Test strip for detecting beta-amyloid protein and application thereof

Abstract

The invention relates to the technical field of immunochromatography, in particular to a test strip for detecting beta-amyloid protein and application of the test strip. The color microspheres are combined with the beta-amyloid protein monoclonal antibody through covalent bonds and then coated on the combination pad, compared with colloidal gold, the color microspheres are more stable and higher in sensitivity, and the color microspheres are bright and rich in color, uniform in particle size, good in monodispersity and high in repeatability of detection results. The test strip provided by the invention has the advantages of simplicity, convenience, rapidness and strong timeliness, no other reagents, instruments or professionals are needed, field operation can be realized, a detection result can be judged within 15 minutes as long as a sample to be detected is dropwise added to a sample adding port of the test strip, the result judgment is vivid, visual, accurate, simple and clear, and the test strip is suitable for large-scale popularization and application. And artificial misjudgments such as false positive and false negative are not easy to occur.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatography, and in particular to a test strip for detecting beta-amyloid protein and application thereof. Background Art

[0002] Studies have shown that patients with mild cognitive impairment (MCI) can be reversed to a normal cognitive 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 can help exclude other types of dementia and improve the accuracy of AD diagnosis. Currently, the diagnosis of markers is mainly based on cerebrospinal fluid and blood samples. These two types of sampling are invasive and are not feasible or universal for home self-testing.

[0003] Urine can also reflect the pathological features of AD, and the presence of beta-amyloid (Aβ) in urine reflects changes in the production, solubility, and clearance of Aβ in the brain. This method serves as a diagnostic and monitoring tool to help monitor and evaluate AD and MCI patients during disease-modifying treatment.

[0004] At present, colloidal gold is generally used as a biomarker for the detection of Aβ in urine. However, the antigen / antibody of the colloidal gold immunochromatography method is easily detached from the surface of the gold particles, resulting in unstable markers and large differences between product batches. In addition, colloidal gold has the problem that the color of the test line and the quality control line will change over time due to oxidation. Therefore, the detection has the problem of relatively poor timeliness and sensitivity. It is difficult to meet the detection needs for AD projects that require high sensitivity. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a test strip for detecting β-amyloid protein and its application. The test strip provided by the present invention has the advantages of simple operation, rapid and accurate detection, high sensitivity, low cost and good stability, can detect urine samples, and realize home self-testing. It is a test strip for batch detection of human β-amyloid protein.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a test strip for detecting beta-amyloid protein, a test strip for detecting beta-amyloid protein, the test strip comprising a bottom plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a conjugation pad 5, a sample pad 6 and a water absorbent pad 7; the bottom plate 1 is provided with a sample pad 6, a conjugation pad 5, a nitrocellulose membrane 2 and a water absorbent pad 7 which are overlapped in sequence, the conjugation pad 5 is coated with a first beta-amyloid protein monoclonal antibody coupled with a colored microsphere; the nitrocellulose membrane 2 is provided with a detection line 3 on one side close to the conjugation pad 5, the nitrocellulose membrane 2 is provided with a control line 4 on one side close to the water absorbent pad 7, the detection line 3 is coated with a second beta-amyloid protein monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first beta-amyloid protein monoclonal antibody and the second beta-amyloid protein monoclonal antibody respectively recognize different antigenic determinant cluster sites of beta-amyloid protein.

[0008] Preferably, the conjugate pad 5 is first coated with a first pretreatment liquid and then coated with the first β-amyloid protein monoclonal antibody coupled to the colored microspheres, wherein the first pretreatment liquid comprises: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5-7wt.%, bovine serum albumin 1wt.% and trehalose 3-5wt.%; the pH value of the Tris buffer is 7.4.

[0009] Preferably, the coating amount of the first pretreatment liquid is 55-65 μL / cm 2 The coating amount of the first β-amyloid monoclonal antibody coupled to the colored microspheres is 3 to 6 μL / cm.

[0010] Preferably, the method for preparing the first β-amyloid protein monoclonal antibody coupled to the colored microspheres comprises: activating the colored microspheres using EDC and NHS, mixing and coupling the activated colored microspheres and the first β-amyloid protein monoclonal antibody, and then blocking with bovine serum albumin to obtain the first β-amyloid protein monoclonal antibody coupled to the colored microspheres.

[0011] Preferably, 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.

[0012] Preferably, the rotation speed of the mixed coupling is 30 r / min and the time is 120 min.

[0013] Preferably, the first β-amyloid protein monoclonal antibody includes β-amyloid protein 1-42 or β-amyloid protein 1-40; the second β-amyloid protein monoclonal antibody includes β-amyloid protein 1-42 or β-amyloid protein 1-40.

[0014] Preferably, the sample pad 2 is coated with a second pretreatment liquid, which includes 0.02M Tris buffer, 2wt.% bovine serum albumin, 1wt.% sucrose, 1wt.% Tween 20, 1wt.% surfactant S17, and 0.05wt.% PC300 antibacterial preservative; the concentration of the second β-amyloid protein monoclonal antibody is 2mg / mL, and the concentration of the IgG polyclonal antibody is 1mg / mL.

[0015] Preferably, the coating amount of the second pretreatment liquid is 55-65 μL / cm 2 ; The coating amount of the second β-amyloid protein monoclonal antibody is 1.0 to 2.0 μL / cm; The coating amount of the IgG polyclonal antibody is 1.0 to 2.0 μL / cm.

[0016] The present invention provides the use of the test strip described in the above technical solution in detecting β-amyloid protein, and the direct purpose of the use is non-diagnosis and non-treatment.

[0017] Beneficial effects:

[0018] The invention provides a test strip for detecting beta-amyloid protein, a test strip for detecting beta-amyloid protein, the test strip comprising a bottom plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a conjugation pad 5, a sample pad 6 and a water absorbent pad 7; the bottom plate 1 is provided with a sample pad 6, a conjugation pad 5, a nitrocellulose membrane 2 and a water absorbent pad 7 which are overlapped in sequence, the conjugation pad 5 is coated with a first beta-amyloid protein monoclonal antibody coupled with a colored microsphere; the nitrocellulose membrane 2 is provided with a detection line 3 on one side close to the conjugation pad 5, the nitrocellulose membrane 2 is provided with a control line 4 on one side close to the water absorbent pad 7, the detection line 3 is coated with a second beta-amyloid protein monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first beta-amyloid protein monoclonal antibody and the second beta-amyloid protein monoclonal antibody respectively recognize different antigenic determinant cluster sites of beta-amyloid protein. The present invention uses colored microspheres to be combined with β-amyloid monoclonal antibodies through covalent bonds and coated on the conjugate pad. Compared with colloidal gold, it is more stable and more sensitive, and the colored microspheres are bright and rich in color, uniform in particle size, monodisperse, and the test results are highly repeatable. The test strip provided by the present invention has the advantages of simplicity, speed and strong timeliness, does not require any other reagents, instruments and professionals, can be operated on site, and the test result can be determined within 15 minutes as long as the sample to be tested is dripped at the sample adding port of the test strip. The result is determined vividly, intuitively, accurately, simply and clearly, and it is not easy to have artificial misjudgments such as false positives and false negatives; in addition, the test strip provided by the present invention has a wide range of applications, which can meet the needs of personnel at different levels, including professional inspections, large-scale population censuses, and home self-tests by non-professional people, etc., which is convenient for promotion and application, and has broad market prospects and obvious economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 The schematic diagram of the structure of the test strip for detecting β-amyloid protein of the present invention; wherein 1 is a base plate, 2 is a nitrocellulose membrane, 3 is a detection line, 4 is a control line, 5 is a binding pad, 6 is a sample pad, and 7 is a water absorbent pad. DETAILED DESCRIPTION

[0021] The present invention provides a test strip for detecting beta-amyloid protein, the test strip comprising a bottom plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a conjugation pad 5, a sample pad 6 and a water absorbent pad 7; the bottom plate 1 is provided with a sample pad 6, a conjugation pad 5, a nitrocellulose membrane 2 and a water absorbent pad 7 which are overlapped in sequence, the conjugation pad 5 is coated with a first beta-amyloid protein monoclonal antibody coupled with a colored microsphere; the nitrocellulose membrane 2 is provided with a detection line 3 on one side close to the conjugation pad 5, the nitrocellulose membrane 2 is provided with a control line 4 on one side close to the water absorbent pad 7, the detection line 3 is coated with a second beta-amyloid protein monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first beta-amyloid protein monoclonal antibody and the second beta-amyloid protein monoclonal antibody respectively recognize different antigenic determinant cluster sites of beta-amyloid protein.

[0022] As an implementation manner, the material of the base plate 1 can be PVC.

[0023] As an embodiment, the material of the sample pad 6 can be a glass fiber membrane. As an embodiment, the sample pad 6 is coated with a second pretreatment liquid, which includes 0.02M Tris buffer, 2wt.% bovine serum albumin, 1wt.% sucrose, 1wt.% Tween 20, 1wt.% surfactant S17, and 0.05wt.% PC300 antibacterial preservative. As an embodiment, the coating amount of the second pretreatment liquid is 55-65μL / cm 2 .

[0024] As an embodiment, the material of the conjugate pad 5 can be a glass fiber membrane. As an embodiment, the conjugate pad 5 is first coated with a first pretreatment liquid and then coated with the first β-amyloid protein monoclonal antibody coupled to the colored microspheres, and the first pretreatment liquid includes: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5-7wt.%, bovine serum albumin 1wt.% and trehalose 3-5wt.%; the pH value of the Tris buffer is 7.4. As an embodiment, the coating amount of the first pretreatment liquid is 55-65μL / cm 2; The coating amount of the first β-amyloid monoclonal antibody coupled to the colored microspheres is 3 to 6 μL / cm. As an embodiment, the first β-amyloid monoclonal antibody includes Aβ monoclonal antibody, β-amyloid protein 1-42 or β-amyloid protein 1-40. As another embodiment, the Aβ monoclonal antibody, β-amyloid protein 1-42 or β-amyloid protein 1-40 can be one or more of Table 1. As another embodiment, the first β-amyloid protein monoclonal antibody includes the antibody with the item number EKN0448A, A09N03-S5, A09N03-T3 or Aβ1-42002 in Table 1.

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

[0026] Antibody factory Part Number Aβ monoclonal antibody Beijing Aibosheng Biotechnology Co., Ltd. K70013C06H01C Aβ1-42 Beijing Aibosheng Biotechnology Co., Ltd. K92011M16F04C Amyloid β 1-40 (Aβ1-40) Antibody Xibao Biotechnology (Shanghai) Co., Ltd. EKN0448A Amyloid β 1-40 (Aβ1-40) Antibody Xibao Biotechnology (Shanghai) Co., Ltd. EKN0447A Aβ1-40 Beijing Baixinyi Biotechnology Co., Ltd. A09N03-S5 Aβ1-40 Beijing Baixinyi Biotechnology Co., Ltd. A09N03-T3 Aβ monoclonal antibody Beijing Baixinyi Biotechnology Co., Ltd. A09N04-A6 Aβ1-42antibody Chengdu Baling Biotechnology Co., Ltd. Aβ1-42002 Aβ1-42antibody Chengdu Baling Biotechnology Co., Ltd. Aβ1-42001

[0027] As an embodiment, the preparation method of the first β-amyloid monoclonal antibody coupled to the colored microspheres includes: activating the colored microspheres with EDC and NHS, mixing and coupling 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 coupled to the colored microspheres. As an embodiment, the mass ratio of the colored microspheres, EDC and NHS is 1000:8:16; the activation speed is 30r / min and the time is 30min. As an embodiment, the mixed coupling speed is 30r / min and the time is 120min.

[0028] As an embodiment, the second amyloid beta monoclonal antibody includes amyloid beta 1-42 or amyloid beta 1-40. As another embodiment, the second amyloid beta monoclonal antibody includes an antibody with the item number EKN0447A, A09N04-A6, Aβ1-42001 or K92011M16F04C in Table 1; as another embodiment, the antibodies are counted by the item number in Table 1, and the antibody combination of the first amyloid beta monoclonal antibody and the second amyloid beta monoclonal antibody includes: EKN0448A and EKN0447A are paired, A09N03-S5 and A09N04-A6 are paired, A09N03-T3 and A09N04-A6 are paired, Aβ1-42002 and Aβ1-42001 are paired, and K70013C06H01C and K92011M16F04C are paired.

[0029] As an embodiment, the concentration of the second β-amyloid protein monoclonal antibody is 2 mg / mL, and the concentration of the IgG polyclonal antibody is 1 mg / mL. As another embodiment, the IgG polyclonal antibody is a goat anti-mouse IgG polyclonal antibody. As another embodiment, the diluent of the second β-amyloid protein monoclonal antibody or the IgG polyclonal antibody can be Tris buffer. As an embodiment, the coating amount of the second β-amyloid protein monoclonal antibody is 1.0 to 2.0 μL / cm; the coating amount of the IgG polyclonal antibody is 1.0 to 2.0 μL / cm.

[0030] As an embodiment, the particle size of the colored microspheres is 300 nm. As another embodiment, the colored microspheres can be one or more of those listed in Table 2.

[0031] Table 2 Sources of colored microspheres

[0032] Microspheres factory Part Number Carboxyl colored microspheres Suzhou Weidu Biotechnology Co., Ltd. DR0300CA Colored microspheres Shenzhen Maifu New Material Technology Co., Ltd. DPR300C-COOH Colored microspheres Beijing Chunda Technology Co., Ltd. AJ30COOH-L1-R2

[0033] The test strip provided by the present invention can use urine as the sample type. The urine sample is non-traumatic and non-invasive, is not affected by risk factors related to sample storage, and enables users to complete sample collection and testing at home and obtain test results.

[0034] When the test strip provided by the present invention is tested, if the sample contains beta-amyloid protein, the beta-amyloid protein will bind to the first beta-amyloid protein monoclonal antibody coupled to the colored microspheres in the binding pad to form an "Aβ-Aβ monoclonal antibody-colored microsphere complex". When the complex is chromatographed upward to the detection area, it will bind to the second beta-amyloid protein monoclonal antibody pre-coated in the detection area to form a color line in the detection area, which is judged as positive; otherwise, it is judged as negative. The sheep anti-mouse IgG polyclonal antibody is coated in the nitrocellulose membrane quality control area. Regardless of whether beta-amyloid protein exists in urine, a line will appear in the quality control area as a basis for the effectiveness of the experiment.

[0035] The present invention provides the use of the test strip described in the above technical solution in detecting beta-amyloid protein, and the direct purpose of the application is non-diagnosis and non-treatment. As an embodiment, the sample for detecting beta-amyloid protein can be urine. The direct purpose of the application of the present invention is non-diagnosis and non-treatment, which is to obtain an intermediate value of whether the sample contains beta-amyloid protein.

[0036] In order to further illustrate the present invention, a test strip for detecting β-amyloid protein and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] A test strip for detecting beta-amyloid protein, such as Figure 1 As shown, the test strip includes a base plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a conjugation pad 5, a sample pad 6 and a water absorbent pad 7; the base plate 1 is provided with a sample pad 6, a conjugation pad 5, a nitrocellulose membrane 2 and a water absorbent pad 7 which are overlapped in sequence, and the conjugation pad 5 is coated with a first β-amyloid protein monoclonal antibody coupled with a colored microsphere; a detection line 3 is provided on the side of the nitrocellulose membrane 2 close to the conjugation pad 5, and a control line 4 is provided on the side of the nitrocellulose membrane 2 close to the water absorbent pad 7, the detection line 3 is coated with a second β-amyloid protein monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first β-amyloid protein monoclonal antibody and the second β-amyloid protein monoclonal antibody respectively recognize different antigenic determinant cluster sites of β-amyloid protein.

[0039] The preparation method of the test strip is as follows:

[0040] 1. Soak the glass fiber membrane in the second pretreatment solution for 10 minutes, dry it in a drying oven at 37°C for 22 hours for use, and obtain the sample pad 6; the second pretreatment solution is composed of the following components in 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.

[0041] 2. Pretreatment of glass fiber wool: Cut the glass fiber wool into 9mm wide strips and apply 60μL / cm 2 The pretreated glass fiber membrane was immersed in the first pretreatment solution for 10 minutes and dried in a drying oven for 22 hours to obtain the pretreated glass fiber membrane; the first pretreatment solution consisted of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5wt.%, bovine serum albumin 1wt.% and trehalose 3wt.%; the pH value of the Tris buffer was 7.4.

[0042] 3. The steps of coupling the colored microspheres with the β-amyloid monoclonal antibody are as follows:

[0043] 1) Take 1 mg of colored microspheres (Beijing Chunda Technology Co., Ltd., catalog number AJ30COOH-L1-R2), add 1 ml of MES buffer and centrifuge, discard the supernatant, repeat the above steps and centrifuge once, resuspend the colored microspheres to 5 mg / ml by ultrasonication, add 8 μg of EDC and 16 μg of NHS respectively for activation, and put them into a mixer for rotation mixing at 30 r / min for 30 min;

[0044] 2) Take out the activated microspheres and wash them twice by centrifugation using 1 ml of MES buffer, resuspend them to 5 mg / ml using MES buffer by ultrasound, add 0.5 mg of Aβ monoclonal antibody (Xibao Bio, catalog number EKN0448A) to the microspheres after ultrasound, and put them into a mixer for rotation mixing at 30 r / min for 120 min;

[0045] 3) Add 1% BSA to the microspheres and mix well, then place in a mixer and rotate at 30 rpm for 30 min to obtain blocked microspheres;

[0046] 4) taking out the blocked microspheres, centrifuging, discarding the supernatant, and washing twice with 1 ml of microsphere preservation solution, wherein the microsphere preservation solution is composed of the following components: 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 value of the Tris buffer is 7.4; using the microsphere preservation solution, ultrasonically resuspending the blocked microspheres to 5 mg / ml to obtain a microsphere-coupled antibody suspension;

[0047] 5) The microsphere-coupled antibody suspension is sprayed onto the pretreated glass fiber membrane obtained in step 2 at a coating amount of 6 μL / cm, and then dried at a constant temperature of 37° C. for 18 h to obtain a conjugate pad 5.

[0048] 4. Dilute Aβ monoclonal antibody (Xibao Bio, catalog number EKN0447A) and goat anti-mouse IgG antibody to 2 mg / ml and 1 mg / ml respectively using Tris diluent, use a coating amount of 1 μl / cm to prepare NC membrane, and dry the prepared NC membrane at 37°C for 24 hours to obtain nitrocellulose membrane 2.

[0049] 5. Cut the absorbent paper of 200×300 mm into 15 mm wide strips with a cutting machine, dry them and set them aside to make the absorbent pad 7.

[0050] 6. The prepared sample pad 6, conjugate pad 5, nitrocellulose membrane 2 and absorbent pad 7 are sequentially attached to a 60×300 mm bottom plate 1, overlapping and connecting from left to right, with an overlapping width of 1 to 2 mm, and then cut into a test strip with a width of 4 mm and a length using a cutting machine to obtain the test strip; for ease of use, the test strip is installed in a plastic card shell with a sample addition hole and an observation window to obtain a test paper card.

[0051] Comparative Example 1

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

[0053] Comparative Example 2

[0054] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, bovine serum albumin 1wt.% and trehalose 3wt.%; the pH value of the Tris buffer is 7.4.

[0055] Comparative Example 3

[0056] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, bovine serum albumin 1wt.% and trehalose 5wt.%; the pH value of the Tris buffer is 7.4.

[0057] Comparative Example 4

[0058] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 3wt.%, bovine serum albumin 1wt.% and trehalose 1wt.%; the pH value of the Tris buffer is 7.4.

[0059] Comparative Example 5

[0060] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5wt.%, bovine serum albumin 1wt.% and trehalose 1wt.%; the pH value of the Tris buffer is 7.4.

[0061] Comparative Example 6

[0062] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 7wt.%, bovine serum albumin 1wt.% and trehalose 1wt.%; the pH value of the Tris buffer is 7.4.

[0063] Comparative Example 7

[0064] A test strip similar to Example 1, except that: the first pretreatment solution consists of the following components in the following concentrations: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 1wt.%, casein 0.5wt.% and trehalose 1wt.%; the pH value of the Tris buffer is 7.4.

[0065] Test Example 1

[0066] The sample diluent and Aβ antigen (Xibao Bio, catalog number EDD0720A) were used in the corresponding proportions to prepare detection limit concentrations of 100, 80, and 50 pg / ml, respectively. The test strips prepared in Comparative Examples 1 to 7 were used for sample addition detection according to the detection method. The color depth of the test strips was observed to analyze the detection limit of each formulation. The detection method is as follows:

[0067] The sample diluent formula is: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 0.1wt.%, bovine serum albumin 1wt.%, trehalose 1wt.% and PC300 antibacterial preservative 0.05wt.%;

[0068] Slowly add 100 μL of the sample to be tested dropwise onto the sample pad of the test strip, wait for 15 minutes, and then check the test line and the control line;

[0069] Result evaluation: Negative (-): only the control line is colored. Positive (+): both the test line and the control line are colored. The results are shown in Table 3.

[0070] Table 3 Test results of different test strips

[0071]

[0072]

[0073] Note: The color depth is determined by naked eye observation, where +++: strongly positive; ++: positive; +: weakly positive; + / -: slight band; -: negative, the same applies to the following table.

[0074] The results showed that the optimal Tween 20 content was 5% to 7%, which was equivalent to 3% to 5% trehalose. BSA was more sensitive than casein, so the protein type, surfactant, and sugar content were set to 1% BSA, 5% to 7% Tween 20, and 3% to 5% trehalose, respectively.

[0075] Test Example 2

[0076] Normal human urine samples (negative urine) and Aβ antigen (Xibao Bio, catalog number EDD0720A) were prepared into 1000, 500, 100, 50, 10pg / ml and negative urine 0pg / ml according to the corresponding ratios, and the test strips prepared in Example 1 were used for sample addition detection according to the detection method, and the color depth of the test strips was observed to analyze the detection limit. The results are shown in Table 4.

[0077] Table 4 Detection results of different antigen concentrations

[0078] Antigen concentration (pg / ml) result 1000 +++ 500 +++ 100 ++ 50 + 10 + / - 5 - 0 -

[0079] It can be seen from Table 3 and Table 4 that the test results of the test strips prepared in Comparative Examples 1 to 7 are + / - at a concentration of 80 to 100 pg / ml, and the test results of the test strips prepared in Example 1 are + / - 10 pg / ml, so the sensitivity of the optimized pretreatment formula can be increased by about 8 to 10 times.

[0080] Test Example 3

[0081] 1. Linear correlation: Add Aβ antigen to negative urine, and use negative urine to dilute it to 0.05ng / ml, 0.1ng / ml, 0.5ng / ml, 1ng / ml, 5ng / ml respectively. And use test strips to detect negative urine. The results show that the color of the T line increases proportionally to the detection result when the Aβ urine test is 0-5ng / ml.

[0082] 2. Detection limit test: Use high-value clinical sample 64pg / ml concentration sample, and dilute it to 40, 30, 20, 10, 8, 6, 5pg / ml respectively with negative urine according to the proportion. The test results are compared with Hunan Qiankang Technology Reagent (whose invention patent CN111656197A clearly states that its detection limit is 40pg / ml) and are shown in Table 5.

[0083] Table 5 Test results of different test strips

[0084] concentration Qiankang Colloidal Gold Test Paper Box Example 1 Test Strip 64pg / ml ++ +++ 40pg / ml + +++ 30pg / ml + / - ++ 20pg / ml - ++ 10pg / ml - + / - 8pg / ml - + / - 6pg / ml - + / - 5pg / ml - -

[0085] It can be seen from the data in the above table that the detection limit of the Qiankang colloidal gold test kit is 40pg / ml, and the detection limit of the test kit of the present invention for Aβ antigen is 8pg / ml; the detection limit of the test kit of the present invention is lower.

[0086] 3. Clinical application:

[0087] The kit of the present invention collected 193 clinical samples, including 66 clinically diagnosed Alzheimer's syndrome patients and 127 normal human urine samples. The test results are shown in Tables 6 and 7.

[0088] Table 6 Clinical diagnosis results

[0089]

[0090] Table 7 Sensitivity and specificity results

[0091] Group Example 1 Test Strip Sensitivity (N=66) 93.85％ Specificity (N=127) 96.92％

[0092] In the above experiment, samples of patients with Alzheimer's syndrome that showed weak positive results when detected by the kit of the present invention were selected and tested using the Qiankang colloidal gold test paper box, and the results were all negative. The experiment proved that the Qiankang colloidal gold test paper box had insufficient sensitivity when detecting weak positive samples, resulting in failure to detect them.

[0093] Comparative Example 8 Color Microsphere Screening Experiment

[0094] A test strip similar to Example 1, except that the colored microspheres in step 3 are replaced by the microspheres in Table 8, respectively, to prepare different test strips.

[0095] Table 8 Sources and particle sizes of different microspheres

[0096] Test strips Microspheres factory Part Number Particle size B1 Carboxyl colored microspheres Suzhou DR0200CA 200nm B2 Carboxyl colored microspheres Suzhou DR0300CA 300nm B3 Carboxyl colored microspheres Suzhou DR0400CA 400nm B4 Carboxyl colored microspheres Suzhou DB0200CA 200nm B5 Carboxyl colored microspheres Suzhou DB0300CA 300nm B6 Carboxyl colored microspheres Suzhou DB0400CA 400nm B7 Red microspheres Beijing Chunda Technology AJ30COOH-L1-B2 300nm B8 (i.e. embodiment 1) Colored microspheres Beijing Chunda Technology AJ30COOH-L1-R2 300nm B9 Colored microspheres Shenzhen Maifu New Materials Technology Co., Ltd. DPR300C-COOH 300nm B10 Colored microspheres Shenzhen Maifu New Materials Technology Co., Ltd. DPR200C-COOH 200nm B11 Colored microspheres Shenzhen Maifu New Materials Technology Co., Ltd. DPR400C-COOH 400nm

[0097] Sample diluent and Aβ antigen (Xibao Bio, catalog number EDD0720A) were used in corresponding proportions to prepare detection limit concentrations of 100, 80, 50, 10 pg / ml and sample diluent 0 pg / ml, respectively. The sample diluent formula was the same as that of Test Example 1.

[0098] Using the test strips in Table 8, the sample addition test was performed according to the detection method of Test Example 1, and the detection limit and specificity of each formulation were analyzed by observing the color depth of the test strips. The test results are shown in Table 9.

[0099] Table 9 Test results of different test strips

[0100] Test strips 500pg / ml 100pg / ml 50pg / ml 10pg / ml Sample diluent B1 +++ ++ + / - + / - - B2 +++ ++ + + / - - B3 +++ ++ + + / - + / - B4 + + / - - - - B5 + + / - - - - B6 ++ + + / - - - B7 ++ + + - - B8 +++ ++ + + / - - B9 +++ ++ + + / - - B10 +++ ++ + / - + / - - B11 +++ ++ + / - + / - + / -

[0101] The results showed that the best test strips were B2, B8 and B9 according to the antigen detection limit and specificity of the reagents, and their colored microspheres were DR0300CA, AJ30COOH-L1-R2 and DPR300C-COOH respectively.

[0102] Microsphere particle size analysis: From the analysis of the test results of B1, B10, B3, and B11 test strips, it can be seen that the B3 and B11 test strips both use 400nm microspheres, and it is found that lines appear in the sample dilution, resulting in a high background value, so the 400nm particle size is not suitable for test strip preparation. B1 and B10 test strips both use 200nm microspheres, and the detection limit is lower than 300nm, and the detection limit is about 50pg / ml.

[0103] From the test results of B4, B5, B6 and B7, it can be seen that the sensitivity of the reagent is affected by the color depth and particle size of the microspheres themselves, and the color depth is positively correlated with the sensitivity. The larger the particle size of the microspheres, the higher the sensitivity. The selection of microsphere raw materials should be screened according to product requirements.

[0104] Comparative Example 9 Paired Antibody Screening Experiment

[0105] A test strip similar to Example 1, except that the first β-amyloid protein monoclonal antibody and the second β-amyloid protein monoclonal antibody used are different, and the paired antibodies are shown in Table 10.

[0106] Table 10 Different antibodies and their sources

[0107]

[0108] Note: The first monoclonal antibody in the table is the first β-amyloid protein monoclonal antibody, and the second monoclonal antibody is the second β-amyloid protein monoclonal antibody.

[0109] Ten normal controls and ten clinically confirmed Alzheimer's disease patients (AD) collected from the hospital were tested using the C1 to C10 test strips in Table 10, and the optimal paired antibodies were screened based on the detection sensitivity and specificity. The test results are shown in Table 11.

[0110] Table 11 Test results of different test strips

[0111]

[0112]

[0113] It can be seen from the results that the detection results of different paired antibodies are used to compare the effects of distinguishing AD from normal. The paired antibodies preferably selected from the pairings of the first β-amyloid monoclonal antibody and the second β-amyloid monoclonal antibody are EKN0448A and EKN0447A, A09N03-S5 and A09N04-A6, A09N03-T3 and A09N04-A6, Aβ1-42002 and Aβ1-42001, K70013C06H01C and K92011M16F04C.

[0114] Experiments have shown that different β-amyloid monoclonal antibodies have different effects after pairing; due to the different characteristics of the antibodies themselves, the amino acid sequences and spatial structures of the antigen binding sites of monoclonal antibodies from different manufacturers are different, which makes their affinity with the antigen different. Therefore, the affinity and antibody activity of paired antibodies from different manufacturers used in the test strip preparation process described in the present invention also show different performance results. The use of monoclonal antibodies must meet the conditions of using clinical diagnosis of AD as a control, and the sensitivity and normal people must reach more than 80% to achieve the detection effect of the present invention.

[0115] In summary, the test strip provided by the present invention has the advantages of simple operation, rapid and accurate detection, high sensitivity, low cost and good stability. It can detect urine samples and realize self-testing at home. It is a test strip for batch detection of human β-amyloid protein.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A test strip for detecting β-amyloid protein, the test strip comprising a base plate (1), a nitrocellulose membrane (2), a detection line (3), a control line (4), a conjugate pad (5), a sample pad (6) and a water absorbent pad (7); the base plate (1) is provided with a sample pad (6), a conjugate pad (5), a nitrocellulose membrane (2) and a water absorbent pad (7) which are overlapped in sequence, characterized in that: The conjugate pad (5) is coated with a first β-amyloid monoclonal antibody coupled to a colored microsphere; a detection line (3) is provided on the side of the nitrocellulose membrane (2) close to the conjugate pad (5), and a control line (4) is provided on the side of the nitrocellulose membrane (2) close to the water absorbent pad (7); the detection 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 cluster 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 liquid and then coated with the first β-amyloid protein monoclonal antibody coupled to the colored microspheres, wherein the first pretreatment liquid comprises: Tris buffer 0.02M, PVP 0.5wt.%, Tween 20 5-7wt.%, bovine serum albumin 1wt.% and trehalose 3-5wt.%; the pH value 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 liquid is 55-65 μL / cm 2 The coating amount of the first β-amyloid monoclonal antibody coupled to the colored microspheres is 3 to 6 μL / cm.

4. The test strip according to any one of claims 1 to 3, characterized in that: The preparation method of the first β-amyloid protein monoclonal antibody coupled to the colored microspheres comprises: activating the colored microspheres by using EDC and NHS, mixing and coupling the activated colored microspheres and the first β-amyloid protein monoclonal antibody, and blocking by using bovine serum albumin to obtain the first β-amyloid protein monoclonal antibody coupled to the colored microspheres.

5. The test strip according to claim 4, 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.

6. The test strip according to claim 4, characterized in that: The rotation speed of the mixed coupling is 30 r / min and the time is 120 min.

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

8. The test strip according to claim 1, characterized in that: The sample pad (2) is coated with a second pretreatment liquid, which comprises 0.02M Tris buffer, 2wt.% bovine serum albumin, 1wt.% sucrose, 1wt.% Tween 20, 1wt.% surfactant S17, and 0.05wt.% PC300 antibacterial preservative; the concentration of the second β-amyloid protein monoclonal antibody is 2mg / mL, and the concentration of the IgG polyclonal antibody is 1mg / mL.

9. The test strip according to claim 8, characterized in that: The coating amount of the second pretreatment liquid is 55-65 μL / cm 2 ; The coating amount of the second β-amyloid protein monoclonal antibody is 1.0 to 2.0 μL / cm; The coating amount of the IgG polyclonal antibody is 1.0 to 2.0 μL / cm.

10. Use of the test strip according to any one of claims 1 to 9 in detecting β-amyloid protein, wherein the direct purpose of the use is non-diagnosis and non-treatment.

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