A test strip for detecting beta-amyloid and use thereof

Abstract

The present application relates to the field of immunochromatography, in particular to a test strip for detecting beta-amyloid and application thereof. The present application adopts color microspheres which are combined with beta-amyloid monoclonal antibodies by covalent bond and are coated on a combination pad. Compared with colloidal gold, the color microspheres are more stable and have higher sensitivity. The color microspheres have bright and rich colors, uniform particle size, good monodispersity and strong repeatability of detection results. The test strip provided by the present application has the advantages of simplicity, rapidness and strong timeliness. Without any other reagents, instruments and professional personnel, the test strip can be operated on site. As long as the sample to be detected is added to the sample adding port of the test strip, the detection result can be determined within 15 minutes. The result determination is visual, intuitive, accurate, simple and clear, and is not easy to appear false positive and false negative and other human misjudgments.

Description

TECHNICAL FIELD

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

[0002] Studies have shown that patients with mild cognitive impairment (MCI) can reverse to normal cognitive state, so early identification and intervention at the MCI stage is an effective strategy for preventing and treating Alzheimer's disease (AD), and early screening and diagnosis of AD in clinical practice can help to exclude other types of dementia and improve the accuracy of AD diagnosis. Currently, the main markers for diagnosis are cerebrospinal fluid and blood samples, both of which are invasive sampling, and are not very feasible and 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 the changes in the production, solubility and clearance of Aβ in the brain. This method as a diagnostic and monitoring tool helps to monitor and evaluate AD and MCI patients during disease-modifying treatment.

[0004] Currently, the detection of Aβ in urine generally uses colloidal gold for biological labeling, and the antigen / antibody of colloidal gold immunochromatography is easy to detach from the surface of gold particles, resulting in unstable markers, large batch-to-batch differences, and the problem that the color of the detection line and the quality control line will change over time due to oxidation, so the detection has the problems of timeliness and relatively poor sensitivity, and it is difficult to meet the detection needs for AD projects that require high sensitivity. SUMMARY

[0005] To solve the above problems, the present application provides a test strip for detecting beta-amyloid and application thereof. The test strip provided by the present application has the advantages of simple operation, rapid and accurate detection, high sensitivity, low cost and good stability, can detect urine samples, realizes home self-testing, and is a test strip for batch detection of human beta-amyloid.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] The application provides a test strip for detecting beta-amyloid protein, which comprises a base plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a binding pad 5, a sample pad 6 and a water absorption pad 7; the sample pad 6, the binding pad 5, the nitrocellulose membrane 2 and the water absorption pad 7 are sequentially overlapped on the base plate 1, the binding pad 5 is coated with a first beta-amyloid protein monoclonal antibody coupled with color microspheres, the detection line 3 is arranged on one side of the nitrocellulose membrane 2 close to the binding pad 5, the control line 4 is arranged on the other side of the nitrocellulose membrane 2 close to the water absorption 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 sites of beta-amyloid protein.

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

[0009] Preferably, the coating amount of the first pretreatment liquid is 55-65μL / cm 2 ; and the coating amount of the first beta-amyloid protein monoclonal antibody coupled with color microspheres is 3-6μL / cm.

[0010] Preferably, the preparation method of the first beta-amyloid protein monoclonal antibody coupled with color microspheres comprises the following steps: activating color microspheres by using EDC and NHS, mixing and coupling the activated color microspheres and the first beta-amyloid protein monoclonal antibody, and blocking by using bovine serum albumin to obtain the first beta-amyloid protein monoclonal antibody coupled with color microspheres.

[0011] Preferably, the mass ratio of the color microspheres, EDC and NHS is 1000:8:16, the rotation speed of the activation is 30r / min, and the activation time is 30min.

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

[0013] Preferably, the first beta-amyloid protein monoclonal antibody comprises beta amyloid 1-42 or beta amyloid 1-40, and the second beta-amyloid protein monoclonal antibody comprises beta amyloid 1-42 or beta amyloid 1-40.

[0014] Preferably, the sample pad 2 is coated with a second pretreatment liquid, the second pretreatment liquid comprising Tris buffer 0.02M, bovine serum albumin 2wt.%, sucrose 1wt.%, Tween 20 1wt.%, surfactant S171wt.% and PC300 bacteriostatic preservative 0.05wt.%; the concentration of the second β-amyloid 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 55-65μL / cm 2 ; the coating amount of the second β-amyloid monoclonal antibody is 1.0-2.0μL / cm; and the coating amount of the IgG polyclonal antibody is 1.0-2.0μL / cm.

[0016] The application provides application of the test strip in detection of β-amyloid protein, and a direct purpose of the application is non-diagnosis and non-treatment.

[0017] Beneficial effects:

[0018] The application provides a test strip for detecting beta-amyloid, which comprises a base plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a binding pad 5, a sample pad 6 and a water absorption pad 7; the sample pad 6, the binding pad 5, the nitrocellulose membrane 2 and the water absorption pad 7 are sequentially overlapped on the base plate 1, the binding pad 5 is coated with a first beta-amyloid monoclonal antibody coupled with color microspheres, the detection line 3 is arranged on one side of the nitrocellulose membrane 2 close to the binding pad 5, the control line 4 is arranged on the other side of the nitrocellulose membrane 2 close to the water absorption pad 7, the detection line 3 is coated with a second beta-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first beta-amyloid monoclonal antibody and the second beta-amyloid monoclonal antibody respectively recognize different antigenic determinant sites of beta-amyloid. The color microspheres are coated on the binding pad by being combined with the beta-amyloid monoclonal antibody through a covalent bond, are more stable and have higher sensitivity compared with colloidal gold, and the color microspheres have bright and rich colors, uniform particle size, good monodispersity and strong repeatability of detection results. The test strip has the advantages of simplicity, rapidness and strong timeliness, can be operated on site without any other reagents, instruments and professional personnel, can determine the detection results within 15 minutes as long as the sample to be detected is added at a sample adding port of the test strip, and has the advantages of visual, intuitive, accurate, simple and clear results and low possibility of false positive and false negative and the like. In addition, the test strip has a wide application range and can meet the needs of different levels of personnel, including professional inspection, mass population screening and home self-testing of non-professional personnel, is convenient for popularization and application, has a broad market prospect and obvious economic and social benefits. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0020] Figure 1 Fig. 1 is a structural schematic diagram of the test strip for detecting beta-amyloid of the application; 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 absorption pad. DETAILED DESCRIPTION

[0021] The application provides a test strip for detecting beta-amyloid, which comprises a base plate 1, a nitrocellulose membrane 2, a detection line 3, a control line 4, a binding pad 5, a sample pad 6 and a water absorption pad 7; the sample pad 6, the binding pad 5, the nitrocellulose membrane 2 and the water absorption pad 7 are sequentially overlapped on the base plate 1, the binding pad 5 is coated with a first beta-amyloid monoclonal antibody coupled with color microspheres, the detection line 3 is arranged on one side of the nitrocellulose membrane 2 close to the binding pad 5, the control line 4 is arranged on the other side of the nitrocellulose membrane 2 close to the water absorption pad 7, the detection line 3 is coated with a second beta-amyloid monoclonal antibody, and the control line 4 is coated with an IgG polyclonal antibody; the first beta-amyloid monoclonal antibody and the second beta-amyloid monoclonal antibody respectively recognize different antigenic determinant sites of beta-amyloid.

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

[0023] As an implementation form, the material of the sample pad 6 can be a glass fiber membrane. As an implementation form, the sample pad 6 is coated with a second pretreatment liquid, and the second pretreatment liquid comprises Tris buffer 0.02M, bovine serum albumin 2wt.%, sucrose 1wt.%, Tween 20 1wt.%, surfactant S171wt.% and PC300 bacteriostatic preservative 0.05wt.%. As an implementation form, the coating amount of the second pretreatment liquid is 55-65μL / cm 2 .

[0024] As an implementation form, the material of the binding pad 5 can be a glass fiber membrane. As an implementation form, the binding pad 5 is coated with a first pretreatment liquid and then coated with the first beta-amyloid monoclonal antibody coupled with color microspheres, and 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. As an implementation form, the coating amount of the first pretreatment liquid is 55-65μL / cm 2; and the first β-amyloid monoclonal antibody coupled with the color microspheres has a coating amount of 3-6 μL / cm. As an embodiment, the first β-amyloid monoclonal antibody includes an Aβ monoclonal antibody, β-amyloid 1-42 or β-amyloid 1-40. As another embodiment, the Aβ monoclonal antibody, β-amyloid 1-42 or β-amyloid 1-40 can be one or more of those in Table 1. As another embodiment, the first β-amyloid monoclonal antibody includes an 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 Manufacturer Catalogue number Aβ monoclonal antibody Beijing Aiboseng Biotechnology Co., Ltd. K70013C06H01C Aβ1-42 Beijing Aiboseng Biotechnology Co., Ltd. K92011M16F04C Aβ1-40 antibody Xibao Biotech (Shanghai) Co., Ltd. EKN0448A Aβ1-40 antibody Xibao Biotech (Shanghai) Co., Ltd. EKN0447A Aβ1-40 Beijing Bainixin Biotechnology Co., Ltd. A09N03-S5 Aβ1-40 Beijing Bainixin Biotechnology Co., Ltd. A09N03-T3 Aβ monoclonal antibody Beijing Bainixin Biotechnology Co., Ltd. A09N04-A6 Aβ1-42 antibody Chengdu Baozero Biotechnology Co., Ltd. Aβ1-42002 Aβ1-42 antibody Chengdu Baozero Biotechnology Co., Ltd. Aβ1-42001

[0027] As an embodiment, the method for preparing the first β-amyloid monoclonal antibody coupled with the color microspheres includes: activating the color microspheres by using EDC and NHS, mixing and coupling the activated color microspheres and the first β-amyloid monoclonal antibody, and blocking by using bovine serum albumin to obtain the first β-amyloid monoclonal antibody coupled with the color microspheres. As an embodiment, the mass ratio of the color microspheres, EDC and NHS is 1000:8:16; the rotation speed of the activation is 30 r / min, and the time is 30 min. As an embodiment, the rotation speed of the mixing and coupling is 30 r / min, and the time is 120 min.

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

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

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

[0031] Table 2 Source of color microspheres

[0032] Microspheres Manufacturer Catalogue number Carboxyl color microspheres Suzhou Weidu Biotechnology Co., Ltd. DR0300CA Color microspheres Shenzhen Mai Fu New Material Technology Co., Ltd. DPR300C-COOH Color microspheres Beijing Chundate Technology Co., Ltd. AJ30COOH-L1-R2

[0033] The test strip provided by the present application can use urine as a sample type, and the urine sample is non-invasive and non-invasive, is not affected by risk factors related to sample storage, and can achieve that the user can collect and detect the sample at home and obtain the detection result.

[0034] When the test strip provided by the present application is detected, if the β-amyloid protein is contained in the sample to be detected, the β-amyloid protein is combined with the first β-amyloid monoclonal antibody coupled with the color microspheres in the combination pad to form an "Aβ-Aβ monoclonal antibody-color microsphere complex", and when the complex is chromatographed upward to the detection area, it will be combined with the second β-amyloid monoclonal antibody coated in the detection area, and a color line is formed at the detection area, which is judged as positive; otherwise, it is judged as negative. The goat anti-mouse IgG polyclonal antibody is coated in the nitrocellulose membrane quality control area, and whether the β-amyloid protein exists in the urine or not, a line will appear in the quality control area, which serves as the basis for the effectiveness of the experiment.

[0035] The present application 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-diagnostic and non-therapeutic. As an embodiment, the sample for detecting β-amyloid protein can be urine. The direct purpose of the use described in the present application is non-diagnostic and non-therapeutic, which is to obtain the intermediate value of whether the sample contains β-amyloid protein.

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

[0037] Example 1

[0038] A test strip for detecting β-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 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 detection line 3 on the side near the conjugate pad 5 and a control line 4 on the side near the 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 sites of β-amyloid protein.

[0039] The test strip is prepared as follows:

[0040] 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.

[0041] 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.

[0042] 3. The conjugation of colored microspheres with β-amyloid monoclonal antibody is performed as follows:

[0043] 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.

[0044] 2) Take out the activated microspheres and wash twice with 1 ml of MES buffer solution, resuspend to 5 mg / ml with the MES buffer solution, add 0.5 mg of Aβ monoclonal antibody (Xibao Biology, item number EKN0448A) to the microspheres after ultrasonic, put into a vortex mixer and rotate at 30 r / min for 120 min;

[0045] 3) Add 1% BSA to the microspheres and mix, put into a vortex mixer and rotate at 30 r / min for 30 min to obtain the blocked microspheres;

[0046] 4) Take out the blocked microspheres, centrifuge, discard the supernatant, wash twice with 1 ml of microsphere preservation solution, which is composed of the following components at the following concentrations: Tris buffer solution 0.02 M, Tween 20 0.1 wt.%, casein 0.5 wt.%, trehalose 5 wt.% and PC300 bacteriostatic preservative 0.05 wt.%; the pH value of the Tris buffer solution is 7.4; resuspend the blocked microspheres to 5 mg / ml with the microsphere preservation solution to obtain a microsphere-antibody suspension;

[0047] 5) Inject the microsphere-antibody suspension onto the pretreated glass fiber membrane obtained in step 2, with a coating amount of 6 μL / cm, and dry at 37°C for 18 h to obtain a binding pad 5.

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

[0049] 5, Cut a 200x300mm size water absorption paper into 15mm wide strips with a cutting machine, dry for standby, which is a water absorption pad 7.

[0050] 6, Attach the prepared sample pad 6, binding pad 5, nitrocellulose membrane 2 and water absorption pad 7 to a 60x300mm base plate 1 in order from left to right, overlap and connect with a stacking width of 1-2mm, then cut into 4mm wide test strips to obtain the test strips; for easy use, the test strips are loaded into a plastic card shell with a sample 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 fluid consists of the following concentrations of components: Tris buffer 0.02 M, PVP 0.5 wt.%, Tween 20 1 wt.%, bovine serum albumin 1 wt.% and trehalose 1 wt.%; the Tris buffer has a pH of 7.4.

[0053] Comparative Example 2

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

[0055] Comparative Example 3

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

[0057] Comparative Example 4

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

[0059] Comparative Example 5

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

[0061] Comparative Example 6

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

[0065] Test Example 1

[0066] Using the sample diluent and the Aβ antigen (Xibao Biology, item number EDD0720A) to prepare the detection limit concentrations of 100, 80, 50 pg / ml respectively according to the corresponding proportion, using the test strips prepared in Comparative Examples 1-7, according to the detection method, sample detection was carried out, and the detection limit of each formula was analyzed by observing the color depth of the test strip. The detection method is as follows:

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

[0068] Slowly add 100 μL of the sample to be tested drop by drop on the sample pad of the test strip, and after standing for 15 min, check the detection line and the control line;

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

[0070] Table 3 Detection results of different test strips

[0071]

[0072]

[0073] Note: The color depth is determined by visual observation, in which +++ represents strong positive, ++ represents positive, + represents weak positive, + / - represents slight band, and - represents negative. The same applies to the following table.

[0074] The results show that the best Tween 20 content is 5%-7%, which is equivalent to 3%-5% trehalose. The sensitivity of BSA is better than that of casein, so the protein type, surfactant and sugar content are set to 1% BSA, 5%-7% Tween 20 and 3%-5% trehalose, respectively.

[0075] Test Example 2

[0076] Using normal human urine samples (negative urine) and Aβ antigen (Xibao Biology, item number EDD0720A) to prepare 1000, 500, 100, 50, 10 pg / ml and negative urine 0 pg / ml respectively according to the corresponding proportion, using the test strip prepared in Example 1, the sample was added and detected according to the detection method, and the detection limit was analyzed by observing the color depth of the test strip. The results are shown in Table 4.

[0077] Table 4 Detection results of different antigen concentrations

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

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

[0080] Test Example 3

[0081] 1. Linear correlation: Add Aβ antigen to negative urine, and use negative urine to dilute it to 0.05 ng / ml, 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 5 ng / ml, and negative urine to detect using the test strip. The results show that the T line color of Aβ urine detection at 0-5 ng / ml presents a positive incremental detection result.

[0082] 2. Detection limit detection: Using a high-value clinical sample of 64 pg / ml concentration, dilute it to 40, 30, 20, 10, 8, 6, 5 pg / ml according to the proportion using negative urine. Compared with Hunan Qiankang Technology reagent (its detection limit is 40 pg / ml in the invention patent CN111656197A), the detection results are shown in Table 5.

[0083] Table 5 Detection results of different test strips

[0084] Concentration Kang Colloidal Gold Test Kit Example 1 test strip 64 pg / ml ++ +++ 40 pg / ml + +++ 30 pg / ml + / - ++ 20 pg / ml - ++ 10 pg / ml - + / - 8 pg / ml - + / - 6 pg / ml - + / - 5 pg / ml - -

[0085] From the above data, it can be seen that the detection limit of Qiankang colloidal gold kit is 40 pg / ml, and the detection limit of the kit of the present application is 8 pg / ml; the detection limit of the kit of the present application is lower.

[0086] 3. Clinical application:

[0087] The kit of the present application collected 193 clinical samples, including 66 cases of clinically diagnosed Alzheimer's syndrome patients and 127 cases of normal urine, and the detection results are shown in Table 6 and Table 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-mentioned test, the sample of Alzheimer's disease patient showing weak positive was selected for detection by using the Jiankang colloidal gold test strip kit, and the result was negative. The experiment proved that the sensitivity of Jiankang colloidal gold test strip kit was insufficient when detecting weak positive sample, which led to the failure of detection.

[0093] Comparative Example 8 Color microsphere screening experiment

[0094] A test strip similar to Example 1, the difference is that the color microspheres in step 3 are replaced by the microspheres in Table 8 respectively, and different test strips are prepared.

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

[0096] Test strip Microspheres Manufacturer Catalogue number Particle size B1 Carboxyl color microspheres Suzhou Weidu DR0200CA 200 nm B2 Carboxyl color microspheres Suzhou Weidu DR0300CA 300 nm B3 Carboxyl color microspheres Suzhou Weidu DR0400CA 400 nm B4 Carboxyl color microspheres Suzhou Weidu DB0200CA 200 nm B5 Carboxyl color microspheres Suzhou Weidu DB0300CA 300 nm B6 Carboxyl color microspheres Suzhou Weidu DB0400CA 400 nm B7 Red microspheres Beijing Chundate Technology AJ30COOH-L1-B2 300 nm B8 (i.e., Example 1) Color microspheres Beijing Chundate Technology AJ30COOH-L1-R2 300 nm B9 Color microspheres Shenzhen Mai Fu New Material Technology DPR300C-COOH 300 nm B10 Color microspheres Shenzhen Mai Fu New Material Technology DPR200C-COOH 200 nm B11 Color microspheres Shenzhen Mai Fu New Material Technology DPR400C-COOH 400 nm

[0097] Using sample diluent and Aβ antigen (Xibao Biology, item number EDD0720A), prepare the detection limit concentration of 100, 80, 50, 10 pg / ml and sample diluent 0 pg / ml according to the corresponding proportion, and the sample diluent formula is the same as that of Test Example 1.

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

[0099] Table 9 Detection results of different test strips

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

[0101] The results show that according to the antigen detection limit and specificity analysis of the reagent, the best test strips are B2, B8 and B9. The color microspheres are DR0300CA, AJ30COOH-L1-R2 and DPR300C-COOH respectively.

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

[0103] From the detection results of B4, B5, B6 and B7, it can be seen that the reagent sensitivity is influenced by the color depth of the microspheres and the particle size of the microspheres, 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 the microsphere raw material should be based on the product requirements.

[0104] Screening experiment of paired antibodies of Comparative Example 9

[0105] A test strip similar to 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.

[0106] Table 10 Different antibodies and sources

[0107]

[0108] 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.

[0109] Ten normal controls collected from hospitals, and ten clinically diagnosed Alzheimer's syndrome patients (AD) were detected using C1-C10 test strips in Table 10, and the optimal paired antibodies were screened according to the detection sensitivity and specificity. The detection results are shown in Table 11.

[0110] Table 11 Detection results of different test strips

[0111]

[0112]

[0113] From the results, it can be seen that the detection results of different paired antibodies are used to compare the effects of distinguishing AD and normal, and the preferred paired antibodies 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 show that different paired β-amyloid monoclonal antibodies have different effects; due to the different characteristics of the antibodies, the amino acid sequences and spatial structures of the antigen binding sites of the monoclonal antibodies of different manufacturers are different, which makes their affinity to the antigen different. Therefore, the affinity of the antibodies to the antigen and the activity of the antibodies also show different results when the antibodies of different manufacturers are applied to the preparation process of the test strip described in the application. The monoclonal antibodies used need to meet the conditions that the sensitivity and the normal person both reach more than 80% for the clinical diagnosis of AD, so as to achieve the detection effect of the application.

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

[0116] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which all belong to the protection scope of the application.

Claims

1. A test strip for detecting β-amyloid, 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 absorption pad (7); the sample pad (6), the conjugate pad (5), the nitrocellulose membrane (2) and the water absorption pad (7) are sequentially overlapped on the base plate (1), characterized in that, The first β-amyloid monoclonal antibody coupled with the color microspheres is coated on the binding pad (5); a detection line (3) is arranged on one side of the nitrocellulose membrane (2) close to the binding pad (5), and a control line (4) is arranged on the other side of the nitrocellulose membrane (2) close to the water absorption pad (7); the second β-amyloid monoclonal antibody is coated on the detection line (3), and the 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; the first β-amyloid monoclonal antibody is β-amyloid 1-40 antibody EKN0448A, and the second β-amyloid monoclonal antibody is β-amyloid 1-40 antibody EKN0447A; both the β-amyloid 1-40 antibody EKN0448A and the β-amyloid 1-40 antibody EKN0447A are from Shanghai Xibao Biotechnology Co., Ltd.; The first pretreatment liquid is coated on the binding pad (5) first, and then the first β-amyloid monoclonal antibody coupled with the color microspheres is coated; 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; the second pretreatment liquid is coated on the sample pad (2), and the second pretreatment liquid comprises Tris buffer 0.02M, bovine serum albumin 2wt.%, sucrose 1wt.%, Tween 20 1wt.%, surfactant S17 1wt.% and PC300 bacteriostatic preservative 0.05wt.%; the concentration of the second β-amyloid monoclonal antibody is 2mg / mL, and the concentration of the IgG polyclonal antibody is 1mg / mL; The particle size of the color microspheres is 300nm.

2. The test strip of claim 1, wherein, 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 with color microspheres is 3-6 μL / cm.

3. The test strip according to claim 1 or 2, characterized in that The preparation method of the first β-amyloid monoclonal antibody coupled with the color microspheres comprises the following steps: activating the color microspheres by using EDC and NHS, mixing and coupling the activated color microspheres and the first β-amyloid monoclonal antibody, and then blocking by using bovine serum albumin to obtain the first β-amyloid monoclonal antibody coupled with the color microspheres.

4. The test strip of claim 3, wherein, The mass ratio of the color microspheres, EDC and NHS is 1000:8:16; the rotation speed of the activation is 30r / min, and the time is 30min.

5. The test strip of claim 3, wherein, The rotation speed of the mixing and coupling is 30r / min, and the time is 120min.

6. The test strip of claim 5, wherein, The coating amount of the second pretreatment liquid is 55-65 μL / cm 2 ; the coating amount of the second β-amyloid monoclonal antibody is 1.0-2.0 μL / cm; and the coating amount of the IgG polyclonal antibody is 1.0-2.0 μL / cm.

7. The application of the test strip in any one of claims 1-6 in the detection of β-amyloid for non-diagnostic purposes.

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