Alzheimer's disease screening marker, antibody, kit and method

Abstract

The invention belongs to the technical field of biotechnology and medical detection, and particularly discloses an Alzheimer's disease screening marker, antibody, kit and method, the screening marker is SRC protein in plasma exosome; the antibody can specifically recognize the SRC protein in the plasma exosome; the Alzheimer's disease screening method comprises the following steps: acquiring a plasma sample of a to-be-detected individual; extracting an exosome from the plasma sample; detecting the expression level of the SRC protein in the exosome by using an antibody; and comparing the expression level with a reference value, and if the expression level is higher than the reference value, prompting that the individual has the risk of Alzheimer's disease. The kit can noninvasively perform early screening on the Alzheimer's disease, is convenient to operate and low in cost, and has high sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and medical testing technology, specifically relating to an Alzheimer's disease screening biomarker, antibody, reagent kit, and method. Background Technology

[0002] Alzheimer's disease (AD) is a progressive, irreversible neurodegenerative disease characterized by memory loss, cognitive decline, and behavioral disturbances. According to the World Health Organization, AD has become one of the leading causes of disability and death among the elderly worldwide, imposing a heavy economic and care burden on patients' families and society. Currently, there is no effective cure for AD; therefore, early screening and intervention are key strategies for slowing disease progression and improving patients' quality of life.

[0003] Currently, clinical diagnosis of Alzheimer's disease (AD) mainly relies on clinical symptom assessment, neuropsychological scale testing, and imaging examinations. However, these methods have significant limitations. Early symptoms of AD are insidious and similar to normal aging or mild cognitive impairment, making accurate differentiation difficult. By the time of diagnosis, patients are often in the middle or late stages, missing the optimal intervention window. Imaging examinations are expensive and complex to operate, and their sensitivity in detecting early, subtle lesions is limited, hindering large-scale screening. Neuropsychological scale testing is highly subjective, and results may vary among different assessors.

[0004] Therefore, developing a non-invasive, easy-to-operate, low-cost, highly sensitive and specific early screening method is a technical problem that urgently needs to be solved in the medical field. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide an Alzheimer's disease screening biomarker, antibody, reagent kit and method. This invention can perform early screening of Alzheimer's disease non-invasively, and is convenient to operate, low in cost, and has high sensitivity and specificity.

[0006] The technical solution of this invention is implemented as follows:

[0007] An Alzheimer's disease screening biomarker, wherein the screening biomarker is SRC protein in plasma exosomes.

[0008] This invention provides an antibody that specifically recognizes the SRC protein in plasma exosomes as described above.

[0009] Furthermore, the antibody is a polyclonal antibody or a monoclonal antibody.

[0010] Furthermore, the antibody is prepared by immunizing animals with an immunogen, wherein the immunogen comprises a partial sequence of the SRC protein, the amino acid sequence of which is shown in SEQ ID NO.1, specifically:

[0011] MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQTGYIPSNY VAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLG.

[0012] The present invention also provides an Alzheimer's disease screening kit, the kit comprising the antibodies described above.

[0013] Furthermore, the kit also includes reagents for plasma secretion extraction and / or auxiliary reagents for immunoassay.

[0014] This invention also provides a method for screening Alzheimer's disease, specifically including the following steps:

[0015] S1: Obtain a plasma sample from the individual to be tested;

[0016] S2: Extracting exosomes from plasma samples;

[0017] S3: Using the antibody described above, detect the expression level of SRC protein in the exosomes;

[0018] S4: Compare the expression level with the reference value. If the expression level is greater than or equal to the reference value, it indicates that the individual has a risk of having Alzheimer's disease.

[0019] Furthermore, in step S3, the detection method is Western blotting, enzyme-linked immunosorbent assay (ELISA), or chemiluminescent immunoassay.

[0020] The application of the screening biomarkers described above in the preparation of Alzheimer's disease screening agents or kits.

[0021] The aforementioned antibodies are used in the preparation of Alzheimer's disease screening agents or kits.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention creatively discovers a strong correlation between SRC protein in plasma exosomes and Alzheimer's disease (AD). This discovery provides a novel liquid biopsy target with high application potential for early screening of AD. Moreover, the SRC protein biomarker is derived from plasma exosomes, which is easy to obtain non-invasively, overcoming the limitations of invasiveness in traditional cerebrospinal fluid testing and high cost of imaging examinations.

[0024] 2. This invention successfully expressed and purified recombinant SRC protein through genetic engineering, producing a high-titer, high-specificity anti-SRC polyclonal antibody (titer ≥ 1:80000). This antibody can accurately recognize SRC protein in natural plasma exosomal tissues, providing a reliable core raw material for the accurate and stable detection of biomarkers.

[0025] 3. This invention establishes a complete immunological detection method (such as WB and ELISA) based on biomarkers and antibodies, and provides the composition scheme of the kit. This method is simple, rapid, and relatively low-cost, making it easy to implement in general laboratories and clinical testing departments. Preliminary clinical validation shows that this system can effectively distinguish AD patients from healthy controls and establish a preliminary diagnostic reference range, laying a solid technical foundation for developing an in vitro diagnostic kit suitable for early screening of large populations. Attached Figure Description

[0026] Figure 1 Transmission electron micrograph of plasma exosomes in AD.

[0027] Figure 2 -WB results of exosome membrane proteins and cellular calcinin in healthy controls.

[0028] Figure 3 -Differential protein heatmap of plasma proteins in NC and AD.

[0029] Figure 4 -Graph of differential protein antigenicity analysis.

[0030] Figure 5 - Target fragment, enzyme digestion and recovery products, colony PCR identification diagram, and recombinant expression plasmid identification diagram.

[0031] Figure 6 - SDS diagram of induced expression under different induction conditions.

[0032] Figure 7 SDS diagram of purified pColdII-SRC truncated recombinant protein.

[0033] Figure 8 -WB identification diagram of plasma exosomal proteins.

[0034] Figure 9 - ELISA standard curve.

[0035] Figure 10 - Distribution of SRC protein concentration in clinical samples.

[0036] Figure 11 -Interval estimation plot of clinical sample data. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0038] I. Extraction and Identification of Plasma Exosomes

[0039] Plasma exosomes are disc-shaped vesicles with a diameter of 40-100 nanometers. They are released into the extracellular matrix after the fusion of intracellular multivesicles with the cell membrane. They are rich in cholesterol, sphingomyelin and various proteins (such as tetraspan membrane proteins CD63, CD81 and annexin). Their shape is mainly spherical or disc-shaped and they have a lipid bilayer membrane structure.

[0040] The specific steps are as follows:

[0041] Step S1: Extract plasma exosomes.

[0042] 10 mL of anticoagulated peripheral blood was collected from multiple healthy controls (NC) and Alzheimer's disease patients (AD). Exosomes were processed by ultracentrifugation as follows: (1) The anticoagulated peripheral blood was mixed with an equal volume of 1×PBS buffer, centrifuged at 10000×g at 4℃ for 30 min to remove cell debris and obtain exosomes; (2) The exosomes were transferred to an ultracentrifuge tube, centrifuged at 100000×g at 4℃ for 120 min to obtain the initial exosome precipitate; (3) The initial exosome precipitate was resuspended with 1×PBS buffer, centrifuged again at 100000×g at 4℃ for 120 min, and then washed to obtain the exosome precipitate; (4) The exosome precipitate was resuspended with 100 μL of 1×PBS buffer to obtain the purified plasma exosome suspension, which was then aliquoted and stored at -80℃.

[0043] Step S2: Identification of plasma exosomes

[0044] Transmission electron microscopy (TEM) observation: 10 µL of plasma exosome suspension was dropped onto the copper grid of the TEM microscope and incubated for 5 min. Excess liquid was absorbed with filter paper. A 1% phosphotungstic acid solution was added for negative staining for 3 min, and the mixture was allowed to air dry before observation under a TEM microscope. The vesicles of the plasma exosomes all exhibited a typical cup-shaped morphology, consistent with the characteristics of exosomes. The TEM image of plasma exosomes from AD patients is shown below. Figure 1 As shown.

[0045] Western blotting (WB) for protein immunoblotting: 20 μL of plasma exosomes quantified by the BCA method were mixed with 5 µL of SDS-PAGE 5× loading buffer (Sangon Biotech-C516031-0005), and incubated at 95°C for 10 minutes for heat denaturation. The mixture was then centrifuged at 10000×g at 4°C for 15 minutes, and the supernatant was collected. The supernatant was accurately loaded into the wells of a pre-prepared SDS-PAGE protein gel. Electrophoresis was performed at a constant current of 18 mA for 90 minutes until the bromophenol blue indicator migrated to the bottom of the gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using a wet transfer method at a constant current of 68 mA for 70 minutes. After transfer, the membrane was immersed in 5% skim milk blocking buffer and gently shaken at room temperature for 1.5 hours to block non-specific binding. After blocking, the membrane was washed three times with TBST buffer, 5 min each time. Then, the membrane was placed in rabbit polyclonal antibody (CD63 Rabbit Polyclonal Antibody) diluted 1:1000 with primary antibody dilution buffer and incubated overnight (14 h) at 4°C with gentle shaking. After incubation, the membrane was washed three times thoroughly with TBST; then, horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) secondary antibody diluted 1:3000 with TBST was added, and the membrane was incubated at room temperature with shaking for 2 h. After washing the membrane again with TBST, the membrane was incubated uniformly with the chemiluminescent substrate BeyoECL Moon working solution for 1 min, and then the signal image was acquired using an E-BLOT contact chemiluminescence imager. After CD63 detection, to detect the negative marker on the same membrane, the membrane was treated with Western primary and secondary antibody removal buffer for 15 min to remove the bound antibodies. After TBST washing, the membrane was blocked, incubated with primary antibody (using 1:1000 diluted Calnexin mouse monoclonal antibody, overnight at 4°C), washed, incubated with secondary antibody (using 1:3000 diluted HRP-labeled goat anti-mouse IgG (H+L), 2 h at room temperature), and finally washed. Finally, the membrane was developed and imaged again using chemiluminescent substrate, thus enabling the joint identification of exosome positive marker (CD63) and negative marker (Calnexin) on the same membrane.

[0046] Figure 2 This is a Western blot (WB) image of exosome membrane proteins and cellular calcinin in healthy controls. Figure 2 (a) Results of WB analysis of exosome membrane proteins. Figure 2(b) is a diagram showing the cellular calcinin results, where NC represents 293T cell exosomes and exo represents plasma exosomes. Figure 2 (a) As can be seen, clear CD63 protein bands appeared at approximately 50 kDa in both lanes 1 and 2, indicating the presence of exosomes in the plasma samples. Figure 2 (b) It can be seen that a clear Calnexin protein band appears at 90 kDa in lane 1, while no Calnexin protein signal is detected in lane 2, indicating that the present invention extracts high-purity plasma exosomes.

[0047] II. Screening of differentially expressed proteins in plasma exosomals and determination of SRC markers

[0048] The specific method for screening differentially expressed proteins in plasma exosomals based on proteomics is as follows:

[0049] (1) Take three plasma exosome samples from the AD group and the NC group respectively;

[0050] (2) Add DTT at a concentration of 5 mM, incubate at 37°C for 1 h, then restore to room temperature, add iodoacetamide at a concentration of 10 mM, and incubate at room temperature in the dark for 45 min.

[0051] (3) Dilute the sample 4 times with 25 mM ammonium bicarbonate, add trypsin at a protein:trypsin mass ratio of 50:1, incubate overnight at 37°C, and add formic acid the next day to stop the enzyme digestion.

[0052] (4) Desalting was performed using a C18 desalting column. The column was activated with 100% acetonitrile, equilibrated with 0.1% formic acid, loaded with the sample, washed, and finally eluted with 70% acetonitrile and lyophilized. Then, LC-MS mass spectrometry analysis was performed. The mobile phase was prepared, the lyophilized powder was dissolved and centrifuged, 1 µg of sample was injected, and mass spectrometry was performed according to the set parameters to generate raw data.

[0053] The raw mass spectrometry data were processed using MaxQuant software, and the UniProt Human protein database was searched. Quantitative analysis was performed on the identified proteins, with the screening criteria being: a fold change ≥ 2.0 between the AD group and the NC group, and a statistically significant p-value < 0.05.

[0054] Differential protein heatmap of plasma proteins between NC and AD as shown in Figure Figure 3 As shown, SRC protein (tyrosine kinase) was significantly upregulated in plasma exosomes of AD patients and was identified as a candidate biomarker. Further analysis of the antigenicity of SRC protein was performed using bioinformatics tools (such as AntibodyEpitope Prediction). Figure 4 ), Figure 4The region within the green box exhibits good antigenicity and hydrophilicity, making it the target sequence for subsequent recombinant protein expression and antibody preparation.

[0055] III. Expression, purification, and preparation of polyclonal antibodies for SRC recombinant proteins

[0056] 3.1 Construction of SRC truncated gene expression vector

[0057] Based on immunogenicity analysis, a partial SRC gene fragment (its amino acid sequence is shown in SEQ ID NO.1) was selected for expression. Cloning primers F1 and F2 containing Kpn I and EcoRI restriction sites were designed. Using pET-28a(+)-SRC plasmid as a template, KOD enzyme PCR was performed for amplification (reaction system shown in Table 1, reaction conditions shown in Table 2). The target fragment was recovered by agarose gel electrophoresis. Next, pCold II plasmid was double-digested with Kpn I and EcoRI (reaction system shown in Table 3), digested at 37℃ for 90 min, and inactivated at 80℃ for 20 min. The product was recovered. The target fragment and the recovered product were then mixed in a seamless cloning system (reaction conditions shown in Table 4), incubated at 50℃ for 20 min, transformed into competent cells, and plated to obtain single colonies of the recombinant expression plasmid pCold II-SRC. After culture expansion by shaking, the plasmid was extracted and verified by restriction enzyme digestion.

[0058] The amino acid sequence corresponding to SEQ ID NO.1 is as follows:

[0059] MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQTGYIPSNY VAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLG.

[0060] The sequence of primer F1 is:

[0061] ATATGGAGCTCGGTACCatgggtagcaacaaatctaaaccga.

[0062] The sequence of primer F2 is:

[0063] AGGTCGACAAGCTTGAATTCacccagtttaacttccagacgc.

[0064]

[0065]

[0066]

[0067]

[0068] The target fragment, enzyme digestion and recovery products, colony PCR identification image, and recombinant expression plasmid identification image are shown below. Figure 5 As shown, and corresponding to 5a, 5b, 5c and 5d respectively. Figure 5 In a, M is the DNA Marker, and 1-2 are electrophoresis diagrams of PCR products; Figure 5 In b, M represents DNA Marker, 1 represents pCold II plasmid, and 2 represents the gel recovery product after double enzyme digestion of pCold II plasmid. Figure 5 In c, M stands for DNA Marker, 1 is the colony PCR control, and 2-5 are identification diagrams of pCold II-SRC truncated colonies. Figure 5 In d, M stands for DNA Marker; 1 is a truncated recombinant plasmid pCold II-SRC; 2 is pCold II plasmid; and 3 is a truncated recombinant expression plasmid pCold II-SRC digested with a single enzyme.

[0069] 3.2 Induction and Condition Optimization of Recombinant Protein Expression

[0070] The pCold II-SRC truncated plasmid was transformed into E. coli competent cells BL21(DE3). To obtain soluble expression, the induction conditions (IPTG concentration, induction temperature and time) were systematically optimized. Figure 6 SDS plots of induced expression under different induction conditions, Figure 6 The induction conditions corresponding to a-6i are shown in Table 5, and Figure 6 In a-6h, M is the protein marker, 1 is the whole bacterial culture before induction, 2 is the whole bacterial culture after induction, 3 is the ultrasonically disrupted and slurried culture after induction, and 4 is the ultrasonically disrupted and precipitated culture after induction. Figure 6 In i, M represents the protein marker, 1 represents the whole bacterial culture before induction, 2 represents the whole bacterial culture after induction, 3 represents the ultrasonically disrupted and precipitated culture after induction, and 4 represents the ultrasonically disrupted and sizing culture after induction; from Figure 6 It can be seen that under the conditions of IPTG concentration of 0.5 mM, induction temperature of 16℃ and induction time of 12 hours, the target protein (predicted molecular weight of about 30 kDa) showed the highest soluble expression level in the supernatant. Therefore, these conditions were used as the optimal induction conditions for large-scale expression.

[0071]

[0072] 3.3 Purification of recombinant proteins

[0073] After high-level expression under optimal induction conditions, bacterial cells were collected, sonicated, and centrifuged to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter and purified by nickel column affinity chromatography using an AKTA system. Gradient elution was performed using elution buffers containing 10 mM, 50 mM, 100 mM, 200 mM, and 500 mM imidazole, and the protein solution at the elution peaks was collected. SDS-PAGE analysis was then performed, and the analysis results are shown in the figure below. Figure 7 As shown in the figure, M represents the protein marker; 1 represents pre-column protein slurry; 2 represents column breakthrough buffer; 3 represents 10 mM imidazole elution buffer; 4 represents 20 mM imidazole elution buffer; 5 represents 50 mM imidazole elution buffer; 6 represents 100 mM imidazole elution buffer; 7 represents 200 mM imidazole elution buffer; and 8 represents 500 mM imidazole elution buffer. Figure 7 As can be seen, the amount of target protein before induction was significantly less than that after induction, indicating successful induction of the target protein. Simultaneously, no target protein was found in the column permeate, while the target protein was clearly present in the elution peaks at 200 mM and 500 mM imidazole, indicating successful purification and elution of the target protein. The high-purity protein fraction was collected, dialyzed to remove imidazole, and concentrated to obtain purified recombinant SRC truncated protein for subsequent immunization.

[0074] 3.4 Preparation and purification of polyclonal antibodies

[0075] Two New Zealand white rabbits were immunized using purified recombinant SRC protein as an immunogen. The immunization procedure was as follows: On day 1, the antigen and Freund's complete adjuvant emulsion were injected subcutaneously at multiple sites on the back of the neck to immunize the two rabbits. Booster immunizations were performed on days 15, 29, and 43 using the antigen and Freund's incomplete adjuvant emulsion in the same manner. On day 53, blood was collected from the carotid artery, exsanguinated overnight at 4°C, and then centrifuged to collect plasma. The plasma was purified using an antigen affinity column to obtain high-purity anti-SRC polyclonal antibodies, which were stored at -20°C. The antibodies obtained from immunization of the two New Zealand white rabbits were PcAb-R1 and PcAb-R2, respectively.

[0076] IV. Identification of the titer and specificity of anti-SRC polyclonal antibodies

[0077] 4.1 Antibody titer detection

[0078] The titer of polyclonal antibodies was detected by enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[0079] (1) Dilute the antigen to 1 μg / mL with carbonate buffer and add 100 μL to each well of a 96-well microplate. Then incubate the microplate at 4°C overnight.

[0080] (2) Discard the coating solution, add 200 μL of blocking solution to each well, and place at 37°C for 1.5 h.

[0081] (3) Discard the blocking solution and perform a series of serial dilutions of the purified anti-SRC polyclonal antibodies (PcAb-R1, PcAb-R2) with the sample dilution solution (starting from 1:1250 and diluting sequentially to 1:80000). Add 100 μL of the diluted antibody sample to each well and place at 37°C for 1 h. At the same time, set up a negative control well and add 100 μL of IgG diluted 1250 times with normal rabbit IgG to the control well.

[0082] (4) Discard the liquid in the wells, wash the plate 5 times with washing solution and pat dry. Add 100 μL of secondary antibody to each well and place at 37°C for 30 min.

[0083] (5) Discard the secondary antibody, wash the plate 5 times with washing solution and pat dry; add 100 μL of TMB chromogenic substrate to each well and place at 37℃ for 15 min; after the sample wells show obvious blue color, add 50 μL of 2M sulfuric acid stop solution to each well to stop the enzymatic reaction, and the color will easily change from blue to yellow.

[0084] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 450 nm.

[0085] The antibody titer test results are shown in Table 6.

[0086]

[0087] Potency determination criteria: usually based on the OD of the sample well. 450 Value (S) and OD of negative control well 450 The highest antibody dilution factor at which the ratio of the antibody titer (S / N) is ≥2.1 is defined as the antibody titer. As can be seen from the table above, when the antibody is diluted to 1:80000, its S / N ratio is still greater than 2.1, indicating that the prepared anti-SRC polyclonal antibody has a titer of not less than 1:80000 and has high reactivity.

[0088] 4.2 Antibody Specificity Validation

[0089] The analysis was performed using Western blotting, and included the following steps:

[0090] (1) Take 20 μL of plasma exosome protein samples from Alzheimer's disease patients (AD group) and healthy individuals (NC group), respectively, and mix them with 5 μL of SDS-PAGE 5× loading buffer (Sangon Biotech-C516031-0005). Then place the mixture in a metal bath at 95℃ for 10 min for heat denaturation, followed by an ice bath for 5 min. Then centrifuge the samples at 10000×g, 4℃ for 15 min to obtain the supernatant. Load the supernatant onto an SDS-PAGE protein gel (Beyotime-P0012A) and electrophores it at a constant current of 18 mA for 90 min to separate the proteins according to their molecular weight.

[0091] (2) After electrophoresis, the proteins separated in the protein gel were transferred to an NC membrane under a constant current of 68 mA for 70 min. After the transfer, the membrane was immersed in 5% skim milk blocking solution for 1.5 h.

[0092] (3) The blocked membrane was washed with 5% TBST, and then the membrane was incubated overnight at 4°C for 14 h with a primary antibody mixture diluted at a ratio of 1:1000.

[0093] (4) Wash with 5% TBST, then add a secondary antibody mixture containing Goat Anti-Rabbit IgG (H+L) diluted 1:3000 with TBST, and incubate at room temperature for 2 h. Wash again with 5% TBST. Incubate the membrane with BeyoECL Moon (Beyotime-P0018FS) chromogenic solution for 1 min. Then image using an E-BLOT (eBLOT-Touch Imager XLi) contact chemiluminescence imager.

[0094] Western blot (WB) identification of plasma exosome proteins is shown below. Figure 8 As shown, with exosomal membrane protein CD63 as a control, SRC was detected in both the NC and AD groups, but the amount of SRC in the NC group was significantly less than that in the AD group, indicating that the antibody can specifically recognize SRC, and there is a significant difference in the expression level of SRC between the AD and NC groups.

[0095] V. Clinical validation and diagnostic efficacy evaluation of SRC as an AD screening biomarker

[0096] 5.1 Clinical Sample Testing

[0097] Clinical sample validation was performed using an ELISA (enzyme-linked immunosorbent assay). Recombinant SRC protein was used as a standard and serially diluted with carbonate buffer (CBS) to obtain a standard curve (see [link to ELISA]). Figure 9The gradient dilution concentrations were: 1000, 500, 250, 125, 62.5, 32.25, 15.625, and 7.8125 pg / mL.

[0098] To expand the clinical sample size (10 cases in the AD group and 17 cases in the NC group), plasma exosomes were extracted. Each plasma sample was diluted 50-fold with carbonate buffer as an antigen. 100 μL of the diluted plasma sample was then added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The plates were then washed with PBST and blotted dry. 200 μL of blocking buffer containing 1% BSA was added to each well, and the plates were blocked at 37°C for 2 h. The plates were then washed with PBST and blotted dry. Anti-SRC monoclonal antibody was diluted 1:1000 using antibody dilution buffer, and 100 μL was added to each well. The plates were incubated at 37°C for 2 h. Discard the primary antibody, wash the plate with PBST and blot dry. Add 100 μL of a secondary antibody mixture of Goat Anti-Rabbit IgG (H+L) diluted 1:3000 with PBST to each well, and incubate at 37°C for 2 h. Discard the secondary antibody, wash the plate with PBST and blot dry. Add 100 μL of TMB chromogenic solution to each well, and incubate at 37°C in the dark for 15 min. Finally, add 50 μL of 2M sulfuric acid stop solution to each well to terminate the reaction. Measure the absorbance of each well at 450 nm using a microplate reader. Multiple replicates were performed for each sample, and the average absorbance value of each sample was taken.

[0099] Substitute the absorbance value of each sample into y = 5.227.2x 2 -227.68x-153.86, thus obtaining the concentration of SRC protein in each plasma exosome sample, the results are as follows. Figure 10 As shown in the figure, there is a significant difference between the AD group and the NC control group (p<0.01), indicating that SRC can screen for early Alzheimer's disease using enzyme-linked immunosorbent assay (ELISA).

[0100] The interval estimation plot of clinical sample data is shown in Figure 11. OD of the NC group samples 450 The mean was 0.317, the standard deviation was 0.035, and the OD of the AD group samples was... 450 The mean was 0.395, and the standard deviation was 0.063. When the OD of the sample to be tested... 450 A value greater than 0.40 can be considered a high risk of AD; when OD 450 A value less than 0.32 indicates low risk of AD, while a value between the two indicates suspected AD.

[0101] Finally, it should be noted that the above embodiments of the present invention are merely illustrative examples and not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. An Alzheimer's disease screening biomarker, characterized in that, The screening biomarker is the SRC protein in plasma exosomes.

2. An antibody that specifically recognizes the SRC protein in plasma exosomes of claim 1.

3. The antibody according to claim 2, characterized in that, The antibody is a polyclonal antibody or a monoclonal antibody.

4. An antibody according to claim 3, characterized in that, The antibody is prepared by immunizing animals with an immunogen, the immunogen comprising a partial sequence of the SRC protein, the specific amino acid sequence of which is shown in SEQ ID NO.

1.

5. An Alzheimer's disease screening kit, characterized in that, The kit comprises the antibody as described in any one of claims 2-4.

6. The Alzheimer's disease screening kit according to claim 5, characterized in that, It also includes reagents for the extraction of plasma secretions and / or auxiliary reagents for immunoassay.

7. A method for screening Alzheimer's disease, characterized in that, Specifically, the following steps are included: S1: Obtain a plasma sample from the individual to be tested; S2: Extracting exosomes from plasma samples; S3: Using the antibody according to any one of claims 2-4, detect the expression level of SRC protein in the exosomes; S4: Compare the expression level with the reference value. If the expression level is greater than or equal to the reference value, it indicates that the individual has a risk of having Alzheimer's disease.

8. The method for screening Alzheimer's disease according to claim 7, characterized in that, In step S3, the detection method is Western blotting, enzyme-linked immunosorbent assay (ELISA), or chemiluminescent immunoassay.

9. The use of the screening biomarker of claim 1 in the preparation of Alzheimer's disease screening agents or kits.

10. The use of the antibody according to any one of claims 2-4 in the preparation of Alzheimer's disease screening agents or kits.

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