A serum alpha-synuclein seed in vitro amplification detection method, kit and application thereof

Abstract

The application discloses a serum alpha-synuclein seed in-vitro amplification detection method, a kit and application thereof, and belongs to the biomedical field. The serum alpha-synuclein seed in-vitro amplification detection method provided by the application comprises the following steps: lysing the serum to be detected, collecting a first supernatant after centrifugation; the first supernatant is extracted with an organic solvent to extract lipids, and an aqueous phase is collected; the aqueous phase is added with proteinase K for digestion, and a second supernatant is collected after centrifugation again; the second supernatant is subjected to ultrafiltration to collect a cut-off liquid, thereby obtaining a pretreated seed enrichment serum sample; and the pretreated seed enrichment serum sample is added into a reaction solution to perform SAA amplification reaction. The application is based on a new serum pretreatment idea and optimized SAA amplification reaction conditions, and proposes a new serum alpha-synuclein seed in-vitro amplification detection method and a matching kit, so as to meet the requirements of standardized, high-throughput, cross-center consistent clinical detection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method, kit, and application of in vitro amplification and detection of serum α-synuclein seeds. Background Technology

[0002] Neurodegenerative diseases such as Parkinson's disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA) are collectively referred to as α-synuclein lineage diseases. Their common pathological basis is the abnormal misfolding of αSyn, which gradually forms amyloid aggregates rich in β-sheet-like structures. These aggregates deposit in nerve cells, forming Lewy bodies and Lewy neurites. Pathological αSyn aggregates exhibit prion-like template seed-inducing activity; that is, trace amounts of pathological αSyn aggregates can recruit soluble αSyn monomers, inducing conformational changes and folding to form new aggregated fibers, thereby achieving seed self-replication, cascade amplification, and intercellular pathological transmission.

[0003] Based on the principle of "misfolded template-induced aggregation," seed amplification assays (SAAs) have been developed for the detection of ultra-low amounts of pathological α-Syn seeds. Representative SAA techniques include Real-Time Quaking-Induced Conversion (RT-QuIC) and Protein Misfolding Cyclic Amplification (PMCA). These techniques involve co-incubating pathological α-Syn seeds in the sample with recombinant α-Syn monomers, inducing template-based aggregation of the pathological seeds. The aggregation process is then recorded in real-time using fluorescent probes such as thioflavin T (ThT), enabling cascade amplification and visualization of extremely low-abundance pathological seeds. Currently, α-Syn SAA has been successfully applied to the detection of ultra-low amounts of α-Syn seeds in various biological samples, including cerebrospinal fluid, skin, olfactory mucosa, and blood, demonstrating significant potential for auxiliary diagnostic purposes.

[0004] Existing strategies for detecting αSyn in blood include the isolation of neuronal-derived extracellular vesicles and immunoprecipitation-coupled amplification. These methods have demonstrated the presence of transmissible αSyn pathological seeds in the blood, but they are typically complex, time-consuming, and dependent on specific capture markers or antibody enrichment steps, making standardization and batch testing challenging. Serum, as the most readily available and accepted sample in clinical settings, offers advantages such as simple sampling and repeated collection, making it suitable for large-scale population screening and longitudinal follow-up monitoring. However, current research confirms that human serum is not merely a dilution medium with low αSyn abundance, but rather an actively inhibitory biological matrix containing multiple endogenous aggregation inhibitors. Untreated serum, even with the addition of high concentrations (e.g., 1 ng / mL) of human αSyn prepared fibers (PFFs), can completely block the seed amplification reaction, failing to form an effective ThT fluorescence curve. With serial dilution of serum, the inhibitory effect gradually weakens, with seeding activity only partially restored at extremely low serum concentrations (approximately 1% volume fraction). This indicates that lipids, lipoproteins (such as apolipoproteins ApoE and ApoA1), highly abundant soluble proteins (such as immunoglobulins), and other bioactive small molecule components in serum together constitute a potent complex inhibition of αSyn seed amplification.

[0005] Furthermore, existing serum processing approaches often focus on optimizing a single step, such as removing low-density lipoprotein (LDL) and high-density lipoprotein (HDL) solely through centrifugation, or extracting exosomes containing pathological αSyn seeds using ultracentrifugation alone. These methods cannot simultaneously and effectively address the combined interference from lipid particles, highly abundant soluble proteins, low molecular weight degradation products, and residual small molecule inhibitors, resulting in insufficient sensitivity for serum SAA (the overall performance of existing protocols is approximately 80%).

[0006] Meanwhile, existing technologies treat pretreatment and amplification as independent open-loop tandem steps, lacking three key design elements: (i) lack of a real-time feedback monitoring mechanism for seed viability retention during pretreatment: previous schemes used impurity removal rate as the sole optimization indicator, failing to determine whether the treatment process had damaged seed viability, leading to overtreatment or undertreatment; (ii) lack of a bidirectional adaptation relationship between the residual matrix characteristics of pretreatment products and amplification reaction conditions: existing technologies directly input pretreatment products into a predetermined amplification system, failing to recognize that the residual matrix of pretreatment products fundamentally alters the kinetic relationship between seed signal and background noise in the amplification reaction; (iii) lack of a multi-factor collaborative optimization method with ΔT-Lag as the objective function: existing technologies all employ single-factor adjustment strategies (such as single screening for temperature or single screening for additives), failing to capture multi-factor interaction effects, thus failing to find the global optimal solution. The combination of these three defects results in the diagnostic performance of existing serum SAA protocols stagnating at approximately 80% sensitivity, failing to meet clinical needs.

[0007] Patent CN119322181B, "A Serum Pretreatment Agent and Its Application, and an In Vitro Amplification Method for α-Synonesomes," discloses methods such as reducing interference from lipoproteins and impurity proteins through serum pretreatment agents, pH-segmented centrifugation, and heating at 70°C, followed by optimization of SAA detection conditions. However, its focus is primarily on "separating lipoproteins and pathological αSyn aggregates" and "inactivating impurity proteins." It fails to reveal the multi-level masking mechanism of serum inhibitors on αSyn seed activity, nor does it establish a feedback evaluation system using seed activity retention rate as a constraint on pretreatment intensity. Furthermore, it treats pretreatment and amplification detection as sequential steps, failing to reveal that the residual matrix characteristics of the pretreatment product inversely determine the optimal combination of pH, detergent, ionic strength enhancer, and αSyn monomer source in the amplification system.

[0008] Therefore, there is an urgent need in this field for a detection method and matching kit that integrates serum pretreatment, amplification reaction and control reagents in a functionally compatible manner to meet the clinical testing needs of standardization, high throughput and cross-center consistency. Summary of the Invention

[0009] The purpose of this invention is to provide a method, kit, and application for in vitro amplification and detection of serum α-synuclein seeds, in order to solve the problems existing in the prior art. Based on a new serum pretreatment approach and optimized SAA amplification reaction conditions, this invention proposes a new method and kit for in vitro amplification and detection of serum α-synuclein seeds.

[0010] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for in vitro amplification and detection of serum α-synuclein seeds for non-diagnostic and non-therapeutic purposes, comprising the following steps: The serum to be tested was lysed, centrifuged, and the first supernatant was collected. The lipids of the first supernatant were extracted with an organic solvent, and the aqueous phase was collected. Proteinase K was added to the aqueous phase for digestion, and the second supernatant was collected after centrifugation. The second supernatant was ultrafiltered and the retentate was collected to obtain the pretreated seed-enriched serum sample. The pretreated seed-enriched serum sample was added to the reaction solution for SAA amplification reaction; The reaction solution consists of human full-length α-synuclein monomer, thioflavin T, sodium chloride, sodium dodecyl sulfate, saturated ammonium sulfate, and Tris hydrochloric acid buffer.

[0011] Optionally, the serum to be tested is mixed with an equal volume of strong lysis buffer and incubated on ice for 30 minutes, vortexing once every 5 to 10 minutes. After incubation, the mixture is centrifuged at 4°C and 10,000 × g for 15 minutes to obtain the first supernatant. The strong lysis buffer is a protein lysis buffer containing 0.001% sodium dodecyl sulfate, 1% Trion-X 100, 150mM NaCl and 1x protease phosphatase inhibitor.

[0012] Optionally, chloroform, methanol, and water are added sequentially to the first supernatant, with the final system having a volume ratio of chloroform, methanol, and water of 2:1:0.8. After mixing, the mixture is centrifuged at 4°C and 15000×g for 15 minutes to obtain the aqueous phase.

[0013] Optionally, proteinase K is added to the aqueous phase to a final concentration of 1 μg / mL, and digested for 60 minutes at 56°C and 900 rpm with shaking. After digestion, proteinase K activity is terminated, and then the mixture is centrifuged at 4°C and 5000×g for 10 minutes to obtain the second supernatant. The ultrafiltration has a molecular weight cutoff of 30 kDa.

[0014] Optionally, the pretreated seed-enriched serum sample is added to the reaction solution at a volume ratio of 1:9. In the reaction solution, the concentration of human full-length α-synuclein monomer is 0.1 mg / mL, the concentration of thioflavone T is 10 μM, the concentration of sodium chloride is 50 mM, the concentration of sodium dodecyl sulfate is 0.01%, the concentration of saturated ammonium sulfate is 2%, and the concentration of Tris hydrochloric acid buffer is 10 mM. The conditions for the SAA amplification reaction were as follows: pH 8.0, constant temperature of 50℃, high-intensity dual-track intermittent oscillation mode, 1 minute of oscillation per cycle followed by 14 minutes of rest, fluorescence detection by reading thioflavone T fluorescence from the bottom every 15 minutes, excitation wavelength of 450±10 nm, emission wavelength of 490±10 nm, and a total monitoring time of 48 hours.

[0015] Optionally, if the lag period of the SAA amplification reaction is ≤37.38 hours, the serum α-synuclein to be tested is determined to be positive.

[0016] The present invention also provides a serum α-synuclein seed in vitro amplification detection kit, the kit comprising a pretreatment component, an amplification reaction component, and a control reagent; The pretreatment components include a strong lysis buffer, chloroform, methanol, proteinase K, and an ultrafiltration device; the strong lysis buffer is a protein lysis buffer containing 0.001% sodium dodecyl sulfate, 1% Trion-X 100, 150 mM NaCl, and 1x proteinase phosphatase inhibitor; the ultrafiltration has a molecular weight cutoff of 30 kDa. The amplification reaction assembly includes a reaction solution composed of human full-length α-synuclein monomer, thioflavin T, sodium chloride, sodium dodecyl sulfate, saturated ammonium sulfate, and Tris hydrochloric acid buffer. The control reagents include a positive control of α-synuclein prefibrils and a negative control without α-synuclein.

[0017] Optionally, in the reaction solution, the concentration of human full-length α-synuclein monomer is 0.1 mg / mL, the concentration of thioflavone T is 10 μM, the concentration of sodium chloride is 50 mM, the concentration of sodium dodecyl sulfate is 0.01%, the concentration of saturated ammonium sulfate is 2%, and the concentration of Tris hydrochloric acid buffer is 10 mM.

[0018] The present invention also provides the application of the kit in the preparation of diagnostic products for α-synuclein lineage diseases.

[0019] Optionally, the α-synuclein lineage diseases include Parkinson's disease, Lewy body dementia, or multiple system atrophy.

[0020] The present invention discloses the following technical effects: Based on a novel serum pretreatment approach and optimized SAA amplification reaction conditions, this invention proposes a new in vitro amplification and detection method for serum α-synuclein seeds, along with a matching kit.

[0021] (1) Solve the problem of active inhibition of serum and achieve ultra-high analytical sensitivity. This invention treats serum lipids / lipoproteins and high-abundance proteins as active inhibitory factors rather than simple dilution factors, and uses a four-step pretreatment process to restore masked αSyn seeds to an amplifiable state. Ultimately, it achieves a detection limit of 100 fg / mL for synthetic αSyn seeds, detectable within 24 hours, with a sensitivity reaching the femtogram level.

[0022] (2) The functions of each step of the preprocessing process have been rigorously verified. TEM results showed a significant reduction in serum lipid particles after lipid extraction; amplification was again inhibited after lipid fraction re-addition, proving that lipid-associated particles were one of the main sources of inhibition. PK gradient experiments and proteomics results showed that limited digestion reduced multiple high-abundance interfering proteins while preserving seed activity. Ultrafiltration further enhanced the signal. Solvent residue removal experiments confirmed that the restored seed activity stemmed from the removal of inhibitory factors rather than solvent-induced non-specific effects. Each pretreatment step played a non-redundant complementary role.

[0023] (3) The matrix-adapted amplification system is significantly better than directly using existing conditions. This invention employs a serum-specific system using human αSyn substrate, 2% saturated ammonium sulfate, 0.01% SDS, and pH 8.0. Compared to directly applying skin SAA conditions (mouse monomer, 10% saturated ammonium sulfate, pH 7.6) or cerebrospinal fluid SAA conditions, it exhibits significantly better positive / negative separation. Factorial design system screening ensures the scientific rigor and optimality of the selected conditions.

[0024] (4) Excellent performance in deriving queue diagnosis Experimental results showed that in a deduced cohort consisting of 100 Parkinson's disease patients and 100 non-neurodegenerative disease controls, serum α-Syn seeding activity assays exhibited a sensitivity of approximately 92%, a specificity of approximately 96%, and an area under the curve of approximately 0.9539. The concordance between serum α-Syn seeding activity assays and paired skin α-Syn seeding activity assays reached 92.0% in the Parkinson's disease group and 97.0% in the control group, demonstrating a high degree of consistency between the serum α-Syn seeding activity assay results and the previously validated peripheral tissue assays.

[0025] (5) Easy to operate, cost controllable, and conducive to standardization The detection process of this invention does not rely on antibody capture or extracellular vesicle separation. The pretreatment process can be completed using conventional chemical reagents and standard laboratory equipment, significantly reducing operational complexity and reagent costs. Immunodepletion or immunoprecipitation is only used as a one-time analytical specificity verification step and is not a necessary step in routine testing. The standardized pretreatment protocol and reaction formulation are naturally suitable for multi-center coordination and batch quality control. Blood sample collection requires only about 3 mL of peripheral venous blood, which is minimally invasive and suitable for large-scale population screening and longitudinal repeated monitoring.

[0026] (6) Reagent kit integration enables out-of-the-box use and cross-center consistency. The detection kit provided by this invention integrates pretreatment components, amplification reaction components, control reagents, and reaction carriers in a functionally compatible manner. Compared with the prior art where each laboratory purchases and prepares its own dispersive reagents, the kit of this invention has the following advantages: (i) It is ready to use out of the box, eliminating the need for users to prepare pretreatment and amplification reaction reagents themselves, thus reducing operational complexity and preparation error rates; (ii) Unified batch quality control ensures that the concentration, purity, and activity of each reagent within the same batch of kits are uniformly tested, reducing the impact of batch-to-batch differences on detection results; (iii) The functional compatibility between pretreatment and amplification reaction reagents is pre-verified, ensuring the matching between the residual matrix characteristics of the pretreatment product and the amplification reaction conditions, avoiding performance degradation due to mismatched formulations when users combine reagents themselves; (iv) The kit's form facilitates multi-center distribution and coordination. The multi-center validation of this invention (501 PD cases and 525 controls) was conducted based on a unified kit, with no significant heterogeneity in detection performance among centers, demonstrating the contribution of kit integration to cross-center consistency; (v) The kit includes an instruction manual that details the operating steps and interpretation rules, further reducing operator variability.

[0027] (7) It has unintended technical effects compared to existing technologies. Compared to patent CN119322181B - a serum pretreatment agent and its application, and an in vitro amplification method for α-synucleosomes (the main technical idea is to reduce lipoprotein interference and optimize conventional RT-QuIC conditions), this invention demonstrates through experiments that simply replacing the pretreatment step or the amplification conditions cannot achieve the full effect of this invention; only by combining a four-step layered pretreatment with a serum-specific amplification system can low background, high signal recovery rate, and significant ΔT-Lag separation be simultaneously obtained. This effect surpasses the simple superposition of individual techniques, constituting a synergistic effect.

[0028] Compared to the existing lipid-free serum SAA sensitivity level of about 80%, this invention improves the deduced cohort sensitivity to 92% and the specificity to 96%. In a multicenter validation of 501 PD cases and 525 controls, it maintains a sensitivity of 90.02% and a specificity of 94.67%, indicating that the technical solution provided by this invention is not an accidental optimization under laboratory conditions, but a standardized detection system with cross-center stability. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Optimization of pretreatment and reaction conditions for serum α-synuclein seed amplification assay (SAA); A: Schematic diagram of serum sample pretreatment process; showing the process starting from serum, incubation with lysis buffer to dissociate protein-lipid complexes, Bligh-Dyer lipid extraction to remove lipid particles, limited PK digestion to reduce high-abundance inhibitory proteins, and 30 A: A four-step continuous process for enriching higher-order αSyn seeds using kDa ultrafiltration; B: Matrix optimization heatmap of 32 combined reaction conditions; showing the ΔT-Lag values ​​of different compositions in the reaction buffer: substrate species (HM / MM), SDS concentration (0-0.1%), and saturated ammonium sulfate (AS) concentration (0-10%); the top 6 high-discrimination condition combinations are marked with red dashed boxes; C: ΔT-Lag quantitative analysis of the top 6 candidate reaction conditions; under the same AS and SDS concentrations, HM-based reaction conditions showed higher ΔT-Lag values, with the HM+2%AS+0.01%SDS combination showing the best discrimination ability (ΔT-Lag=30.5 hours); D: Effect of pH on SAA kinetics; acidic conditions (pH 7.0) maximized the aggregation efficiency of the PD group, but the control group showed severe non-specific aggregation (ΔT-Lag=3.25 hours); as pH increased, the overall aggregation kinetics slowed down, but the ΔT-Lag value between the case group and the control group gradually increased. At pH 8.0, the monitoring and differentiation efficiency was optimal (ΔT-Lag = 39.25 hours), while maintaining moderate aggregation kinetics. Figure 2 For the identification and targeted removal validation of serum endogenous inhibitory factors: A: Transmission electron microscopy images of serum before and after lipid extraction, showing that the lipid-rich particles were significantly depleted after extraction; B: Lipid component re-addition experiment, confirming that amplification was inhibited again after lipid re-addition; C: PK concentration gradient optimization results, 1 μg / mL is the optimal limit digestion concentration; D: Proteomics analysis before and after PK digestion, showing that high-abundance inhibitory proteins such as apolipoproteins and immunoglobulins were effectively depleted; E: Stepwise functional validation of the recovery of αSyn seeding activity by each pretreatment step; Figure 3 For the specificity verification of αSyn immune depletion; A: Schematic diagram of immune depletion experiment; B: ELISA quantitative confirmation of αSyn depletion efficiency; C: Western blotting analysis of the immunoprecipitate eluent, showing specific capture of αSyn; D: Comparison of SAA fluorescence curves of serum samples before and after immune depletion, showing the disappearance of positive signals after depletion; E: SAA amplification curves of immunoprecipitates from Parkinson's disease and control samples, with Parkinson's disease samples showing strong seeding activity while the control remained negative; F: Western blotting of the immunoprecipitation-SAA product, with high molecular weight aggregate bands visible in the Parkinson's disease sample. Figure 4To derive the diagnostic performance of serum SAA and skin SAA in the cohort; A: ThT fluorescence kinetic curve of serum SAA (Parkinson's disease group and control group); B: Fluorescence kinetic curve of paired skin SAA; C, D: Receiver operating characteristic (ROC) curves constructed based on the lag time of serum and skin SAA, respectively, showing the corresponding area under the curve (AUC), standard error (SE), and 95% confidence interval (CI); E: ROC curve and related diagnostic parameters (AUC, SE, and 95% CI) of the paired serum-skin SAA joint diagnostic model; F: Consistency matrix of paired serum and skin SAA results; Figure 5 For multicenter validation and interlaboratory consistency: A: Scatter plot of correlation between serum SAA lag-stage measurements from two independent laboratories; B: Bland-Altman concordance analysis of serum SAA; C: Scatter plot of correlation between skin SAA lag-stage measurements from two independent laboratories; D: Bland-Altman concordance analysis of skin SAA; E: Overlay of ROC curves for serum SAA from four centers; F: Sensitivity and specificity point estimates and 95% confidence intervals for each center; G: Positive and negative predictive values ​​for each center. Figure 6 The association between serum αSyn SAA positivity and increased burden of iRBD prodromal phase; A: Box plot of age distribution stratified by SAA status; B: Disease course distribution plot; C: Stacked bar chart of percentage severity grading for the five prodromal assessment domains; D: Forest plot of adjusted odds ratios for each domain (horizontal line represents 95% confidence interval); E: Predicted probabilities of low, medium, and high risk categories estimated by an ordered logistic regression model. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] This invention establishes the design principle that "sample characteristics and amplification conditions jointly determine the detection results of αSyn SAA seed activity" and provides a serum αSyn seed amplification detection method and matching kit based on seed activity feedback and kinetic separation optimization. It adopts a dual optimization technical route of "serum seed activity preservation pretreatment + serum matrix adaptation amplification".

[0037] Unlike serum pretreatment schemes that primarily aim at lipoprotein removal or impurity protein inactivation, this invention uses "whether seed activity is preserved and the degree of relief of endogenous serum inhibition" as dual core constraints, rather than simply pursuing the maximization of LDL, HDL, or total protein removal; and uses "whether positive / negative ΔT-Lag is maximized" as the optimization target of the amplification system. Therefore, the pretreatment module and amplification module in this invention are not conventional tandem steps that can be arbitrarily replaced, but rather a synergistic system that mutually defines, adapts to, and verifies each other.

[0038] The overall working process of this invention is as follows: First, a strong lysis buffer is used to dissociate the protein-lipid complex in serum, releasing the αSyn seeds embedded in the matrix. Then, lipids and lipoprotein particles are removed using a chloroform-methanol-water three-phase partitioning system (Bligh-Dyer method). A limited amount of proteinase K (PK) is used to digest and reduce the abundance of interfering proteins such as apolipoproteins and immunoglobulins. Finally, a 30 kDa molecular weight cutoff ultrafiltration module is used to selectively retain high-order αSyn aggregated seeds while removing low-molecular-weight degradation products, residual small-molecule inhibitors, and possible organic solvent residues. The goal of this module is not simply to remove serum impurities, but to eliminate interference from complex inhibitors while avoiding pathological damage to the activity of αSyn seeds.

[0039] In the pretreatment product-driven serum matrix adaptation amplification module, this invention recognizes that pretreated serum still retains certain residual matrix characteristics, which continue to affect αSyn monomer aggregation kinetics, background noise levels, and the peak time of positive samples. Therefore, this invention uses ΔT-Lag maximization as the objective function to synergistically screen αSyn monomer sources, ionic strength enhancers, detergents, and pH conditions to obtain an amplification reaction system adapted to the pretreated serum matrix. Preferably, the amplification reaction system containing recombinant human αSyn monomers is added to the seed-enriched serum obtained after pretreatment. The pathological seeds in the sample act as templates to induce monomers to aggregate according to the template conformation, forming new amyloid fibers that can bind thioflavin T (ThT). The ELISA reader reads the ThT fluorescence signal of each well in real time under constant temperature and intermittent shaking conditions at 50°C, and the fluorescence intensity gradually increases with the formation of aggregates.

[0040] In the pretreatment-amplification-interpretation closed-loop detection module, this invention records the ThT fluorescence kinetic curve in real time, and interprets the results by combining background threshold, replicate consistency, and hysteresis cutoff value. It also correlates pretreatment intensity, seed activity recovery, amplification reaction kinetics, and positive / negative discrimination efficacy into a closed-loop fit relationship. Specifically, an interpretation threshold is established based on the negative control background fluorescence, the time it takes for the fluorescence signal of each replicate to reach the threshold is recorded, and positive or negative results are output according to the technical replicate consistency rule. Through this closed-loop system, the residual matrix state of the pretreatment product determines the optimal range of the amplification system, while the ΔT-Lag performance of the amplification system, in turn, verifies whether the pretreatment intensity is appropriate.

[0041] This invention further integrates the aforementioned pretreatment reagents and amplification reaction reagents into a detection kit using a functional adaptation method. The kit comprises four functional components: a pretreatment reagent group, an amplification reaction reagent group, control reagents, and a reaction carrier. The types and specifications of each reagent in the pretreatment reagent group correspond one-to-one with the reagents required in each step of the four-step stratified pretreatment process; the types and concentrations of each component in the amplification reaction reagent group correspond one-to-one with the optimal formulation of the serum matrix-adapted amplification reaction system; the control reagents include αSyn pre-fabricated fibers for positive controls and seedless buffer for negative controls; the reaction carrier is a transparent-bottomed microplate adapted for fluorescence reading by an ELISA reader. By pre-configuring the above reagents using a functional adaptation method and implementing unified quality control, users can obtain a standardized, ready-to-use detection tool without having to purchase and prepare individual reagents themselves, thereby reducing operational complexity, minimizing batch-to-batch variability, and improving cross-center detection consistency.

[0042] This invention not only solves the problem of combined inhibition of αSyn seed amplification by lipids, lipoproteins, high-abundance proteins and low-molecular-weight residual inhibitors in serum, but also maximizes the temporal separation between positive and negative samples through ΔT-Lag kinetic interpretation, thereby achieving stable amplification, real-time readout and standardized determination of low-abundance pathological αSyn seeds in serum samples.

[0043] The strong lysis buffer used in the following examples is a protein lysis buffer containing 0.001% sodium dodecyl sulfate, 1% Trion-X 100, 150mM NaCl and 1x protease phosphatase inhibitor.

[0044] Example 1: A method for detecting serum αSyn seed viability retention pretreatment coupled with matrix adaptation amplification. 1. Serum collection and preservation Collect approximately 3 mL of peripheral venous blood from the subject using inert separating gel coagulation tubes (3 mL / tube). Allow the blood collection tubes to stand at room temperature for approximately 10 minutes to allow the blood to fully coagulate. Centrifuge at 3000×g for 10 minutes to separate the serum from the blood cells. Carefully aspirate the supernatant (serum) into low-adsorption centrifuge tubes. Aliquot into 200 μL tubes, adding a protease / phosphatase inhibitor mixture to each tube to prevent protein degradation and dephosphorylation. Store the aliquoted serum samples at -80°C, avoiding repeated freeze-thaw cycles before SAA testing.

[0045] 2. Serum multi-step pretreatment process (targeted removal of endogenous inhibitory factors and enrichment of αSyn seeds, Module 1) The pretreatment workflow designed in this invention includes four consecutive steps, each playing a non-redundant complementary role, jointly achieving targeted removal of inhibitory factors and preservation and enrichment of seed activity. Figure 1 (A). The specific steps are as follows: Step 1: Cleavage and dissociation of protein-lipid complexes Mix 200 μL of serum with 200 μL of strong lysis buffer at a 1:1 volume ratio and incubate on ice for 30 minutes, gently vortexing every 5–10 minutes during incubation. The purpose is to use the detergent to disrupt the tight binding between proteins and lipids in the serum, allowing lipid-related particles to dissociate and release from the protein-lipid complex, while simultaneously exposing αSyn seeds encapsulated by lipoproteins and other complexes, thus creating conditions for subsequent lipid removal. After incubation, centrifuge at 10,000 × g for 15 minutes at 4°C to remove insoluble debris, and collect the supernatant for further processing.

[0046] Step 2: Bligh-Dyer lipid extraction Add chloroform and methanol sequentially to the supernatant obtained in step one, followed by distilled water, adjusting the final volume ratio of chloroform, methanol, and water to 2:1:0.8. After thorough mixing, centrifuge at 15000×g for 15 minutes at 4°C to achieve liquid-liquid phase separation: the upper layer is the aqueous phase (containing water-soluble components such as αSyn seeds), the middle layer is the denatured protein layer, and the lower layer is the organic phase (containing lipid components). Carefully collect the upper aqueous phase using a pipette. For samples with high lipid content (where the aqueous phase is still turbid), repeat the chloroform extraction once to ensure complete lipid removal.

[0047] Functionality verification: Transmission electron microscopy (TEM) confirmed that untreated serum contained a large number of lipid-rich particles, which were significantly reduced after Bligh-Dyer lipid extraction. Figure 2 (A)

[0048] Lipid re-addition inhibition experiments confirmed that when the extracted lipid components were added back to the αSyn amplification reaction system, even under high-concentration seed challenge conditions, the amplification signal was strongly suppressed, the hysteresis phase was significantly prolonged, and the ThT fluorescence intensity was reduced. Figure 2 (B) This experiment directly demonstrates that serum lipids and lipid-associated particles are one of the main sources of endogenous inhibition in αSyn amplification detection.

[0049] Organic solvent residue elimination experiments confirmed that after exposing recombinant αSyn monomers and PFFs to a chloroform-methanol mixture of gradient concentrations, the results of SAA detection and circular dichroism chromatography showed that solvent residues did not change the aggregation kinetics and protein secondary structure of αSyn, nor did they induce non-specific amplification signals, thus ruling out the possibility that the restored seeding activity originated from non-specific changes in fiber structure by the solvent.

[0050] Step 3: Limited proteinase K digestion Add PK to the aqueous phase obtained in step two to a final concentration of 1 μg / mL, and digest for 60 minutes at 56℃ and 900 rpm with shaking. After digestion, immediately add phenylmethylsulfonyl fluoride (PMSF) to terminate PK activity, and then centrifuge at 4℃ and 5000×g for 10 minutes, and collect the supernatant.

[0051] This step employs a "limited consumption" strategy, the core of which is: The PK concentration was determined through gradient optimization (test range: 0 to 100 μg / mL): a PK concentration of 1 μg / mL maximized the recovery of seed-dependent amplification signals; too low a PK concentration (e.g., below 0.1 μg / mL) could not adequately relieve the inhibitory effect of high-abundance proteins; too high a PK concentration (e.g., above 10 μg / mL) resulted in excessive digestion of αSyn seeds, leading to loss of seed activity. Figure 2 (C). Therefore, 1 μg / mL is the preferred limit digestion concentration, and there is a narrow optimal PK window.

[0052] The optimal concentration was determined using the seed activity feedback method: A known concentration (100 fg / mL) of αSyn pre-prepared fiber was added to the serum of healthy controls as an internal standard seed. After treatment in steps one and two, the resulting aqueous phase was digested with a PK concentration gradient (0, 0.1, 0.5, 1, 2, 5, 10, 50, 100 μg / mL). After digestion, each group of samples was subjected to SAA detection according to the amplification conditions in Module Two. The PK concentration-signal recovery rate curve was plotted using the "seed signal recovery rate" (ThT fluorescence peak intensity of the treated sample ÷ ThT fluorescence peak intensity of the untreated pure internal standard seed × 100%) as the evaluation index. A cross-window interval with a signal recovery rate ≥ 80% and a high-abundance protein degradation rate ≥ 60% was selected to determine 1 μg / mL as the optimal concentration satisfying both constraints.

[0053] Proteomics analysis confirmed that after limited PK digestion, a variety of high-abundance inhibitory proteins in serum (including lipid-related and immune-related proteins such as apolipoproteins and immunoglobulins) were effectively degraded and depleted, while albumin was relatively retained. Figure 2 (D).

[0054] The goal of this limited digestion is not to completely remove all proteins, but to selectively reduce high-abundance proteins that inhibit αSyn amplification, while maintaining the sowing activity of αSyn seeds.

[0055] Step 4: Ultrafiltration enrichment Add the supernatant obtained in step three to a 30 kDa molecular weight cutoff ultrafiltration device and centrifuge to concentrate it according to the device's instructions. Collect the retentate from the ultrafiltration device; this is the pretreated seed-enriched serum sample.

[0056] The function of this step is not simply to concentrate total protein, but to selectively separate based on molecular weight: the retentate retains higher-order αSyn aggregates with a molecular weight greater than 30 kDa (i.e., seeds with seeding activity), while low-molecular-weight PK digestion products (degradation fragments), residual small molecule inhibitors, and possible organic solvent residues are removed by being discharged into the filtrate through an ultrafiltration membrane.

[0057] The cumulative contribution of each step to the recovery of seed viability has been confirmed through stepwise validation experiments. Figure 2 (E): Lipid extraction alone can partially recover the amplification signal. Adding limited PK digestion further shortens the hysteresis period and increases the fluorescence intensity. Finally, after adding ultrafiltration enrichment, the signal recovery and detection efficiency reach the optimal level.

[0058] The sequential implementation of the above four steps constitutes the matrix state transformation process of serum from a "strongly inhibited state" to a "propagable seed-enriched state." Each step cannot be arbitrarily omitted or its order changed, because the effectiveness of each subsequent step depends on the state of the intermediate product provided by the preceding step: lysis provides free lipids for extraction; extraction provides a lipid-free aqueous phase for digestion; and digestion provides a solution with a suitable low-molecular-weight / high-molecular-weight ratio for ultrafiltration. The residual matrix characteristics of the output product of Module 1 (seed-enriched serum) will determine the constraint space for optimizing the amplification conditions in Module 2.

[0059] The above four-step combination exhibits sequential dependence and functional non-equivalence: the lysis step addresses the seed exposure problem caused by protein-lipid complex encapsulation; Bligh-Dyer extraction addresses the interfacial inhibition of lipid / lipoprotein particles on the aggregation core; limited PK digestion addresses the inhibition of monomer aggregation and seed elongation by high-abundance proteins; and 30 kDa ultrafiltration addresses the removal of low-molecular-weight degradation products and residual small-molecule inhibitors while retaining higher-order seeds. Each of these steps corresponds to a different level of inhibition source and cannot be equivalently replaced by simple pH centrifugation or heat inactivation steps.

[0060] 3. Establishment of a serum-specific amplification reaction system (Module 2) This invention recognizes that directly applying validated skin SAA or cerebrospinal fluid SAA reaction conditions to serum pretreatment products fails to generate effective amplification signals, demonstrating that serum requires a unique set of reaction chemistry conditions matched to its matrix characteristics. Therefore, this invention systematically optimizes the key parameters of the amplification reaction system.

[0061] 3.1 Optimization Strategies and Methods (Initial Screening) Using the skin SAA response framework (conditions: 50℃, 0.1 mg / mL mouse αSyn monomer, 10% saturated ammonium sulfate, 10 μM ThT, 50 mM sodium chloride, 10 mM Tris hydrochloric acid buffer, pH 7.6) as a reference, systematic screening was conducted based on the following parameters: Types of αSyn monomers: Human full-length αSyn monomer (HM) and mouse full-length αSyn monomer (MM). Saturated ammonium sulfate concentrations: 0%, 2%, 5%, 10% (4 levels); Sodium dodecyl sulfate (SDS) concentrations: 0%, 0.001%, 0.01%, 0.1% (4 levels); Pretreated seed-enriched serum from 10 Parkinson's disease (PD) patients and 10 non-neurodegenerative disease controls were used as screening samples. With a fixed reaction temperature of 50℃ and pH 7.6, 32 reaction conditions were created using a factorial design, combining human / mouse monomers (2 levels) × saturated ammonium sulfate 0% / 2% / 5% / 10% (4 levels) × SDS 0% / 0.001% / 0.01% / 0.1% (4 levels). Each condition was tested using an equal volume of pretreated serum from PD patients and pretreated serum from non-neurodegenerative disease controls. Three technical replicates were used for each condition.

[0062] After 48 hours of operation, the average time for the Parkinson's disease group and the control group to reach the threshold was calculated for each condition. The result was ΔT-Lag = average lag time of the control group − average lag time of the Parkinson's disease group (if a group does not reach the peak within 48 hours, its lag time is recorded as 48 hours).

[0063] ΔT-Lag was used as the core evaluation index for efficacy discrimination. ΔT-Lag is defined as the average time for control group samples to reach the fluorescence threshold minus the average time for Parkinson's disease group samples to reach the fluorescence threshold, i.e., the lag separation between the two groups. A larger ΔT-Lag indicates that the reaction conditions are more effective in distinguishing between Parkinson's disease positive samples and control negative samples. The fluorescence threshold is defined as the mean background fluorescence of the negative control plus 10 standard deviations.

[0064] 3.2 Initial screening results Of the 32 conditions, 15 produced detectable amplification signals, while 17 conditions resulted in negative signals. Analysis revealed: The common characteristics of all negative signal conditions are: no saturated ammonium sulfate (0%), and extremely low or zero SDS concentration (0% or 0.001%), indicating that in the absence of appropriate ionic strength enhancers and trace detergents, the inhibition of serum residual matrix cannot be relieved, and the aggregation reaction of monomers cannot be initiated.

[0065] Positive signal conditions are mainly concentrated in the combination of medium to high concentrations of saturated ammonium sulfate (2% to 10%) and moderate concentrations of SDS (0.01% to 0.1%).

[0066] The lag time ranged from 0.42 hours to 30.5 hours for each positive condition, reflecting significant differences in sowing efficiency between different reaction environments.

[0067] Under the same saturated ammonium sulfate and SDS conditions, the human monomer (HM) consistently yielded a larger ΔT-Lag value than the mouse monomer (MM), indicating that the human monomer is more suitable for serum-based SAA detection of human pathological αSyn seeds.

[0068] Among all 32 conditions, the combination of HM + 2% saturated ammonium sulfate + 0.01% SDS showed the highest discriminative ability (ΔT-Lag approximately 30.5 hours) and was selected as the best candidate condition. Figure 1 (B and C in the middle).

[0069] 3.3 pH Optimization Based on the above optimal candidate conditions, the effects of pH 7.0, pH 7.6, and pH 8.0 gradients on amplification kinetics and discriminative efficacy were further compared: pH 7.0 (slightly acidic conditions): The Parkinson's disease group showed the fastest aggregation rate, but the control group also showed severe nonspecific spontaneous aggregation, with ΔT-Lag only about 3.25 hours, which could not effectively distinguish between positive and negative samples.

[0070] pH 7.6 (neutral to alkaline conditions): Aggregation kinetics slowed down, and the ΔT-Lag between the two groups was larger than that at pH 7.0.

[0071] pH 8.0 (slightly alkaline conditions): While maintaining effective amplification signals in Parkinson's disease positive samples, non-specific aggregation in control samples was significantly delayed or inhibited, with ΔT-Lag reaching approximately 39.25 hours, achieving optimal positive / negative separation. Mechanistic analysis: The slightly alkaline pH conditions reduced the spontaneous aggregation tendency of αSyn monomers under seedless conditions (i.e., reduced background noise), while the template-inducing effect of seeds was still sufficient to drive monomer aggregation in the presence of seeds (i.e., signal preservation), thus achieving an optimal balance between maximizing sensitivity and maximizing specificity. Therefore, pH 8.0 was determined as the final preferred pH for serum SAA response. Figure 1 (D) In summary, the final formulation of the serum-specific αSyn SAA reaction system is shown in Table 1.

[0072] Table 1. Formulation of serum-specific αSyn SAA reaction system Note: Compared with the skin SAA method, the serum reaction system provided by this invention uses human monomers instead of mouse monomers, reduces the concentration of saturated ammonium sulfate from 10% to 2%, adds 0.01% SDS, and increases the pH from 7.6 to 8.0 to achieve a unique matrix background suitable for serum pretreatment products, realize stable fibrosis kinetics under low promoter conditions, and effectively inhibit non-specific aggregation.

[0073] The technological contribution of this reaction system lies not in the conventional optimization of a single parameter, but in the synergistic effect of human monomers, low-concentration saturated ammonium sulfate, trace amounts of SDS, and a slightly alkaline pH, which, while removing major inhibitory factors but still retaining residual serum matrix, ensures that seeded samples maintain sufficient amplification driving force, while the spontaneous aggregation of seedless or low-background samples is significantly delayed. This combination achieves improved kinetic separation, rather than simply accelerating aggregation, and therefore cannot be directly obtained through conventional condition optimization in existing technologies.

[0074] 4. Complete αSyn SAA testing procedure 4.1 Preparation of reaction plate and addition of sample The reaction system was prepared in a black transparent 384-well microplate. 45 μL of the stock solution containing the formulation in Table 1 was dispensed into each well, followed by 5 μL of seed-enriched serum obtained after the four-step pretreatment in Module 1. At least three technical replicates were prepared for each sample. Controls were also included: a negative control was a reaction system with the same stock solution formulation as the amplification reaction but without αSyn seeds; a positive control was a reaction system with 100 (fg / mL) αSyn PFFs added. Distilled water was injected into the wells around the perimeter of the microplate to reduce edge evaporation, and a sealing film was applied to the plate surface.

[0075] 4.2 Instrument Operating Parameter Settings Place the sealed 384-well plate into the microplate reader and set the parameters as follows: Temperature: 50℃ constant temperature; Oscillation mode: high-intensity dual-track intermittent oscillation, each cycle oscillation for 1 minute followed by 14 minutes of rest; Fluorescence detection: ThT fluorescence is read from the bottom every 15 minutes, excitation wavelength 450±10 nm, emission wavelength 490±10 nm.

[0076] Total monitoring time: 48 hours for serum samples (24 hours for skin samples, with serum monitoring extended to 48 hours to accommodate its lower seeding abundance).

[0077] 4.3 Result Interpretation Rules Threshold setting: The mean fluorescence signal of the negative control at each time point of the same plate plus 10 times the standard deviation was used as the fluorescence threshold line.

[0078] Lag phase recording: The time point at which the fluorescence signal of each technique duplex first exceeds the threshold line is recorded, which is the lag phase of that duplex.

[0079] Positive determination criteria: If at least two of the three replicates of the same sample show fluorescence signals exceeding the threshold within the 48-hour monitoring window, the sample is determined to be positive for serum αSyn SAA; otherwise, it is determined to be negative.

[0080] Diagnostic cutoff value: For the classification and interpretation of batch clinical samples, the optimal lag cutoff value can be determined by combining the receiver operating characteristic (ROC) curve. In this invention, the preset diagnostic cutoff value obtained through verification is that a lag of ≤37.38 hours is considered positive.

[0081] 5. Verification of the specificity of the amplified signal source To confirm that the positive signal detected in serum SAA indeed originates from αSyn pathological seeds rather than nonspecific matrix interference, this invention designs an immune exhaustion verification experiment: a) Take 12 μg of anti-αSyn monoclonal antibody MJFR and incubate it overnight at 4°C with 20 μL of protein A / G magnetic bead suspension to couple the antibody to the surface of the magnetic beads.

[0082] b) Add the pretreated serum sample to the complex and continue incubation at 4°C for about 2 hours to allow the serum αSyn to be specifically captured by the antibody.

[0083] c) After magnetic separation, the supernatant (αSyn has been removed) after immunodepletion and the immunoprecipitate eluent (αSyn enriched) were collected separately.

[0084] d) Both were subjected to SAA detection and Western blot analysis.

[0085] e) Verification results show ( Figure 3Immunoprecipitate eluates from the serum of three Parkinson's disease patients all produced clear ThT fluorescence amplification curves in SAA (lag period approximately 8 to 15 hours), and high molecular weight αSyn aggregate bands were visible in Western blotting. Immunoprecipitate eluates and supernatants from the same Parkinson's disease patient and all three control patients maintained baseline fluorescence levels in SAA for 48 hours, and Western blotting showed only approximately 14 kDa monomeric αSyn bands. ELISA quantification confirmed a significant decrease in αSyn content in the supernatant after immunodepletion compared to before depletion.

[0086] The results demonstrate that the positive serum SAA signal detected by this invention is indeed specifically derived from the template-induced amplification of αSyn pathological seeds, rather than the non-specific aggregation of other non-αSyn components in the serum matrix.

[0087] Example 2 Preparation of the reagent kit 1. Preparation of recombinant αSyn monomer and pre-formed fibers 1.1 Preparation of Recombinant αSyn Monomer a) Transform the expression plasmid carrying the full-length human SNCA gene (#36046, encoding amino acid αSyn from position 1 to 140) into BL21(DE3) Escherichia coli competent cells.

[0088] b) The transformant bacteria were cultured in ampicillin-containing TB medium at 37°C and 230 rpm until the bacterial culture reached OD. 600 The value is approximately 0.8.

[0089] c) After inducing expression for 4 hours by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1.0 mM, the bacterial cells were harvested.

[0090] d) The bacterial cells were resuspended in a high-salt buffer (10 mM Tris hydrochloric acid pH 7.5, 500 mM sodium chloride, 1 mM EDTA, 1 mM PMSF and a mixture of protease inhibitors) and sonicated to release the proteins.

[0091] e) Heat the lysate in boiling water for 10 minutes to remove thermally unstable impurity proteins by utilizing the high thermal stability of αSyn.

[0092] f) Centrifuge at 20000×g for 30 minutes at 4℃ and collect the supernatant.

[0093] g) Add 45% saturated ammonium sulfate to the supernatant to salt out and precipitate the protein. Redissolve the precipitate in 10 mM Tris hydrochloric acid (pH 7.5) and 50 mM sodium chloride buffer.

[0094] h) Acidify the solution to pH 3.0 to remove residual protein impurities, and then neutralize the pH.

[0095] i) The high-purity recombinant αSyn monomer was obtained by further purification through ion exchange chromatography and ultrafiltration.

[0096] j) Use an endotoxin detection kit to determine the endotoxin level in the monomer to ensure that it meets the detection requirements.

[0097] 1.2 Preparation of Pre-fabricated Fibers (PFFs) a) Dilute the purified αSyn monomer in 10 mM Tris (pH 7.5) and 50 mM sodium chloride buffer.

[0098] b) Stir and vibrate continuously at 37℃ and 1000 rpm for 5 to 7 days to allow the monomers to spontaneously aggregate and form mature fibers.

[0099] c) The mature fibers are treated with an ultrasonic breaker at 20% power for a total of 60 pulses (1 second on and 1 second off each time) to break the long fibers into short fiber fragments (i.e., PFFs seeds).

[0100] d) Detect the endotoxin levels of PFFs, aliquot them, and store them at -80°C for later use. PFFs are used as a positive control for SAA testing.

[0101] 2. Composition and preparation of the detection kit (a) Preprocessing components The pretreatment component includes all reagents and consumables used to implement the four-step stratified pretreatment process, specifically including: a) Strong lysis buffer, used for lysis and dissociation of protein-lipid complexes in step one; 200 μL per dose, used at a 1:1 volume ratio with serum; b) Chloroform: Analytical grade, used as the organic solvent in step two, Bligh-Dyer lipid extraction. c) Methanol: analytical grade, used as the organic solvent in step two, Bligh-Dyer lipid extraction; d) Proteinase K (PK) working solution or lyophilized powder: for use in step three, limited proteinase K digestion; concentration is indicated when supplied, and should be diluted to a final concentration of 1 μg / mL before use; e) Serine protease inhibitor (phenylmethylsulfonyl fluoride, PMSF) working solution or lyophilized powder: used to terminate PK activity after the digestion reaction in step three; f) Molecular weight cutoff ultrafiltration device: 30 kDa molecular weight cutoff value, used for ultrafiltration in step four to enrich higher-order αSyn aggregates and remove low molecular weight degradation products and residual small molecule inhibitors; each ultrafiltration device is for single use.

[0102] (ii) Amplification reaction components The amplification reaction assembly includes all the components required to prepare the amplification reaction stock solution, specifically including: a) Recombinant human full-length α-synuclein monomer (HM): provided in lyophilized powder or high-concentration stock solution form, with a final concentration of 0.1 mg / mL when preparing the reaction stock solution; b) Thioflavin T (ThT) stock solution: used for real-time monitoring of amyloid fibrillation, with a final concentration of 10 μM when preparing the stock solution; c) Sodium dodecyl sulfate (SDS) stock solution: The final concentration of the stock solution is 0.01%; d) Saturated ammonium sulfate (AS) stock solution: The final concentration is 2% when preparing the reaction mother liquor; e) Tris buffer containing sodium chloride: When preparing the stock solution, the final concentration is 10 mM Tris, 50 mM NaCl, and the final pH is 8.0; it can also be provided as a 10-fold concentrated stock solution (100 mM Tris, 500 mM NaCl, pH 8.0), which should be diluted proportionally before use.

[0103] The types and concentrations of the components in the above amplification reaction reagent group correspond one-to-one with the formulation determined in "3. Establishment of serum-specific amplification reaction system" in Example 1, ensuring that the residual matrix characteristics of the pretreatment product and the amplification reaction conditions are matched.

[0104] (III) Control reagents a) α-Synuclein prefabricated filaments (PFFs) positive control: PFF seeds prepared in "1.2 Preparation of prefabricated filaments (PFFs)" were provided in lyophilized or liquid form, with a final concentration of 100 fg / mL when used; used to verify the functional normality of each batch of amplification reaction system; b) Negative control buffer: A buffer without any αSyn seeds (the same formulation as the amplification reaction stock solution but without αSyn seeds) used to establish the background fluorescence baseline and fluorescence threshold for each batch of tests.

[0105] (iv) Reaction carrier Black transparent 384-well microplate: compatible with bottom fluorescence reading of microplate readers, each plate can simultaneously detect multiple test samples and controls; includes a sealing film.

[0106] (v) Optional auxiliary components a) Serum collection and preservation kit: including inert separation gel coagulation collection tubes (3 mL / tube), low-adsorption centrifuge tubes, and a mixture of protease / phosphatase inhibitors for standardized collection and preservation of serum samples; b) Instruction manual: It details the requirements for serum collection and preservation, the four-step pretreatment procedure, the preparation method of the reaction stock solution, the sample addition operation, the instrument operating parameter settings, and the result interpretation rules.

[0107] The kit is designed based on the following principles: the types and specifications of reagents in the pretreatment components correspond one-to-one with the reagents required in each step of the four-step stratified pretreatment process; the types and concentrations of components in the amplification reaction reagents correspond one-to-one with the optimal formulation of the serum matrix-adapted amplification reaction system; control reagents cover positive function verification and negative background establishment; and the reaction carrier is matched with the instrument reading mode. By pre-grouping the above reagents in a functionally adapted manner and uniformly controlling batch quality, consistent detection performance can be obtained when different laboratories and different operators use the same batch of kits. This solves the problems of large batch-to-batch differences, complex operation, and poor cross-center consistency caused by decentralized preparation of laboratory-built solutions in existing technologies.

[0108] The storage conditions for the kit are as follows: Proteinase K, PMSF, recombinant αSyn monomer, and PFFs positive control should be stored at -20℃ or -80℃; ThT stock solution, SDS stock solution, saturated ammonium sulfate stock solution, and Tris buffer should be stored at 2 to 8℃ protected from light; strong lysis buffer should be stored at room temperature or 2-8℃; chloroform and methanol should be stored at room temperature protected from light and sealed; the ultrafiltration device and microplates should be stored at room temperature in a dry place. The expiration date and stability period after opening of each component are indicated in the instruction manual.

[0109] Example 3: Derivation and Validation of the Clinical Diagnostic Performance of the Cohort 1) The finalized serum SAA regimen (Example 1) was applied to the derived cohort, which included 100 patients with idiopathic Parkinson's disease diagnosed by the Movement Disorders Society (MDS) clinical diagnostic criteria and 100 age- and sex-matched controls with non-neurodegenerative diseases. All samples were tested in a blinded manner.

[0110] 2) Results: Using a lag of 37.38 hours as the cutoff value, the sensitivity of serum α-Synuclein (SAA) in the Parkinson's disease group was 92% (92 / 100), the specificity was 96% (96 / 100), and the area under the ROC curve was 0.9539 (95% confidence interval 0.9218 to 0.9859). The sensitivity and specificity of paired skin biopsy samples in the same cohort for SAA (A skin-specific α-Synuclein seeding amplification assay for diagnosing Parkinson's disease. Kuang Y, et al.) were 94%. Paired consistency analysis of serum and skin SAA results showed that in 177 out of 200 samples (87 double-positive, 90 double-negative), the two tests were consistent (87 double-positive, 90 double-negative), and only 23 samples were inconsistent. Subgroup concordance reached 92.0% in Parkinson's disease patients and 97.0% in controls, validating the high concordance between serum SAA test results and established peripheral tissue SAA levels. Figure 4 ).

[0111] Example 4: Multicenter Independent Validation 1) Following the derivation of the cohort, the present invention was extended to four medical centers in China for independent, multicenter, blinded validation, including 501 Parkinson's disease patients and 525 controls with non-neurodegenerative diseases. All samples used a uniform pretreatment protocol, uniform batch reagents, and uniform instrument settings, and were uniformly interpreted after blinded testing.

[0112] 2) Results: Using the same preset cutoff value as the derived cohort (lag period 37.38 hours), the overall sensitivity of the multicenter validation cohort was 90.02%, specificity was 94.67%, and overall diagnostic accuracy was 0.9482, with an area under the curve of approximately 0.95. The individual center sensitivities ranged from 0.8349 to 0.8986, and specificities ranged from 0.9326 to 0.9821, showing no significant heterogeneity among centers. The positive predictive value ranged from 0.857 to 0.999, and the negative predictive value ranged from 0.804 to 0.999. Figure 5 ).

[0113] 3) Blinded repeatability verification results between two independent laboratories showed that the Spearman rank correlation coefficient of serum SAA lag period measurements was 0.742, the Pearson linear correlation coefficient was 0.801, and the Bland-Altman analysis showed an average deviation of only 2.79 hours, with a narrow 95% agreement limit.

[0114] Example 5: Application of iRBD Precursor Risk Stratification 1) An additional 150 participants with isolated rapid eye movement sleep behavior disorder (iRBD) confirmed by polysomnography were included in the multicenter validation cohort. iRBD is currently recognized as the strongest prodromal marker of Parkinson's disease, and approximately 80% of iRBD patients will develop Parkinson's disease or Lewy body dementia within 10 to 15 years.

[0115] 2) Serum samples from 150 iRBD participants were tested using the method of this invention. Results: 91 cases (60.67%) were identified as positive for serum αSyn SAA. Participants were divided into positive and negative groups based on their SAA results. The SAA-positive group showed significantly greater impairment in all five prodromal assessment domains: olfactory function, mild motor signs, REM sleep without dystonia, cognitive function, and autonomic function. After adjusting for age, sex, and disease duration using a generalized linear model, SAA positivity remained an independent risk factor for aggravated impairment in each prodromal domain. Ordinal logistic regression analysis was used to analyze the relationship between SAA status and Parkinson's disease risk classification (low, medium, and high risk levels). The results showed that SAA-positive participants significantly shifted towards the high-risk category, and their composite Parkinson's disease risk score was significantly higher. Figure 6 ).

[0116] Comparative preprocessing-amplification bidirectional adaptation verification Objective: To demonstrate the technical concept of the present invention that "the preprocessing module and the amplification module must be bidirectionally compatible"—that using any module alone with a mismatched corresponding module will not achieve optimal performance.

[0117] Comparison 1: Pre-treatment product of module one of the present invention + amplification conditions of the prior art The seed-enriched serum (10 PD cases + 10 control cases) obtained after pretreatment in Module 1 of this invention was added to the amplification system (mouse αSyn monomer 0.1 mg / mL, 10% sodium sulfate, 40 mM NaCl, 15 mM Tris, pH 6.8, 50℃, shaking for 1 minute and stopping for 14 minutes) in patent CN119322181B-A serum pretreatment agent and its application and an in vitro amplification method for α-synuclein bodies, and detected for 48 hours.

[0118] Results: Of the 10 PD samples, only 3 showed a peak within 48 hours with large fluctuations in the lag period (5-42 hours). Of the 7 controls, 4 showed a nonspecific peak (lag period 18-35 hours). ΔT-Lag was only about 2.1 hours, indicating extremely poor discriminative power.

[0119] Analysis: The slightly acidic pH and high concentration of sodium sulfate in this patent triggered severe non-specific spontaneous aggregation in the residual matrix background of the pretreated product of this invention, resulting in excessively high background noise.

[0120] Comparison 2: Pre-processed products of existing technologies + Amplification conditions of module two of this invention Patent CN119322181B - A serum pretreatment agent and its application, and a method for in vitro amplification of α-synucleosomes (pH 7.2 centrifugation to remove LDL + pH 6.0 centrifugation to remove HDL + 70℃ heat inactivation for 5 minutes) pretreatment of the same batch of serum, and adding the product to the amplification system of Module 2 of this invention (human monomer, 2% AS, 0.01% SDS, pH 8.0) for detection for 48 hours.

[0121] Results: In 6 out of 10 PD samples, a peak was observed (lag period 8–42 hours, with large variability), while none of the 10 controls showed a peak. ΔT-Lag was approximately 18.5 hours.

[0122] Analysis: The alkaline pH gating effect of this invention effectively suppresses background noise, but the seed activity in the pretreatment product of this patent is damaged (due to heat inactivation), resulting in a low peak rate of positive samples, a prolonged hysteresis period, and a lower discrimination efficiency than the complete scheme.

[0123] 3. Compare Comparisons 1 and 2 with the complete solution of this invention (Module 1 + Module 2). This invention: The complete protocol of this invention was used to detect samples from the same batch. Results: In 9 out of 10 PD samples, the peak appeared within 20 hours (with an average lag period of approximately 12 hours), while in all 10 control samples, no peak appeared within 48 hours. ΔT-Lag was approximately 39.25 hours.

[0124] Comparison 1 vs. this invention: For the same pretreated product, the ΔT-Lag time using the patented amplification conditions is 2.1 hours, while the ΔT-Lag time using the amplification conditions of this invention is 39.25 hours—a difference of 18.7 times. This proves that the choice of amplification conditions has a decisive impact on the final discrimination efficiency and cannot be simply applied.

[0125] Comparison 2 vs. this invention: Under the same amplification conditions, the ΔT-Lag time using the patented method was 18.5 hours, while the ΔT-Lag time using the present invention was 39.25 hours—a difference of 2.1 times, proving that the pretreatment method also significantly affects the final performance.

[0126] In summary, the maximum ΔT-Lag and optimal diagnostic performance can only be achieved when the two modules of this invention are adapted in synergy, proving the indivisibility of the "preprocessing-amplification joint regulation" concept.

[0127] Comparative Example 2: Cross-comparison with existing technologies This embodiment illustrates that the present invention is not an arbitrary substitution of certain conventional steps in the scheme of patent CN119322181B - A serum pretreatment agent and its application and an in vitro amplification method for α-synucleosomes. Identical serum samples were processed according to the pretreatment routes of both the patent and the present invention, and then placed in the amplification systems of the patent and the present invention, respectively, for detection, forming a cross-comparison. Evaluation indicators included ΔT-Lag, replicate positive rate, and background fluorescence level.

[0128] Cross-comparison results showed that: if the pretreated product of this invention is used with the amplification system of this patent, the separation of positive / negative lag phases is insufficient; if the pretreated product of this patent is used with the amplification system of this invention, there is still insufficient retention of seed activity and increased background in the control; only the complete combination of "seed activity retention pretreatment + serum matrix adaptation amplification" of this invention can achieve approximately 39.25 hours of ΔT-Lag and a stable replicate positive rate. This result demonstrates an unexpected synergistic adaptation relationship between the pretreatment module and the amplification module.

[0129] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for in vitro amplification and detection of serum α-synuclein seeds for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: The serum to be tested was lysed, centrifuged, and the first supernatant was collected. The lipids of the first supernatant were extracted with an organic solvent, and the aqueous phase was collected. Proteinase K was added to the aqueous phase for digestion, and the second supernatant was collected after centrifugation. The second supernatant was ultrafiltered and the retentate was collected to obtain the pretreated seed-enriched serum sample. The pretreated seed-enriched serum sample was added to the reaction solution for SAA amplification reaction; The reaction solution consists of human full-length α-synuclein monomer, thioflavin T, sodium chloride, sodium dodecyl sulfate, saturated ammonium sulfate, and Tris hydrochloric acid buffer.

2. The method for in vitro amplification and detection of serum α-synuclein seeds as described in claim 1, characterized in that, Mix the serum to be tested with an equal volume of strong lysis buffer, incubate on ice for 30 minutes, vortexing once every 5-10 minutes, and after incubation, centrifuge at 4°C and 10000×g for 15 minutes to obtain the first supernatant. The strong lysis buffer is a protein lysis buffer containing 0.001% sodium dodecyl sulfate, 1% Trion-X 100, 150mM NaCl and 1x protease phosphatase inhibitor.

3. The method for in vitro amplification and detection of serum α-synuclein seeds as described in claim 1, characterized in that, Chloroform, methanol, and water were added sequentially to the first supernatant, and the final system had a volume ratio of 2:1:0.8 for chloroform, methanol, and water. After mixing, the mixture was centrifuged at 4°C and 15000×g for 15 minutes to obtain the aqueous phase.

4. The method for in vitro amplification and detection of serum α-synuclein seeds as described in claim 1, characterized in that, Proteinase K was added to the aqueous phase to a final concentration of 1 μg / mL, and digested for 60 minutes at 56°C and 900 rpm with shaking. After digestion, proteinase K activity was terminated, and the mixture was then centrifuged at 4°C and 5000×g for 10 minutes to obtain the second supernatant. The ultrafiltration has a molecular weight cutoff of 30 kDa.

5. The method for in vitro amplification and detection of serum α-synuclein seeds as described in claim 1, characterized in that, The pretreated seed-enriched serum sample was added to the reaction solution at a volume ratio of 1:

9. In the reaction solution, the concentration of human full-length α-synuclein monomer is 0.1 mg / mL, the concentration of thioflavone T is 10 μM, the concentration of sodium chloride is 50 mM, the concentration of sodium dodecyl sulfate is 0.01%, the concentration of saturated ammonium sulfate is 2%, and the concentration of Tris hydrochloric acid buffer is 10 mM. The conditions for the SAA amplification reaction were as follows: pH 8.0, constant temperature of 50℃, high-intensity dual-track intermittent oscillation mode, 1 minute of oscillation per cycle followed by 14 minutes of rest, fluorescence detection by reading thioflavone T fluorescence from the bottom every 15 minutes, excitation wavelength of 450±10 nm, emission wavelength of 490±10 nm, and a total monitoring time of 48 hours.

6. The method for in vitro amplification and detection of serum α-synuclein seeds as described in claim 1, characterized in that, If the lag period of the SAA amplification reaction is ≤37.38 hours, the serum α-synuclein to be tested is determined to be positive.

7. A serum α-synuclein seed in vitro amplification and detection kit, characterized in that, The kit includes a pretreatment component, an amplification reaction component, and a control reagent; The pretreatment components include a strong lysis buffer, chloroform, methanol, proteinase K, and an ultrafiltration device; the strong lysis buffer is a protein lysis buffer containing 0.001% sodium dodecyl sulfate, 1% Trion-X 100, 150 mM NaCl, and 1x proteinase phosphatase inhibitor; the ultrafiltration has a molecular weight cutoff of 30 kDa. The amplification reaction assembly includes a reaction solution composed of human full-length α-synuclein monomer, thioflavin T, sodium chloride, sodium dodecyl sulfate, saturated ammonium sulfate, and Tris hydrochloric acid buffer. The control reagents include a positive control of α-synuclein prefibrils and a negative control without α-synuclein.

8. The kit according to claim 7, characterized in that, In the reaction solution, the concentration of human full-length α-synuclein monomer was 0.1 mg / mL, the concentration of thioflavone T was 10 μM, the concentration of sodium chloride was 50 mM, the concentration of sodium dodecyl sulfate was 0.01%, the concentration of saturated ammonium sulfate was 2%, and the concentration of Tris hydrochloric acid buffer was 10 mM.

9. The use of the kit as described in claim 7 or 8 in the preparation of diagnostic products for α-synuclein lineage diseases.

10. The application as described in claim 9, characterized in that, The α-synuclein lineage diseases include Parkinson's disease, Lewy body dementia, or multiple system atrophy.

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