Saliva exosome physical examination test kit for auxiliary screening of Alzheimer disease and preparation method of saliva exosome physical examination test kit

Abstract

The invention relates to the technical field of biological detection, and discloses a saliva exosome detection kit for non-invasive auxiliary screening of Alzheimer's disease and a preparation method of the saliva exosome detection kit. The specific exosome enrichment system composed of the anti-L1CAM / CD63 double antibody coated capture plate and the anti-MUC1 / KRT19Fab fragment blocking agent is constructed for the first time, about 85% of oral cavity source exosomes can be effectively removed, and the capture purity of neuron source exosomes is remarkably improved; the special saliva preserving fluid containing DTT and a specific enzyme inhibitor is developed, the technical bottlenecks that saliva RNA is easy to degrade and a sample is uneven in viscosity are overcome, and the room temperature stability is prolonged to 8 hours; an RT-qPCR detection system taking saliva exosome miRNA-135a as a core and taking internal reference miRNA-16-5p standardization is established, and clinical sample verification shows that the kit can effectively distinguish the Alzheimer's disease patient from a healthy control, shows excellent inter-group distinguishing ability, and provides a stable, efficient, reliable and complete technical scheme for solving the problem of AD non-invasive early screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a saliva exosome detection kit for auxiliary screening of Alzheimer's disease and a preparation method thereof. BACKGROUND

[0002] Early screening of Alzheimer's disease currently faces major challenges. Existing clinical diagnosis mainly relies on cerebrospinal fluid detection and imaging examination, but these methods have obvious technical bottlenecks. First, the invasive sampling method limits its application. The detection method based on cerebrospinal fluid amyloid beta 42 (Aβ42) and human phosphorylated tau protein (p-tau) requires lumbar puncture, resulting in poor patient compliance and a high clinical rejection rate of 65%, so it is not suitable for large-scale screening. Second, the reliability of blood detection is insufficient. Peripheral blood miRNA detection is easily affected by hemolysis, and the proportion of brain-derived exosomes in blood is less than one thousandth due to the limitation of the blood-brain barrier, resulting in a detection sensitivity of only 52-58% for preclinical stage Alzheimer's disease. Third, the potential of saliva exosomes has not been fully utilized.

[0003] Although there are hundreds of millions to billions of exosomes per milliliter in saliva, the existing saliva diagnostic technology still has three key problems that have not been solved: first, the cell origin of exosomes cannot be effectively distinguished, and oral epithelial cell exosomes and neuron-derived exosomes cannot be separated; second, the degradation of miRNA by saliva proteases cannot be overcome, and the degradation rate of the sample reaches 70% after two hours at room temperature; third, a specific exosome miRNA marker combination suitable for saliva samples has not been established. Therefore, there is an urgent need to develop a new detection method that can specifically capture neuron-derived exosomes from saliva exosomes and stably detect miRNA-135a with auxiliary diagnostic value for Alzheimer's disease. SUMMARY

[0004] The purpose of the present application is to provide a saliva exosome detection kit for auxiliary screening of Alzheimer's disease.

[0005] The kit of the present application comprises a capture plate, an oral epithelial exosome blocking agent, a special saliva preservative, an exosome lysis solution, an RT-qPCR (reverse transcription quantitative polymerase chain reaction) premix and a standard:

[0006] The capture plate is coated with mouse-derived anti-human L1 cell adhesion molecule (L1CAM) monoclonal antibody and mouse-derived anti-human CD63 monoclonal antibody;

[0007] The oral epithelial exosome blocking agent comprises an anti-mucin 1 (MUC1) antigen binding (Fab) fragment and an anti-cytokeratin 19 (KRT19) Fab fragment;

[0008] The special saliva preservative solution comprises a protease inhibitor, an RNase inhibitor, and a reducing agent.

[0009] The RT-qPCR premix solution comprises specific primers and probes for detecting miRNA-135a and a reference miRNA.

[0010] The application also provides a preparation method of a saliva exosome detection kit for auxiliary screening of Alzheimer's disease, specifically comprising the following steps:

[0011] (8) Preparation of the capture plate: take a 96-well enzyme-labeled plate, and coat the plate with antibodies by physical adsorption; dilute mouse-derived anti-human L1CAM monoclonal antibodies and mouse-derived anti-human CD63 monoclonal antibodies in PBS buffer with pH 7.4, respectively, and add 50 μL of the antibody solution to each well, and incubate at 4°C for 12 hours; discard the liquid in the wells, and wash the wells with PBST buffer containing 0.05% Tween-20 for 3 times; then add 200 μL of PBS solution containing 2% bovine serum albumin (BSA) to each well for blocking, and incubate at 37°C for 2 hours; discard the blocking solution, dry the plate in a sterile environment, vacuum-seal the plate, and store the plate at 4°C for standby;

[0012] (9) Preparation of the oral epithelial exosome blocking agent: dissolve the anti-MUC1 Fab fragment and the anti-KRT19 Fab fragment in PBS buffer with pH 7.2, respectively, and adjust the concentration to 50 μg / mL; mix the two solutions in equal volumes to form a ready-to-use mixed blocking agent solution; divide the solution into 100 μL per tube, and store the solution at -20°C for freezing;

[0013] (10) Preparation of the special saliva preservative solution: dissolve 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 1 U / μL SUPERase-In RNase inhibitor, 5 mM dithiothreitol (DTT), and 0.5% phenylmethylsulfonyl fluoride in sterile RNase-free water, respectively; adjust the pH value of the mixed solution to 6.8 by using hydrochloric acid or sodium hydroxide solution; filter the solution through a 0.22 μm filter membrane to remove bacteria, divide the solution into 1.5 mL per tube in sterile centrifuge tubes, and store the solution at 4°C;

[0014] (11) Preparation of exosome lysis buffer: Take 1 mL of Triton X-100, 0.771 g of DTT and 0.186 g of disodium ethylenediaminetetraacetate (EDTA-2Na), dissolve them thoroughly in 40 mL of 50 mM Tris-HCl buffer (pH 7.6) and bring the volume up to 50 mL to obtain a lysis buffer containing 1% Triton X-100, 0.1 M DTT and 10 mM EDTA; after mixing thoroughly at room temperature, dispense 1 mL into each tube and store at 4 °C protected from light.

[0015] (12) Preparation of RT-qPCR premix: Using 2×One-Step RT-qPCR Buffer as the basic reaction system, deoxyribonucleoside triphosphates (dNTPs) were added sequentially to achieve a final concentration of 0.4 mM. Magnesium chloride (MgCl2) was added to adjust the total magnesium ion concentration to 4.5 mM. Reverse transcriptase and 0.05 U / μL HotStart Taq were then added. DNA polymerase was then added; subsequently, specific stem-loop primers, PCR primers, and TaqMan probes designed for miRNA-135a with a target sequence of 5′-GCUAUGGCUUUUUAUUCCUA-3′ and internal control miRNA-16-5p with a target sequence of 5′-UAGCAGCACGUAAAUAUUGGCG-3′ were added to bring the final concentration of each nucleic acid to 0.4 μM; finally, 2% glycerol and 0.2 mg / mL BSA were added as reaction stabilizers, and the volume was adjusted to the target volume with RNase-free water. After thorough mixing, the mixture was aliquoted as needed and stored at -20°C.

[0016] (13) Preparation of standards: Full-length miRNA-135a standard molecules were prepared by chemical synthesis and purified by high performance liquid chromatography; the standards were diluted to 10 with RNase-free water. 8 As a central storage solution, copies / μL were used to construct 10 copies / μL of the solution through a 10-fold serial dilution. 7 Up to 10 2 A series of standard samples in copies / μL; all dispensed samples should be stored at -80℃ for long-term storage.

[0017] (14) Assembly of the kit: Put the above-prepared capture plate, oral epithelial exosome blocking agent, saliva preservation solution, exosome lysis buffer, RT-qPCR premix and standard into the outer packaging box of the kit along with the product instructions, clearly label it and store it at -20℃.

[0018] Furthermore, the concentration of the mouse-derived anti-human L1CAM monoclonal antibody in step (1) is 5 μg / mL.

[0019] Furthermore, the concentration of the mouse-derived anti-human CD63 monoclonal antibody in step (1) is 2 μg / mL.

[0020] Furthermore, the final concentration of the reverse transcriptase in step (5) is 0.5 U / μL.

[0021] Furthermore, the final concentration of the HotStart Taq DNA polymerase in step (5) is 0.05 U / μL.

[0022] The present invention also provides the use of a salivary exosome detection kit for Alzheimer's disease auxiliary screening in the preparation of products for Alzheimer's disease auxiliary screening:

[0023] Furthermore, the sample of the product is human saliva;

[0024] Furthermore, the product enables auxiliary screening by detecting the expression level of miRNA-135a in neuronal-derived exosomes in human saliva samples.

[0025] The advantages of this invention are:

[0026] 1. This invention achieves, for the first time, high-purity enrichment of brain-derived exosomes in saliva samples by combining L1CAM / CD63 dual antibody synergistic capture with an oral-specific blocking agent.

[0027] 2. This invention develops a dedicated preservation solution system containing DTT, simultaneously solving the two major problems of easy degradation and uneven viscosity of salivary RNA, extending room temperature stability to 8 hours, and laying the foundation for standardized detection.

[0028] 3. The salivary exosome detection platform constructed in this invention can efficiently enrich neuronal-derived exosomes and accurately detect their contents. Under this platform, miRNA-135a showed excellent potential as an AD biomarker, with its expression upregulation (>2-fold) significantly better than that of the commonly used biomarker miR-132, highlighting the unique advantages of this platform in biomarker screening and validation. Attached Figure Description

[0029] Figure 1 This is a characterization of the capture efficiency of the salivary exosome detection kit prepared in this invention.

[0030] Figure 2 This is a characterization of the blocking efficiency of the kit of the present invention.

[0031] Figure 3 This is a characterization of the capture specificity of the kit of the present invention.

[0032] Figure 4 This is a characterization of the stability of saliva samples in the kit of the present invention.

[0033] Figure 5 This is a characterization of the screening performance of the kit of the present invention. Detailed Implementation

[0034] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0035] Example 1: Preparation of a salivary exosome detection kit

[0036] (1) Preparation of capture plate: Take a 96-well microplate and coat it with antibody by physical adsorption method; dilute mouse anti-human L1CAM monoclonal antibody to 5 μg / mL and mouse anti-human CD63 monoclonal antibody to 2 μg / mL with PBS buffer at pH 7.4, and add 50 μL of the above antibody solution to each well, and incubate at 4℃ for 12 h; discard the liquid in the well and wash 3 times with PBST buffer containing 0.05% Tween-20; then add 200 μL of PBS solution containing 2% BSA to each well for blocking, and incubate at 37℃ for 2 h; discard the blocking solution, air dry the plate under sterile conditions, vacuum seal it and store it at 4℃ for later use;

[0037] (2) Preparation of oral epithelial exosome blocking agent: Anti-MUC1 Fab fragment and anti-KRT19 Fab fragment were dissolved in PBS buffer at pH 7.2, and the concentration was adjusted to 50 μg / mL. They were mixed in equal volumes to form a ready-to-use mixed blocking agent solution. The solution was dispensed into 100 μL tubes and stored frozen at -20℃.

[0038] (3) Preparation of special saliva preservation solution: Dissolve 50 mM HEPES, 0.1 mM AEBSF, 1 U / μL SUPERase-In RNase inhibitor, 5 mM DTT and 0.5% benzyl sulfonyl fluoride in sterile, RNase-free ultrapure water in sequence. Adjust the pH of the mixture to 6.8 using hydrochloric acid or sodium hydroxide solution. After sterilization by filtration through a 0.22 μm filter membrane, dispense 1.5 mL into sterile centrifuge tubes and store at 4°C.

[0039] (4) Preparation of exosome lysis buffer: Take 1 mL of Triton X-100, 0.771 g of DTT and 0.186 g of EDTA-2Na, dissolve them thoroughly in 40 mL of 50 mM pH 7.6 Tris-HCl buffer and bring the volume up to 50 mL to obtain a lysis buffer containing 1% Triton X-100, 0.1 M DTT and 10 mM EDTA; mix well at room temperature and dispense into 1 mL tubes, and store at 4 °C in the dark.

[0040] (5) Preparation of RT-qPCR premix: Using 2×One-Step RT-qPCR Buffer as the basic reaction system, dNTPs were added sequentially to achieve a final concentration of 0.4 mM. MgCl2 was added to adjust the total magnesium ion concentration to 4.5 mM. Reverse transcriptase and HotStart Taq were added to a final concentration of 0.5 U / μL and 0.05 U / μL, respectively. DNA polymerase was then added; subsequently, specific stem-loop primers, PCR primers, and TaqMan probes designed for miRNA-135a with a target sequence of 5′-GCUAUGGCUUUUUAUUCCUA-3′ and internal control miRNA-16-5p with a target sequence of 5′-UAGCAGCACGUAAAUAUUGGCG-3′ were added to bring the final concentration of each nucleic acid to 0.4 μM; finally, 2% glycerol and 0.2 mg / mL BSA were added as reaction stabilizers, and the volume was adjusted to the target volume with RNase-free water. After thorough mixing, the mixture was aliquoted as needed and stored at -20°C.

[0041] (6) Preparation of standards: Full-length miRNA-135a standard molecules were prepared by chemical synthesis and purified by high performance liquid chromatography; the standards were diluted to 10 with RNase-free water. 8 As a central storage solution, copies / μL were used to construct 10 copies / μL of the solution through a 10-fold serial dilution. 7 Up to 10 2 A series of standard samples in copies / μL; all dispensed samples should be stored at -80℃ for long-term storage.

[0042] (7) Assembly of the kit: Put the above-prepared capture plate, oral epithelial exosome blocking agent, saliva preservation solution, exosome lysis buffer, RT-qPCR premix and standard into the outer packaging box of the kit along with the product instructions, clearly label it and store it at -20℃.

[0043] Comparative Example 1: Preparation of a salivary exosome detection kit without anti-L1CAM antibody

[0044] The difference between this comparative example and Example 1 is that in step (1), only mouse-derived anti-human CD63 monoclonal antibody was coated on the ELISA plate, and mouse-derived anti-human L1CAM monoclonal antibody was not coated.

[0045] Comparative Example 2: Preparation of a salivary exosome detection kit without oral epithelial exosome blocking agent

[0046] The difference between this comparative example and Example 1 is that no oral epithelial exosome blocking agent was added.

[0047] Comparative Example 3: Preparation of a salivary exosome detection kit based on a commercially available general-purpose preservation solution

[0048] The difference between this comparative example and Example 1 is that a commercially available general-purpose preservation solution was used instead of a specially prepared saliva preservation solution.

[0049] Comparative Example 4: Preparation of a salivary exosome detection kit without preservation solution

[0050] The difference between this comparative example and Example 1 is that no preservation solution was used.

[0051] Experimental Example 1: Characterization of the capture efficiency of the salivary exosome detection kit prepared in Example 1

[0052] To quantitatively evaluate the exosome capture efficiency of the kit prepared in Example 1, standard exosomes of known concentration were precisely added to saliva samples from healthy individuals as test samples. These samples were then divided into two parallel groups: the experimental group underwent exosome enrichment using the capture plate of the kit prepared in Example 1; the control group was processed using conventional ultracentrifugation. Finally, the exosomes obtained by both methods were quantitatively detected using a CD63 ELISA kit, and the capture efficiency of the two methods was directly compared by calculating the exosome recovery rate.

[0053] The results are as follows Figure 1 As shown, the average recovery rate of exosomes by the capture plate of the kit prepared in Example 1 reached 85.3%, significantly higher than the 56.6% recovery rate of the conventional ultracentrifugation method. This result fully demonstrates that the capture plate based on the double antibody sandwich method of the kit prepared in Example 1 has extremely high exosome capture efficiency.

[0054] Experimental Example 2: Characterization of the capture specificity of the salivary exosome detection kit prepared in Example 1

[0055] To verify the specificity of the kit prepared in Example 1 in capturing neuronal-derived exosomes and the effectiveness of the oral epithelial exosome blocking agent, enzyme-linked immunosorbent assay (ELISA) was used to quantitatively characterize the capture specificity and blocking agent efficiency of the kit. The same mixed saliva sample was treated with capture plates from Example 1 and Comparative Example 2, respectively. After exosome enrichment and washing, HRP-labeled mouse anti-human L1CAM, CD63, and MUC1 detection antibodies were added sequentially for incubation. After TMB substrate color development, the absorbance values ​​of each well were measured at 450 nm using an ELISA reader. The blocking efficiency was calculated using the formula: MUC1 removal rate (%) = [1 - (OD value of Example 1 group / OD value of Comparative Example 2 group)] × 100%. Furthermore, the capture purity of neuronal exosomes was assessed by the ratio of L1CAM to CD63 OD values. A higher ratio directly reflects the relative proportion of neuronal-derived exosomes in the total captured exosomes; a higher ratio indicates stronger capture specificity.

[0056] The results are shown in Table 1 and Figure 2 As shown, in terms of blocking efficiency, the signal intensity of the oral epithelial marker MUC1 in its captured product was significantly reduced compared to Comparative Example 2, with a calculated removal rate as high as 85.9%; in terms of capture specificity, the results are as follows: Figure 3 As shown, the L1CAM / CD63 signal ratio in Example 1 reached 0.78, significantly higher than the 0.41 in Comparative Example 2. These data fully demonstrate that the present invention can effectively remove approximately 86% of non-target exosomes using a specific blocking agent and significantly improve the relative capture purity of neuronal-derived exosomes by over 90%, laying a solid foundation for subsequent high-specificity biomarker analysis.

[0057] Table 1 shows the average OD values ​​of each antibody specifically captured using the salivary exosome detection kit prepared in Example 1.

[0058]

[0059] Experimental Example 3: Characterization of the stability of the salivary exosome detection kit prepared in Example 1 using saliva samples.

[0060] To evaluate the stability of the saliva preservation solution for the kit prepared in Example 1, saliva samples from Alzheimer's patients and healthy controls were selected and divided into three groups: Group A used the kit prepared in Example 1, Group B used the kit prepared in Comparative Example 3, and Group C used the kit prepared in Comparative Example 4. All samples were stored at room temperature (0, 2, 4, 8 h) and 4°C (24, 48, 72 h), respectively, and the Ct value of miRNA-135a was detected by RT-qPCR at each predetermined time point to assess its stability.

[0061] The results are as follows Figure 4As shown, in group A, the Ct values ​​of miRNA-135a changed by less than 1.5 under the conditions of 8 hours at room temperature and 72 hours at 4°C, indicating that its content remained highly stable. In contrast, the Ct values ​​of group B changed significantly under the same conditions, while in group C, the Ct value was found to be drastically delayed after 2 hours at room temperature. Therefore, the saliva preservation solution of the kit prepared in Example 1 can effectively resist enzymatic degradation in saliva and maintain the long-term stability of miRNA.

[0062] Experimental Example 4: Screening performance characterization of the salivary exosome detection kit prepared in Example 1

[0063] To evaluate the kit's ability to distinguish between AD patients and healthy individuals, we collected saliva samples from 15 clinically diagnosed AD patients and 15 age-matched healthy controls. All samples were uniformly processed using the kit prepared in Example 1: neuronal-derived and total exosomes in the saliva were specifically captured by the L1CAM and CD63 antibodies coated on the kit. After lysis, the miRNA-135a content was detected using the RT-qPCR premix provided in the kit, and normalized to the internal reference gene miRNA-16-5p. Finally, the ΔCt value (ΔCt = Ct_miRNA-135a - Ct_miRNA-16-5p) was calculated. The ΔCt value is inversely proportional to the relative expression level of the target gene; that is, the smaller the ΔCt value, the higher the expression level of miRNA-135a.

[0064] To make a direct comparison between groups, the ΔCt value is expressed using Formula 2. -ΔCt The data were converted to relative expression levels for statistical analysis. The results are as follows: Figure 5 As shown, the relative expression level of miRNA-135a in salivary exosomes of AD patients showed a highly significant upregulation trend compared with that of healthy controls, with the average expression level in the AD group (2... -ΔCt The level was 2.1 times that of the control group. This indicates that the kit prepared in Example 1 can efficiently and specifically capture and detect changes in AD-related biomarkers from saliva samples, and the discovered significantly high expression pattern of miRNA-135a is highly correlated with the pathological progression of AD.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A salivary exosome detection kit for auxiliary screening of Alzheimer's disease, characterized in that, The kit contains a capture plate, an oral epithelial exosome blocking agent, a special saliva preservation solution, an exosome lysis buffer, an RT-qPCR premix, and standards. The capture plate is coated with mouse-derived anti-human L1CAM monoclonal antibody and mouse-derived anti-human CD63 monoclonal antibody. The oral epithelial exosome blocking agent comprises an anti-MUC1Fab fragment and an anti-KRT19Fab fragment; The special saliva preservation solution contains a protease inhibitor, an RNase inhibitor, and a reducing agent; The RT-qPCR premix contains specific primers and probes for detecting miRNA-135a and internal reference miRNA.

2. A method for preparing a salivary exosome detection kit for auxiliary screening of Alzheimer's disease, characterized in that, The preparation method includes the following steps: (1) Preparation of capture plate: Antibody coating was performed by physical adsorption method. Diluted mouse anti-human L1CAM monoclonal antibody and mouse anti-human CD63 monoclonal antibody were added to 96-well plates and incubated at 4°C for 12 h. Then, the plates were washed with PBST buffer containing 0.05% Tween-20. Subsequently, PBS solution containing 2% BSA was added to each well for blocking and incubated at 37°C for 2 h. The blocking solution was discarded, the plates were dried under sterile conditions, vacuum sealed and stored at 4°C for later use. (2) Preparation of oral epithelial exosome blocking agent: The anti-MUC1Fab fragment and the anti-KRT19Fab fragment were dissolved in PBS buffer, and the mixture was mixed in equal volumes to form a ready-to-use mixed blocking agent solution, which was then aliquoted and frozen for storage. (3) Preparation of special saliva preservation solution: Dissolve 50 mM HEPES, 0.1 mM MAEBSF, 1 U / μL SUPERase-In RNase inhibitor, 5 mM DTT and 0.5% benzyl sulfonyl fluoride in sterile RNase-free ultrapure water in sequence, and adjust the pH to 6.8; filter the solution through a 0.22 μm filter membrane for sterilization and then aliquot it into sterile centrifuge tubes for storage; (4) Preparation of exosome lysis buffer: Take 1 mL of Triton X-100, 0.771 g of DTT and 0.186 g of EDTA-2Na, dissolve them thoroughly in 50 mM Tris-HCl buffer at pH 7.6 and bring the volume up to 50 mL to obtain a lysis buffer containing 1% Triton X-100, 0.1 M DTT and 10 mM EDTA; mix well at room temperature, aliquot and store in the dark. (5) Preparation of RT-qPCR premix: 2×One-Step RT-qPCR Buffer was used as the basic reaction system. dNTPs were added sequentially to bring the final concentration to 0.4 mM. MgCl2 was added to adjust the total final concentration of magnesium ions to 4.5 mM. Reverse transcriptase and HotStart Taq DNA polymerase were added. Then, specific stem-loop primers, PCR primers and TaqMan probes designed for miRNA-135a with target sequence 5′-GCUAUGGCUUUUUAUUCCUA-3′ and internal control miRNA-16-5p with target sequence 5′-UAGCAGCACGUAAAUAUUGGCG-3′ were added to bring the final concentration of each nucleic acid to 0.4 μM. Finally, 2% glycerol and 0.2 mg / mL BSA were added as reaction stabilizers. The mixture was then diluted with RNase-free water, aliquoted and frozen for storage. (6) Preparation of standards: Full-length miRNA-135a standard molecules were prepared by chemical synthesis and purified by high performance liquid chromatography; Dilute the standard to 10% using RNase-free water. 8 As a central storage solution, copies / μL were used to construct 10 copies / μL of the solution through a 10-fold serial dilution. 7 Up to 10 2 A series of standard samples in copies / μL; all dispensed samples should be stored at -80℃ for long-term storage. (7) Assembly of the kit: Put the above-prepared capture plate, oral epithelial exosome blocking agent, saliva preservation solution, exosome lysis buffer, RT-qPCR premix and standard into the outer packaging box of the kit along with the product instructions, clearly label it and store it at -20℃.

3. The method for preparing the reagent kit according to claim 2, characterized in that, The concentration of the mouse-derived anti-human L1CAM monoclonal antibody in step (1) is 5 μg / mL.

4. The method for preparing the reagent kit according to claim 2, characterized in that, The concentration of the mouse-derived anti-human CD63 monoclonal antibody in step (1) is 2 μg / mL.

5. The method for preparing the reagent kit according to claim 2, characterized in that, The final concentration of the reverse transcriptase in step (5) is 0.5 U / μL.

6. The method for preparing the reagent kit according to claim 2, characterized in that, The final concentration of HotStartTaq DNA polymerase in step (5) is 0.05 U / μL.

7. Use of the kit as described in any one of claims 1-7 in the preparation of a product for auxiliary screening of Alzheimer's disease.

8. The use as described in claim 7, characterized in that, The sample of the product is human saliva.

9. The use as described in claim 7, characterized in that, The product enables auxiliary screening by detecting the expression level of miRNA-135a in neuronal-derived exosomes in human saliva samples.

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