An immunochromatographic kit for combined detection of Aβ1-42, NfL, and UCH-L1

Abstract

The present invention provides a rapid, intuitive, simple and low-cost immunochromatographic kit for the combined detection of Aβ1‑42, NfL and UCH‑L1. The kit comprises an immunochromatographic test strip, the test strip comprises a sample pad, a conjugate pad and a coating membrane, and the conjugate pad comprises a microsphere-labeled Aβ1‑42, NfL and UCH‑L1 antibody conjugate. The three indicators of Aβ1‑42, NfL and UCH‑L1 are combined for detection to increase the accuracy of early diagnosis of Alzheimer's disease or brain health diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to an immunochromatographic kit for the combined detection of Aβ1-42, NfL, and UCH-L1 and its application in the early diagnosis of Alzheimer's disease or brain health. Background Art

[0002] Senile plaques are one of the most prominent pathological features in the brains of patients with Alzheimer's disease (AD). They are structures in which insoluble proteins abnormally aggregate to form amyloid fibers with rich β-sheet structures and accumulate in the intercellular spaces of the brain parenchyma. The latest research has found that the detection of Aβ1-42 levels in the plasma of AD patients has the same significance as the detection of Aβ1-42 in cerebrospinal fluid (CSF). The plasma Aβ42 levels in AD patients or mild cognitive impairment (MCI) patients who are positive for amyloid protein are also reduced. This non-invasive test is more easily accepted by patients and easier to promote in clinical practice.

[0003] UCH-L1 (Ubiquitin C-terminal Hydrolase-L1) is a neuron-specific protease. It plays an important role in the metabolism and cell death of neurons. Recent studies have shown that UCH-L1 is of great significance in the diagnosis and treatment of Alzheimer's disease (AD).

[0004] NfL (Neurofilament Light) is a marker of axonal injury. Neurofilaments (NFs) are one of the structural proteins unique to neurons, and are composed of five subunits: neurofilament light chain (NfL), neurofilament medium chain (NFM), neurofilament heavy chain (NFH), α-filament annexin (INA) and peripherin. Neurofilaments play an important role in the growth of axons and dendrites, nerve conduction and synaptic transmission, thereby maintaining a stable speed of nerve signal transduction. Compared with neurofilament medium chain and neurofilament heavy chain, neurofilament light chain can achieve self-assembly, and its abnormal transport and assembly can affect the function of neuronal axons, and neuronal axoplasmic transport dysfunction is one of the main pathological manifestations of neurodegenerative diseases. After axonal injury or neuronal degeneration, NfL is released into the intercellular space, and the expression of NfL in Alzheimer's disease is significantly higher than that in normal groups. The level of NfL can not only reflect the degree of neuronal damage in the course of Alzheimer's disease, but also reflect the disease process and clinical treatment effect.

[0005] The existing technology for diagnosing Alzheimer's disease mainly relies on blood or cerebrospinal fluid, which is traumatic to the body. The present invention conducts urine testing and adopts the combined detection of three indicators, Aβ1-42, NfL, and UCH-L1, to increase the accuracy of Alzheimer's disease diagnosis.

[0006] Immunochromatography is based on the principle of antigen-antibody reaction. It detects the target substance through the specific binding of the antibody to the target analyte. Immunochromatography usually uses a test strip or a test card to fix the antibody on a solid phase carrier, such as a nitrocellulose membrane or a microplate. When the target analyte in the sample binds to the antibody on the solid phase carrier, the color development or signal generation of the marker (such as colloidal gold) can be used to visually observe and judge the test results. However, many complexes in biological fluids may affect the test results, and in order to detect changes in biomarkers as early as possible, a highly sensitive detection method is required. Summary of the invention

[0007] The purpose of the present invention is to provide a rapid, intuitive, simple and low-cost immunochromatographic kit for the combined detection of Aβ1-42, NfL and UCH-L1.

[0008] The invention provides an immunochromatographic kit for combined detection of Aβ1-42, NfL and UCH-L1, comprising an immunochromatographic test strip, wherein the immunochromatographic test strip comprises a sample pad, a conjugation pad and a coating membrane, wherein the conjugation pad comprises an Aβ1-42, NfL and UCH-L1 antibody conjugate labeled with microspheres, wherein the sample pad is treated with a treatment solution, wherein the sample pad treatment solution is a PBS buffer solution containing 0.5%-1.0% (w / v%) BSA, 0.4%-0.8% (w / v%) Tween-20, 0.1%-0.5% (w / v%) trehalose and 0.02%-0.1% (w / v%) calcium chloride; wherein the conjugation pad is treated with an immersion solution, wherein the immersion solution is a PBS buffer solution containing 0.8%-1.2% (w / v%) BSA, 1.5%-2% (w / v%) sucrose and 0.1%-0.2% (w / v%) calcium chloride. (w / v%) Tween-20, 0.2%-0.5% (w / v%) MES in PBS buffer.

[0009] The inventors of the present application have found that after adding BSA, Tween-20, trehalose and calcium chloride to the treatment solution to treat the sample pad, the antigen detection of Aβ1-42, NfL and UCH-L1 is more effective, and the interference of the detection background signal can be effectively reduced, thereby improving the sensitivity of the three detections. After the binding pad is treated with the soaking solution, the stability of the detection kit is improved. In particular, MES is added to the soaking solution, and the dispersion stability of the particles is ensured by using MES, which effectively prevents the coagulation of the particles from affecting the detection signal, thereby ensuring the accuracy of the detection results.

[0010] In some embodiments, the concentration of the PBS buffer of the sample pad treatment solution is 10 mM, and the pH is 7.0-7.5. Preferably, the pH is preferably 7.2.

[0011] In some embodiments, the sample pad treatment solution is a PBS buffer containing 0.5% (w / v%) BSA, 0.5% (w / v%) Tween-20, 0.2% (w / v%) trehalose, and 0.05% (w / v%) calcium chloride.

[0012] In some embodiments, the soaking solution is a PBS buffer containing 1% (w / v%) BSA, 2% (w / v%) sucrose, 0.1% (w / v%) Tween-20, and 0.3% (w / v%) MES.

[0013] In some embodiments, the PBS buffer of the conjugate pad soaking solution has a concentration of 10 mM and a pH of 7.0-7.5. Preferably, the pH is preferably 7.2.

[0014] In some embodiments, the conjugate pad further comprises a microsphere-labeled quality control antibody conjugate, wherein the microsphere-labeled quality control antibody conjugate is selected from microsphere-labeled rabbit IgG antibody, chicken IGY antibody or mouse IgG antibody. Preferably, the microsphere-labeled quality control antibody conjugate is selected from microsphere-labeled rabbit IgG antibody conjugate.

[0015] In some embodiments, the coating membrane is a nitrocellulose membrane.

[0016] In some embodiments, the coating membrane includes Aβ1-42, NfL, UCH-L1 antibodies for detection and quality control antibodies for detection.

[0017] In some embodiments, the Aβ1-42, NfL, and UCH-L1 antibodies for detection specifically bind to the Aβ1-42, NfL, and UCH-L1 antibody conjugates labeled with microspheres, respectively. The Aβ1-42, NfL, and UCH-L1 antibodies for detection are anti-Aβ1-42 antibodies, anti-NfL antibodies, and anti-UCH-L1 antibodies.

[0018] In some embodiments, the detection quality control antibody is selected from goat anti-rabbit IgG antibody, goat anti-chicken IGY antibody, goat anti-mouse IgG antibody. Preferably, the detection quality control antibody is selected from goat anti-rabbit IgG antibody.

[0019] In some embodiments, the microspheres are colored latex microspheres, colloidal gold, colloidal carbon, gold nanorods, magnetic nanoparticles or fluorescent microspheres. Preferably, the microspheres are selected from colored latex microspheres.

[0020] In some embodiments, the particle size of the microsphere is 100 to 500 nm. Preferably, the particle size of the microsphere is 200 to 300 nm. Further, the microsphere is selected from 100 nm latex microspheres and 300 nm microspheres, and the weight ratio of the two is 3:1 to 5:1 (preferably 4:1). By selecting microspheres of different particle sizes to prepare Aβ1-42, NfL, and UCH-L1 microsphere conjugates, the technical problem that microspheres with small particle sizes can detect high concentrations of antigens but cannot be detected at low concentrations, and microspheres with large particle sizes can detect low concentrations of antigens but cannot be detected at high concentrations is effectively solved. The technical solution provided by the present invention selects two particle size microspheres to simultaneously detect low and high concentrations of antigens, and the detection range can reach a minimum of 0.1 ng / mL and a maximum of 1000 ng / mL.

[0021] Furthermore, preferably, the microspheres are aminodextran coated microspheres. Compared with uncoated microspheres, aminodextran treated microsphere surfaces can effectively eliminate nonspecific binding, increase the signal-to-noise ratio of the detection signal, and improve stability.

[0022] In some embodiments, the microsphere-labeled Aβ1-42, NFL, and UCH-L1 antibody conjugates are dissolved in a PBS buffer containing 0.5% (w / v%) BSA and 0.5% (w / v%) Tween-20 to form a microsphere mixed solution with a concentration of 0.01% to 0.2% (w / v%). Preferably, a microsphere mixed solution with a concentration of 0.1% (w / v%) is formed. The inventors found during the experiment that the sensitivity was not achieved below 0.01% (w / v%); the microsphere release effect was poor and the precision was poor when the concentration was higher than 0.2% (w / v%).

[0023] Further, the microsphere mixed solution is sprayed on the conjugate pad and dried. Preferably, the spraying amount is 30-50 μL / cm. More preferably, the spraying amount is 40 μL / cm.

[0024] In some embodiments, the drying temperature is 35-40° C., preferably, the drying temperature is 37° C., and the drying time is 16 to 24 hours.

[0025] In some embodiments, the coating membrane is coated with three detection lines of Aβ1-42, NfL, and UCH-L1 antibodies; or the coating membrane is coated with any one or two detection lines of Aβ1-42, NfL, and UCH-L1 antibodies, and the coating membranes including different detection lines are installed together to achieve combined detection.

[0026] In some embodiments, the preparation steps of the microsphere-labeled (eg, Aβ1-42, NFL, UCH-L1, rabbit IgG antibody) antibody conjugate include: reacting the microspheres with the above antibodies, blocking and then sonicating.

[0027] In some embodiments, the steps of preparing the microsphere-labeled (eg, Aβ1-42, NfL, UCH-L1) antibody conjugate include:

[0028] The microspheres were dissolved in MES buffer solution, centrifuged, and the supernatant was removed;

[0029] Add MES buffer solution, add NHS and / or EDC activator, react, centrifuge, and remove the supernatant;

[0030] Dissolve in PBS buffer, add antibody to react, centrifuge, and remove supernatant;

[0031] Add blocking reagent, perform blocking treatment, centrifuge, and remove the supernatant;

[0032] A resolubilization reagent is added for resolubilization to obtain a microsphere-labeled antibody conjugate.

[0033] In some embodiments, in step 2), the activator is selected from NHS and / or EDC; the final concentration of NHS is 0.02-0.1 mg / mL, preferably the final concentration of NHS is 0.05 mg / mL; the final concentration of EDC is 0.02-0.1 mg / mL, preferably the final concentration of EDC is 0.05 mg / mL.

[0034] In some embodiments, in step 2), the microspheres react with the activator in the dark for 20-60 min, preferably 30 min.

[0035] In some embodiments, in step 3), the microspheres are reacted with the antibody for 2-5 hours, preferably 3 hours.

[0036] In some embodiments, in step 4), the blocking reagent is a 10 mM PBS buffer with a pH of 7.2 containing 0.5% (w / v%) glycine. The inventors of the present application also tried a PBS buffer without 0.5% (w / v%) glycine in the blocking reagent during the experiment. In general, adding a PBS solution containing glycine to terminate the activation reaction effectively avoids the occurrence of nonspecific reactions and ensures the stability and activity of Aβ1-42, NfL, and UCH-L1 antibodies.

[0037] In some embodiments, in step 5), the reconstitution reagent is a PBS buffer containing 0.5% BSA. The pH of the PBS buffer is 7-7.5, preferably 7.2. The inventors of the present application have also tried to use different ratios of BSA in the reconstitution reagent during the experiment, wherein the content of BSA is too high, such as 2%, and the signal value is generally not as good as the effect of low concentration.

[0038] Preferably, in step 2), after adding the MES buffer solution, aminodextran is added first, and then the activator of NHS and / or EDC is added, reacted, centrifuged, and the supernatant is removed. The aminodextran-coated microspheres can effectively eliminate nonspecific binding, improve the signal-to-noise ratio of the detection signal, and improve stability.

[0039] In some embodiments, the steps of preparing the microsphere-labeled (eg, Aβ1-42, NfL, UCH-L1) antibody conjugate include:

[0040] 1) Take 0.1 mL of microspheres with a solid content of 0.1%-20% (W / V), add 0.9 mL of 25 mM MES buffer solution with a pH of 6.0, vortex mix, centrifuge at 10,000-12,000 rpm for 30 minutes, and remove the supernatant;

[0041] 2) Add 1 mL of 25 mM MES buffer solution with a pH of 6.0 to step 1), mix by ultrasonication at 100 W, add EDC and NHS to a final concentration of 0.05 mg / mL; vortex mix, react for 30 minutes in the dark; centrifuge at 12000-15000 rpm for 30 minutes, and remove the supernatant;

[0042] 3) Add 1 mL of 10 mM PBS buffer with a pH of 7.2 to step 2), mix by ultrasonication at 100W, then add 0.1 mg of antibody, vortex mix, let react for 3 hours, centrifuge the tube at 10,000-12,000 rpm for 30 minutes, and remove the supernatant;

[0043] 4) Add 1 mL of 10 mM PBS with a pH of 7.2 and 0.5% (w / v%) glycine to the mixture in step 3); vortex to mix, and allow to react for 30 min; centrifuge at 12,000-15,000 rpm for 30 min;

[0044] 5) Resuspend the microspheres in 1 mL of 10 mM PBS buffer (pH 7.2) containing 0.5% BSA to obtain the microsphere-labeled antibody conjugate.

[0045] On the other hand, the present invention also provides the use of the above immunochromatographic kit in detecting antigens in samples, wherein the antigens are preferably proteins, such as Aβ1-42, NFL and UCH-L1. The samples are human blood, urine, cerebrospinal fluid, feces, sputum or tissue fluid.

[0046] Beneficial effects:

[0047] (1) The immunochromatographic kit of the present invention can rapidly detect multiple antigens simultaneously within a short detection time. It not only has high detection sensitivity, but also can realize one-step sample loading operation, greatly improving the precision and accuracy of the operation, reducing errors caused by human operation, making the operation simpler and conducive to industrial production.

[0048] (2) The present invention combines the detection of Aβ1-42, NfL, and UCH-L1. By pre-treating the sample pad and the binding pad, the mutual interference between the three components is reduced, the detection background signal is low, the sensitivity is high, and the stability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the structure of the immunochromatographic test strip for combined detection of Aβ1-42, NfL, and UCH-L1 of the present invention;

[0050] Figure 2 This is a schematic diagram of another bottom plate of the immunochromatographic test strip for combined detection of Aβ1-42, NfL, and UCH-L1 of the present invention.

[0051] Reference numerals:

[0052] 1. Sample pad; 2. Binding pad; 3. Bottom plate; 4. Aβ1-42 detection line; 5. NfL detection line; 6. UCH-L1 detection line; 7. Quality control line (C line); 8. Coating membrane; 9. Absorbent paper. DETAILED DESCRIPTION

[0053] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0055] Unless otherwise specified, % in this article refers to mass volume percentage.

[0056] Abbreviations:

[0057] MES: 2-(N-morpholino)ethanesulfonic acid; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; BSA: bovine serum albumin.

[0058] Example 1 Immunochromatographic Kit for Combined Detection of Aβ1-42, NfL, and UCH-L1

[0059] (1) Preparation of latex microsphere-labeled antibody conjugates

[0060] The commercially available Aβ1-42, NFL, and UCH-L1 monoclonal antibodies 2 were labeled with colored latex microspheres to prepare Aβ1-42 monoclonal antibody 2, NFL monoclonal antibody 2, and UCH-L1 monoclonal antibody 2 latex immune microspheres (T-line immune latex microspheres); rabbit IgG was labeled with latex microspheres to prepare rabbit IgG latex microspheres (used as C-line, i.e., quality control line). The specific operations are as follows:

[0061] Take 0.1 mL of latex microspheres with a diameter of 200-300 nm and a solid content of 0.1%-20% (W / V), add 0.9 mL of 25 mM MES buffer solution with a pH of 6.0, vortex mix, centrifuge at 10000-12000 rpm for 30 minutes, and remove the supernatant;

[0062] Add 1 mL of 25 mM MES buffer solution with a pH of 6.0 to the centrifuge tube in the previous step, mix by ultrasonication at 100 W, add EDC and NHS with a concentration of 0.05 mg / mL; vortex mix, react for 30 minutes in the dark; centrifuge at 12000-15000 rpm for 30 minutes, and remove the supernatant;

[0063] Add 1 mL of 10 mM PBS buffer at pH 7.2 to the centrifuge tube in the previous step, mix by ultrasonication at 100W, then add 0.1 mg of Aβ1-42 monoclonal antibody 2, vortex mix, and let stand for reaction for 3 hours; centrifuge at 10000-12000 rpm for 30 minutes, remove the supernatant, add 10 mM PBS at pH 7.2 containing 0.5% (w / v%) glycine; vortex mix, let stand for reaction for 30 minutes; centrifuge at 12000-15000 rpm for 30 minutes;

[0064] The microspheres were resuspended in 1 mL of 10 mM PBS buffer (pH 7.2) containing 0.5% BSA to obtain Aβ1-42 immune latex microspheres.

[0065] The above preparation process was used to replace 0.1 mg of Aβ1-42 monoclonal antibody 2 with 0.1 mg of NFL monoclonal antibody 2, 0.1 mg of UCH-L1 monoclonal antibody 2, and 0.1 mg of rabbit IgG to prepare NFL latex microspheres, UCH-L1 latex microspheres, and rabbit IgG latex microspheres, respectively.

[0066] The Aβ1-42, NFL, UCH-L1, and rabbit IgG latex microspheres prepared above were diluted with 10 mM PBS buffer (pH 7.2) containing 0.5% (w / v%) BSA and 0.5% (w / v%) Tween-20 to prepare a latex mixed microsphere solution with a concentration of 0.1% (w / v%).

[0067] (2) Preparation of conjugate pad

[0068] The conjugate pad soaking solution was prepared as follows: 10 mM PBS buffer, pH 7.2, containing 1% (w / v%) BSA, 2% (w / v%) sucrose, 0.1% (w / v%) Tween-20, and 0.3% (w / v%) MES.

[0069] The glass fiber pad was immersed in the soaking solution in advance, and the soaking treatment was carried out at 200 rpm for 30 minutes, and then dried; the mixed solution of Aβ1-42, NFL, UCH-L1, and rabbit IgG latex microspheres prepared in step (1) was sprayed on the soaked glass fiber pad at a spraying amount of 40 μL / cm, and dried in an oven at 37° C. for 16 to 24 hours to prepare a conjugate pad containing four kinds of immune latex microspheres.

[0070] (3) Preparation of coating membrane

[0071] The commercially available Aβ1-42 monoclonal antibody 1, NFL monoclonal antibody 1, UCH-L1 monoclonal antibody 1 and goat anti-rabbit IgG monoclonal antibody were diluted to 1.0 mg / mL with 10 mM PBS buffer at pH 7.2, sprayed on nitrocellulose membrane as the test line (T line) and quality control line (C line), respectively, and placed in a 37°C oven for drying for 16 to 24 hours to prepare a coating membrane. Figure 1 As shown, the Aβ1-42 detection line (4), NFL detection line (5), UCH-L1 detection line (6), and quality control line (7) can be sprayed on the coating film in sequence.

[0072] Or, if Figure 2 As shown, a coating membrane containing an Aβ1-42 detection line (4) and a quality control line (7), a coating membrane containing an NFL detection line (5) and a quality control line (7), and a coating membrane containing an UCH-L1 detection line (6) and a quality control line (7) are prepared respectively, and then the coating membranes including different detection lines are installed together to achieve combined detection.

[0073] (4) Preparation of sample pad

[0074] Prepare the sample pad treatment solution: 10 mM PBS solution (pH 7.2), the PBS solution contains 0.5% (w / v%) BSA, 0.5% (w / v%) Tween-20, 0.2% (w / v%) trehalose, and 0.05% (w / v%) calcium chloride.

[0075] The glass fiber pad was immersed in the sample pad treatment solution at 200 rpm for 30 min, and then dried in a 37° C. oven for 16 to 24 hours to obtain a treated sample pad.

[0076] (5) Assembly of latex immunoassay strips

[0077] like Figure 1 As shown, the coating membrane (8) is first fixed to the bottom plate (3) by an overlapping method, and then the sample pad (1) and the binding pad (2) are fixed to one side of the coating membrane (8) in sequence, and the absorbent pad (9) is fixed to the other side of the coating membrane (8); specifically, the sample pad (1) presses the binding pad (2) by 1-2 mm, the binding pad (2) presses the coating membrane (8) by 1-2 mm, and the absorbent pad (9) presses the coating membrane by 1-2 mm; then, it is cut into a test strip with a width of 4 mm, that is, a latex immunochromatographic test strip for the combined detection of Aβ1-42 / NFL / UCH-L1.

[0078] (6) Card Group

[0079] The test strip in step (5) is placed into the corresponding cartridge to form a detection kit.

[0080] Example 2 Immunochromatographic Kit for Combined Detection of Aβ1-42, NfL, and UCH-L1

[0081] The same as Example 1, except for the preparation of latex microsphere-labeled antibody conjugate in step (1). In this example, the surface of the immune latex microspheres is modified (aminoglycan-coated latex microspheres), and the preparation process is as follows:

[0082] Take 0.1 mL of latex microspheres with a diameter of 200-300 nm and a solid content of 0.1%-20% (W / V), add 0.9 mL of 25 mM MES buffer solution with a pH of 6.0, vortex mix, centrifuge at 10000-12000 rpm for 30 minutes, and remove the supernatant;

[0083] Add 1 mL of 25 mM MES buffer solution with a pH of 6.0 to the centrifuge tube in the previous step, mix with ultrasound at 100w, add 1 mL of 10% aminodextran, and add EDC and NHS with a concentration of 0.05 mg / mL; vortex mix, and react for 30 minutes in the dark; centrifuge at 12000-15000 rpm for 30 minutes, and remove the supernatant;

[0084] Add 1 mL of 10 mM PBS buffer at pH 7.2 to the centrifuge tube in the previous step, mix by ultrasonication at 100W, then add 0.1 mg of Aβ1-42 monoclonal antibody 2, vortex mix, and let stand for reaction for 3 hours; centrifuge at 10000-12000 rpm for 30 minutes, remove the supernatant, add 10 mM PBS at pH 7.2 containing 0.5% (w / v%) glycine; vortex mix, let stand for reaction for 30 minutes; centrifuge at 12000-15000 rpm for 30 minutes;

[0085] The microspheres were resuspended in 1 mL of 10 mM PBS buffer (pH 7.2) containing 0.5% BSA to obtain Aβ1-42 immune latex microspheres.

[0086] The above preparation process was used to replace 0.1 mg of Aβ1-42 monoclonal antibody 2 with 0.1 mg of NFL monoclonal antibody 2, 0.1 mg of UCH-L1 monoclonal antibody 2, and 0.1 mg of rabbit IgG to prepare NFL latex microspheres, UCH-L1 latex microspheres, and rabbit IgG latex microspheres, respectively.

[0087] The Aβ1-42, NFL, UCH-L1, and rabbit IgG latex microspheres prepared above were diluted with 10 mM PBS buffer (pH 7.2) containing 0.5% (w / v%) BSA and 0.5% (w / v%) Tween-20 to prepare a latex mixed microsphere solution with a concentration of 0.1% (w / v%).

[0088] Test Example 1 Determination of samples

[0089] When the sample (such as urine) contains antigens such as Aβ1-42, NFL, and UCH-L1, when the sample solution passes through the binding pad, the corresponding immune latex microspheres in the binding pad are re-dissolved and specifically bind to the specific antibodies on the immune latex microspheres (such as Aβ1-42 monoclonal antibody 2). When the sample solution continues to flow forward, it will be captured by the corresponding capture monoclonal antibody 1 (such as Aβ1-42 monoclonal antibody 1, i.e., anti-Aβ1-42 monoclonal antibody) on the T line to form a latex microsphere antibody 2-antigen-antibody 1 sandwich complex, and a red stripe will appear in the detection area (T); when it flows to the quality control area (C), the rabbit IgG antibody labeled with the latex microspheres binds to the goat anti-rabbit IgG antibody in the quality control area, and a red stripe will appear in the quality control area.

[0090] Test Example 2 Detection Limit Detection

[0091] Different concentrations of Aβ1-42, NFL, and UCH-L1 were prepared using negative urine samples:

[0092] Aβ1-42: 0 ng / mL; 0.5 ng / mL; 5 ng / mL;

[0093] NFL: 0 ng / mL; 0.5 ng / mL; 5 ng / mL;

[0094] UCH-L1: 0 ng / mL; 0.5 ng / mL; 5 ng / mL;

[0095] 75 μL of sample was added to the sample pad of the kit of Example 1, and 10 replicates were set for each concentration. The results are shown in Table 1:

[0096] Table 1 Detection limit test results

[0097] Test items Test results 0 ng / mL Aβ1-42 All 10 tests are: - / - / - 0.5 ng / mL Aβ1-42 All 10 tests were: + / - / - 5 ng / mL Aβ1-42 All 10 tests were: + / - / - 0 ng / mL NFL All 10 tests are: - / - / - 0.5 ng / mL NFL All 10 tests are: - / + / - 5 ng / mL NFL All 10 tests are: - / + / - 0 ng / mL UCH-L1 All 10 tests are: - / - / - 0.5 ng / mL UCH-L1 All 10 tests are: - / - / + 5 ng / mL UCH-L1 All 10 tests are: - / - / +

[0098] Note: - indicates negative result, + indicates positive result

[0099] From the above structure, it can be seen that the antigen detection limit of the Aβ1-42, NFL, and UCH-L1 combined detection kit of the present invention is no higher than 0.5 ng / mL.

[0100] Test Example 3 Effect of sample pad treatment solution on the performance of the detection kit

[0101] According to the method of use of the above detection kit, the sample pad treatment solution was optimized, and other reagents and conditions were the same as in Example 1.

[0102] Use negative urine samples to prepare Aβ1-42, NFL, and UCH-L1 at a concentration of 0.5 ng / mL: Add 75 μL of sample to the sample pad of the above kit, set 10 replicates for each concentration, and the results are as follows:

[0103] Table 2 Test results of the effect of sample pad treatment solution on the performance of the test kit

[0104]

[0105] Note: - indicates negative result, + indicates positive result

[0106] Test Example 4 Effect of the Conjugate Pad Soaking Solution on the Stability of the Detection Kit

[0107] According to the method of use of the above detection kit, the conjugate pad soaking solution was optimized, and other reagents and conditions were the same as in Example 1. The test results after being placed at 45° C. for 7 days were tested to evaluate the stability.

[0108] Use negative urine samples to prepare Aβ1-42, NFL, and UCH-L1 at a concentration of 0.5 ng / mL: Add 75 μL of sample to the sample pad of the above kit, set 10 replicates for each concentration, and the results are as follows:

[0109] Table 3 Test results of the stability of the detection kit by the pad soaking solution

[0110]

[0111] Note: - indicates negative result, + indicates positive result

[0112] It can be seen from the data in the above table that by optimizing the conjugate pad soaking solution, the kit of the present invention including the Aβ1-42, NFL, and UCH-L1 triple detection test strips has good stability.

[0113] Test Example 5: Clinical application of the Aβ1-42, NFL, and UCH-L1 combined detection kit of the present invention

[0114] Urine samples from 129 healthy people under the age of 40 were tested, and more than 90% of the healthy people had negative results. 124 positive samples from people diagnosed with Alzheimer's disease or cognitive impairment were tested, and more than 80% of the results were positive. The test results of the three items were separately or combined and statistically analyzed as follows:

[0115] Table 4 Aβ1-42 detection results

[0116]

[0117] Among them, the test results of Aβ1-42 were: positive compliance rate: 63.7%, negative compliance rate: 98.4%, and total compliance rate: 81.4%.

[0118] Table 5 NFL test results

[0119]

[0120] Among them, the NFL's test results were: positive compliance rate: 59.7%, negative compliance rate: 99.2%, and total compliance rate: 79.8%.

[0121] Table 6 UCH-L1 test results

[0122]

[0123] Among them, the test results of UCH-L1 were: positive compliance rate: 67.7%, negative compliance rate: 98.4%, and total compliance rate: 83.4%.

[0124] Table 7 Results of combined detection of Aβ1-42, NFL and UCH-L1

[0125]

[0126] Among them, the combined detection results of Aβ1-42, NFL, and UCH-L1 were: positive compliance rate: 88.7%, negative compliance rate: 96.1%, and total compliance rate: 92.5%.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. An immunochromatographic kit for the combined detection of Aβ1-42, NfL, and UCH-L1, characterized in that: The kit comprises an immunochromatographic test strip, which comprises a sample pad, a conjugation pad and a coating membrane, wherein the conjugation pad comprises Aβ1-42, NfL and UCH-L1 antibody conjugates labeled with microspheres, the sample pad is treated with a treatment solution, wherein the sample pad treatment solution is a PBS buffer solution containing 0.5%-1.0% BSA by weight volume, 0.4%-0.8% Tween-20 by weight volume, 0.1%-0.5% trehalose by weight volume and 0.02%-0.1% calcium chloride by weight volume; the conjugation pad is treated with an immersion solution, wherein the immersion solution is a PBS buffer solution containing 0.8%-1.2% BSA by weight volume, 1.5%-2% sucrose by weight volume, 0.1%-0.2% Tween-20 by weight volume and 0.2%-0.5% MES by weight volume.

2. The immunochromatographic kit for combined detection of Aβ1-42, NfL and UCH-L1 according to claim 1, characterized in that: The treatment solution is a PBS buffer solution containing 0.5% BSA by volume, 0.5% Tween-20 by volume, 0.2% trehalose by volume, and 0.05% calcium chloride by volume.

3. An immunochromatographic kit for combined detection of Aβ1-42, NfL, and UCH-L1 according to claim 1 or 2, characterized in that: The concentration of the PBS buffer in the treatment solution is 10 mM, and the pH is 7.0-7.

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4. The immunochromatographic kit for combined detection of Aβ1-42, NfL and UCH-L1 according to claim 1, characterized in that: The soaking solution is a PBS buffer solution containing 1% BSA by volume, 2% sucrose by volume, 0.1% Tween-20 by volume, and 0.3% MES by volume.

5. An immunochromatographic kit for combined detection of Aβ1-42, NfL, and UCH-L1 according to claim 1 or 4, characterized in that: The PBS buffer of the soaking solution has a concentration of 10 mM and a pH of 7.0-7.

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6. The immunochromatographic kit for combined detection of Aβ1-42, NfL and UCH-L1 according to claim 1, characterized in that: The coating membrane is a nitrocellulose membrane.

7. The immunochromatographic kit for combined detection of Aβ1-42, NfL and UCH-L1 according to claim 1, characterized in that: The conjugate pad also contains a microsphere-labeled quality control antibody conjugate, and the microsphere-labeled quality control antibody conjugate is selected from microsphere-labeled rabbit IgG antibody, chicken IGY antibody or mouse IgG antibody.

8. The immunochromatographic kit for combined detection of Aβ1-42, NfL, and UCH-L1 according to claim 1, characterized in that: The microspheres are colored latex microspheres, colloidal gold, colloidal carbon, gold nanorods, magnetic nanoparticles or fluorescent microspheres.

9. The immunochromatographic kit for the combined detection of Aβ1-42, NfL, and UCH-L1 according to claim 1, characterized in that: The coating membrane is coated with three detection lines of Aβ1-42, NfL, and UCH-L1 antibodies; or the coating membrane is coated with any one or two detection lines of Aβ1-42, NfL, and UCH-L1 antibodies, and the coating membranes including different detection lines are installed together to realize joint detection.

10. The immunochromatographic kit for combined detection of Aβ1-42, NfL, and UCH-L1 according to claim 1, characterized in that: The preparation steps of the microsphere-labeled Aβ1-42, NfL, and UCH-L1 antibody conjugates include: 1) Dissolve the microspheres in MES buffer solution, centrifuge, and remove the supernatant; 2) Add MES buffer solution, add NHS and / or EDC activator, react, centrifuge, and remove the supernatant; 3) Dissolve in PBS buffer, add antibody to react, centrifuge, and remove the supernatant; 4) Add blocking reagent, perform blocking treatment, centrifuge, and remove the supernatant; 5) Add the reconstitution reagent to reconstitute and obtain the microsphere-labeled antibody conjugate.

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