nitrocellulose membrane in the modification solution, oscillate at 25-35°C for 2-3 h, take out the nitrocellulose membrane, wash with water, and dry at 37°C;

[0012] (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 to the detection T line and the C line coating solution to the quality control C line, dry, and obtain a coated nitrocellulose membrane.

[0013] The application provides a test strip for Alzheimer-related neurofilament protein AD7c-NTP, which is improved by the preparation method of the nitrocellulose membrane, so that the test strip has significant improvement in sensitivity, accuracy and stability, and provides a more reliable tool for the diagnosis and screening of Alzheimer's disease. Specifically, by adding pullulan to the coating liquid, the stability of mouse anti-AD7c-NTP monoclonal antibody II and goat anti-mouse IgG polyclonal antibody can be enhanced, antibody degradation can be effectively avoided, and the detection stability and detection repeatability of the kit can be improved; trehalose and BSA can help to protect the activity of the antibody and reduce the influence of external factors on the antibody; Tween-80 can reduce the surface tension, so that the antibody is better dispersed in the diluent, and the coating effect is ensured. In addition, by using the ring-opening reaction of epichlorohydrin under alkaline conditions to modify the nitrocellulose membrane, more hydroxyl groups are introduced onto the surface of the nitrocellulose membrane, which can form hydrogen bonds with the amino groups of the antibody, so that the antibody can be fully and stably fixed on the surface of the nitrocellulose membrane, thereby increasing the number of antigen-antibody complexes captured by the T line and improving the fluorescence signal intensity, so that the detection sensitivity and accuracy are significantly improved. In summary, the optimization of the nitrocellulose membrane in the application makes the test strip more effectively identify the target protein in urine with low concentration when detecting Alzheimer-related neurofilament protein AD7c-NTP, reduces non-specific interference, reduces the probability of false positives, provides a more reliable screening tool for the detection of early AD patients, and better meets the needs of rapid detection in primary medical institutions.

[0014] Optionally, the spraying amount of the T-line coating liquid and the C-line coating liquid is 0.8-1 μl / cm.

[0015] Optionally, the preparation method of the binding pad comprises the following steps: activating the latex microspheres, then adding mouse anti-AD7c-NTP monoclonal antibody I for labeling, blocking, resolubilizing, diluting, then spraying onto the surface of the pretreated binding pad, and drying to obtain a binding pad containing mouse anti-AD7c-NTP monoclonal antibody I labeled with latex microspheres.

[0016] Optionally, the pretreatment method of the binding pad is as follows: soaking the binding pad in a binding pad pretreatment liquid for 30-60 min, and drying to obtain a pretreated binding pad.

[0017] The binding pad pretreatment liquid 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‰ Proclin300, and the rest is PBS buffer with pH 7.2-7.4 and 8-12 mM.

[0018] In the present application, the binding pad is pretreated by using a pretreatment liquid, sucrose in the pretreatment liquid can be used as a protective agent to maintain the structural stability of the membrane; sodium caseinate as an anti-interference component can inhibit the degradation of the antibody by protease in urine; BSA can reduce non-specific adsorption; trehalose has a protective and stabilizing effect; the molecular structure of BIO-TERGE AS-40 contains hydrophilic sulfonic acid groups and hydrophobic alkyl chains, and the surface activity thereof can reduce the surface tension of the binding pad material, change the surface of the binding pad from hydrophobic to hydrophilic, accelerate the diffusion and penetration of the sample in the binding pad, and improve the reaction efficiency of the labeled antibody and the antigen; Proclin 300 can inhibit microbial growth and prevent the membrane from being contaminated; after the pretreatment of the binding pad, the anti-interference ability, stability and detection accuracy of the test strip can be significantly improved.

[0019] Optionally, the dilution liquid used for dilution comprises the following components: 0.5-1.5% BSA, 3-7% trehalose, 0.5-2% PVP-40, 0.1-0.3% PEG-20000, and the rest is Tris-Hcl buffer solution with pH 8.0 and 50mM.

[0020] In the dilution liquid provided by the present application, the pyrrolidone groups (-NCO-) on the PVP-40 molecular chain have polarity and hydrophilicity, and the hydroxyl groups (-OH) at both ends of the PEG-20000 molecule have strong hydrophilicity. Both of the above components can be combined with water through hydrogen bonds to form a layer of "hydration film" on the surface of the latex microspheres, reduce the surface tension of the microspheres, reduce the van der Waals force between particles, avoid the agglomeration of the labeled antibody latex microspheres due to charge attraction or hydrophobic effect, ensure uniform distribution when sprayed onto the binding pad later, and improve the stability of the detection signal.

[0021] Optionally, the distance between the detection T line and the quality control C line is 4±0.5mm.

[0022] Optionally, the preparation of the sample pad comprises the following steps: placing the sample pad in a tray, using a pipette to absorb the sample pad treatment liquid to wet the sample pad, soaking for 20-30min and then drying to obtain the sample pad.

[0023] The sample pad treatment liquid comprises the following components: 0.3-0.7% Triton X-100, 0.5-1.5% casein, 0.2-0.6‰ Proclin 300, 0.05-0.2% EDTA-2Na, and the rest is PBS buffer solution with pH 7.2-7.4 and 8-12mM.

[0024] In a second aspect, the present application provides an Alzheimer-related neurofilament protein AD7c-NTP detection kit, which comprises an Alzheimer-related neurofilament protein AD7c-NTP test strip.

[0025] In summary, the application has the following beneficial effects:

[0026] The application provides a test strip for Alzheimer-related neurofilament protein AD7c-NTP. Through optimization of a nitrocellulose membrane and a binding pad, the test strip can effectively identify a low concentration of target protein in urine when detecting Alzheimer-related neurofilament protein AD7c-NTP, reduce non-specific interference, and reduce the probability of false positives, thereby providing a reliable screening tool for early AD patient detection and better meeting the needs of rapid detection in primary medical institutions. DETAILED DESCRIPTION

[0027] The application provides a test strip for Alzheimer-related neurofilament protein AD7c-NTP, which comprises a PVC base plate. A sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC base plate. The binding pad contains latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I. 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.

[0028] In the embodiments of the application, the latex microspheres are purchased from Shenzhen Maifu New Material Technology Co., Ltd., with the item number DPR300C; the nitrocellulose membrane is purchased from Tianli Membrane Technology (Shaoxing) Co., Ltd., with the item number Equ LFN 120, 20mmx100m; the binding pad is purchased from Guangzhou Yuyi Biological Technology Instrument Co., Ltd., with the item number IVDT8975; the sample pad is purchased from Shanghai Jieyi Biological Technology Co., Ltd., with the item number NJ-Y8; the mouse anti-AD7c-NTP monoclonal antibody I is purchased from Beijing Boao Sen Biological Technology Co., Ltd., with the item number V3003; the mouse anti-AD7c-NTP monoclonal antibody II is purchased from Beijing Boao Sen Biological Technology Co., Ltd., with the item number V3005; the goat anti-mouse IgG polyclonal antibody is purchased from Feipeng Biological; the pullulan has a CAS number of 9057-02-7; the sodium caseinate has a CAS number of 9005-46-3; the BSA is bovine serum albumin, which is purchased from SIGMA, with the item number V900933; the BIO-TERGE AS-40 is a S6 surfactant, which is purchased from Jinan Yili; the PEG-20000 has a CAS number of 25322-68-3; the reagents, solvents and materials used in the application can be obtained by commercial purchase.

[0029] The application will be further described in detail in combination with the following examples and performance test experiments.

[0030] Example 1

[0031] Embodiment 1 provides a test strip of Alzheimer-related neurofilament protein AD7c-NTP.

[0032] The preparation method of the test strip comprises the following steps:

[0033] (1) Preparation of the conjugate pad:

[0034] (1-1) Antibody labeling:

[0035] Microsphere activation: 25 μL of latex microspheres (10 mg / mL) were dissolved in 375 μL of activation solution (50 mM, pH 6.0 MES buffer containing 0.05% Tween-20); 8 μl of EDC at a concentration of 15 mg / mL and 8 μl of NHS at a concentration of 15 mg / mL were added, and incubated at 37°C with shaking for 40 min; centrifuged at 12000g for 15 min, and the supernatant was discarded;

[0036] Labeling: 400 μL of 10 mM, pH 7.5-7.7 borax-boric acid buffer was added to the activated microspheres, and 50 μg of mouse anti-AD7c-NTP monoclonal antibody I was added, and the reaction was shaken for 60 min;

[0037] Blocking: 40 ul of 10% BSA solution was added, and the reaction was shaken for 30 min, centrifuged for 10 min, and the supernatant was discarded;

[0038] Resuspension: The blocked antibody was resuspended with 200 μL of 10 mM, pH 7.6 borax-boric acid buffer to obtain latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I;

[0039] The preparation method of the borax-boric acid buffer is as follows: first, weigh 0.9535 g of borax (Na2B4O7·10H2O) and dissolve it in 50 mL of pure water to obtain a 0.05 M borax solution; weigh 0.6185 g of boric acid (H3BO3) and dissolve it in 50 mL of pure water to obtain a 0.2 M boric acid solution; mix 1.5 mL of 0.05 M borax solution with 8.5 mL of 0.2 M boric acid solution, adjust the pH, and dilute 20 times to obtain a 10 mM, pH 7.6 borax-boric acid buffer.

[0040] (1-2) Pre-treatment of the conjugate pad: the conjugate pad was soaked in the conjugate pad pre-treatment solution for 30 min, and then dried at 37±3°C to obtain the pre-treated conjugate pad;

[0041] The conjugate pad pre-treatment solution comprises the following components in the following amounts: 2% sucrose, 0.2% sodium caseinate, 1% BSA, 0.5% trehalose, 0.3% BIO-TERGE AS-40, 0.3‰ Proclin300, and the balance is PBS buffer at pH 7.4 and 10 mM.

[0042] (1-3)Dilution and spraying: the latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I was diluted with a diluent at a volume ratio of 1:2, sprayed onto the pretreated conjugate pad at 4 μl / cm, and dried at 37±3°C to obtain the conjugate pad;

[0043] The diluent comprises the following components: 1% BSA, 5% trehalose, 1% PVP-40, 0.2% PEG-20000, and the rest is Tris-Hcl buffer solution with pH 8.0 and 50 mM.

[0044] (2) Preparation of nitrocellulose membrane:

[0045] (1) Preparation of T-line coating solution and C-line coating solution: the mouse anti-AD7c-NTP monoclonal antibody II was diluted with a coating diluent to obtain a T-line coating solution with a dilution concentration of 1.2 mg / ml; the goat anti-mouse IgG polyclonal antibody was diluted with a coating diluent to obtain a C-line coating solution with a dilution concentration of 0.8 mg / ml;

[0046] The coating diluent comprises 1% trehalose, 1% BSA, 0.5% pullulan, 0.2% Tween-80, and the rest is PBS buffer solution with pH 7.4 and 10 mM;

[0047] (2) Modification of nitrocellulose membrane: the nitrocellulose membrane was soaked in anhydrous ethanol for 12 min, then washed with water for 3 times, drained, and obtained a pretreated nitrocellulose membrane; the epichlorohydrin was mixed with 5 wt% sodium hydroxide solution at a volume ratio of 1:5, stirred uniformly, and adjusted to pH 10.0±0.2 to obtain a modification solution; the pretreated nitrocellulose membrane was soaked in the modification solution, and oscillated at 30°C for 2 h, then the nitrocellulose membrane was taken out, washed with water, and dried at 37°C;

[0048] (3) T-line and quality control C-line were drawn on the surface of the modified nitrocellulose membrane; the T-line coating solution was sprayed onto the detection T-line by using a gold spraying membrane drawing instrument, and the C-line coating solution was sprayed onto the quality control C-line, the spraying amount of the T-line coating solution and the C-line coating solution was 1 μl / cm, and the coated nitrocellulose membrane was obtained after drying.

[0049] (3) Preparation of sample pad:

[0050] The sample pad was placed in a tray, and the sample pad was wetted by using a pipette to suck the sample pad treatment solution, soaked for 20-30 min, and dried at 37±3°C to obtain the sample pad;

[0051] The sample pad treatment solution comprises the following components: 0.5% Triton X-100, 1% casein, 0.4‰ Proclin 300, 0.1% EDTA-2Na, and the rest is PBS buffer with pH 7.4 and 10 mM.

[0052] (4) Assembly of the test strip for Alzheimer-related neurofilament protein AD7c-NTP: Under the conditions of 20-30% humidity and 20-25°C temperature, the prepared sample pad, the conjugate pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the PVC base plate with an overlap of 2±1 mm, and then placed in a cutting machine to obtain the test strip for Alzheimer-related neurofilament protein AD7c-NTP with a width of 4±0.1 mm.

[0053] Examples 2-6

[0054] Examples 2-6 respectively provide a test strip for Alzheimer-related neurofilament protein AD7c-NTP.

[0055] The above examples differ from Example 1 in that the component contents of the coating diluent used in the preparation of the T-line coating solution and the C-line coating solution are as shown in Table 1 below.

[0056] Comparative Examples 1-3

[0057] Examples 1-3 respectively provide a test strip for Alzheimer-related neurofilament protein AD7c-NTP.

[0058] The above comparative examples differ from Example 1 in that the component contents of the coating diluent used in the preparation of the T-line coating solution and the C-line coating solution are as shown in Table 1 below.

[0059] Table 1 Component and content of the coating solution used in Examples 1-6 and Comparative Examples 1-3

[0060]

[0061] Examples 7-11

[0062] Examples 7-11 respectively provide a test strip for Alzheimer-related neurofilament protein AD7c-NTP.

[0063] The above examples differ from Example 1 in that the component contents of the coating diluent used in the preparation of the T-line coating solution and the C-line coating solution are as shown in Table 1 below.

[0064] Comparative Examples 4-5

[0065] Comparative Examples 4-5 respectively provide a test strip for Alzheimer-related neurofilament protein AD7c-NTP.

[0066] The above comparative example differs from Example 1 in that the contents of the components in the pretreatment solution of the conjugate pad are as shown in Table 2 below.

[0067] Table 2 Contents of components in the pretreatment solution of the conjugate pad in Example 1, Examples 7-11, Comparative Examples 4-5

[0068] Comparative Example 6

[0069] Comparative Example 6 provides a test strip for detecting Alzheimer-associated neurofilament protein AD7c-NTP.

[0070] The above comparative example differs from Example 1 in that (1-3) in the dilution and spraying step, the dilution solution used for dilution comprises the following contents of components: 1% BSA, 5% trehalose, and the balance is Tris-Hcl buffer solution with pH 8.0, 50 mM.

[0071] Comparative Example 7

[0072] Comparative Example 7 provides a test strip for detecting Alzheimer-associated neurofilament protein AD7c-NTP.

[0073] The above comparative example differs from Example 1 in that the nitrocellulose membrane is not subjected to modification treatment.

[0074] Performance detection test

[0075] (I) Accuracy

[0076] The test strips for detecting Alzheimer-associated neurofilament protein AD7c-NTP prepared using Examples 1-11 and Comparative Examples 1-7 were used to detect the samples to be tested, and the detection process was as follows, and the results are shown in Table 3 below.

[0077] 1. Detection method: use a pipette to suck urine and drop 3-4 drops into the sample addition hole of the test card, stand for 10-15 min; then observe whether the detection T line and the quality control C line of the test strip appear a band with the naked eye, and determine the positive and negative of the sample to be tested; the determination rules are as follows:

[0078] The quality control C line appears a band, the detection T line does not appear a band, and the determination result is negative;

[0079] The quality control C line appears a band, and the detection T line appears a band, and the determination result is positive;

[0080] The quality control C line does not appear a band, and the determination result is invalid.

[0081] 2. According to the above detection method, urine (sample to be tested ①, sample to be tested ②) was detected, 30 groups of each sample were detected in parallel, the positive and negative of the sample to be tested were obtained, and the results are shown in Table 3.

[0082] Note: The sample ① to be tested is a urine sample of an Alzheimer's patient, and the content of the Alzheimer-related neurofilament protein AD7c-NTP is high; the sample ② to be tested is a urine sample of a healthy person, and the content of the Alzheimer-related neurofilament protein AD7c-NTP is low.

[0083] Table 3 Accuracy detection results of each Alzheimer-related neurofilament protein AD7c-NTP test strip

[0084]

[0085]

[0086] According to the detection results in Table 3, the urine positive sample and the urine negative sample were detected by using the Alzheimer-related neurofilament protein AD7c-NTP test strips provided in Examples 1-11, and the accuracy of the detection results was 93.33-100%; while the urine positive sample and the urine negative sample were detected by using the Alzheimer-related neurofilament protein AD7c-NTP test strips provided in Comparative Examples 1-5, and the accuracy of the detection results was 66.67-96.67%. Therefore, it is shown that by using a specific coating diluent to dilute the antibodies of the T line and the C line, a specific conjugate pad pretreatment liquid to soak the conjugate pad, and modifying the nitrocellulose membrane, the Alzheimer-related neurofilament protein AD7c-NTP test strip prepared by the application can stably and accurately detect AD7c-NTP in the sample, and realize the diagnosis and screening of Alzheimer's disease.

[0087] (ii) Sensitivity

[0088] The sensitivity of the Alzheimer-related neurofilament protein AD7c-NTP test strips provided in Examples 1-11 and Comparative Examples 1-7 was investigated, and the specific process was as follows:

[0089] The AD7c-NTP recombinant antigen was gradiently diluted to obtain linear dilutions with concentrations of 50 ng / ml, 10 ng / ml and 4 ng / ml, respectively; the Alzheimer-related neurofilament protein AD7c-NTP test strips of Examples 1-11 and Comparative Examples 1-7 were used to detect the above linear dilutions, and each linear dilution was detected in parallel for 30 groups to obtain the positive and negative samples of the sample to be tested, and the results are shown in Table 4.

[0090] Table 4 Sensitivity detection results of each Alzheimer-related neurofilament protein AD7c-NTP test strip

[0091]

[0092]

[0093] The detection results of Table 4 show that the accuracy of the test strips for Alzheimer-related neurofilament protein AD7c-NTP provided by Examples 1-11 for detecting AD7c-NTP reference product dilutions with a concentration as low as 4 ng / ml is 93.33-100.00% (≥ 90%); while the accuracy of the test strips for Alzheimer-related neurofilament protein AD7c-NTP provided by Comparative Examples 1-7 for detecting AD7c-NTP reference product dilutions with a concentration as low as 4 ng / ml is only 0-6.67%. Therefore, it is shown that the test strips for Alzheimer-related neurofilament protein AD7c-NTP provided by the present application have good detection sensitivity, and for samples containing Alzheimer-related neurofilament protein AD7c-NTP with a content as low as 4 ng / ml, a very high detection accuracy can also be obtained.

[0094] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, are within the scope of the present application claimed.

Claims

1. A test strip for detecting Alzheimer's-related neurofilament protein AD7c-NTP, characterized in that, The application relates to a rapid detection kit for AD7c-NTP, which comprises a PVC base plate; a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad are arranged on the PVC base plate; the binding pad contains latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I; 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 comprises the following steps: (1) preparing T line coating liquid and C line coating liquid: mouse anti-AD7c-NTP monoclonal antibody II is diluted by using a coating diluent, and the dilution concentration is 1.0-1.5 mg / ml, so as to obtain the T line coating liquid; goat anti-mouse IgG polyclonal antibody is diluted by using the coating diluent, and the dilution concentration is 0.5-1.0 mg / ml, so as to obtain the C line coating liquid; The coating diluent comprises 0.5-1.5% trehalose, 0.5-1.5% BSA, 0.3-1% pullulan, 0.1-0.3% Tween-80, and the rest is PBS buffer solution with pH 7.2-7.4 and 10 mM; (2) modifying the nitrocellulose membrane: the nitrocellulose membrane is soaked in anhydrous ethanol for 10-15 min, then is washed with water for 2-3 times, is drained, and a pretreated nitrocellulose membrane is obtained; epoxy chloropropane and 5-10 wt% sodium hydroxide solution are mixed in a volume ratio of 1: (4-6), are stirred uniformly, are adjusted to pH 10.0+ / -0.2, and a modified solution is obtained; the pretreated nitrocellulose membrane is soaked in the modified solution, is oscillated at 25-35 DEG C for 2-3 h, is taken out, is washed with water, and is dried at 37 DEG C; (3) drawing the detection T line and the quality control C line on the surface of the modified nitrocellulose membrane; the T line coating liquid is sprayed to the detection T line by using a gold spraying membrane drawing instrument, the C line coating liquid is sprayed to the quality control C line, is dried, and a coated nitrocellulose membrane is obtained; The preparation method of the binding pad comprises the following steps: the latex microspheres are activated, then mouse anti-AD7c-NTP monoclonal antibody I is added for labeling, is blocked, is redissolved, is diluted, is then sprayed to the surface of the pretreated binding pad, and is dried, so as to obtain the binding pad containing the latex microsphere-labeled mouse anti-AD7c-NTP monoclonal antibody I; The pretreatment method of the binding pad is as follows: the binding pad is soaked in a binding pad pretreatment liquid for 30-60 min, is dried, and a pretreated binding pad is obtained; The binding pad pretreatment liquid comprises the following components in the following contents: 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‰ Proclin300, and the rest is PBS buffer solution with pH 7.2-7.4 and 8-12 mM; The diluent used for dilution comprises the following components: 0.5-1.5% BSA, 3-7% trehalose, 0.5-2% PVP-40, 0.1-0.3% PEG-20000, and the rest is Tris-Hcl buffer solution with pH 8.0 and 50mM.

2. The test strip for Alzheimer-related neurofilament protein AD7c-NTP according to claim 1, characterized by, The spraying amount of the T-line coating solution and the C-line coating solution is 0.8-1ul / cm.

3. The test strip for Alzheimer-related neurofilament protein AD7c-NTP according to claim 1, wherein, The distance between the detection T-line and the quality control C-line is 4±0.5mm.

4. The test strip for Alzheimer-related neurofilament protein AD7c-NTP according to claim 1, wherein, The preparation of the sample pad comprises the following steps: placing the sample pad in a tray, using a pipette to suck the sample pad treatment solution to wet the sample pad, drying after soaking for 20-30min, and obtaining the sample pad. The sample pad treatment solution comprises the following components: 0.3-0.7% Triton X-100, 0.5-1.5% casein, 0.2-0.6‰ Proclin 300, 0.05-0.2% EDTA-2Na, and the rest is PBS buffer solution with pH 7.2-7.4 and 8-12mM.

5. A test kit for Alzheimer-associated neurofilament protein AD7c-NTP, characterized by, The test strip comprises the Alzheimer-related neurofilament protein AD7c-NTP according to any one of claims 1-4.

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