An alzheimer's disease marker detection kit and application thereof

Abstract

This invention relates to an Alzheimer's disease biomarker detection kit and its application. The kit includes a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease biomarkers. 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 includes microspheres conjugated with a second antibody and a second buffer solution, wherein the second antibody includes pTau217 antibody and / or pTau181 antibody. The kit provided by this invention can highly enrich brain-derived exosomes and can detect Alzheimer's disease biomarkers (Tau217 antibody and / or pTau181 antibody) with high sensitivity and high specificity to assist in the diagnosis of AD.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic testing technology, and in particular to an Alzheimer's disease biomarker detection kit and its application. Background Technology

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system. Traditional AD diagnostic methods are costly, highly dependent on professionals, and even somewhat invasive, making them difficult to implement on a large scale in clinical practice. Blood sample collection is minimally invasive, rapid, and inexpensive, and some blood biomarkers show significant changes before the appearance of clinical symptoms in AD, making it an ideal method for screening early-stage AD patients. However, due to the blood-brain barrier, the abundance of relevant biomarkers in plasma samples is significantly lower than in cerebrospinal fluid, and peripheral blood contains more metabolic interfering substances, leading to inaccurate test results.

[0003] Exosomes are nanoscale extracellular vesicle structures that facilitate intercellular communication. The composition and content of various bioactive molecules they carry vary depending on the originating and recipient cells. With the deepening research into neurodegenerative diseases, researchers have discovered that exosomes secreted by all cells in the brain (including neurons, microglia, and astrocytes) play a crucial role in cell communication and participate in the entire process of AD development.

[0004] Exosomes can carry relevant biomarkers across the blood-brain barrier bidirectionally without altering their surface composition. However, their biological origins are widespread, and exosomes expressed in multiple organs exist in blood samples. Without differentiation, the signals of highly specific but low-abundance biomarkers can be submerged in complex background signals, which is highly detrimental to biomarker detection. To enrich exosomes from specific cell sources, researchers often use immunoaffinity capture techniques for exosome purification. Traditional immunoaffinity capture techniques are highly dependent on the antibodies used. Inappropriate antibody selection can introduce bias, resulting in a lower proportion of target exosomes captured, and can also affect immunoaffinity capture efficiency, leading to a low number of captured target exosomes that cannot meet the detection requirements of downstream low-abundance biomarkers (such as pTau217). Summary of the Invention

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

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] On one hand, the present invention provides an Alzheimer's disease biomarker detection kit, the kit comprising a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease biomarkers;

[0008] 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.

[0009] 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.

[0010] Preferably, the first antibody is composed of LINGO1 antibody and OMGP antibody.

[0011] Preferably, the molar ratio of the LINGO1 antibody to the OMGP antibody is 0.1 to 10:1.

[0012] Preferably, the preparation method of the first reagent includes the following steps:

[0013] (1) In the presence of a coupling buffer, the activated naked microspheres were coupled with the first antibody;

[0014] (2) Mix the microspheres conjugated with the first antibody obtained in step (1) with the first blocking solution;

[0015] (3) Mix the sealed microspheres obtained in step (2) with the first buffer solution;

[0016] 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.

[0017] Preferably, 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.

[0018] Preferably, the pH of the first sealing solution is 6 to 7.0.

[0019] Preferably, 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.

[0020] 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, and 50-150 mM PBS.

[0021] 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, and 20-100 mM Tris-HCl.

[0022] Preferably, the surface of the bare microspheres is modified with one or more groups selected from carboxyl, amino, toluenesulfonyl, epoxy, streptavidin, and protein A / G.

[0023] Preferably, the test sample type of the kit is derived from one or more of blood, body fluids, saliva, or urine.

[0024] On the one hand, the present invention provides the application of the reagent kit described in any of the above claims in the preparation of detection products for Alzheimer's disease biomarkers.

[0025] The effects of the invention

[0026] The Alzheimer's disease biomarker detection kit provided by this invention can specifically capture brain-derived exosomes and quantitatively analyze their Alzheimer's disease biomarkers (pTau181 and pTau217), exhibiting high specificity and high sensitivity. Using this kit eliminates the influence of plasma-free pTau181 and pTau217, as well as pTau181 and pTau217 in exosomes from other organs, on detection, thereby improving the ability to identify and assess the risk of AD in patients. Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the particle size and concentration of exosomes captured using the Co-Capture antibody-exosome capture reagent.

[0028] Figure 2 Morphological images of exosomes captured by the Co-Capture antibody-exosome capture reagent for observation using transmission electron microscopy.

[0029] Figure 3 This is a gel image of WB markers on the membrane surface of exosomes captured using exosome capture reagents coated with different antibodies.

[0030] Figure 4 ROC curves for identifying AD using the kit provided by this invention to test pTau181 protein levels in clinical samples.

[0031] Figure 5 ROC curves for identifying AD using the kit provided by this invention to test pTau271 protein levels in clinical samples.

[0032] Figure 6 ROC curves were obtained for identifying AD using the kit provided by this invention to detect pTau181 and pTau271 protein levels in clinical test samples. Detailed Implementation

[0033] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may 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 meanings as those in the technical field to which this application pertains.

[0034] pTau217 is abundant in cerebrospinal fluid samples from AD patients, but due to the blood-brain barrier, it cannot directly cross the barrier to enter blood samples in large quantities, resulting in extremely low levels of pTau217 in blood samples, rendering conventional detection methods ineffective. Exosomes, on the other hand, can cross the blood-brain barrier bidirectionally without altering their surface composition. Therefore, surface markers can be used to capture exosomes of neural cell origin, identify their cellular origin, and further detect the levels of AD markers in the identified brain-derived exosomes to improve the accuracy of AD detection.

[0035] In view of this, the inventors of this invention, through extensive and in-depth research and numerous experiments, provide a kit for detecting brain-derived exosomes in blood to aid in the diagnosis of Alzheimer's disease. Specifically, a Co-Capture antibody combination is constructed by selecting a combination of LINGO1 and / or OMGP specific antibodies to capture brain-derived exosomes (exosomes derived from nerve cells and oligodendrocytes). The levels of Tau protein-related markers (including pTau217 and / or pTau181) in the exosomes are further detected to identify and determine the risk of patients having AD. Based on this, the present invention is completed.

[0036] On one hand, the present invention provides an Alzheimer's disease biomarker detection kit, the kit comprising a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease biomarkers;

[0037] 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.

[0038] 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.

[0039] In some embodiments, the first antibody consists of a LINGO1 antibody and an OMGP antibody.

[0040] In some embodiments, the molar ratio of the LINGO1 antibody to the OMGP antibody is 0.1 to 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, or 10:1.

[0041] In some embodiments, the molar ratio of the LINGO1 antibody to the OMGP antibody is 0.5 to 5:1.

[0042] In some embodiments, the molar ratio of the LINGO1 antibody to the OMGP antibody is 0.5 to 3:1.

[0043] In some embodiments, the preparation method of the first reagent includes the following steps:

[0044] (1) In the presence of a coupling buffer, the activated naked microspheres were coupled with the first antibody;

[0045] (2) Mix the microspheres conjugated with the first antibody obtained in step (1) with the first blocking solution;

[0046] (3) Mix the sealed microspheres obtained in step (2) with the first buffer solution;

[0047] 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.

[0048] In some embodiments, the casein content in the first blocking solution is 0.3wt%, 0.4wt%, 0.5wt%, or 0.6wt%.

[0049] In some embodiments, the BSA content in the first sealing liquid is 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, or 15 wt%.

[0050] In some embodiments, the content of Tween 20 in the first sealing liquid is 1 vol%, 1.5 vol%, 2 vol%, 2.5 vol%, 3 vol%, 3.5 vol%, 4 vol%, 4.5 vol%, and 5 vol%.

[0051] In some embodiments, the content of P300 in the first sealing liquid 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%, and 0.5 vol%.

[0052] In some embodiments, the PBS concentration in the first blocking solution is 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, or 150mM.

[0053] In some embodiments, 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.

[0054] In some embodiments, the first blocking solution comprises 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20, 0.1 vol% P300, and 100 mM PBS.

[0055] In some embodiments, the pH of the first sealing solution is 6.0 to 7.0, for example 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0056] In some embodiments, the pH of the first sealing solution is 6.5.

[0057] 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, and 50–150 mM PBS.

[0058] In some embodiments, the NaCl content in the first buffer solution is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.5wt%, or 2.0wt%.

[0059] In some embodiments, the BSA content in the first buffer solution 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%, or 5 wt%.

[0060] In some embodiments, the Tween 20 content in the first buffer solution 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%, and 0.5 vol%.

[0061] In some embodiments, the content of P300 in the first buffer solution 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%, and 0.5 vol%.

[0062] In some embodiments, the PBS concentration in the first buffer solution 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, or 150 mM.

[0063] In some embodiments, the first buffer solution comprises 0.5-1 wt% NaCl, 0.5-2 wt% BSA, 0.05-0.2 vol% Tween 20, 0.05-0.2 vol% P300, and 80-120 mM PBS.

[0064] In some implementations, the pH of the first buffer solution is 7.4.

[0065] In some embodiments, 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, and 20-100 mM Tris-HCl.

[0066] In some embodiments, the NaCl content in the second buffer solution is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.5wt%, or 2.0wt%.

[0067] In some embodiments, the trehalose content in the second buffer solution is 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%.

[0068] In some embodiments, the BSA content in the second buffer solution 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%, or 5 wt%.

[0069] In some embodiments, the Tween 20 content in the second buffer solution is 0.005 vol%, 0.006 vol%, 0.007 vol%, 0.008 vol%, 0.009 vol%, 0.01 vol%, 0.012 vol%, 0.014 vol%, 0.016 vol%, 0.018 vol%, 0.02 vol%, 0.025 vol%, 0.03 vol%, 0.035 vol%, 0.04 vol%, 0.045 vol%, and 0.05 vol%.

[0070] In some embodiments, the content of P300 in the second buffer solution 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%, and 0.5 vol%.

[0071] In some embodiments, the Tris-HCl concentration in the second buffer solution 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, or 100 mM.

[0072] In some embodiments, the second buffer solution comprises 0.9 wt% NaCl, 2 wt% trehalose, 1 wt% BSA, 0.01 vol% Tween 20, 0.1 vol% P300, and 50 mM Tris-HCl.

[0073] In some implementations, the second buffer solution has a pH of 7.5.

[0074] In some 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) of MES.

[0075] In some embodiments, the coupling buffer is 30-60 mM MES.

[0076] In some embodiments, the pH of the coupling buffer is 5.0.

[0077] In some embodiments, the surface of the bare microspheres is modified with one or more groups selected from carboxyl (COOH), amino (NHS), toluenesulfonyl (Tysol), epoxy (Epoxy), streptavidin (SA), and protein A / G (Protein-A / G).

[0078] In some implementations, the test sample type of the kit is derived from one or more of blood, body fluids, saliva, or urine.

[0079] On the one hand, the present invention provides the application of the reagent kit described in any of the above claims in the preparation of detection products for Alzheimer's disease biomarkers.

[0080] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents volume percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit). Example

[0081] This invention provides an Alzheimer's disease biomarker detection kit, the kit comprising a first reagent for enriching brain-derived exosomes and a second reagent for detecting the concentration of Alzheimer's disease biomarkers in brain-derived exosomes, wherein the capture antibody for enriching brain-derived exosomes is selected from LINGO1 antibody and / or OMGP antibody; the Alzheimer's disease biomarker is pTau217 antibody and / or pTau181 antibody.

[0082] Preparation method of the first reagent:

[0083] (1) In the presence of a coupling buffer, the activated naked microspheres were coupled with the first antibody;

[0084] (2) Mix the microspheres conjugated with the first antibody obtained in step (1) with the first blocking solution;

[0085] (3) Mix the sealed microspheres obtained in step (2) with the first buffer solution to obtain the first reagent;

[0086] The coupling buffer formulation in this embodiment is: 50 mM MES, pH 5.0;

[0087] The formulation of the first blocking solution in this embodiment is as follows: 100mM PBS, 0.5wt% casein, 10wt% BSA, 2vol% Tween 20, 0.1vol% P300; pH is 6.5.

[0088] The formulation of the first buffer solution (magnetic bead dilution solution) in this embodiment is as follows: 100mM PBS, 0.9wt% NaCl, 1wt% BSA, 0.1vol% Tween 20, 0.1vol% P300, pH 7.4;

[0089] The first antibody is a combination of LINGO1 antibody and OMGP antibody, with a molar ratio of LINGO1 antibody to OMGP antibody of 1:1.

[0090] Preparation method of the second reagent:

[0091] (1) In the presence of a coupling buffer, the activated naked microspheres were coupled with pTau217 antibody and / or pTau181 antibody;

[0092] (2) Mix the microspheres conjugated with pTau217 antibody and / or pTau181 antibody obtained in step (1) with the second blocking solution;

[0093] (3) Mix the sealed microspheres obtained in step (2) with the second buffer solution to obtain the second reagent;

[0094] The coupling buffer formulation in this embodiment is: 50 mM MES, pH 5.0;

[0095] The formulation of the second blocking solution in this embodiment is: 25 mM Tris-HCl, 0.75 wt% NaCl, 0.4 wt% casein, 0.01 vol% Tween 20 and 0.1 vol% P300, with a pH of 8.0;

[0096] The second buffer solution in this embodiment is formulated as follows: 50 mM Tris-HCl, 0.9 wt% NaCl, 2 wt% trehalose, 1 wt% BSA, 0.01 vol% Tween 20 and 0.1 vol% P300, with a pH of 7.5.

[0097] Example 1

[0098] Take 100 μL of 100 mg / mL basic magnetic beads (10 mg magnetic beads), add 1 mL (10 times the volume) of coupling buffer (50 mL MME S, pH 5.0), perform magnetic separation washing, and replace the coupling buffer 3 times, then remove the supernatant. Add 100 μL of 10 mg / mL EDC solution and 100 μL of 10 mg / mL NHS to the magnetic beads; seal the centrifuge tube with sealing film, place it in a biochemical incubator at 25°C for activation, and rotate it at 80 rpm for 30 min on a circumferential mixer; add 1 mL (10 times the volume) of coupling buffer, perform magnetic separation washing 3 times; finally, resuspend the coupling buffer. Add the antibody to be coupled according to the feed ratio of 40 μg antibody / mg magnetic beads (the molar ratio of LINGO1 antibody to OMGP antibody is 1:1), shake well, mix at room temperature, and rotate it at 80 rpm for 2 h. Add 0.1 mL of blocking buffer (100 mM PBS, 0.5 wt% casein, 10 wt% BSA, 2 vol% Tween 20, 0.1 vol% P300; pH 6.5), mix circumferentially at room temperature, and rotate at 80 rpm for 1.5 h to block unreacted activated carboxyl groups. Magnetic separation is performed on the blocked magnetic beads, the supernatant is discarded, and the beads are washed three times with 1 mL of magnetic bead dilution buffer (100 mM PBS, 0.9 wt% NaCl, 1 wt% BSA, 0.1 vol% Tween 20, 0.1 vol% P300; pH 7.4). Finally, resuspend the magnetic beads in 1 mL of magnetic bead dilution buffer to prepare a 10 mg / mL first reagent; store at 4 °C.

[0099] Simultaneously, following the above method, magnetic beads are coated with a single antibody (OMGP, LINGO1, or L1CAM) to obtain exosome capture reagents coated with OMGP antibody, exosome capture reagents coated with LINGO1 antibody, and exosome capture reagents coated with L1CAM antibody.

[0100] Example 2

[0101] The first reagent prepared according to Example 1 captures brain-derived exosomes in plasma samples, and the steps are as follows:

[0102] 2.1 Take 500 μL of plasma and 5 μL of purified thrombin, mix well at room temperature for 5 min, centrifuge at 10000 rpm for 5 min, and transfer the supernatant for later use.

[0103] 2.2 Separate serum / plasma samples were centrifuged at 3000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new tube, and the samples were centrifuged again at 10000g for 20 min at 4℃ to remove impurities. The supernatant was transferred to a new tube for later use.

[0104] 2.3 Use a 0.22 μm filter to filter the plasma.

[0105] 2.4 Co-Capture antibody exosome capture:

[0106] 2.4.1 After mixing the magnetic beads, take 120 μL of magnetic beads (exosome capture reagent) and wash the magnetic beads 3 times with washing buffer (100 mM PBS, 0.02 vol% Tween 20, 0.1 vol% P300, pH 6.5);

[0107] 2.4.2 The plasma sample processed in step 2.3 was added to the magnetic beads and mixed well, and then swirled at 37°C for 2 hours;

[0108] 2.4.3 The magnetic bead-plasma complex was washed three times with washing buffer;

[0109] 2.4.4 Resuspend the magnetic beads in 100 μL of sample preservation solution and store the sample temporarily at 4℃ for later use.

[0110] The exosomes obtained above were evaluated by electron microscopy, particle size, and concentration.

[0111] (1) The concentration and particle size of the exosomes obtained above were determined by nanoparticle tracking analysis (NTA). See the results below. Figure 1 And Table 1, by Figure 1 The concentration of exosomes was 14.31e+9 particles / mL, and the measured average particle size was 126.5±24.3 nm.

[0112] (2) Observe the morphology of exosomes using transmission electron microscopy (TEM). Figure 2 As shown, the typical morphology of exosomes—biconcave disc-shaped—can be observed under a TEM electron microscope.

[0113] (3) Western blot was used to detect membrane surface markers of exosomes, and the results are as follows: Figure 3 As shown, by Figure 3It can be seen that positive membrane protein markers of brain-derived exosomes in plasma samples captured by magnetic beads coated with Co-Capture antibody, OMGP antibody, LINGO1 antibody, or L1CAM antibody can be detected, while the negative marker protein Calnexin protein was not detected.

[0114] Table 1

[0115]

[0116] As shown in Table 1, the exosomes captured by the Co-Capture antibody and those captured by L1CAM, LINGO1, and OMGP exhibit good consistency in particle size and concentration, but show significant differences in purity. The exosomes captured by the magnetic beads coated with the Co-Capture antibody provided in this invention have higher purity.

[0117] Example 3

[0118] Magnetic beads coated with pTau217 antibody or pTau181 antibody were prepared according to the preparation method of Example 1. The difference is that different coating antibodies were used. In this example, pTau217 antibody or pTau181 antibody was used to coat the magnetic beads. The blocking agent was formulated as follows: 25mM Tris-HCl, 0.75wt% NaCl, 0.4wt% casein, 0.01vol% Tween 20, 0.1vol% P300, and pH 8.0.

[0119] The magnetic bead preservation buffer solution in this embodiment has the following formulation: 50mM Tris-HCl, 0.9wt% NaCl, 2wt% trehalose, 1wt% BSA, 0.01vol% Tween 20, 0.1vol% P300, and pH 7.5.

[0120] In this embodiment, magnetic beads coated with pTau217 antibody at a concentration of 10 mg / mL and magnetic beads coated with pTau181 antibody at a concentration of 10 mg / mL were prepared.

[0121] Simultaneously, AE-labeled Tau antibodies (AE-labeled pTau217 antibody and AE-labeled pTau181 antibody) were prepared. The operation steps are as follows:

[0122] Equilibrate the commercially available anti-inflammatory agent (AE) by allowing it to stand at room temperature for 30 minutes. Pipette 0.1 mL of antibody (1 mg / mL) into an EP tube. Add 4 μL of AE and mix well. Seal the EP tube and vortex at room temperature for 1 hour (50-70 rpm). Add 0.104 mL of quencher and mix well. Seal the EP tube and vortex at room temperature for 30 minutes (50-70 rpm). Take a G25 desalting column (purchased from Thermo Fisher Scientific), and incubate it vertically at room temperature for 30 minutes to equilibrate. Add 4 mL of PBS equilibration buffer, allowing the equilibration buffer to flow out naturally until no more liquid flows out. Repeat this step twice. Completely add the AE-labeled antibody to the G25 desalting column, and bring the volume to 450 μL with PBS equilibration buffer. Allow the equilibration buffer to flow out naturally until no more liquid flows out. Add 0.5 mL of PBS equilibration buffer, collecting the sample in 3 drops / tubes into clean EP tubes until no more liquid flows out. Number the tubes according to the collection order; at least 15 tubes should be collected. Store temporarily in the dark. Take 1 μL of the collected sample from each tube and dilute it to 2 mL with PBS equilibration buffer. Load the samples onto a magnetic microparticle chemiluminescence immunoassay analyzer (Venus100S) according to the collection order, from smallest to largest. Detect and record the luminescence values ​​of the diluted samples. Record the receiving segment of the first luminescence peak, discard the segments at both ends of the luminescence peak, mix the remaining segments, and record the mixed volume. Take an equal volume of AE antibody preservation solution and mix it with a vortex mixer to obtain AE-labeled Tau antibody (AE-labeled pTau217 antibody or AE-labeled pTau181 antibody).

[0123] The quencher formulation in this embodiment is: 50mM CB buffer, 5wt% glycine, pH 8.0.

[0124] The AE preservation solution in this embodiment is formulated as follows: 20 mM PB buffer, 10 wt% BSA and 50 vol% glycerol.

[0125] Example 4

[0126] Brain-derived exosomes prepared in Example 2, pTau217 antibody-coated magnetic beads and pTau181 antibody-coated magnetic beads prepared in Example 3, and AE-labeled Tau antibody were used. During the detection reaction using the kit, the Tau antibody and pTau-217 antibody / pTau181 antibody can form a stable double-antibody sandwich immune complex with pTau-217 / pTau181 in the sample. This further forms a double-antibody sandwich immune complex consisting of the coated antibody, pTau-217 / pTau181 in the sample, and the labeled antibody, which is used for automated detection of pTau-217 / pTau181 and output of detection signals / data in a fully automated chemiluminescence immunoassay analyzer.

[0127] Take 100 μL of sample and mix with 200 μL of pTau217 antibody-magnetic microparticles and / or pTau181 antibody-magnetic microparticle conjugate complex, incubate at 37℃ for 20 min, and wash three times with separation washing buffer; take 200 μL of Tau antibody-AE complex and mix, incubate at 37℃ for 20 min, wash three times with separation washing buffer, add 150 μL of luminescent substrate solution, and the fully automated chemiluminescence immunoassay analyzer will automatically detect the luminescence signal 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.

[0128] Calibration curve: Seven antigen standards with concentrations from low to high were selected, and the luminescence values ​​of the pTau181 / pTau217 signal of the calibrators were detected to fit the standard curve during the detection process. The detection information of the calibrators is shown in Table 2.

[0129] Table 2

[0130]

[0131] Following the method in Example 3, magnetic beads coated with a single antibody were used to capture brain-derived exosomes in the blood for the detection of Alzheimer's disease markers (pTau217 / pTau181). The results are shown in Table 3.

[0132] Table 3

[0133]

[0134] As shown in Table 3, there are significant differences in total protein purity, pTau181 and pTau217 protein levels between exosomes captured by Co-Capture antibody and those captured by L1CAM, LINGO1, and OMGP. Exosomes captured by Co-Capture antibody have higher exosome purity and pTau181 / pTau217 levels. It can be inferred that in the same volume of plasma sample, exosomes captured by Co-Capture antibody have higher purity, and pTau181 / pTau217 is more easily detected, thus significantly improving detection sensitivity.

[0135] Example 5

[0136] Plasma samples were collected from 26 AD patients at clinical institutions, all with clear clinical diagnostic results (scale assessment and / or PET-CT imaging); 27 CO control plasma samples were collected from healthy individuals. Brain-derived exosomes were captured from the plasma samples using the method described in Example 2, and the pTau181 / pTau217 protein levels in the samples were detected using the method described in Example 4. The pTau181 / pTau217 protein concentrations in the samples were obtained based on the standard curve. The above 53 samples were tested, and the results were statistically analyzed.

[0137] 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.

[0138] Example 6

[0139] In this embodiment, blocking solutions prepared by screening different concentrations of casein and buffer systems 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.

[0140] 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.

[0141] Table 4

[0142]

[0143] 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.

[0144] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A reagent kit for detecting Alzheimer's disease biomarkers, characterized in that, 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 reagent kit according to claim 1, characterized in that, The first antibody consists of LINGO1 antibody and OMGP antibody.

3. The reagent kit according to claim 2, characterized in that, The molar ratio of the LINGO1 antibody to the OMGP antibody is 0.1 to 10:

1.

4. The reagent kit according to claim 1, characterized in that, 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 reagent kit according to claim 4, characterized in that, 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 reagent kit according to claim 5, characterized in that, 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 reagent kit according to claim 4, characterized in that, 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 reagent kit according to claim 1, characterized in that, 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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