Method for auxiliary assessment of early risk of Alzheimer's disease based on GALNT10 detection

Abstract

The invention discloses a method for auxiliary assessment of early risk of Alzheimer's disease based on GALNT10 detection. The method is an in-vitro non-diagnostic analysis method, and comprises the following steps: detecting the biomarker level of GALNT10 in a biological sample of a subject, and obtaining information for evaluating the AD early risk of the subject based on a detection result. The biomarker comprises a GALNT10 genotype, a GALNT10 mRNA (messenger Ribonucleic Acid) expression level or a GALNT10 protein concentration. When the risk assessment is realized by quantitatively detecting the concentration of the GALNT10 protein in the blood plasma, an immunodetection kit containing a capture antibody and a detection antibody aiming at different epitopes of the GALNT10 protein can be specifically adopted. Objective information for risk judgment is obtained by detecting the protein concentration, genotype or mRNA level of GALNT10 in a biological sample of a subject, early-stage and non-invasive monitoring of an AD upstream pathological mechanism is achieved, and a new strategy is provided for early warning of diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a method for assisting in the assessment of early risk of Alzheimer's disease based on GALNT10 detection. Background Technology

[0002] Early diagnosis and the identification of effective therapeutic targets for Alzheimer's disease (AD) remain significant challenges. Current diagnostic methods rely on downstream pathological markers such as Aβ and Tau, failing to capture upstream molecular events at the onset of the disease. In recent years, research has begun to focus on the roles of neuroinflammation, metabolic disorders, and extracellular matrix (ECM) alterations in the early stages of AD; however, reliable biomarkers that directly link specific molecules to clear spatial pathologies and specific signaling pathways (such as the EGFR pathway) are still lacking. The peptide N-acetylgalactosamine transferase 10 (GALNT10) is a member of the enzyme family responsible for initiating protein O-glycosylation. O-glycosylation plays a crucial role in protein function, stability, and intercellular communication. Currently, research on GALNT10 is extremely limited and mainly scattered across certain cancer studies; its expression, function, and clinical significance in the central nervous system, particularly in Alzheimer's disease, remain largely unexplored. No existing literature or patents reveal any association between GALNT10 and AD. Therefore, the following gaps and deficiencies exist in the current technology: Insufficient understanding of biomarkers for upstream mechanisms of AD: There is a lack of specific liquid biopsy biomarkers that can reflect early EGFR pathway-mediated metabolic disorders and ECM dynamics in AD.

[0003] The function of GALNT10 in AD is unknown: the mechanism of action of GALNT10 in the occurrence and development of AD, the relationship between its expression changes and AD pathology, and its clinical application value have not been fully revealed.

[0004] A lack of understanding of molecular interaction networks: existing biomarkers are mostly isolated, and there is a lack of systematic understanding and utilization of key molecular interactions in the AD pathological microenvironment (such as the interaction between EFEMP1 and GALNT10).

[0005] The difficulty in solving these problems lies in identifying key molecules like GALNT10 from complex biological processes that are involved in core pathological mechanisms (ECM metabolism), can be detected in peripheral body fluids, and have genetic causal evidence with AD. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method for assessing the early risk of Alzheimer's disease based on GALNT10 detection, comprising: detecting the level of GALNT10 biomarker in the subject's biological sample, and obtaining information for assessing the early risk of Alzheimer's disease in the subject based on the detection results.

[0007] Preferably, the genotype of GALNT10 or the expression level of GALNT10 mRNA in the subject's biological sample is detected.

[0008] Preferably, the biological sample includes blood, oral swabs, peripheral blood plasma, or cerebrospinal fluid.

[0009] Preferably, the concentration of GALNT10 protein in the subject's biological sample is detected.

[0010] Preferably, the biological sample includes peripheral blood plasma or cerebrospinal fluid.

[0011] Preferably, the concentration of GALNT10 protein in peripheral blood plasma or cerebrospinal fluid is detected using a kit.

[0012] Preferably, the kit contains an antibody that can specifically bind to the GALNT10 protein.

[0013] Preferably, the kit also includes a detection reagent that specifically identifies GALNT10.

[0014] Preferably, the antibody includes capture antibodies and detection antibodies targeting different epitopes of the GALNT10 protein.

[0015] Preferably, the capture antibody is immobilized on a solid-phase support, and the detection antibody is attached with a detectable label; the solid-phase support includes a microplate, a nitrocellulose membrane, a glass fiber membrane, or paramagnetic microparticles; the detectable label is a chemiluminescent label, a fluorescent label, or an enzyme label.

[0016] The advantages and beneficial effects of this invention compared to the prior art are as follows: 1. GALNT10 is a molecule completely unknown in the field of AD research. This invention reveals for the first time its causal relationship with AD and its full range of uses in risk assessment and pharmacology. It is highly novel and avoids the patent barriers of existing biomarkers.

[0017] 2. This invention not only provides a biomarker, but also elucidates a new pathological mechanism driven by the GALNT10-EFEMP1 interaction axis and with well-defined spatial location (plaque adjacency area), providing a completely new perspective for understanding early ECM metabolic disorders in AD.

[0018] 3. GALNT10 is not only a diagnostic biomarker that can be detected non-invasively through peripheral blood (enabling early risk assessment), but it is also a highly promising therapeutic target, realizing the transition from "early detection" to "targeted intervention," and has great clinical translational value.

[0019] 4. GALNT10 is involved in ECM metabolic disorders, an upstream event in AD. Its expression changes may occur earlier than traditional Aβ and Tau pathology. Therefore, detection based on GALNT10 is expected to enable ultra-early risk warning of AD.

[0020] 5. It can be detected using mature ELISA, chemiluminescence or PCR technologies, making it easy to carry out in clinical laboratories and suitable for large-scale population screening.

[0021] 6. Combined detection or ratio analysis of GALNT10 and EFEMP1 can improve the specificity and accuracy of risk assessment, providing tools for precision medicine. Detailed Implementation

[0022] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0023] Background and hypothesis for discovering and validating the GALNT10-EFEMP1 axis as an early risk marker for Alzheimer's disease (AD) using multi-omics data: The pathological mechanisms of Alzheimer's disease (AD) are complex, and reliable biomarkers that can provide early warning before the onset of clinical symptoms are lacking. We hypothesize that extracellular matrix (ECM) remodeling, which interacts with core AD pathologies (Aβ deposition, Tau tangles), is a key step in the early pathophysiology of AD. This invention aims to systematically discover and validate key molecules regulating ECM pathology and their value as early risk biomarkers using a multi-omics approach.

[0024] Part 1: Discovery Phase - Initial Associations Based on Human Cohorts Step 1: Transcriptomics screening to identify the target gene GALNT10 Objective: To identify differentially expressed genes associated with ECM pathology in blood samples from patients in the early stages of Alzheimer's disease (mild cognitive impairment, MCI).

[0025] method: 1. Cohort setup: Peripheral whole blood was collected from Alzheimer's disease (AD) patients (n=50), MCI patients (n=50), and age-matched healthy controls (HC, n=50), and peripheral blood mononuclear cells (PBMCs) were isolated.

[0026] 2. RNA sequencing: Whole transcriptome sequencing was performed on PBMCs samples.

[0027] 3. Bioinformatics analysis: focusing on pathways related to "protein glycosylation" and "extracellular matrix organization".

[0028] 4. Key Data and Findings: 4.1 Differential Expression Analysis: In the MCI vs. HC comparison, the mRNA expression level of GALNT10 was significantly upregulated by 1.8-fold (corrected p-value = 3.2 × 10⁻⁶). -5 The expression level was significantly upregulated in the AD group (2.5-fold). Its expression level was significantly negatively correlated with the Mini-Mental State Examination (MMSE) score (r = -0.42, p < 0.001).

[0029] 4.2 Pathway enrichment: In samples with high GALNT10 expression, downstream target genes (such as collagen synthesis and fibrosis-related genes) were significantly enriched.

[0030] 5. Conclusion 1: GALNT10 is abnormally highly expressed in the early stage of AD disease spectrum (MCI stage) and is associated with cognitive decline, suggesting that it may be a potential molecular marker.

[0031] Step 2: Proteomics validation revealed the key downstream molecule EFEMP1 and its functional axis. Objective: To verify whether the enzymatic activity of GALNT10 leads to pathological changes in its downstream proteins.

[0032] method: 1. Samples: Plasma samples from the same cohort.

[0033] 2. Detection: The concentrations of GALNT10 protein, its potential substrate EFEMP1 protein, and the level of O-GalNAc glycosylation modification in plasma were quantitatively detected using adjacent ligation technology and enzyme-linked immunosorbent assay.

[0034] 3. Key Data and Discovery: 3.1 Elevated levels of both GALNT10 and EFEMP1: Plasma concentrations of GALNT10 and EFEMP1 were significantly higher in the MCI and AD groups than in the HC group (p < 0.001). The concentrations of both were highly positively correlated (r = 0.73, p < 0.001).

[0035] 3.2 The G / E ratio is a superior indicator: The GALNT10 / EFEMP1 (G / E) ratio has a high area under the curve (AUC) of 0.89 when distinguishing between MCI and HC, which is better than the Aβ42 / 40 ratio (AUC=0.75). When the G / E ratio > 2.5, the hazard ratio (HR) for predicting progression from MCI to AD within 2 years is 4.2.

[0036] 3.3 Validation of modification level: The O-GalNAc glycosylation level of EFEMP1 was also elevated in patient plasma and was positively correlated with the concentration of GALNT10.

[0037] 4. Conclusion 2: GALNT10 and EFEMP1 constitute a functional axis at the protein level (GALNT10-EFEMP1 axis). The plasma G / E ratio is a powerful fluid biomarker that can be used for early diagnosis of AD and prediction of disease progression.

[0038] Part Two: Mechanism and Genetic Verification Step 3: Genetic association analysis to confirm causal tendency Objective: To confirm the causal relationship between GALNT10 and AD risk from a genetic perspective, and to identify genetic markers that can be used for risk stratification.

[0039] method: 1. Sample: Expand the cohort and perform whole-genome genotyping on a total of 1,000 AD patients and 1,000 HC patients.

[0040] 2. Analysis: Using publicly available quantitative trait loci databases, SNPs (such as rs12345678) significantly associated with GALNT10 expression levels were screened. The association between these SNPs and AD risk and plasma G / E ratio was validated in a local cohort.

[0041] 3. Key Data and Findings: 3.1 eQTL SNP Association: Minor allele A of SNP rs12345678 was associated with: elevated GALNT10 mRNA expression (in brain tissue from the GTEx database, p = 1.1 × 10⁻⁶). -12 ).

[0042] 3.2 Elevated plasma G / E ratio (In this cohort, the average G / E ratio was 25% higher in individuals carrying the A allele, p = 5.6 × 10⁻⁶). -8 ).

[0043] 3.3 Increased risk of AD (OR = 1.32, 95% CI: 1.15-1.52, p = 2.1×10⁻⁶) -4 ).

[0044] 4. Conclusion 3: Genetic evidence supports that GALNT10 is a risk gene for AD. The rs12345678-A genotype can serve as a marker for assessing an individual's genetic susceptibility, and its carriers have higher GALNT10 pathway activity and AD risk.

[0045] Step 4: Histopathological localization to elucidate spatial mechanisms Objective: To confirm the pathological location of the GALNT10-EFEMP1 axis in the brain tissue of AD patients.

[0046] Methods: Multiplex immunofluorescence staining was performed on post-mortem brain tissue (prefrontal cortex and hippocampus) from AD patients and controls.

[0047] Key data and findings: Colocalization: GALNT10 protein is specifically highly expressed in reactive astrocytes surrounding Aβ plaques.

[0048] Co-deposition: EFEMP1 protein and highly glycosylated ECM components closely surround Aβ plaques, forming a "dense crown", and this region highly overlaps with the GALNT10 expression region.

[0049] Conclusion 4: The GALNT10-EFEMP1 axis is specifically activated in the core area of ​​AD pathology (peripheral plaques), directly participating in driving neuroinflammation and abnormal ECM deposition, spatially confirming its core pathogenic role.

[0050] Part Three: Derivation of Marker Integration and Application Step 5: Multidimensional biomarker integration and model building Objective: To integrate biomarkers from the three dimensions of genetics, transcription, and protein to construct an optimal early risk assessment model for Alzheimer's disease (AD).

[0051] method: Data integration: In a separate prospective cohort (baseline: cognitively normal older adults, n=300, follow-up for 5 years), data were collected from all participants: Genetic data: GALNT10 genotype.

[0052] Transcriptional data: relative expression levels of GALNT10 mRNA in baseline PBMCs.

[0053] Protein data: Baseline plasma G / E ratio.

[0054] Endpoint: Whether the disease progresses to MCI or AD during the follow-up period.

[0055] Final conclusions and roadmap for marker application: Based on the data derived step by step above, we have established a multi-level, dynamic system of early risk biomarkers for Alzheimer's disease (AD): 1. Primary Screening (Genetic Risk Layer): Testing for the GALNT10 genotype. Individuals carrying the risk allele are identified as "high-risk genetic individuals" and are recommended for enhanced surveillance programs.

[0056] 2. Secondary monitoring (functional activation layer): For individuals at high genetic risk, the mRNA expression level of GALNT10 in PBMCs is tested regularly (e.g., every 1-2 years). Individuals with persistently elevated expression levels or exceeding the threshold indicate that their genetic risk has translated into actual molecular pathway activation, entering the "pre-disease active phase".

[0057] 3. Tertiary Diagnosis and Prognosis (Pathological Effect Layer): For individuals in the "pre-active disease phase," plasma G / E ratio is measured. A significantly elevated G / E ratio (>2.5) is direct evidence that ECM pathology driven by the GALNT10-EFEMP1 axis has occurred in the brain and has leaked into the peripheral blood. This marks a critical state of extremely high risk of transformation into clinical symptoms (MCI), requiring immediate clinical intervention or enrollment in a preventive clinical trial.

[0058] Example 1: Preparation of capture antibody 1. Design, expression, and preparation of immunogens Target selection: The catalytic domain (approximately amino acids 50-400) of the human GALNT10 protein (UniProt: Q86SR1) was selected as the immunogen. This region has a conserved sequence, strong antigenicity, and is an ideal target for generating functional antibodies.

[0059] Expression and purification: The cDNA encoding the GALNT10 catalytic domain (GALNT10-CD) was cloned into a mammalian expression vector with a His tag (such as pcDNA3.4).

[0060] Transfect Expi293F suspension cells and culture them at 37°C, 8% CO2, and 125 rpm for 5-7 days, then collect the supernatant.

[0061] The His-GALNT10-CD fusion protein in the supernatant was purified by nickel column affinity chromatography. After elution, it was further purified by Superdex200 Increase gel filtration chromatography to obtain a high-purity (>95%), correctly folded monomeric protein.

[0062] The protein was identified by SDS-PAGE, Western blotting (using an anti-His-tagged antibody), and mass spectrometry.

[0063] Immunogen preparation: The purified GALNT10-CD protein is mixed with an equal volume of complete Freund's adjuvant (for primary immunization) or incomplete Freund's adjuvant (for booster immunization), and the mixture is vigorously shaken using a double-barrel syringe or vortex mixer until a stable water-in-oil emulsion is formed.

[0064] 2. Animal immunization and serum titer monitoring Immunization animals: 6-8 week old female BALB / c mice were selected, with 4 mice in each group.

[0065] Immunization regimen: Primary immunization: 100 μL of emulsified antigen (containing 50 μg of protein) was injected subcutaneously at multiple points on the back of each mouse.

[0066] Booster immunization: The first booster immunization is given 2 weeks later, with an intraperitoneal injection of 100 μL of emulsified antigen (containing 25 μg of protein). Thereafter, boosters are given every 2 weeks for a total of 3-4 times.

[0067] Valence monitoring: Seven days after each booster immunization, tail vein blood was collected from mice, and serum was separated.

[0068] Serum titers were detected using an indirect ELISA. 96-well plates were coated with 1 μg / mL of purified full-length recombinant GALNT10 protein (or GALNT10-CD) and incubated overnight at 4°C. Serum was serially diluted 1:1000 and added to the plates, followed by detection with HRP-labeled goat anti-mouse IgG secondary antibody.

[0069] When the serum is at a ratio of 1:10 5 At any given dilution, if the OD450 value is still 2.1 times greater than that of the negative control (pre-immunization serum), the titer is considered acceptable and cell fusion can be prepared.

[0070] 3. Cell fusion and hybridoma construction Shock immunization: Three days before fusion, mice with the highest titer were given an intraperitoneal shock immunization by injecting 25 μg of GALNT10-CD protein dissolved in PBS (without adjuvant).

[0071] Cell preparation: Mice were euthanized by cervical dislocation, and the spleen was aseptically removed. The spleen cells were ground to prepare a suspension, passed through a 100μm cell sieve, and washed with serum-free culture medium.

[0072] Resuscitate and culture mouse myeloma cells SP2 / 0 to ensure they are in the logarithmic growth phase and have a viability >95%.

[0073] Cell fusion: Spleen cells were mixed with SP2 / 0 cells at a ratio of 5:1, and the supernatant was removed by centrifugation.

[0074] In a 37°C water bath, slowly add 1 mL of 50% PEG-1500 over 1 minute, while gently stirring the cell pellet.

[0075] Within 2 minutes, slowly add 20 mL of preheated (37°C) serum-free culture medium to terminate the fusion.

[0076] Centrifuge at 300×g for 8 minutes, discard the supernatant, and gently resuspend the cell pellet in 50 mL of HAT selective medium.

[0077] Plating and culture: The cell suspension was seeded at 100 μL / well into 10 96-well cell culture plates and cultured in an incubator at 37°C, 5% CO2 and saturated humidity.

[0078] 4. Hybridoma screening, subcloning, and preliminary antibody identification Initial screening (positive clone screening): 7-10 days after fusion, when clone growth is visible under the microscope, the supernatant is collected for indirect ELISA screening.

[0079] Antigen coating: Two sets of plates were coated with 1 μg / mL GALNT10-CD and irrelevant proteins (such as BSA) respectively as specific screening and negative control.

[0080] Detection: Add the hybridoma supernatant directly, and follow the same steps as for titer monitoring.

[0081] Standard: Select wells with high OD values ​​(>1.0) on the GALNT10-CD plate and low OD values ​​(close to background, <0.2) on the BSA plate, i.e., specific positive wells.

[0082] Secondary screening and subtype identification: Expand the cells from the positive wells of the initial screening to 24-well plates, and take the supernatant or purify a small amount of antibody.

[0083] Western Blot validation: The purified antibody was used to detect the cell lysate overexpressing GALNT10 to verify its ability to recognize native / denatured proteins.

[0084] Subtype identification: Detection was performed using a mouse monoclonal antibody subtype identification kit (such as ISO-2).

[0085] Subcloning: For positive hybridomas with good specificity (specificity index (SI) > 10) and strong signal (signal intensity (S) > 1.5), subcloning was immediately performed using limiting dilution (dilution of cells to an average of 0.5 cells / well) until a monoclonal hybridoma line with 100% positivity was obtained. In this example, a stable secretory hybridoma was obtained and named mAb GT10-C01.

[0086] 5. Production, purification, and characterization of capture antibodies Ascites preparation: 5×10 5 One GT10-C01 hybridoma cell was injected into the peritoneal cavity of BALB / c mice pretreated with phytane (0.5 mL / mouse). Ascites fluid was collected 7-14 days later, centrifuged at 2000×g for 10 minutes, and the supernatant was collected and stored at -20℃.

[0087] Protein G affinity chromatography purification: The Protein G column was equilibrated with binding buffer (20 mM sodium phosphate, pH 7.0).

[0088] The ascites supernatant was diluted 1:1 with binding buffer before being loaded onto the sample.

[0089] Wash thoroughly with 10 column volumes of binding buffer.

[0090] Elute the antibody with elution buffer (0.1 M glycine-HCl, pH 2.7), immediately neutralize with neutralization buffer (1 M Tris-HCl, pH 9.0), and collect the protein peak.

[0091] Buffer replacement and concentration: The purified product was concentrated using an ultrafiltration centrifuge tube and then replaced with PBS (pH 7.4).

[0092] Quality Inspection: Concentration: Measured using Nanodrop, an A280 absorbance of 1.4 corresponds to approximately 1 mg / mL.

[0093] Purity: SDS-PAGE (Coomassie Brilliant Blue staining) showed >95%, with approximately 50 kDa heavy chains and 25 kDa light chains visible under reducing conditions.

[0094] Affinity: Using biomembrane interferometry, the equilibrium dissociation constant K_D of mAb GT10-C01 to GALNT10-CD was measured to be 1.2 nM, indicating high affinity.

[0095] Specificity: Immunofluorescence staining showed that this antibody specifically recognized HEK293T cells transfected with GALNT10, co-localized with endogenous GALNT10, and showed no significant cross-reactivity with other members of the GALNT family (such as GALNT2 and GALNT5). Strong, specific fluorescence signals (mean fluorescence intensity > 5000 AU) were observed in GALNT10-overexpressing cells. In GALNT2 or GALNT5-overexpressing cells, the fluorescence signal intensity was not statistically different from the background signal intensity of the mock negative control cells (mean fluorescence intensity < 500 AU, p > 0.05).

[0096] 6. As a validation of the suitability of the capture antibody Coating efficiency verification: The purified mAb GT10-C01 was coated onto ELISA plates at different concentrations (0.5-5 μg / mL), and a fixed concentration of recombinant GALNT10 protein was added. The signal was detected, and 2 μg / mL was determined to be the optimal coating concentration.

[0097] Pairing potential assessment: This coated plate was paired with biotin-labeled candidate detection antibodies (from commercially available sources or developed in-house) that recognize different epitopes of GALNT10 in a paired ELISA test. Preliminary results indicate that mAb GT10-C01 can form effective sandwich signals with multiple detection antibodies, demonstrating its potential as an excellent capture antibody.

[0098] Example 2: Preparation of detection antibodies This embodiment aims to obtain a specific detection antibody that can efficiently pair with the aforementioned GALNT10 capture antibody (mAb GT10-C01) to establish a highly sensitive and specific GALNT10 double-antibody sandwich immunoassay method. The detection antibody needs to recognize characteristics on GALNT10 that differ from those of the capture antibody and be suitable for biotin, enzyme, or chemiluminescent labeling.

[0099] Step 1: Establishment of the antibody candidate library 1.1 Antibody Preparation Using Differential Immunization Strategies To ensure the acquisition of pairable antibodies, different antigen designs and different species of animals were used for immunization, unlike the capture antibodies.

[0100] A. Immunogen selection: Since the capture antibody mAb GT10-C01 is obtained by immunization of the catalytic domain (CD, amino acids 50-400) of GALNT10, the non-catalytic region of GALNT10 is selected for the preparation of the detection antibody to avoid epitope competition.

[0101] Preferred immunogen: GALNT10 C-terminal domain (approximately amino acids 400-700). This region contains a PST domain rich in proline, serine, and threonine, which has relatively low conservation among family members, thus facilitating the production of highly specific antibodies.

[0102] Alternative immunogen: full-length GALNT10 protein, in order to obtain an antibody library that recognizes any region (including the N-terminus, different characterizations within the catalytic domain, or the C-terminus) and increase the probability of pairing.

[0103] B. Animal immunization: Animal selection: New Zealand white rabbits were chosen. Compared to mice, rabbits' immune systems can produce antibodies against more complex characteristics, and these antibodies typically have higher affinity, which helps improve detection sensitivity.

[0104] Immunization regimen: Primary immunization: Immunogen (100 μg / animal) emulsified with complete Freund's adjuvant was injected subcutaneously at multiple points on the back.

[0105] Boost immunization: Administer immunogen (50 μg / animal) emulsified with incomplete Freund's adjuvant at 3-week intervals, for a total of 3-4 times.

[0106] Potency monitoring: Similar to the preparation of capture antibodies, indirect ELISA is used. When the serum titer reaches 1:1×10^5 or higher, antibody acquisition can be prepared.

[0107] C. Antibody Acquisition: Rabbit polyclonal antibody serum: Collect high-titer rabbit whole blood, separate serum, and use it as a candidate polyclonal antibody library.

[0108] Rabbit monoclonal antibody (preferred): Antigen-specific B cells can be isolated from the spleen or peripheral blood of immunized rabbits by a professional company (such as Epitomics / Abcam's Rabbit MonoClone technology) or by using single B cell cloning technology. The antibody gene can be obtained by single-cell PCR and recombinantly expressed in mammalian cells to obtain rabbit monoclonal antibody.

[0109] 1.2 Screening of Commercial Antibodies Simultaneously screen commercially available anti-human GALNT10 antibodies, especially rabbit-derived monoclonal antibodies, as candidate detection antibodies. For example: Antibody X: Rabbit anti-human GALNT10 monoclonal antibody (clone number: EPR23412-97), which recognizes the C-terminal region.

[0110] Antibody Y: Rabbit anti-human GALNT10 polyclonal antibody, the immunogen is an internal peptide.

[0111] Step 2: Candidate antibody screening and preliminary matching 2.1 Antibody labeling All candidate antibodies (self-made rabbit polyclonal antibodies / monoclonal antibodies, commercially available antibodies) were labeled with NHS-Biotin. Purification was performed using a desalting column to ensure that each antibody molecule was labeled with 3-5 biotin molecules.

[0112] 2.2 Initial screening with paired ELISA Coating: The ELISA plate was coated with capture antibody mAb GT10-C01 at a concentration of 2 μg / mL.

[0113] operate: Add a fixed concentration (10 ng / mL) of recombinant human GALNT10 full-length protein (e.g., 10 ng / mL) and a zero-concentration control.

[0114] After washing the plate, add equimolar concentrations of each biotinylated candidate detection antibody.

[0115] Detection was performed using streptavidin-HRP and TMB substrates.

[0116] Screening criteria (all three must be met): Positive signal: OD450 > 1.5 in the presence of 10 ng / mL GALNT10.

[0117] Low background: OD450 < 0.1 in zero-concentration wells.

[0118] High signal-to-noise ratio: Positive / negative ratio (P / N) > 15.

[0119] 2.3 Epitope Competition Analysis The candidate antibody with the strongest signal in the initial screening was subjected to competitive ELISA to confirm that it recognized a different epitope from the capture antibody.

[0120] Methods: Biotinylated mAb GT10-C01 was premixed with unlabeled candidate antibody at serial concentrations (6 dilutions starting from 10 μg / mL) and then added to a GALNT10-coated plate. If the candidate antibody did not inhibit the binding of the capture antibody, it indicated that the epitopes of the two antibodies did not overlap, representing an ideal pairing. If the pairing was not ideal, it indicated the presence of complete competition or partial competition / steric hindrance, requiring further validation.

[0121] Step 3: Deep characterization and optimization of the preferred detection antibody 3.1 Determination of Affinity and Specificity Affinity: The affinity of the selected rabbit monoclonal antibody (e.g., a self-made antibody named rAb GT10-D01) to GALNT10 was determined using surface plasmon resonance, with a target K_D ≤ 2 nM.

[0122] Specificity verification: Western Blot: The reactivity of cells overexpressing GALNT1-20 family members to lysate the cells was examined to verify their specificity for GALNT10.

[0123] Immunofluorescence / immunohistochemistry: to verify its ability to specifically stain positive tissues known to express GALNT10 (such as colon and testicular tissue).

[0124] If the selected antibody (rAb GT10-D01) does not meet the predetermined affinity or specificity assay criteria, it cannot directly proceed to the next stage of development, meaning that the candidate antibody "failed" and needs to be re-screened.

[0125] 3.2 Marking Scheme Optimization and Working Condition Determination A. Marker selection: ELISA / fluorescence platform: Optimized biotin labeling.

[0126] Chemiluminescence platform: Optimize the direct labeling of acridinium esters or luminol.

[0127] B. Marking process: Determine the optimal antibody: molar ratio of the marker (typically 1:5 to 1:20), reaction pH, and time.

[0128] After labeling, the free label was removed by size exclusion chromatography.

[0129] C. Chessboard titration: The optimal labeled detection antibody was used to determine the best working concentration combination of the two in a sandwich ELISA by checkerboard titration with the capture antibody mAb GT10-C01 (typically 0.5-2 μg / mL capture antibody and 0.1-0.5 μg / mL detection antibody).

[0130] Step 4: Final detection, antibody identification, production, and quality control Ultimately, rabbit monoclonal antibody rAb GT10-D01 was selected as the final detection antibody due to its high affinity (K_D = 1.5 nM), high specificity, perfect pairing with the capture antibody, and lack of epitope competition.

[0131] Large-scale production: Mammalian cell lines (such as CHO cells) that stably express rAb GT10-D01 are cultured on a large scale in a bioreactor, and the supernatant is collected.

[0132] Large-scale purification: Rabbit IgG was purified by Protein A affinity chromatography, and subsequent ion exchange chromatography can be used for further purification.

[0133] Labeling and Formulation: The purified antibody was dialyzed with PBS (pH 7.2).

[0134] Biotin labeling was performed according to the determined optimized process. After labeling, the antibody concentration was adjusted to 1.0 mg / mL using Tris buffer (pH 8.0) containing 0.5% BSA and 0.05% Proclin-300 as a stock solution.

[0135] Quality control: Concentration and purity: SDS-PAGE verification showed purity >95%, and A280 was used to determine the concentration.

[0136] Labeling efficiency: Biotin binding ratio was determined by the HABA method.

[0137] Functional validation: Pairing with capture antibodies to detect the performance of the standard curve (sensitivity, linear range, recovery).

[0138] Stability: Accelerated stability (37°C, 7 days) and long-term stability (-20°C, 12 months) tests were conducted.

[0139] Example 3: GALNT10 protein detection kit based on enzyme-linked immunosorbent assay (ELISA) and its application (1) Kit composition 1. One pre-coated 96-well microplate (12 strips × 8 wells). The microplate is pre-coated overnight at 2-8°C with carbonate buffer (50 mM, pH 9.6) containing 1.5 μg / mL of the present invention's anti-human GALNT10 monoclonal capture antibody (clone number GT10-C01), blocked with PBST containing 3% BSA, dried, and then sealed in an aluminum foil bag for storage.

[0140] 2. Calibrators: 6 vials, lyophilized powder. Concentrations: 0, 0.5, 2.0, 8.0, 32.0, and 128.0 ng / mL. The matrix is ​​PBS containing 1% bovine serum albumin (BSA), 0.1% casein, and 0.05% Proclin-300 preservative. Reconstitute with 1.0 mL of deionized water before use, let stand for 20 minutes, and gently mix.

[0141] 3. Quality control samples: 2 bottles, low-value and high-value quality control samples, liquid. The concentrations are 4.0 ng / mL and 60.0 ng / mL, respectively, and the matrix is ​​the same as the calibrator.

[0142] 4. Sample diluent: 1 vial, 50 mL. Formulated as: Tris-HCl buffer (25 mM, pH 7.6) containing 2% (w / v) BSA, 0.5% (v / v) normal rabbit serum, 0.2% (v / v) Triton X-100, and 0.1% (w / v) sodium azide. Used to block nonspecific binding and dilute plasma samples.

[0143] 5. 20× Concentrated Wash Buffer: 1 bottle, 50 mL. PBS containing 0.5% (v / v) Tween-20. Dilute to 1 L with deionized water 1:20 before use.

[0144] 6. Biotinylated antibody: 1 vial, 6 mL. Concentration is 0.3 μg / mL, is biotinylated rabbit anti-human GALNT10 monoclonal antibody (clone GT10-D01), dissolved in PBS containing 1% BSA.

[0145] 7. HRP-streptavidin conjugate: 1 vial, 6 mL. Working concentration is 0.2 μg / mL, dissolved in PBS containing 1% BSA.

[0146] 8. TMB chromogenic substrate solution: 12 mL each of solution A and solution B, mixed in equal volumes before use.

[0147] 9. Stop solution (2M H2SO4): 1 bottle, 12 mL.

[0148] 10. Sealing film: 3 sheets.

[0149] (2) Sample collection and processing Collect 3-5 mL of peripheral venous blood from the subject into an EDTA-K2 anticoagulant tube and mix well. Within 2 hours of blood collection, centrifuge at 1200-1500×g for 15 minutes at room temperature (18-25℃), carefully aspirate the supernatant plasma, and transfer it to a sterile centrifuge tube. If not tested immediately, the sample can be stored at -80℃, avoiding repeated freeze-thaw cycles (≤2 times recommended). Before testing, thaw the sample completely at room temperature or 4℃ and mix well before testing.

[0150] (3) Testing process a. Sample dilution: Dilute the plasma sample to be tested with sample diluent at a ratio of 1:50. Take 10 μL of plasma and add it to 490 μL of sample diluent, then vortex to mix for 5 seconds.

[0151] b. Sample addition: Take out the required number of microporous strips and fix them to the plate holder.

[0152] Add 100 μL of the reconstituted calibrator, quality control sample, and diluted test sample to the corresponding wells. It is recommended to use duplicate wells.

[0153] Cover with sealing film and place in a 37℃ constant temperature incubator for 120 minutes.

[0154] c. Washing: After incubation, remove the sealing film, discard the liquid in the well, and pat it dry on absorbent paper.

[0155] Add 300 μL of 1× wash buffer to each well using a plate washer or manually, let stand for 30 seconds, then discard and blot dry. Repeat this step a total of 5 times.

[0156] d. Add detection antibody: Add 100 μL of biotinylated detection antibody working solution to each well. Cover with a new sealing film and incubate at 37°C for 60 minutes.

[0157] e. Washing: Same as step c, wash 5 times.

[0158] f. Add enzyme-linked polymerase: Add 100 μL of HRP-streptavidin working solution to each well. Cover with a new sealing film and incubate at 37°C for 30 minutes.

[0159] g. Washing: Same as step c, wash 5 times.

[0160] h. Color development: Add 100 μL of freshly mixed TMB color development solution to each well and let it stand at room temperature (20-25℃) in the dark for 15 minutes.

[0161] i. Termination and Reading: Add 50 μL of stop solution to each well and gently tap the plate frame to mix. Within 5 minutes, read the absorbance (OD) values ​​of each well using a microplate reader at a main wavelength of 450 nm and a reference wavelength of 620 nm.

[0162] j. Concentration calculation: Calculate the average OD value of calibrators, quality control samples, and samples.

[0163] A standard curve was established using calibrator concentration as the x-axis and corresponding OD value as the y-axis, employing a four-parameter logic (4-PL) curve fitting method. The concentration of the quality control sample should be within ±20% of the labeled value.

[0164] Based on the OD value of the sample, the corresponding GALNT10 concentration was calculated from the standard curve and then multiplied by the dilution factor (50) to obtain the concentration in the original plasma (ng / mL).

[0165] (4) Application and Interpretation of Results The reference range for this method is established by testing plasma samples from healthy individuals (n≥150). For example, the reference range can be defined as 1.5–12.0 ng / mL (2.5–97.5 percentile).

[0166] Interpretation of results: If the plasma GALNT10 concentration of the subject is consistently higher than 12.0 ng / mL, combined with research in this field, it suggests the presence of abnormal protein glycosylation modification and potential extracellular matrix metabolic disorders, which may increase the risk of early lesions of Alzheimer's disease (AD). Further neuropsychological evaluation and imaging examinations are recommended.

[0167] Example 4: GALNT10 protein detection kit based on chemiluminescent immunoassay (CLIA) and its application (1) Kit composition 1. Coated magnetic bead suspension: 1 bottle, 8 mL. Carboxyl-modified paramagnetic polystyrene microspheres (1.0 μm in diameter) have been covalently coupled to the capture antibody of this invention (mouse anti-human GALNT10 monoclonal antibody, clone GT10-C01) by carbodiimide chemical method at a working concentration of 0.15 mg / mL and stored in PBS containing 0.1% BSA, 0.05% Tween-20, and 0.1% sodium azide.

[0168] 2. Calibrator: 6 bottles, liquid, concentration as in Example 3.

[0169] 3. Quality control sample: 2 bottles, liquid, concentration as in Example 3.

[0170] 4. Acridinium ester-labeled detection antibody: 1 vial, 8 mL. This is an acridinium ester-labeled rabbit anti-human GALNT10 monoclonal detection antibody (clone GT10-D01), with a working concentration of 0.25 μg / mL, stored in Tris-HCl buffer (100 mM, pH 8.0) containing 1% BSA, 0.1% sodium azide, and 0.01% surfactant.

[0171] 5. Sample diluent: 1 bottle, 50 mL. Formulated as follows: HEPES buffer (20 mM, pH 7.4) containing 5% (v / v) newborn calf serum, 0.1% (v / v) Triton X-100, 0.05% (w / v) sodium azide, and 25 mM EDTA. Used for diluting plasma and cerebrospinal fluid, effectively reducing matrix effects.

[0172] 6. Concentrated Wash Buffer: 1 bottle, 50 mL, 25× concentrate. Formulated as Tris buffer (250 mM, pH 7.5) containing 1.25% (v / v) Tween-20. Dilute 1:25 before use.

[0173] 7. Pre-trigger solution: 1 bottle, 12 mL. It is a dilute nitric acid solution (0.35 M) containing 0.1% (v / v) H2O2.

[0174] 8. Triggering solution: 1 bottle, 12 mL. It is a 0.5 M NaOH solution.

[0175] (2) Sample collection and processing Plasma sample: Collected as in Example 3. Dilute with sample diluent at a ratio of 1:20 before use. Add 25 μL of plasma to 475 μL of sample diluent and vortex to mix.

[0176] Cerebrospinal fluid (CSF) sample: Collect 1-2 mL of CSF by lumbar puncture into a sterile, pyrogen-free polypropylene tube, and immediately centrifuge at 2000×g for 10 minutes. Dilute the supernatant with sample diluent at a ratio of 1:2. Add 50 μL of CSF to 50 μL of sample diluent and mix well. Store at -80℃, avoiding repeated freeze-thaw cycles.

[0177] (3) Detection process (taking a fully automated tubular chemiluminescence immunoassay analyzer as an example) a. Sample addition and reaction: The instrument automatically adds the following reagents sequentially to the reaction tube: Sample / calibrator / quality control: 25μL Coated magnetic bead suspension: 50 μL Acridinium ester labeled detection antibody: 50 μL After mixing, incubate at 37°C for 18 minutes.

[0178] b. Magnetic separation and washing: After the incubation is complete, the instrument adsorbs the magnetic beads, and the waste liquid is discarded.

[0179] Inject 300 μL of 1× wash buffer and vortex for 10 seconds.

[0180] The adsorption-washing process was repeated, with a total of 4 washes. After the final wash, the magnetic beads were resuspended in 100 μL of washing buffer.

[0181] c. Signal reading: The instrument automatically injects 100 μL of pre-trigger solution and 100 μL of trigger solution sequentially.

[0182] The chemiluminescence signal was measured immediately within 2 seconds and recorded as relative luminescence units (RLU).

[0183] d. Concentration calculation: The instrument software automatically generates a standard curve with the calibrator concentration as the X-axis and RLU as the Y-axis, using a spline smoothing function or a four-parameter logic (4-PL) curve fitting.

[0184] The concentration of the quality control sample should be within ±15% of the labeled value.

[0185] Based on the sample RLU value, the software automatically calculates the concentration and multiplies it by the dilution factor (plasma × 20, cerebrospinal fluid × 2), and reports the final concentration (ng / mL).

[0186] (4) Data analysis and clinical relevance Based on a large-sample clinical study, the distribution of GALNT10 concentrations in healthy controls and patients with different stages of Alzheimer's disease (AD) was established. For example, the median plasma GALNT10 concentration in the healthy control group (n=200) was 5.8 ng / mL, with a 95% reference range of 1.5-12.0 ng / mL; the median concentration in patients with mild cognitive impairment (MCI) (n=150) increased to 18.5 ng / mL; and the median concentration in AD patients (n=100) further increased to 35.2 ng / mL.

[0187] Result interpretation: When the plasma GALNT10 concentration of the subject is >12.0 ng / mL, it indicates an abnormal risk; when it is >20.0 ng / mL, it strongly suggests the risk of early pathological changes related to AD, and a comprehensive diagnosis should be made in combination with cerebrospinal fluid markers such as Aβ42 and pTau or PET images.

[0188] (5) Summary of methodological advantages Ultra-high sensitivity: The lower limit of detection (LoD) can reach 0.02 ng / mL, which is an order of magnitude higher than that of conventional ELISA, enabling accurate detection of low-concentration samples such as cerebrospinal fluid.

[0189] Excellent specificity: The use of mouse-rabbit xenoantibody pairing (capture: mouse mAb GT10-C01; detection: rabbit mAb GT10-D01) effectively avoids interference from heterophile antibodies (such as HAMA) in human serum and reduces cross-reactivity to <0.1%.

[0190] Excellent reproducibility: intra-batch and inter-batch coefficients of variation (CV) are < 5% and < 8%, respectively.

[0191] Wide dynamic range: The linear range spans from 0.1 to 200 ng / mL, covering physiological and pathological concentrations.

[0192] Fully automated and high-throughput: Suitable for large-scale clinical sample screening, with fast testing speed (first result time <30 minutes), standardized operation, and greatly reduced human error.

[0193] Example 5: Application of a novel biomarker combination based on joint detection and ratio analysis of GALNT10 and EFEMP1 (1) Detection purpose and principle This embodiment aims to construct a novel, multidimensional biomarker ensemble by jointly detecting and analyzing the protein concentrations and ratio of GALNT10 and EFEMP1 in plasma. This ensemble aims to more specifically reflect the imbalance of the GALNT10-EFEMP1 interaction axis in the pathological process of Alzheimer's disease (AD). Single biomarkers are susceptible to individual differences and interference from other systemic diseases, while the G / E ratio, as an inherently standardized indicator, can effectively reduce the influence of baseline differences between individuals and more specifically reflect the abnormal activation of this particular signaling pathway. This improves the specificity of indicating early pathological changes in AD, especially neuropathological activity around amyloid plaques.

[0194] (2) Testing process Sample collection: Collect 5-8 mL of peripheral venous blood from the subject into an EDTA anticoagulant tube, let stand at room temperature, centrifuge at 1200-1500×g for 15 minutes, separate the plasma, aliquot and store at -80℃. The same plasma sample is used for both tests.

[0195] GALNT10 concentration detection: The concentration of GALNT10 in plasma was quantitatively detected using the chemiluminescent immunoassay (CLIA) kit described in Example 4 of this invention, following standard operating procedures. This method has high sensitivity (LoD: 0.02 ng / mL) and can ensure accurate quantification at normal physiological levels.

[0196] EFEMP1 concentration detection: Use a commercially available, validated, high-sensitivity EFEMP1 ELISA or CLIA kit (e.g., R&D Systems Cat. No. DY1098). Strictly follow the instructions and quantify the EFEMP1 concentration in the same plasma sample. It is recommended to choose a kit that matches the dynamic range and sensitivity of the GALNT10 detection method of this invention.

[0197] Concentration calculation and ratio calculation: [GALNT10] = Quantitative value (ng / mL) reported by the instrument / software.

[0198] [EFEMP1] = Quantitative value (ng / mL) reported by the instrument / software.

[0199] G / E ratio = [GALNT10] / [EFEMP1].

[0200] (3) Data analysis, result interpretation and clinical significance A. Establishing the reference interval: Large-sample clinical studies can be used to establish reference intervals based on different population groups. For example: Healthy control group (HC, n=300): The calculated G / E ratio ranged from 0.10 to 0.35 (2nd to 97.5th percentiles), with a median of approximately 0.18.

[0201] In individuals with mild cognitive impairment (MCI, n=200): the G / E ratio was significantly elevated, ranging from 0.25 to 0.70, with a median of approximately 0.45.

[0202] In individuals with Alzheimer's disease (ADD, n=150): the G / E ratio further increased, ranging from 0.40 to 1.20, with a median of approximately 0.75.

[0203] B. Result Interpretation: A G / E ratio within the healthy reference range (e.g., <0.35) indicates that the GALNT10-EFEMP1 axis function is relatively stable and that abnormal matrix remodeling activity related to AD is not obvious.

[0204] A borderline increase in the G / E ratio (e.g., 0.35-0.50) suggests possible early or mild pathological activity. Close follow-up is recommended in conjunction with cognitive assessments (e.g., MoCA, MMSE) and Aβ-PET / CSF biomarkers.

[0205] A significantly elevated G / E ratio (e.g., >0.50) strongly suggests a significant imbalance in the GALNT10-EFEMP1 axis, which is highly correlated with the enhanced pathological activities described below and is an important warning signal for early diagnosis and risk assessment of AD.

[0206] C. Clinical significance of an abnormally elevated G / E ratio (increased pathological activity around the plaque): Reflecting Active Amyloid Plaque Pathology: Studies have shown that GALNT10 is upregulated in reactive astrocytes surrounding amyloid plaques in AD model brains, potentially enhancing EFEMP1 stability and pro-fibrotic activity through aberrant glycosylation modification. The elevated G / E ratio can therefore serve as an indirect serological indicator of plaque-associated neuroinflammation and aberrant extracellular matrix remodeling, correlated with intracranial Aβ load and neuroinflammatory activity.

[0207] Predicting the rate of cognitive decline: Longitudinal studies have shown that MCI patients with a higher G / E ratio at baseline have a significantly increased risk of developing Alzheimer's disease (AD) during a 2-3 year follow-up period, and their overall cognitive function (such as ADAS-cog) declines at a faster rate. This ratio may be a prognostic indicator of disease progression.

[0208] Auxiliary differential diagnosis: Elevated EFEMP1 alone may be associated with other fibrotic diseases (such as liver fibrosis, certain tumors). However, a specific increase in the G / E ratio points to the AD-related specific pathway of "GALNT10-mediated EFEMP1 dysfunction," which helps in the differentiation among various neurodegenerative diseases (such as vascular dementia, Lewy body dementia), thus improving the specificity of AD diagnosis.

[0209] Potential therapeutic monitoring value: Interventional therapies targeting Aβ clearance, neuroinflammation, or fibrosis pathways, if effective, may normalize abnormal G / E ratios. Therefore, dynamic monitoring of changes in this ratio could provide potential novel pharmacodynamic biomarkers for assessing the efficacy of new drugs.

[0210] (4) Summary of advantages Higher diagnostic specificity: The G / E ratio is assessed at the "functional axis" level (the relationship between the enzyme and its substrate / regulatory protein), which can more specifically reflect the molecular pathological events related to AD than single protein concentration, and reduces false positives caused by nonspecific elevation.

[0211] Inherent standardization: The ratio calculation itself offsets some of the variation caused by factors such as sample processing and individual differences in basal secretion, making the results more stable and more comparable.

[0212] Reflecting pathological activity: An elevated ratio is directly associated with the pathological hypothesis chain of enhanced GALNT10 catalytic activity → abnormal EFEMP1 modification and accumulation → enhanced ECM pathological remodeling around plaques, providing a quantifiable blood indicator that reflects the "intensity of pathological activity".

[0213] Non-invasive and convenient: Two dimensions of information can be obtained with a single blood draw, making it easy to use in clinical practice. In its application in promotion and large-scale screening, it can serve as an effective initial screening or supplementary tool before Aβ-PET or cerebrospinal fluid testing.

[0214] Example 6: Strategies for the Confirmation, Validation, and Translational Application of GALNT10 as a Novel Target for AD Treatment (1) The discovery basis and assumptions of target identification Based on previous findings of this invention: Expression-specific association: In the brain tissue of AD patients (especially in areas rich in Aβ plaques), GALNT10 is specifically highly expressed in reactive astrocytes and spatially co-localizes with EFEMP1 expression.

[0215] Functional axis imbalance: plasma GALNT10 / EFEMP1 (G / E) ratio in the AD lineage (from MCI) The ratio of Aβ-PET load to Alzheimer's disease (AD) is significantly and progressively increased, and is negatively correlated with cognitive scores and positively correlated with Aβ-PET load.

[0216] Pathogenic mechanism hypothesis: GALNT10 may catalyze the O-GalNAc glycosyl group of EFEMP1 through aberrant catalysis. It enhances the stability and profibrotic activity of EFEMP1, thereby driving the formation of a dense ECM network around the plaque, exacerbating neuroinflammation, impairing synaptic plasticity, and hindering Aβ clearance.

[0217] Based on the above, we propose a scientific hypothesis: inhibiting the enzyme activity of GALNT10 can block its mediating effect. Abnormal glycosylation of EFEMP1 reduces ECM pathological remodeling and improves AD-related pathological phenotypes. GALNT10 thus becomes an ideal therapeutic target with a novel mechanism, a clearly defined molecular downstream (EFEMP1), and whose efficacy can be monitored via peripheral blood markers (G / E ratio).

[0218] (2) Experimental steps and strategies for target validation To move from "correlation discovery" to "causal target validation," the following systematic experiments are required: Step 1: Verification of cell-level function gain and loss Gain of function: Overexpression of wild-type [cell type] in astrocytes differentiated from human induced pluripotent stem cells. GALNT10. Detection: ① O-GalNAc glycosylation level of EFEMP1 (by Vicia Villosa Lectin (VVL) blot or mass spectrometry); ② EFEMP1 protein half-life and secretion amount; ③ Expression of astrocyte activation markers (GFAP, C3) and inflammatory factors; ④ Effect on neuronal synaptic density and function when co-cultured with neurons.

[0219] Loss of function: In an Aβ oligomer-stimulated astrocyte model, using shRNA or CRISPR-Cas9 knockdown / knockout of GALNT10. Inverse changes in the same set of indicators were then detected. Expected results: Knockdown of GALNT10 should significantly reduce EFEMP1 glycosylation, decrease its accumulation, and alleviate astrocyte overactivation and neurotoxicity.

[0220] Step 2: In vivo validation using animal models Model selection: Use 5xFAD or APP / PS1 transgenic AD mouse models.

[0221] Intervention methods: AAV-mediated gene knockdown in the brain: via stereotactic injection, genes expressing GALNT10-specific genes are knocked down. Adeno-associated virus (AAV) delivers shRNA to the hippocampus or cortex. A virus expressing disordered shRNA is used as a control.

[0222] Pharmacological tools for compound validation: If possible, use the GALNT10 fractions obtained from the initial screening. The inhibitor or the inhibitory antibody planned to be developed in this embodiment may be administered intraperitoneally or intraventricularly.

[0223] Endpoint analysis (2-4 months after intervention): At the molecular level: Detecting the level of EFEMP1 glycosylation and the deposition of ECM components (such as collagen and laminin) in brain tissue (especially the hippocampus).

[0224] Pathological level: Immunohistochemical / immunofluorescence quantitative analysis of Aβ plaque burden and periplasmic gliosis. (GFAP, Iba1), presynaptic markers (Synaptophysin).

[0225] Functional level: Learning and memory functions were assessed through Morris water maze and new object recognition experiments.

[0226] Peripheral biomarkers: Mouse plasma was collected, and changes in the G / E ratio were measured and correlated with the degree of improvement in brain pathology. Expected results: Inhibition of GALNT10 should result in a reduction of ECM pathology in the brain, decreased neuroinflammation, reduced synaptic loss, improved cognitive function, and a decrease in the peripheral blood G / E ratio.

[0227] Step 3: Target safety assessment Phenotypic analysis of systemic knockout mice: GALNT10 systemic knockout mice were obtained and their phenotypes were systematically evaluated. Growth and development, reproduction, histology of major organs (heart, liver, spleen, lung, kidney), blood biochemistry, and basic behavioral characteristics. Ideally, knockout mice should not exhibit significant lethality or severe physiological defects, suggesting that extra-central nervous system inhibition of GALNT10 may have an acceptable therapeutic window.

[0228] Brain tissue-specific analysis: In AD model treatment experiments, body weight, activity level, and other parameters were closely monitored. Neurotoxicity.

[0229] (3) Target transformation and application: from target identification to product development Once GALNT10 is confirmed as an effective and safe target, it can be applied through the following multiple pathways: Application Direction 1: Development of Therapeutic Inhibitors Inhibitory monoclonal antibodies: Development: Using the GALNT10 catalytic domain as an antigen, screen for antigens that can occupy its active pocket or undergo allosteric transformations. Neutralizing monoclonal antibodies that regulate and thus inhibit the enzyme activity. Using phage display or hybridoma technology, the core screening criterion is "inhibition of the glycosylation activity of GALNT10 on substrate peptides (such as Mucin peptide)".

[0230] Advantages: Antibody drugs have high specificity, long half-life, and can be engineered (e.g., reducing...). (Fc effect function, increased blood-brain barrier penetration) optimized into a central nervous system drug.

[0231] Applications: Can be used alone or in combination therapy with Aβ antibodies, anti-inflammatory drugs, etc., to "clear" The multi-pathway synergistic treatment of AD involves "pathogenic proteins" and "improving the pathological microenvironment".

[0232] Small molecule inhibitors: Development: High-throughput screening (HTS) of compound libraries targeting the GALNT10 catalytic center, or structure-based drug design (SBDD).

[0233] Advantages: Can be taken orally, easily crosses the blood-brain barrier, and has low production costs.

[0234] Application Area Two: Expansion of Diagnostic and Companion Diagnostic Products PET tracer development: Development of positron emission tomography (PET) tracers targeting GALNT10 In vivo brain imaging can directly visualize the distribution and expression level of GALNT10 in the brains of AD patients, enabling "in vivo pathological staging," and can be used to screen patients most likely to benefit from GALNT10 inhibitors (those with high target expression).

[0235] Highly Sensitive Companion Diagnostic Kit: Based on the CLIA detection method of this invention, a kit has been developed and approved by the State Food and Drug Administration. Precise companion diagnostic kits are used in clinical trials and future clinical applications to screen patients (e.g., those with a G / E ratio above a specific threshold), monitor drug efficacy (whether the G / E ratio decreases during treatment), and predict treatment outcomes.

[0236] Application Direction 3: Innovation in Intervention Strategies Gene therapy: Designing shRNA or CRISPR inhibitory elements targeting GALNT10, via AAV The target can be delivered intrabrain via vectors to achieve long-term target knockdown.

[0237] Targeted degradation: Develop a proteolytic targeted chimera and a lysosomal targeted chimera to specifically recruit the GALNT10 protein to E3 ubiquitin ligases or lysosomes for degradation.

[0238] Application Direction 4: Mechanism Research and Drug Repositioning In-depth mechanism exploration: Utilizing developed inhibitors or gene tools to elucidate the mechanisms underlying GALNT10. A complete signal network could potentially lead to the discovery of more new therapeutic targets.

[0239] Drug repositioning screening: Utilizing established GALNT10 enzyme activity detection or cell phenotype screening platforms, Screening existing drug libraries to find "old drugs" that unexpectedly inhibit GALNT10 activity could accelerate their clinical translation for AD treatment.

[0240] Example 7: GALNT10 Genotyping and Genetic Risk Assessment (1) Sample collection and genomic DNA extraction Sample collection: Collect 2-3 mL of peripheral venous blood from the subject into an EDTA-K2 anticoagulant tube. Gently shake. Mix thoroughly 8-10 times to ensure adequate anticoagulation. Samples can be stored at 2-8℃ for short periods not exceeding 72 hours, or at -20℃ to -80℃ for long periods.

[0241] DNA extraction: Extraction method: A commercially available column membrane extraction kit (such as the Qiagen QIAamp DNA Blood Mini Kit) was used. Genomic DNA extraction. Brief steps: After treating whole blood samples with red blood cell lysis buffer, proteinase K and binding buffer are added to lyse the cells. The lysis buffer is transferred to a DNA purification column, impurities are removed with washing buffer, and finally, high-purity gDNA is obtained by elution with elution buffer (such as Tris-EDTA buffer or sterile water).

[0242] Quality and Quantification: DNA concentration and purity were determined using a micro-spectrophotometer (such as Nanodrop).

[0243] A qualified DNA sample should have an A260 / A280 ratio between 1.7 and 2.0, and the concentration is recommended to be no less than 20 ng / μL. DNA integrity should be checked using agarose gel electrophoresis (clear main band, no severe degradation).

[0244] Storage: After dispensing, store at -20℃ or -80℃, avoiding repeated freeze-thaw cycles.

[0245] (2) Detection method: TaqMan probe method for genotyping (specific steps) A. Target SNP selection: Based on publicly available databases of quantitative expression trait loci and / or quantitative protein trait loci, and Validate functional SNP sites that are significantly associated with AD risk and GALNT10 expression levels. For example, select: rs12345678: Located in the promoter region of the GALNT10 gene, it corresponds to plasma levels of GALNT10 protein. The expression of allele A was associated with high expression (pQTL p < 5×10^-8).

[0246] rs98765432: Located in the 3'UTR region of the GALNT10 gene, associated with GALNT10 mRNA in brain tissue. Expression levels were significantly correlated (eQTL p < 5×10^-8), and allele C was associated with high expression.

[0247] B. TaqMan probe-based typing process: Primers and probes: TaqMan SNP genotyping premixes targeting the above SNP sites were commissioned for synthesis. The kit contains specific PCR primer pairs for this site, as well as allele-specific probes labeled with VIC and FAM fluorescent dyes respectively (e.g., VIC-labeled allele 1 and FAM-labeled allele 2).

[0248] Reaction system preparation (performed on 96-well or 384-well plates): TaqMan Genotyping Master Mix (2×): 5μL Target SNP TaqMan Assay (20×): 0.5 μL Genomic DNA template (~20 ng / μL): 2 μL Nuclease-free water: 2.5 μL Total volume: 10μL qPCR amplification and typing: Run the program on the computer: Pre-denaturation: 95°C for 10 minutes (1 cycle) Amplification cycle: 95°C for 15 seconds → 60°C for 60 seconds (40 cycles in total, during the annealing / extension step). (Collect fluorescence signals) After the operation is complete, the genotyping software (such as Applied PCR) provided with the real-time quantitative PCR instrument is used. The analysis was performed using the Genotyping module of Biosystems QuantStudio Real-Time PCR Software.

[0249] Result Interpretation: The software will perform polymerization based on the fluorescence signal intensity of each well in the VIC and FAM channels. The cluster analysis automatically classifies samples into homozygous (VIC / VIC or FAM / FAM), heterozygous (VIC / FAM), or template-free controls (NTC). Manual checks of the cluster diagram's separation ensure accurate and reliable genotyping.

[0250] (3) Genetic risk assessment and information provision A. Establish a reference genotype-risk level database: Based on large-sample case-control association studies (e.g., including healthy controls, MCI patients, and AD patients) (Several hundred cases each), statistically analyze the distribution of different SNP genotype combinations in the population and their corresponding odds ratios or relative risks. Establish a pre-defined genotype-risk level mapping table.

[0251] Example 8: Detection of GALNT10 gene expression level (qRT-PCR method) This embodiment provides a detection method based on real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for accurately quantifying the mRNA expression level of the GALNT10 gene in human peripheral blood mononuclear cells (PBMCs), specific blood cell subsets (such as B cells and monocytes), or tissue samples. This detection directly reflects the gene activity of GALNT10 at the transcriptional level and can serve as a dynamic indicator of functional status. It complements information on protein levels (Examples 3 and 4) and genetic risk (Example 5), providing molecular evidence for assessing the activity of the GALNT10 pathway in an individual and monitoring disease progression or treatment response.

[0252] (1) Kit composition 1. Total RNA Extraction Kit: 1 set, including red blood cell lysis buffer, cell lysis buffer, DNase I, RNA washing buffer, RNase-free water, RNA purification column and collection tube.

[0253] 2. First-strand cDNA synthesis premix: 1 tube (enough for 40 reactions), containing M-MLV reverse transcriptase, RNase inhibitor, Oligo(dT) and random primer mixture, dNTPs and reaction buffer.

[0254] 3. qPCR premix: 1 tube (enough for 100 reactions, 2× concentration), containing Hot Start Taq DNA polymerase, dNTPs, MgCl2, SYBR Green I fluorescent dye, and reaction buffer.

[0255] 4. GALNT10 gene-specific primer mixture: 1 tube, containing: Forward primer (10 μM): 5'-GGAGCCTGAGAAGGACCTGT-3' (designed for the conserved exon linker region of GALNT10 mRNA) Reverse primer (10 μM): 5'-CTCCTTGATGCCGTAGTCGT-3' (The amplified product is 108 bp in length and was verified as a single peak by melting curve analysis).

[0256] 5. Internal reference gene (β-Actin / ACTB) specific primer mixture: 1 tube, used for standardized loading volume, sequence is universal.

[0257] 6. RNase / DNase-free water: 1 tube, 1.5 mL.

[0258] 7. Positive template control: 1 tube, containing cDNA from cells known to highly express GALNT10 (such as the human colon cancer cell line HCT116).

[0259] 8. Template-free control: 1 tube, nuclease-free water.

[0260] (2) Sample collection and preprocessing A. Isolation of peripheral blood mononuclear cells (PBMCs) or specific cell subsets: Collect 8-10 mL of peripheral venous blood from the subject into EDTA anticoagulant tubes or PAXgene Blood RNA tubes. PAXgene tubes can stabilize RNA in situ and are suitable for long-distance transport.

[0261] Routine separation: PBMCs were separated using Ficoll density gradient centrifugation (the method is the same as in the EFEMP1 mRNA detection example).

[0262] Specific cell sorting (optional, to improve specificity): CD19+ B cells or CD14+ monocytes are isolated from PBMCs using immunomagnetic bead sorting or flow cytometry sorting. Previous studies have suggested that these cell subsets may exhibit more specific changes in GALNT10 expression under AD pathological conditions.

[0263] B. Total RNA extraction: Add an appropriate amount of lysis buffer to the cell pellet (≥5×10^5 cells) and mix thoroughly by pipetting.

[0264] Subsequent steps were strictly followed in accordance with the instructions of the selected RNA extraction kit, including on-column DNase I digestion to completely remove genomic DNA contamination.

[0265] RNA was eluted with 30-50 μL of RNase-free water, and its concentration and purity were determined (A260 / A280 ~2.0, A260 / A230 >2.0). RNA integrity was assessed using a microfluidic chip (such as the Agilent Bioanalyzer), and the RIN value should be ≥7.0.

[0266] (3) Reverse transcription and qPCR detection process A. cDNA first-strand synthesis: Prepare the reaction mixture (total volume 20 μL) on ice: Total RNA: 500 ng (volume calculated based on concentration) First-strand cDNA synthesis premix: 10 μL Make up to 20 μL with nuclease-free water.

[0267] Reaction procedure: 25℃ for 10 minutes → 50℃ for 60 minutes → 85℃ for 5 minutes → Store at 4℃. The product can be used immediately or stored at -20℃.

[0268] B. Real-time quantitative PCR: Prepare the reaction system (total volume 20 μL / well) in a 96-well optical plate: 2× qPCR premix: 10 μL GALNT10 (or ACTB) primer mixture (10 μM each): 0.8 μL Template cDNA: 2 μL (usually after diluting the reverse transcription product 5 times before use) Nuclease-free water: 7.2 μL Set up duplicate wells: Technical duplicate wells (n=2) are required for the GALNT10 and ACTB tests of each sample.

[0269] At the same time, a positive control, a template-free control, and a reverse transcription negative control were set up (water was used instead of the reverse transcriptase product to confirm the absence of genomic DNA contamination).

[0270] Run the program on the computer (using Applied Biosystems QuantStudio series as an example): Pre-denaturation: 95℃ for 30 seconds (1 cycle) PCR amplification: 95℃ for 5 seconds → 60℃ for 30 seconds → plate reading (collecting fluorescence signal) (40 cycles in total) Melting curve analysis: 95℃ for 15 seconds → 60℃ for 60 seconds → 95℃ for 15 seconds (continuous acquisition) to confirm the specificity of the amplification product.

[0271] (4) Data analysis, standardization and result interpretation A. Obtaining and quality control of the cycle threshold (Ct value): The instrument software performs automatic analysis, and the Ct value threshold is uniformly set at the midpoint of the linear growth during the exponential phase of the amplification curve.

[0272] Quality control standards: Ct values ​​for NTC and reverse transcription negative controls should be ≥ 40 or have no Ct value; Ct values ​​for positive controls should be within the expected range; Ct value differences between replicates should be < 0.5; melting curves should be single peaks.

[0273] B. Relative quantitative analysis: The relative expression level of GALNT10 mRNA was calculated using the ΔΔCt method.

[0274] Calculate the difference in Ct values ​​between GALNT10 and the internal reference gene ACTB for each sample: ΔCt(sample) = Ct(GALNT10) - Ct(ACTB).

[0275] The average ΔCt value of the healthy control group was selected as the calibration benchmark (ΔCt(calibration)).

[0276] Calculate the ΔΔCt value: ΔΔCt = ΔCt(sample) - ΔCt(calibration).

[0277] The relative expression level of GALNT10 mRNA was calculated as: relative expression level = 2^(-ΔΔCt). The mean expression level of the healthy control group was defined as 1.0.

[0278] C. Interpretation of Results and Clinical Relevance: Normal range: A reference range is established based on healthy individuals (n≥100), for example, relative expression levels between 0.5 and 2.0 (5th-95th percentile).

[0279] Expression upward adjustment: Mild upregulation (2.0 - 4.0): suggests enhanced GALNT10 gene transcriptional activity, which may be associated with early AD risk or mild pathological conditions. A comprehensive assessment combining plasma G / E ratio (Example 5) is recommended.

[0280] Significant upregulation (>4.0): strongly suggests significant activation of the GALNT10 pathway. In diagnosed MCI or AD patients, its expression level may be positively correlated with disease severity (e.g., CDR score), and is a potential marker of disease activity. In asymptomatic high-risk individuals, it is a signal requiring close monitoring and early intervention.

[0281] Downregulation (< 0.5): This is relatively rare, and its pathological significance needs to be analyzed in conjunction with the specific clinical context.

[0282] (5) Technical advantages and application scenarios of this embodiment Technical advantages: High sensitivity and specificity: It can detect several copies of mRNA in a single cell, and specific primers and melting curve analysis ensure reliable results.

[0283] Direct functional readouts: directly reflect the real-time transcriptional activity of genes, responding to physiological and pathological changes faster than protein detection.

[0284] Sample flexibility: Not limited to blood, it can also be applied to cerebrospinal fluid cell sedimentation, frozen brain tissue or biopsy samples, providing flexibility for scientific research and clinical testing.

[0285] Application scenarios: Precise dynamic monitoring of high-risk populations: For individuals carrying high-risk genotypes (Example 5) or abnormal G / E ratios, the level of GALNT10 mRNA in PBMCs can be monitored regularly (e.g., annually) to dynamically assess whether their genetic risk has been transformed into functional pathway activation, thus achieving ultra-early warning.

[0286] Patient stratification and prognostic assessment: In AD patients, high mRNA expression may define a subtype of “high GALNT10 activity”, which may have unique pathological features (such as more severe ECM pathology) and a faster rate of progression, and can be used for clinical trial enrollment stratification and prognostic assessment.

[0287] Pharmacokinetic biomarkers: In clinical trials of GALNT10-targeting inhibitors (see Target Examples), monitoring changes in GALNT10 mRNA levels in patients' PBMCs before and after treatment can serve as important molecular pharmacodynamic indicators for assessing whether the drug is on target and whether it induces feedback regulation.

[0288] Mechanism study: Combined with EFEMP1 mRNA detection, the coordination of their expression can be studied at the transcriptional level, allowing for in-depth exploration of their regulatory network.

[0289] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for assessing early risk of Alzheimer's disease based on GALNT10 detection, characterized in that, include: The levels of the GALNT10 biomarker in the subjects' biological samples were detected, and based on the detection results, information was obtained to assess the early risk of Alzheimer's disease in the subjects.

2. The method according to claim 1, characterized in that, The genotype of GALNT10 or the expression level of GALNT10 mRNA in the subjects' biological samples were detected.

3. The method according to claim 2, characterized in that, Biological samples include blood, oral swabs, peripheral blood plasma, or cerebrospinal fluid.

4. The method according to claim 1, characterized in that, The concentration of GALNT10 protein in the subjects' biological samples was detected.

5. The method according to claim 4, characterized in that, Biological samples include peripheral blood plasma or cerebrospinal fluid.

6. The method according to claim 5, characterized in that, The concentration of GALNT10 protein in peripheral blood plasma or cerebrospinal fluid was detected using a kit.

7. The method according to claim 6, characterized in that, The kit includes an antibody that specifically binds to the GALNT10 protein.

8. The method according to claim 7, characterized in that, The kit also includes a detection reagent that specifically identifies GALNT10.

9. The method according to claim 7, characterized in that, The antibodies include capture antibodies and detection antibodies targeting different epitopes of the GALNT10 protein.

10. The method according to claim 9, characterized in that, The capture antibody is immobilized on a solid-phase support, and the detection antibody is attached with a detectable label; the solid-phase support includes a microplate, a nitrocellulose membrane, a glass fiber membrane, or paramagnetic microparticles; the detectable label is a chemiluminescent label, a fluorescent label, or an enzyme label.