Test strip for detecting β-amyloid protein and use thereof

Abstract

The present application relates to the technical field of immunochromatography, and in particular, to a test strip for detecting β-amyloid protein and the use thereof. In the present application, colored microspheres are conjugated to a β-amyloid monoclonal antibody via covalent bonds, and then the conjugate is coated onto a conjugate pad. Compared with colloidal gold, the colored microspheres exhibit greater stability and higher sensitivity. Additionally, the colored microspheres feature bright and diverse colors, uniform particle sizes, good monodispersity, and strong reproducibility of detection results. The test strip provided by the present application has the advantages of simplicity, rapidity and high timeliness, requires no additional reagents, instruments and professional personnel, and supports on-site operation. By means of simply applying a to-be-detected sample to a sample loading port of the test strip, a detection result can be interpreted within 15 min. The result interpretation is visual, intuitive, accurate and straightforward, with a low risk of human errors such as false positives and false negatives.

Description

A test strip for detecting β-amyloid protein and its application

[0001] This application claims priority to Chinese Patent Application No. CN202411957662.8, filed on December 27, 2024, entitled "A test strip for detecting β-amyloid protein and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of immunochromatographic technology, and in particular to a test strip for detecting β-amyloid protein and its application. Background Technology

[0003] Studies have shown that patients with mild cognitive impairment (MCI) can revert to a cognitively normal state. Therefore, early identification and intervention at the MCI stage is an effective strategy for preventing and treating Alzheimer's disease (AD). In clinical practice, early screening and diagnosis of AD helps to rule out other types of dementia, thereby improving the accuracy of AD diagnosis. Currently, the main diagnostic markers are cerebrospinal fluid and blood samples. Both of these are invasive sampling methods, making home self-testing less feasible and universally applicable.

[0004] Urine can also reflect the pathological features of Alzheimer's disease (AD). The presence of urinary β-amyloid (Aβ) reflects changes in the production, solubility, and clearance of Aβ in the brain. This method, as a diagnostic and monitoring tool, helps in the monitoring and assessment of AD and MCI patients during disease-modifying therapy.

[0005] Currently, the detection of Aβ in urine generally uses colloidal gold for biolabeling. However, in colloidal gold immunochromatography, the antigen / antibody is prone to detachment from the gold particle surface, leading to unstable labeling and significant batch-to-batch variability. Furthermore, colloidal gold is susceptible to oxidation, causing the color of the detection line and control line to change over time. Therefore, the detection has relatively poor timeliness and sensitivity, making it difficult to meet the detection requirements of AD items that require high sensitivity. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a test strip for detecting β-amyloid protein and its application. The test strip provided in this application has the advantages of simple operation, rapid and accurate detection, high sensitivity, low cost, and good stability. It can detect urine samples, enabling home self-testing, and is a test strip for batch detection of human β-amyloid protein.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] This application provides a test strip for detecting β-amyloid protein. The test strip includes a base plate 1, a nitrocellulose membrane 2, a test line 3, a control line 4, a conjugate pad 5, a sample pad 6, and an absorbent pad 7. The sample pad 6, conjugate pad 5, nitrocellulose membrane 2, and absorbent pad 7 are sequentially overlapped on the base plate 1. The conjugate pad 5 is coated with a first β-amyloid monoclonal antibody conjugated to colored microspheres. The nitrocellulose membrane 2 has a test line 3 on the side near the conjugate pad 5 and a control line 4 on the side near the absorbent pad 7. The test line 3 is coated with a second β-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody. The first and second β-amyloid monoclonal antibodies respectively recognize different antigenic determinant sites of β-amyloid protein.

[0009] Preferably, the conjugation pad 5 is first coated with a first pretreatment solution and then coated with the first β-amyloid monoclonal antibody conjugated to the colored microspheres. The first pretreatment solution comprises: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5-7wt.%, bovine serum albumin 1wt.%, and trehalose 3-5wt.%; the pH of the Tris buffer is 7.4.

[0010] Preferably, the coating amount of the first pretreatment solution is 55–65 μL / cm. 2 The amount of the first β-amyloid monoclonal antibody conjugated with the colored microspheres is 3-6 μL / cm.

[0011] Preferably, the method for preparing the first β-amyloid monoclonal antibody conjugated with the colored microspheres includes: activating the colored microspheres using EDC and NHS, conjugating the activated colored microspheres with the first β-amyloid monoclonal antibody, and blocking them with bovine serum albumin to obtain the first β-amyloid monoclonal antibody conjugated with the colored microspheres.

[0012] Preferably, the mass ratio of the colored microspheres, EDC, and NHS is 1000:8:16; the activation rotation speed is 30 r / min, and the activation time is 30 min.

[0013] Preferably, the rotational speed of the hybrid coupling is 30 r / min, and the time is 120 min.

[0014] Preferably, the first β-amyloid monoclonal antibody comprises β-amyloid 1-42 or β-amyloid 1-40; the second β-amyloid monoclonal antibody comprises β-amyloid 1-42 or β-amyloid 1-40.

[0015] Preferably, the sample pad 6 is coated with a second pretreatment solution, which includes 0.02M Tris buffer, 2 wt.% bovine serum albumin, 1 wt.% sucrose, 1 wt.% Tween 20, 1 wt.% surfactant S17, and 0.05 wt.% PC300 antibacterial preservative; the concentration of the second β-amyloid monoclonal antibody is 2 mg / mL, and the concentration of the IgG polyclonal antibody is 1 mg / mL.

[0016] Preferably, the coating amount of the second pretreatment solution is 55–65 μL / cm. 2 The amount of the second β-amyloid monoclonal antibody applied is 1.0–2.0 μL / cm; the amount of the IgG polyclonal antibody applied is 1.0–2.0 μL / cm.

[0017] This application provides the application of the test strip described in the above technical solution in the detection of β-amyloid protein, the direct purpose of which is non-diagnostic and non-therapeutic. Beneficial effects:

[0018] This application provides a test strip for detecting β-amyloid protein. The test strip includes a base plate 1, a nitrocellulose membrane 2, a test line 3, a control line 4, a conjugate pad 5, a sample pad 6, and an absorbent pad 7. The sample pad 6, conjugate pad 5, nitrocellulose membrane 2, and absorbent pad 7 are sequentially overlapped on the base plate 1. The conjugate pad 5 is coated with a first β-amyloid monoclonal antibody conjugated to colored microspheres. The nitrocellulose membrane 2 has a test line 3 on the side near the conjugate pad 5 and a control line 4 on the side near the absorbent pad 7. The test line 3 is coated with a second β-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody. The first and second β-amyloid monoclonal antibodies respectively recognize different antigenic determinant sites of β-amyloid protein. This application utilizes colored microspheres covalently bonded to β-amyloid monoclonal antibodies and coated onto a conjugation pad. Compared to colloidal gold, this method is more stable and sensitive. Furthermore, the colored microspheres exhibit vibrant and rich colors, uniform particle size, good monodispersity, and strong reproducibility of test results. The test strip provided by this application offers advantages such as simplicity, speed, and timeliness. No other reagents, instruments, or professional personnel are required; it can be operated on-site. Simply add the sample to be tested to the sample application port of the test strip, and the test result can be determined within 15 minutes. The result interpretation is vivid, intuitive, accurate, and straightforward, minimizing the risk of false positives and false negatives. In addition, the test strip provided by this application has a wide range of applications, meeting the needs of different levels of personnel, including professional testing, large-scale population screening, and home self-testing for non-professionals. It is easy to promote and apply, possessing broad market prospects and significant economic and social benefits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 is a schematic diagram of the structure of the test strip for detecting β-amyloid protein in this application; wherein, 1 is the base plate, 2 is the nitrocellulose membrane, 3 is the detection line, 4 is the control line, 5 is the conjugation pad, 6 is the sample pad, and 7 is the absorbent pad. Detailed Implementation

[0021] This application provides a test strip for detecting β-amyloid protein. The test strip includes a base plate 1, a nitrocellulose membrane 2, a test line 3, a control line 4, a conjugate pad 5, a sample pad 6, and an absorbent pad 7. The sample pad 6, the conjugate pad 5, the nitrocellulose membrane 2, and the absorbent pad 7 are sequentially overlapped on the base plate 1. The conjugate pad 5 is coated with a first β-amyloid monoclonal antibody conjugated to colored microspheres. The nitrocellulose membrane 2 has a test line 3 on the side near the conjugate pad 5 and a control line 4 on the side near the absorbent pad 7. The test line 3 is coated with a second β-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody. The first β-amyloid monoclonal antibody and the second β-amyloid monoclonal antibody respectively recognize different antigenic determinant sites of β-amyloid protein.

[0022] In one embodiment, the base plate 1 can be made of PVC.

[0023] In one embodiment, the sample pad 6 can be made of glass fiber membrane. In another embodiment, the sample pad 6 is coated with a second pretreatment solution, which comprises 0.02M Tris buffer, 2 wt.% bovine serum albumin, 1 wt.% sucrose, 1 wt.% Tween 20, 1 wt.% surfactant S17, and 0.05 wt.% PC300 antibacterial preservative. In another embodiment, the coating amount of the second pretreatment solution is 55–65 μL / cm². 2 .

[0024] In one embodiment, the conjugate pad 5 can be made of glass fiber membrane. In another embodiment, the conjugate pad 5 is first coated with a first pretreatment solution and then coated with the first β-amyloid monoclonal antibody conjugated to the colored microspheres. The first pretreatment solution comprises: Tris buffer 0.02M, PVP 0.5 wt.%, Tween 20 5-7 wt.%, bovine serum albumin 1 wt.%, and trehalose 3-5 wt.%; the pH of the Tris buffer is 7.4. In another embodiment, the coating volume of the first pretreatment solution is 55-65 μL / cm². 2 The coating amount of the first β-amyloid monoclonal antibody conjugated with the colored microspheres is 3–6 μL / cm. In one embodiment, the first β-amyloid monoclonal antibody includes Aβ monoclonal antibody, β-amyloid 1-42, or β-amyloid 1-40. In another embodiment, the Aβ monoclonal antibody, β-amyloid 1-42, or β-amyloid 1-40 can be one or more of those listed in Table 1. In yet another embodiment, the first β-amyloid monoclonal antibody includes antibodies with catalog numbers EKN0448A, A09N03-S5, A09N03-T3, or Aβ1-42002 listed in Table 1.

[0025] Table 1. Sources of different β-amyloid monoclonal antibodies

[0026] In one embodiment, the preparation method of the first β-amyloid monoclonal antibody conjugated with colored microspheres includes: activating the colored microspheres using EDC and NHS; mixing and conjugating the activated colored microspheres with the first β-amyloid monoclonal antibody; and blocking with bovine serum albumin to obtain the first β-amyloid monoclonal antibody conjugated with the colored microspheres. In one embodiment, the mass ratio of the colored microspheres, EDC, and NHS is 1000:8:16; the activation rotation speed is 30 r / min, and the time is 30 min. In another embodiment, the mixing and conjugation rotation speed is 30 r / min, and the time is 120 min.

[0027] In one embodiment, the second β-amyloid monoclonal antibody includes β-amyloid 1-42 or β-amyloid 1-40. In another embodiment, the second β-amyloid monoclonal antibody includes antibodies with catalog numbers EKN0447A, A09N04-A6, Aβ1-42001, or K92011M16F04C listed in Table 1. In yet another embodiment, based on the catalog numbers in Table 1, the antibody combinations of the first and second β-amyloid monoclonal antibodies include: EKN0448A and EKN0447A, A09N03-S5 and A09N04-A6, A09N03-T3 and A09N04-A6, Aβ1-42002 and Aβ1-42001, and K70013C06H01C and K92011M16F04C.

[0028] In one embodiment, the concentration of the second β-amyloid monoclonal antibody is 2 mg / mL, and the concentration of the IgG polyclonal antibody is 1 mg / mL. In another embodiment, the IgG polyclonal antibody is a goat anti-mouse IgG polyclonal antibody. In yet another embodiment, the diluent for the second β-amyloid monoclonal antibody or the IgG polyclonal antibody can be Tris buffer. In one embodiment, the coating volume of the second β-amyloid monoclonal antibody is 1.0–2.0 μL / cm; the coating volume of the IgG polyclonal antibody is 1.0–2.0 μL / cm.

[0029] In one embodiment, the colored microspheres have a particle size of 300 nm. In another embodiment, the colored microspheres can be one or more of the types listed in Table 2.

[0030] Table 2 Sources of colored microspheres

[0031] The test strip provided in this application can use urine as a sample type. Urine samples are non-invasive and not affected by risk factors related to sample storage. Users can collect and test samples at home and obtain test results themselves.

[0032] When the test strip provided in this application detects the presence of β-amyloid protein in the sample, the β-amyloid protein binds to the first β-amyloid monoclonal antibody conjugated to colored microspheres in the conjugate pad, forming an "Aβ-Aβ monoclonal antibody-colored microsphere complex." This complex, upon chromatography up to the detection area, binds to the second β-amyloid monoclonal antibody pre-coated in the detection area, forming a colored line in the detection area, indicating a positive result; conversely, a negative result indicates a negative result. A goat anti-mouse IgG polyclonal antibody is coated in the nitrocellulose membrane control area. Regardless of the presence of β-amyloid protein in the urine, a line will appear in the control area, serving as evidence of the test's validity.

[0033] This application provides the application of the test strip described above in the detection of β-amyloid protein, the direct purpose of which is non-diagnostic and non-therapeutic. As one embodiment, the sample for detecting β-amyloid protein can be urine. The direct purpose of the application described in this application is non-diagnostic and non-therapeutic, aiming to obtain an intermediate value indicating whether the sample contains β-amyloid protein.

[0034] To further illustrate this application, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a test strip for detecting β-amyloid protein and its application, but these descriptions should not be construed as limiting the scope of protection of this application.

[0035] Example 1

[0036] A test strip for detecting β-amyloid protein, as shown in Figure 1, includes a base plate 1, a nitrocellulose membrane 2, a test line 3, a control line 4, a conjugate pad 5, a sample pad 6, and an absorbent pad 7. The sample pad 6, conjugate pad 5, nitrocellulose membrane 2, and absorbent pad 7 are sequentially overlapped on the base plate 1. The conjugate pad 5 is coated with a first β-amyloid monoclonal antibody conjugated to colored microspheres. The nitrocellulose membrane 2 has a test line 3 on the side near the conjugate pad 5 and a control line 4 on the side near the absorbent pad 7. The test line 3 is coated with a second β-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody. The first and second β-amyloid monoclonal antibodies recognize different antigenic determinant sites of β-amyloid protein.

[0037] The test strip is prepared as follows:

[0038] 1. Soak the glass fiber membrane in the second pretreatment solution for 10 min, and dry it in a drying oven at 37℃ for 22 h for later use, which is the sample pad 6; the second pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, bovine serum albumin 2wt.%, sucrose 1wt.%, Tween 20 1wt.%, surfactant S17 1wt.%, and PC300 antibacterial preservative 0.05wt.%; the pH value of the Tris buffer is 7.4.

[0039] 2. Pretreatment of glass fiber wool: Cut the glass fiber wool into strips 9mm wide and apply at a rate of 60μL / cm. 2 The glass fiber membrane was soaked in the first pretreatment solution for 10 minutes and then dried in a drying oven for 22 hours to obtain the pretreated glass fiber membrane. The first pretreatment solution consisted of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5wt.%, bovine serum albumin 1wt.%, and trehalose 3wt.%. The pH of the Tris buffer was 7.4.

[0040] 3. The conjugation of colored microspheres with β-amyloid monoclonal antibody is performed as follows:

[0041] 1) Take 1 mg of colored microspheres (Beijing Chunda Technology Co., Ltd., product number AJ30COOH-L1-R2) and add 1 ml of MES buffer. Centrifuge, discard the supernatant, and repeat the above steps once. After resuspending the colored microspheres with sonication to 5 mg / ml, add 8 μg of EDC and 16 μg of NHS to activate them, and then mix them in a mixer at 30 r / min for 30 min.

[0042] 2) Take out the activated microspheres and centrifuge and wash them twice with 1ml MES buffer. Resuspend them in MES buffer by sonication to 5mg / ml. Add 0.5mg Aβ monoclonal antibody (Xibao Biotechnology, catalog number EKN0448A) to the microspheres after sonication and mix them in a mixer at 30r / min for 120min.

[0043] 3) Add 1% BSA to the microspheres and mix well. Then, place the mixture in a mixer and rotate it at 30 r / min for 30 min to obtain the sealed microspheres.

[0044] 4) Remove the blocked microspheres, centrifuge, discard the supernatant, and wash twice with 1 ml of microsphere preservation solution. The microsphere preservation solution consists of the following components at the following concentrations: Tris buffer 0.02 M, Tween 20 0.1 wt.%, casein 0.5 wt.%, trehalose 5 wt.%, and PC300 antibacterial preservative 0.05 wt.%; the pH of the Tris buffer is 7.4; resuspend the blocked microspheres in the microsphere preservation solution by sonication to 5 mg / ml to obtain a microsphere-conjugated antibody suspension.

[0045] 5) Spray the microsphere-conjugated antibody suspension onto the pretreated glass fiber membrane obtained in step 2 at a coating amount of 6 μL / cm, and dry it at a constant temperature of 37°C for 18 hours to obtain conjugate pad 5.

[0046] 4. Aβ monoclonal antibody (Xibao Biotechnology, catalog number EKN0447A) and goat anti-mouse IgG antibody were diluted to 2 mg / ml and 1 mg / ml respectively with Tris dilution buffer. NC membranes were prepared using a coating volume of 1 μl / cm. The prepared NC membranes were dried at 37℃ for 24 h to obtain nitrocellulose membrane 2.

[0047] 5. Cut the 200×300mm absorbent paper into 15mm wide strips using a cutter, dry them and set them aside. This is the absorbent pad 7.

[0048] 6. The prepared sample pad 6, conjugate pad 5, nitrocellulose membrane 2 and absorbent pad 7 are sequentially attached to a 60×300mm base plate 1, overlapping from left to right with an overlap width of 1-2mm. Then, the sample pad 6, conjugate pad 5, nitrocellulose membrane 2 and absorbent pad 7 are cut into test strips with a width of 4mm to obtain the test strips. For ease of use, the test strips are placed in a plastic card with a sample application hole and an observation window to obtain the test card.

[0049] Comparative Example 1

[0050] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, bovine serum albumin 1wt.%, and trehalose 1wt.%; the pH of the Tris buffer is 7.4.

[0051] Comparative Example 2

[0052] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, bovine serum albumin 1wt.%, and trehalose 3wt.%; the pH of the Tris buffer is 7.4.

[0053] Comparative Example 3

[0054] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, bovine serum albumin 1wt.%, and trehalose 5wt.%; the pH of the Tris buffer is 7.4.

[0055] Comparative Example 4

[0056] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 3wt.%, bovine serum albumin 1wt.%, and trehalose 1wt.%; the pH of the Tris buffer is 7.4.

[0057] Comparative Example 5

[0058] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5wt.%, bovine serum albumin 1wt.%, and trehalose 1wt.%; the pH of the Tris buffer is 7.4.

[0059] Comparative Example 6

[0060] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 7wt.%, bovine serum albumin 1wt.%, and trehalose 1wt.%; the pH of the Tris buffer is 7.4.

[0061] Comparative Example 7

[0062] A test strip similar to that of Example 1, except that: the first pretreatment solution consists of the following components at the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, casein 0.5wt.%, and trehalose 1wt.%; the pH of the Tris buffer is 7.4.

[0063] Test Example 1

[0064] Sample diluent and Aβ antigen (Xibao Biotechnology, catalog number EDD0720A) were used to prepare test strips with detection limits of 100, 80, and 50 pg / ml, respectively, according to the corresponding ratios. Test strips prepared using comparative examples 1 to 7 were tested according to the detection method. The detection limits of each formulation were analyzed by observing the color intensity of the test strips. The detection method is as follows:

[0065] The sample diluent formulation is as follows: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 0.1wt.%, bovine serum albumin 1wt.%, trehalose 1wt.%, and PC300 antibacterial preservative 0.05wt.%.

[0066] Slowly add 100 μL of the sample to be tested drop by drop to the sample pad of the test strip, let it stand for 15 minutes, and then check the test line and the control line.

[0067] Results interpretation: Negative (-): Only the control line shows color. Positive (+): Both the test line and the control line show color. See Table 3 for results.

[0068] Table 3. Detection results of different test strips

[0069] Note: The color intensity is determined by visual observation. +++ indicates strong positive; ++ indicates positive; + indicates weak positive; + / - indicates slight band; - indicates negative. The same applies to the table below.

[0070] The results showed that the optimal Tween 20 content was 5%–7%, which was comparable to 3%–5% trehalose. BSA showed better sensitivity than casein, so the protein type, surfactant, and sugar content were set to 1% BSA, 5%–7% Tween 20, and 3%–5% trehalose, respectively.

[0071] Test Example 2

[0072] Normal human urine samples (negative urine) and Aβ antigen (Xibao Biotechnology, catalog number EDD0720A) were prepared at corresponding ratios of 1000, 500, 100, 50, 10 pg / ml and negative urine 0 pg / ml, respectively. Test strips prepared in Example 1 were used, and samples were added for testing according to the detection method. The detection limit was analyzed by observing the color intensity of the test strips. The results are shown in Table 4.

[0073] Table 4. Detection results at different antigen concentrations

[0074] As shown in Tables 3 and 4, the test strips prepared in Comparative Examples 1 to 7 showed a + / - concentration of 80 to 100 pg / ml, while the test strip prepared in Example 1 showed a + / - concentration of 10 pg / ml. Therefore, the optimized pretreatment formula can improve the sensitivity by about 8 to 10 times.

[0075] Test Example 3

[0076] 1. Linear correlation: When Aβ antigen was added to negative urine, it was serially diluted to 0.05 ng / ml, 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 5 ng / ml, and negative urine, respectively, and the test strips were used for detection. The results showed that the T line color of the Aβ urine test at 0-5 ng / ml showed a positive increase in the detection result.

[0077] 2. Limit of Detection: High-value clinical samples with a concentration of 64 pg / ml were diluted with negative urine to 40, 30, 20, 10, 8, 6, and 5 pg / ml respectively. The detection results were compared with those of Hunan Qiankang Technology Reagent (whose limit of detection is specified as 40 pg / ml in its invention patent CN111656197A), as shown in Table 5.

[0078] Table 5. Detection results of different test strips

[0079] As can be seen from the data in the table above, the detection limit of the Qiankang colloidal gold reagent kit is 40 pg / ml, while the detection limit of the reagent kit of this application for Aβ antigen is 8 pg / ml; the detection limit of the reagent kit of this application is even lower.

[0080] 3. Clinical applications:

[0081] The kit in this application will collect 193 clinical samples; including 66 clinically diagnosed Alzheimer's disease patients and 127 normal urine samples. The test results are shown in Tables 6 and 7.

[0082] Table 6 Clinical Diagnostic Results

[0083] Table 7 Sensitivity and Specificity Results

[0084] In the above experiment, Alzheimer's disease patient samples that showed weak positive results in the test kit of this application were selected and tested using the QianKang colloidal gold test kit. The results were all negative. The experiment proved that the sensitivity of the QianKang colloidal gold test kit was insufficient when detecting weak positive samples, resulting in failure to detect them.

[0085] Comparative Example 8: Colored Microsphere Screening Experiment

[0086] A test strip similar to Example 1 is prepared by replacing the colored microspheres in step 3 with the microspheres in Table 8, resulting in different test strips.

[0087] Table 8. Sources and particle sizes of different microspheres

[0088] The sample diluent and Aβ antigen (Xibao Biotechnology, catalog number EDD0720A) were prepared in the corresponding proportions to achieve detection limit concentrations of 100, 80, 50, and 10 pg / ml, and the sample diluent was prepared at 0 pg / ml. The sample diluent formulation was the same as that in test example 1.

[0089] Using the test strips in Table 8, samples were added for testing according to the detection method in Test Example 1. The detection limit and specificity of each formulation were analyzed by observing the color intensity of the test strips. The test results are shown in Table 9.

[0090] Table 9. Detection results of different test strips

[0091] The results showed that, based on the reagent antigen detection limit and specificity analysis, the optimal test strips were B2, B8, and B9, respectively. Their colored microspheres were DR0300CA, AJ30COOH-L1-R2, and DPR300C-COOH, respectively.

[0092] Microsphere size analysis: Analysis of the test results of test strips B1, B10, B3, and B11 shows that test strips B3 and B11, which use 400nm microspheres, exhibited lines in the sample dilution solution, leading to a higher background value. Therefore, 400nm microspheres are not suitable for test strip preparation. Test strips B1 and B10, which use 200nm microspheres, have a lower detection limit than 300nm microspheres, approximately 50 pg / ml.

[0093] The test results for B4, B5, B6, and B7 show that the reagent sensitivity is affected by both the color intensity and particle size of the microspheres, with a positive correlation between color intensity and sensitivity. Larger microsphere particle sizes result in higher sensitivity, and the selection of microsphere raw materials should be based on product requirements.

[0094] Comparative Example 9: Paired Antibody Screening Experiment

[0095] A test strip similar to that in Example 1, except that the first β-amyloid monoclonal antibody and the second β-amyloid monoclonal antibody used are different, and the paired antibodies are shown in Table 10.

[0096] Table 10 Different Antibodies and Their Sources

[0097] Note: The first monoclonal antibody in the table is the first β-amyloid monoclonal antibody, and the second monoclonal antibody is the second β-amyloid monoclonal antibody.

[0098] Ten healthy controls and ten clinically diagnosed Alzheimer's disease (AD) patients were collected from the hospital. Test strips C1-C10 (Table 10) were used for sample addition and testing. The optimal paired antibody was selected based on the test sensitivity and specificity. The test results are shown in Table 11.

[0099] Table 11 Detection results of different test strips

[0100] The results show that the detection results of different paired antibodies are effective in distinguishing AD from normal. The preferred paired antibodies are EKN0448A and EKN0447A, A09N03-S5 and A09N04-A6, A09N03-T3 and A09N04-A6, Aβ1-42002 and Aβ1-42001, K70013C06H01C and K92011M16F04C.

[0101] Experiments show that different β-amyloid monoclonal antibodies exhibit varying effects after pairing. Due to differences in the inherent properties of the antibodies, the amino acid sequences and spatial structures of the antigen-binding sites of monoclonal antibodies from different manufacturers differ, resulting in variations in their affinity for the antigen. Therefore, when paired antibodies from different manufacturers are used in the test strip preparation process described in this application, their affinity for the antigen and antibody activity also show different results. The use of monoclonal antibodies must meet the following conditions: with clinical diagnosis of AD as a control, the sensitivity and normal levels must both reach at least 80% to achieve the detection effect described in this application.

[0102] In summary, the test strip provided in this application has the advantages of simple operation, rapid and accurate detection, high sensitivity, low cost, and good stability. It can detect urine samples, enabling home self-testing, and is a test strip for batch detection of human β-amyloid protein.

[0103] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.

Claims

1. A test strip for detecting β-amyloid protein, the test strip comprising a base plate (1), a sample pad (6), a conjugation pad (5), a nitrocellulose membrane (2), and an absorbent pad (7) sequentially overlapped on the base plate (1), and further comprising a detection line (3) and a control line (4), characterized in that, The conjugate pad (5) is coated with a first β-amyloid monoclonal antibody conjugated to colored microspheres; a detection line (3) is provided on the side of the nitrocellulose membrane (2) near the conjugate pad (5), and a control line (4) is provided on the side of the nitrocellulose membrane (2) near the absorbent pad (7); a second β-amyloid monoclonal antibody is coated on the detection line (3), and an IgG polyclonal antibody is coated on the control line (4); the first β-amyloid monoclonal antibody and the second β-amyloid monoclonal antibody respectively recognize different antigenic determinant sites of β-amyloid protein.

2. The test strip according to claim 1, characterized in that, The conjugate pad (5) is first coated with a first pretreatment solution and then coated with the first β-amyloid monoclonal antibody conjugated to the colored microspheres. The first pretreatment solution includes: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5-7wt.%, bovine serum albumin 1wt.%, and trehalose 3-5wt.%. The pH of the Tris buffer is 7.

4.

3. The test strip according to claim 2, characterized in that, The coating amount of the first pretreatment solution is 55–65 μL / cm. 2 The amount of the first β-amyloid monoclonal antibody conjugated with the colored microspheres is 3-6 μL / cm.

4. The test strip according to claim 1 or 2, characterized in that, The material of the bonding pad (5) includes a glass fiber membrane.

5. The test strip according to any one of claims 1 to 3, characterized in that, The method for preparing the first β-amyloid monoclonal antibody conjugated with the colored microspheres includes: activating the colored microspheres using EDC and NHS, conjugating the activated colored microspheres with the first β-amyloid monoclonal antibody, and blocking them with bovine serum albumin to obtain the first β-amyloid monoclonal antibody conjugated with the colored microspheres.

6. The test strip according to claim 5, characterized in that, The mass ratio of the colored microspheres, EDC, and NHS is 1000:8:16; the activation speed is 30 r / min and the activation time is 30 min.

7. The test strip according to claim 5, characterized in that, The rotational speed of the hybrid coupling was 30 r / min, and the time was 120 min.

8. The test strip according to claim 1, characterized in that, The first β-amyloid monoclonal antibody includes Aβ monoclonal antibody, β-amyloid 1-42 or β-amyloid 1-40; the second β-amyloid monoclonal antibody includes Aβ monoclonal antibody, β-amyloid 1-42 or β-amyloid 1-40.

9. The test strip according to claim 8, characterized in that, The Aβ monoclonal antibody, β-amyloid 1-42, or β-amyloid 1-40 includes one or more of the following:

10. The test strip according to claim 9, characterized in that, The first β-amyloid monoclonal antibody includes antibodies with catalog numbers EKN0448A, A09N03-S5, A09N03-T3, or Aβ1-42002; the second β-amyloid monoclonal antibody includes antibodies with catalog numbers EKN0447A, A09N04-A6, Aβ1-42001, or K92011M16F04C.

11. The test strip according to claim 9 or 10, characterized in that, The antibody combinations paired with the first β-amyloid monoclonal antibody and the second β-amyloid monoclonal antibody include antibody combinations with the following catalog numbers: EKN0448A and EKN0447A, A09N03-S5 and A09N04-A6, A09N03-T3 and A09N04-A6, Aβ1-42002 and Aβ1-42001, and K70013C06H01C and K92011M16F04C.

12. The test strip according to claim 1, characterized in that, The sample pad (6) is coated with a second pretreatment solution, which includes 0.02M Tris buffer, 2 wt.% bovine serum albumin, 1 wt.% sucrose, 1 wt.% Tween 20, 1 wt.% surfactant S17, and 0.05 wt.% PC300 antibacterial preservative.

13. The test strip according to claim 12, characterized in that, The coating amount of the second pretreatment solution is 55–65 μL / cm. 2 .

14. The test strip according to claim 1 or 12, characterized in that, The sample pad (6) is made of glass fiber membrane.

15. The test strip according to claim 1, characterized in that, The concentration of the second β-amyloid monoclonal antibody is 2 mg / mL, and the concentration of the IgG polyclonal antibody is 1 mg / mL; the amount of the second β-amyloid monoclonal antibody applied is 1.0–2.0 μL / cm; and the amount of the IgG polyclonal antibody applied is 1.0–2.0 μL / cm.

16. The test strip according to claim 15, characterized in that, The solvent for the second β-amyloid monoclonal antibody or the IgG polyclonal antibody includes Tris buffer.

17. The test strip according to claim 1 or 15, characterized in that, The IgG polyclonal antibody is a goat anti-mouse IgG polyclonal antibody.

18. The test strip according to claim 1, characterized in that, The colored microspheres have a particle size of 300 nm.

19. The test strip according to claim 1 or 18, characterized in that, The colored microspheres include one or more of the following:

20. The test strip according to claim 1, characterized in that, The base plate (1) is made of PVC.

21. A test strip for detecting β-amyloid protein, characterized in that, It is composed of a test strip as described in any one of claims 1 to 20 and a plastic casing; the plastic casing is provided with a sample application hole and an observation window; the test strip is located inside the plastic casing, the sample application hole is located above the sample pad (6) on the test strip, and the observation window is located above the nitrocellulose membrane (2) on the test strip.

22. The use of the test strip according to any one of claims 1 to 20 or the test card according to claim 21 in the detection of β-amyloid protein.

23. A method for detecting β-amyloid protein, characterized in that, Includes the following steps: Add the sample to be tested to the sample pad (6) of the test strip, let it stand for 15 minutes, and observe the detection line (3) and control line (4) on the test strip; the test strip is the test strip according to any one of claims 1 to 20 or the test strip in the test strip card according to claim 21; Result evaluation: Negative (-): Only the control line (4) shows color; Positive (+): Both the test line (3) and the control line (4) show color.

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