Alzheimer's disease marker detection kit and application thereof

Abstract

The invention relates to an Alzheimer's disease marker detection kit and application thereof. The kit comprises a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease markers, the first reagent comprises microspheres coupled with a first antibody and a first buffer solution, and the first antibody comprises a LINGO1 antibody and / or an OMGP antibody; the second reagent comprises microspheres coupled with a second antibody and a second buffer solution, and the second antibody comprises a pTau217 antibody and / or a pTau181 antibody. The kit provided by the invention can highly enrich brain-derived exosomes, and can detect Alzheimer's disease markers (a Tau217 antibody and / or a pTau181 antibody) with high sensitivity and high specificity so as to assist in diagnosing AD (Alzheimer's disease).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnostic detection, in particular to an Alzheimer's disease marker detection kit and application thereof. BACKGROUND

[0002] Alzheimer disease (AD) is a degenerative disease of the central nervous system. Traditional AD diagnosis schemes are costly, highly dependent on professionals, and even have certain invasiveness, which are difficult to promote on a large scale in clinical practice. Blood sample collection has the characteristics of microtrauma, rapidness, and economy, and some blood biomarkers have changed significantly before the appearance of AD clinical symptoms, which is an ideal means for screening early AD patients. Limited by the blood-brain barrier, the abundance of related markers in plasma samples is significantly lower than that in cerebrospinal fluid, and there are many peripheral blood metabolic interferents, resulting in inaccurate detection results.

[0003] Exosomes are a kind of nanoscale extracellular vesicle structure for information communication and exchange between cells, and the components and contents of various bioactive molecules carried by the exosomes vary according to the origin cells and receptor cells. With the deepening of the research on neurodegenerative diseases, researchers have found that exosomes secreted by all cells in the brain, including neurons, microglia and astrocytes, play a key role in cell communication and are involved in the whole process of AD development.

[0004] Exosomes can carry related markers to cross the blood-brain barrier in both directions, and the surface components do not change, but the biological sources are extensive, and there are exosomes expressed by multiple organs in blood samples. If not distinguished, the signals of those markers with high specificity but low abundance will be submerged in the complex background signals, which is very unfavorable for the detection of the markers. In order to enrich exosomes from specific cells, researchers often use immunoaffinity capture technology for exosome purification. The traditional immunoaffinity capture technology is highly dependent on the antibodies used. If the antibodies are not properly selected, on the one hand, it will introduce bias and cause the target captured exosomes to account for a small proportion, and on the other hand, it will affect the immunoaffinity capture efficiency, resulting in a small number of target exosomes captured, which cannot meet the detection needs of low-abundance markers such as pTau217. SUMMARY

[0005] In view of the defects of the prior art mentioned above, the present application provides an Alzheimer's disease marker detection kit containing a first reagent capable of highly enriching brain-derived exosomes in blood and a second reagent for detecting the concentration of Alzheimer's disease markers (Tau217 antibody and / or pTau181 antibody) carried by the exosomes, which has the advantages of high sensitivity, high specificity, high accuracy, safety and non-invasiveness.

[0006] To solve the above technical problems, the application adopts the following technical solutions: In one aspect, the application provides an Alzheimer's disease marker detection kit, which comprises a first reagent for enriching brain-derived exosomes, and a second reagent for detecting the concentration of an Alzheimer's disease marker; The first reagent comprises microspheres coupled with a first antibody and a first buffer, and the first antibody comprises a LINGO1 antibody and / or an OMGP antibody. The second reagent comprises microspheres coupled with a second antibody and a second buffer, and the second antibody comprises a pTau217 antibody and / or a pTau181 antibody.

[0007] Preferably, the first antibody consists of a LINGO1 antibody and an OMGP antibody.

[0008] Preferably, the molar ratio of the LINGO1 antibody and the OMGP antibody is 0.1-10:1.

[0009] Preferably, the preparation method of the first reagent comprises the following steps: (1) coupling activated bare microspheres with a first antibody in the presence of a coupling buffer; (2) mixing the microspheres coupled with the first antibody obtained in step (1) with a first blocking solution; (3) mixing the blocked microspheres obtained in step (2) with a first buffer; In step (2), the first blocking solution comprises 0.3-0.6wt% casein, 5-15wt% BSA, 1-5vol% Tween 20, 0.05-0.5vol% P300, and 50-150mM PBS.

[0010] Preferably, the first blocking solution comprises 0.3-0.6wt% casein, 8-12wt% BSA, 1-3vol% Tween 20, 0.05-0.3vol% P300, and 80-120mM PBS.

[0011] Preferably, the pH of the first blocking solution is 6-7.0.

[0012] Preferably, the first blocking solution comprises 0.5wt% casein, 10wt% BSA, 2vol% Tween 20, 0.1vol% P300, and 100mM PBS, and the pH of the first blocking solution is 6.5.

[0013] Preferably, the first buffer solution comprises 0.1-2 wt% NaCl, 0.5-5 wt% BSA, 0.05-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 50-150 mM PBS.

[0014] Preferably, the second buffer solution comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0015] Preferably, the bare microspheres are modified with one or more of carboxyl, amino, tosyl, epoxy, streptavidin and protein A / G groups.

[0016] Preferably, the detection sample type of the kit is derived from one or more of blood, body fluid, saliva or urine.

[0017] In one aspect, the present application provides use of any of the above kits in the preparation of an Alzheimer's disease marker detection product.

[0018] Effects of the application The Alzheimer's disease marker detection kit provided by the present application can specifically capture brain-derived exosomes and quantitatively analyze the Alzheimer's disease markers (pTau181 and pTau217) thereof, and has high specificity and high sensitivity. Using the kit of the present application can exclude the influence of plasma free pTau181 and pTau217 and pTau181 and pTau217 in exosomes from other organs on detection, thereby improving the ability to identify and judge the risk of patients suffering from AD. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Schematic diagram of particle size and concentration of exosomes captured by Co-Capture antibody exosome capture reagent.

[0020] Figure 2 Morphology diagram of exosomes captured by Co-Capture antibody exosome capture reagent observed by transmission electron microscope.

[0021] Figure 3 WB gel diagram of exosome membrane surface markers captured by different antibody exosome capture reagents.

[0022] Figure 4 ROC curve of pTau181 protein level in clinical detection samples identified by the kit provided by the present application for AD.

[0023] Figure 5 The ROC curve for identifying AD by using the kit provided by the present application to identify the pTau271 protein level of the clinical detection sample.

[0024] Figure 6 The ROC curve for identifying AD by using the kit provided by the present application to identify the pTau181 combined with pTau271 protein level of the clinical detection sample. DETAILED DESCRIPTION

[0025] In order to make the technical solutions and beneficial effects of the present application more obvious and easy to understand, the following will be described in detail by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the present application belongs.

[0026] pTau217 exists in large quantities in the cerebrospinal fluid sample of AD patients, but due to the influence of the blood-brain barrier, pTau217 cannot directly pass through the blood-brain barrier in large quantities into the blood sample, resulting in that the direct content of pTau217 in the blood sample is extremely low, and the conventional detection method cannot achieve effective detection effect. Exosomes can bidirectionally cross the blood-brain barrier (BBB), and the surface components do not change. Therefore, surface markers can be used to capture exosomes of neural cell origin, identify their cell origin, and further detect the level of AD markers in the identified brain-derived exosomes to improve the accuracy of detecting AD.

[0027] In view of this, the present inventors provide a kit for detecting brain-derived exosomes in blood to assist in the diagnosis of Alzheimer's disease through extensive and in-depth research and a large number of experiments, specifically selecting LINGO1 and / or OMGP specific antibody combination to construct Co-Capture antibody combination, capturing brain-derived exosomes (neural cell and oligodendrocyte-derived exosomes), further detecting the level of Tau protein-related markers (including pTau217 and / or pTau181) in the exosomes, and then identifying and judging the risk of patients suffering from AD, based on which, the present application is completed.

[0028] In one aspect, the present application provides an Alzheimer's disease marker detection kit, which comprises a first reagent for enriching brain-derived exosomes, and a second reagent for detecting the concentration of Alzheimer's disease markers. The first reagent comprises microspheres coupled with a first antibody and a first buffer, and the first antibody comprises a LINGO1 antibody and / or an OMGP antibody. The second reagent includes microspheres coupled with a second antibody and a second buffer, the second antibody includes a pTau217 antibody and / or a pTau181 antibody.

[0029] In some embodiments, the first antibody consists of a LINGOl antibody and an OMGP antibody.

[0030] In some embodiments, the molar ratio of the LINGOl antibody and the OMGP antibody is 0.1-10:1, for example, 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1.

[0031] In some embodiments, the molar ratio of the LINGOl antibody and the OMGP antibody is 0.5-5:1.

[0032] In some embodiments, the molar ratio of the LINGOl antibody and the OMGP antibody is 0.5-3:1.

[0033] In some embodiments, the preparation method of the first reagent includes the following steps: (1) coupling activated bare microspheres with a first antibody in the presence of a coupling buffer; (2) mixing the microspheres coupled with the first antibody obtained in step (1) with a first blocking solution; (3) mixing the blocked microspheres obtained in step (2) with a first buffer; In step (2), the first blocking solution includes 0.3-0.6wt% casein, 5-15wt% BSA, 1-5vol% Tween 20, 0.05-0.5vol% P300, 50-150mM PBS.

[0034] In some embodiments, the content of casein in the first blocking solution is 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%.

[0035] In some embodiments, the content of BSA in the first blocking solution is 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%.

[0036] In some embodiments, the content of Tween 20 in the first blocking solution is 1vol%, 1.5vol%, 2vol%, 2.5vol%, 3vol%, 3.5vol%, 4vol%, 4.5vol%, 5vol%.

[0037] In some embodiments, the first sealing solution comprises 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.15 vol%, 0.20 vol%, 0.25 vol%, 0.3 vol%, 0.35 vol%, 0.4 vol%, 0.45 vol%, 0.5 vol% P300.

[0038] In some embodiments, the first sealing solution comprises 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM PBS.

[0039] In some embodiments, the first sealing solution comprises 0.3-0.6 wt% casein, 8-12 wt% BSA, 1-3 vol% Tween 20, 0.05-0.3 vol% P300, 80-120 mM PBS.

[0040] In some embodiments, the first sealing solution comprises 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20, 0.1 vol% P300, 100 mM PBS.

[0041] In some embodiments, the first sealing solution has a pH of 6.0-7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.

[0042] In some embodiments, the first sealing solution has a pH of 6.5.

[0043] In some embodiments, the first buffer solution comprises 0.1-2 wt% NaCl, 0.5-5 wt% BSA, 0.05-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 50-150 mM PBS.

[0044] In some embodiments, the first buffer solution comprises 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt% NaCl.

[0045] In some embodiments, the concentration of BSA in the first buffer is 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%.

[0046] In some embodiments, the concentration of Tween 20 in the first buffer is 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.12 vol%, 0.14 vol%, 0.16 vol%, 0.18 vol%, 0.2 vol%, 0.25 vol%, 0.3 vol%, 0.35 vol%, 0.4 vol%, 0.45 vol%, 0.5 vol%.

[0047] In some embodiments, the concentration of P300 in the first buffer is 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.1 vol%, 0.15 vol%, 0.20 vol%, 0.25 vol%, 0.3 vol%, 0.35 vol%, 0.4 vol%, 0.45 vol%, 0.5 vol%.

[0048] In some embodiments, the concentration of PBS in the first buffer is 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM.

[0049] In some embodiments, the first buffer comprises 0.5-1 wt% NaCl, 0.5-2 wt% BSA, 0.05-0.2 vol% Tween 20, 0.05-0.2 vol% P300, 80-120 mM PBS.

[0050] In some embodiments, the pH of the first buffer is 7.4.

[0051] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0052] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0053] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0054] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0055] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0056] In certain embodiments, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, 20-100 mM Tris-HCl.

[0057] In certain embodiments, the Tris-HCl concentration in the second buffer is 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM.

[0058] In certain embodiments, the second buffer comprises 0.9 wt% NaCl, 2 wt% trehalose, 1 wt% BSA, 0.01 vol% Tween 20, 0.1 vol% P300, 50 mM Tris-HCl.

[0059] In certain embodiments, the pH of the second buffer is 7.5.

[0060] In certain embodiments, the coupling buffer is 20-80 mM (e.g., 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM) MES.

[0061] In certain embodiments, the coupling buffer is 30-60 mM MES.

[0062] In certain embodiments, the pH of the coupling buffer is 5.0.

[0063] In certain embodiments, the bare microspheres are modified with one or more of carboxyl (COOH), amino (NHS), tosyl (Tysol), epoxy (Epoxy), streptavidin (SA), and Protein-A / G.

[0064] In certain embodiments, the test sample type of the kit is derived from one or more of blood, bodily fluid, saliva, or urine.

[0065] In one aspect, the present application provides use of any of the above kits in the preparation of a detection product for an Alzheimer's disease marker.

[0066] The present application is further described by the following examples. Unless otherwise specified, "%" means volume percent. The materials and reagents used in the following examples are conventional in the art and are commercially available or synthesized by known methods, unless otherwise specified. Unless otherwise noted, the experimental methods in the following examples were performed under conventional experimental conditions or according to the conditions recommended by the manufacturer of the relevant reagent (kit). Examples

[0067] The application provides an Alzheimer's disease marker detection kit, which comprises a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of an Alzheimer's disease marker in the brain-derived exosomes, and the capture antibody for enriching the brain-derived exosomes is selected from a LINGO1 antibody and / or an OMGP antibody; the Alzheimer's disease marker is a pTau217 antibody and / or a pTau181 antibody.

[0068] A preparation method of the first reagent is as follows: (1) coupling the activated bare microspheres with the first antibody in the presence of a coupling buffer; (2) mixing the microspheres coupled with the first antibody obtained in step (1) with a first blocking solution; (3) mixing the blocked microspheres obtained in step (2) with a first buffer, namely the first reagent; The formula of the coupling buffer in the embodiment is 50 mM MES, pH 5.0; The formula of the first blocking solution in the embodiment is 100 mM PBS, 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20 and 0.1 vol% P300, and the pH is 6.5; The formula of the first buffer (magnetic bead diluent) in the embodiment is 100 mM PBS, 0.9 wt% NaCl, 1 wt% BSA, 0.1 vol% Tween 20 and 0.1 vol% P300, and the pH is 7.4; The first antibody is a combination of a LINGO1 antibody and an OMGP antibody, and the molar ratio of the LINGO1 antibody to the OMGP antibody is 1:1.

[0069] A preparation method of the second reagent is as follows: (1) coupling the activated bare microspheres with a pTau217 antibody and / or a pTau181 antibody in the presence of a coupling buffer; (2) mixing the microspheres coupled with the pTau217 antibody and / or the pTau181 antibody obtained in step (1) with a second blocking solution; (3) mixing the blocked microspheres obtained in step (2) with a second buffer, namely the second reagent; The formula of the coupling buffer in the embodiment is 50 mM MES, pH 5.0; The formula of the second blocking solution in the embodiment is 25 mM Tris-HCl, 0.75 wt% NaCl, 0.4 wt% casein, 0.01 vol% Tween 20 and 0.1 vol% P300, and the pH is 8.0; The formulation of the second buffer in this embodiment is: 50 mM Tris-HCl, 0.9 wt% NaCl, 2 wt% trehalose, 1 wt% BSA, 0.01 vol% Tween 20 and 0.1 vol% P300, and the pH is 7.5.

[0070] Example 1

[0071] Take 100 μL of 100 mg / mL base magnetic beads (10 mg of magnetic beads), add 1 mL (10 times the volume) of coupling buffer (50 mM MES, pH 5.0), magnetically separate and wash, and replace the coupling buffer 3 times, and remove the supernatant. Add 100 μL of 10 mg / mL EDC solution to the magnetic beads, and add 100 μL of 10 mg / mL NHS; seal the centrifuge tube with a sealing film, and place it in a biochemical incubator at 25°C for activation, and rotate on a circumferential mixer at 80 rpm for 30 min; add 1 mL (10 times the volume) of coupling buffer, magnetically separate and wash 3 times; and finally resuspend in coupling buffer. According to the feeding ratio of 40 μg of antibody / mg of magnetic beads, add the antibody to be coupled (the ratio of LINGO1 antibody to OMGP antibody is 1:1 in molar ratio), shake well, mix at room temperature, and rotate at 80 rpm for 2 h. Add 0.1 mL of blocking solution (100 mM PBS, 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20, 0.1 vol% P300; pH 6.5), and rotate at 80 rpm for 1.5 h at room temperature to block the unreacted activated carboxyl groups. Magnetically separate the blocked magnetic beads, discard the supernatant, and wash with 1 mL of magnetic bead diluent (100 mM PBS, 0.9 wt% NaCl, 1 wt% BSA, 0.1 vol% Tween 20, 0.1 vol% P300; pH 7.4) for 3 times, and finally resuspend the magnetic beads in 1 mL of magnetic bead diluent to prepare a first reagent with a concentration of 10 mg / mL; store at 4°C.

[0072] Meanwhile, according to the above method, magnetic beads are coated with single antibodies (OMGP, LINGO1, or L1CAM) to obtain an OMGP antibody-coated exosome capture reagent, a LINGO1 antibody-coated exosome capture reagent, and a L1CAM antibody-coated exosome capture reagent.

[0073] Example 2 The first reagent prepared according to Example 1 is used to capture brain-derived exosomes in a plasma sample, and the steps are as follows: 2.1 Take 500 μL of plasma and 5 μL of purified thrombin, mix at room temperature for 5 min, centrifuge at 10,000 rpm for 5 min, and then remove the supernatant for use.

[0074] 2.2 Separate the serum / plasma sample, centrifuge at 3000g for 10 min at 4°C to remove cell debris, transfer the supernatant to a new tube, centrifuge the sample again at 10000g for 20 min at 4°C to remove impurities, and transfer the supernatant to a new tube for use.

[0075] 2.3 Filter the plasma using a 0.22 μm filter.

[0076] 2.4 Co-Capture antibody exosome capture: 2.4.1 After mixing the magnetic beads, 120 μL of magnetic beads (exosome capture reagent) are taken, and the magnetic beads are washed 3 times with washing buffer (100 mM PBS, 0.02 vol% Tween 20, 0.1 vol% P300, pH 6.5); 2.4.2 The plasma sample treated in step 2.3 is added to the magnetic beads and mixed, and the mixture is mixed at 37°C for 2 h; 2.4.3 The magnetic bead-plasma complex is washed 3 times with washing buffer; 2.4.4 The magnetic beads are resuspended with 100 μL of sample preservation solution, and the sample is temporarily stored at 4°C for use.

[0077] The exosomes obtained above are subjected to electron microscopy, particle size, and concentration evaluation: (1) The concentration and particle size of the exosomes obtained above are measured by nanoparticle tracking analysis (NTA), and the results are shown in Figure 1 and Table 1, from which it can be seen that the concentration of the exosomes is 14.31e+9 particles / mL, and the average particle size is 126.5 ± 24.3 nm. Figure 1

[0078] (2) The morphology of the exosomes is observed by transmission electron microscopy (TEM), as shown in Figure 2 Under the TEM, the typical morphology of the exosomes, i.e., a double-concave disc shape, can be observed.

[0079] (3) The membrane surface markers of the exosomes are detected by Western blot, and the results are shown in Figure 3 From Figure 3 it can be seen that the positive membrane protein markers of the brain-derived exosomes captured by the Co-Capture antibody, OMGP antibody, LINGO1 antibody, or L1CAM antibody-coated magnetic beads can be detected, and the negative marker protein Calnexin protein is not detected.

[0080] Table 1

[0081] ​As can be seen from Table 1, the Co-Capture antibody has better consistency with the exosomes captured by L1CAM, LINGO1 and OMGP in terms of particle size and particle concentration, but has significant difference in purity. The Co-Capture antibody coated magnetic beads provided by the application can capture exosomes with higher purity.

[0082] Example 3 The pTau217 antibody or pTau181 antibody coated magnetic beads were prepared according to the preparation method of Example 1, except that the coating antibody used was different. In this embodiment, the pTau217 antibody or pTau181 antibody was used to coat the magnetic beads, and the formula of the blocking agent used was: 25 mM Tris-HCl, 0.75 wt% NaCl, 0.4 wt% casein, 0.01 vol% Tween 20, 0.1 vol% P300, and pH 8.0.

[0083] The formula of the magnetic bead storage buffer in this embodiment was: 50 mM Tris-HCl, 0.9 wt% NaCl, 2 wt% trehalose, 1 wt% BSA, 0.01 vol% Tween 20, 0.1 vol% P300, and pH 7.5.

[0084] The pTau217 antibody coated magnetic beads with a concentration of 10 mg / mL and the pTau181 antibody coated magnetic beads with a concentration of 10 mg / mL were prepared in this embodiment.

[0085] Meanwhile, AE labeled Tau antibodies (AE labeled pTau217 antibody and AE labeled pTau181 antibody) were prepared, and the operation steps were as follows: Take the commercially available AE and stand for 30 min at room temperature, and balance the AE. Take 0.1 mL of antibody (concentration of 1 mg / mL) into an EP tube. Add 4 μL of AE and mix well. Seal the EP tube and mix in a vortex mixer at room temperature for 1 hour (rotation speed of 50-70 rpm). Add 0.104 mL of quenching agent and mix well. Seal the EP tube and mix in a vortex mixer at room temperature for 30 min (rotation speed of 50-70 rpm). Take a G25 desalting column (purchased from Thermo Fisher) and stand vertically at room temperature for 30 min. Add 4 mL of PBS balancing buffer and let the balancing liquid flow out naturally until no liquid flows out. Repeat this step twice to completely pour the AE-labeled antibody into the G25 desalting column. Add PBS balancing buffer to make up the volume to 450 μL. Let the balancing liquid flow out naturally until no liquid flows out. Add 0.5 mL of PBS balancing buffer and receive it into clean EP tubes according to the order of 3 drops per tube. At least 15 tubes should be received according to the order of receiving. Store them in the dark temporarily. Take 1 μL of the received sample from each tube, dilute it with PBS balancing buffer to 2 mL, and load it on a magnetic microparticle chemiluminescence immunoassay analyzer (Venus 100S) according to the order of receiving from small to large. Detect the luminescence value of the diluted sample and record it. Record the receiving flow section of the first luminescence value peak, discard the samples in the flow section on both sides of the luminescence value peak, mix the remaining flow section samples, and record the mixed volume. Take an equal volume of AE antibody storage solution, mix it with a vortex mixer, and obtain AE-labeled Tau antibody (AE-labeled pTau217 antibody or AE-labeled pTau181 antibody).

[0086] The formula of the quenching agent in this example is: 50 mM CB buffer, 5 wt% glycine, pH 8.0.

[0087] The formula of the AE storage solution in this example is: 20 mM PB buffer, 10 wt% BSA, and 50 vol% glycerol.

[0088] Example 4 The brain-derived exosomes prepared in Example 2, the pTau217 antibody-coated magnetic beads and the pTau181 antibody-coated magnetic beads prepared in Example 3, and the AE-labeled Tau antibody are used in the detection reaction of the kit. During the detection reaction, the Tau antibody, the pTau-217 antibody / pTau181 antibody can form a stable double antibody sandwich immune complex with pTau-217 / pTau181 in the sample, and further form a double antibody sandwich immune complex of coated antibody-pTau-217 / pTau181 in the sample-labeled antibody for the automatic detection of pTau-217 / pTau181 on the full-automatic chemiluminescence immunoassay analyzer and the output of detection signal / data.

[0089] Take 100 μL sample and mix with 200 μL pTau217 antibody-magnetic microparticle and / or pTau181 antibody-magnetic microparticle conjugate complex, incubate at 37°C for 20 min, and wash three times with separation and washing solution; take 200 μL Tau antibody-AE complex mixture, incubate at 37°C for 20 min, wash three times with separation and washing solution, add 150 μL luminescent substrate solution, and automatically detect the luminescent signal by the automatic chemiluminescence immunoassay analyzer, and automatically calculate and output the signal value and concentration value of pTau181 / pTau217 in the sample according to the calibration curve; the overall detection time is less than 40 min.

[0090] Calibration curve: select seven low to high concentration antigen standard samples, then detect the luminescent value of pTau181 / pTau217 signal value of the calibration sample, which is used for fitting the standard curve in the detection process, and the detection information of the calibration sample is shown in Table 2.

[0091] Table 2

[0092] According to the method of Example 3, the brain-derived exosomes captured by the magnetic beads coated with single antibody in the blood are used for detecting the concentration of Alzheimer's disease marker (pTau217 / pTau181), and the results are shown in Table 3.

[0093] Table 3

[0094] As can be seen from Table 3, the exosomes captured by Co-Capture antibody and L1CAM, LINGO1, and OMGP have significant differences in total protein purity, pTau181 and pTau217 protein levels. The exosomes captured by Co-Capture antibody have higher exosome purity and pTau181 / pTau217 levels, which can be inferred that in the same volume of plasma sample, after capturing exosomes by Co-Capture antibody, the exosome purity is higher, and pTau181 / pTau217 is more easily detected, and the detection sensitivity is significantly improved.

[0095] Example 5 Twenty-six AD patient plasma samples were collected from a clinical institution, and the samples all had clear clinical diagnosis results (scale evaluation and / or PET-CT image examination); 27 CO control plasma samples (healthy people). The brain-derived exosomes in the plasma samples were captured by the method of Example 2, and the pTau181 / pTau217 protein level in the samples was detected by the method of Example 4, and the pTau181 / pTau217 protein concentration of the samples was obtained according to the standard sample curve. The above 53 samples were detected, and the sample detection results were statistically analyzed.

[0096] Statistical analysis of the tested sample data showed that the area under the ROC curve for pTau181 protein levels was 0.871 ( ). Figure 4 The area under the ROC curve for pTau217 protein level was 0.892. Figure 5 The area under the ROC curve for the combined analysis of pTau181 and pTau217 protein levels was 0.953. Figure 6 The brain-derived exosome capture system of this invention shows a very significant gradient difference in pTau181 and pTau217 protein levels between healthy individuals and Alzheimer's disease patients, demonstrating excellent clinical diagnostic and screening value for AD.

[0097] Example 6 In this embodiment, blocking solutions prepared by screening different concentrations of casein and buffer system were used to block magnetic beads. The background luminescence value of the blocked magnetic beads was detected by a magnetic microparticle chemiluminescence immunoassay analyzer to evaluate their non-specific adsorption capacity in complex samples.

[0098] In this embodiment, the exosome capture reagent coated with Co-Capture antibody was prepared in the same manner as in Example 1, except that the composition of the blocking solution was different. In addition, the methods of Examples 3 and 4 were used to capture brain-derived exosomes from the blood using magnetic beads of Co-Capture antibody obtained with different blocking solutions for the detection of the Alzheimer's disease marker (pTau217). The blocking effect of each group and the results of pTau217 background detection are shown in Table 4. The concentrations of the buffer system PBS, MES, and Tris-HCl were all 100 mM, 100 mM, and 100 mM, respectively. In Table 4, "%" for casein and BSA represents mass percentage, and "%" for Tween 20 and P300 represents volume percentage.

[0099] Table 4

[0100] As shown in Table 4, different blocking solutions have a significant impact on the detection results. Only casein within a certain range (0.3~0.6wt%) has the best blocking effect, which greatly reduces non-specific adsorption based on the antibody IgG structure, improves the immunoaffinity capture efficiency, and makes the immunoaffinity-captured exosomes more specific.

[0101] It should be understood that the above examples are exemplary and are not intended to limit the scope of the claims encompassing all possible embodiments. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the present disclosure. Similarly, various technical features of the above examples can be combined arbitrarily to form additional embodiments of the present application that can not be explicitly described. Therefore, the above examples merely express several embodiments of the present application and do not limit the scope of protection of the present patent.

Claims

1. An Alzheimer's disease marker detection kit, characterized by comprising: The kit includes a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease markers; The first reagent includes microspheres conjugated with a first antibody and a first buffer solution, wherein the first antibody includes LINGO1 antibody and / or OMGP antibody. The second reagent comprises microspheres conjugated with a second antibody and a second buffer, wherein the second antibody comprises pTau217 antibody and / or pTau181 antibody.

2. The kit of claim 1, wherein The first antibody consists of LINGO1 antibody and OMGP antibody.

3. The kit of claim 2, wherein The molar ratio of the LINGO1 antibody to the OMGP antibody is 0.1 to 10:

1.

4. The kit of claim 1, wherein The preparation method of the first reagent includes the following steps: (1) In the presence of a coupling buffer, the activated naked microspheres were coupled with the first antibody; (2) Mix the microspheres conjugated with the first antibody obtained in step (1) with the first blocking solution; (3) Mix the sealed microspheres obtained in step (2) with the first buffer solution; In step (2), the first blocking solution includes 0.3-0.6 wt% casein, 5-15 wt% BSA, 1-5 vol% Tween 20, 0.05-0.5 vol% P300, and 50-150 mM PBS.

5. The kit of claim 4, wherein The first blocking solution comprises 0.3–0.6 wt% casein, 8–12 wt% BSA, 1–3 vol% Tween 20, 0.05–0.3 vol% P300, and 80–120 mM PBS; And / or, the pH of the first sealing solution is 6.0 to 7.

0.

6. The kit of claim 5, wherein The first blocking solution comprises 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20, 0.1 vol% P300, and 100 mM PBS; the pH of the first blocking solution is 6.

5.

7. The kit according to claim 1 or 4, characterized in that, The first buffer solution comprises 0.1–2 wt% NaCl, 0.5–5 wt% BSA, 0.05–0.5 vol% Tween 20, 0.05–0.5 vol% P300, and 50–150 mM PBS; And / or, the second buffer comprises 0.1-2 wt% NaCl, 1-5 wt% trehalose, 0.5-5 wt% BSA, 0.001-0.5 vol% Tween 20, 0.05-0.5 vol% P300, and 20-100 mM Tris-HCl.

8. The kit of claim 4, wherein The surface of the bare microspheres is modified with one or more groups selected from carboxyl, amino, toluenesulfonyl, epoxy, streptavidin, and protein A / G.

9. The kit of claim 1, wherein The test sample types for the kit are derived from one or more of blood, body fluids, saliva, or urine.

10. The use of the kit according to any one of claims 1 to 9 in the preparation of detection products for Alzheimer's disease biomarkers.

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