Methods, kits and uses thereof for semi-quantitatively detecting lewy body dementia pathological seed load in a biological sample

Abstract

The application belongs to the technical field of biological medicine, and discloses a method and kit for semi-quantitative detection of Lewy fold type alpha-synuclein pathological seed load in a biological sample and application thereof. The alpha-synuclein seed amplification technology is upgraded from qualitative detection to semi-quantitative detection for the first time: firstly, positive sample preliminary screening is carried out, then matrix-matched serial dilution is carried out on the preliminary screening positive sample, combined with long-term seed amplification reaction, the total number of positive samples of all dilution degrees is counted, and Lewy fold pathological score is obtained. The LFP score is positively correlated with the Hoehn & Yahr stage of Parkinson's disease patients, the MDS-UPDRS III motor score, and is negatively correlated with the MoCA cognitive score, and can be used for auxiliary diagnosis, auxiliary evaluation of severity, progress monitoring and auxiliary evaluation of drug efficacy. The method can also be used for detecting alpha-synuclein seeds in samples of Alzheimer's disease patients, and provides a reference for co-pathological evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method, kit, and application for semi-quantitative detection of Lewy foldable α-synuclein pathological seed load in biological samples. Background Technology

[0002] Misfolding of α-Syn exhibits a "seeding" property: a small number of pathologically conformed α-Syn aggregates (seeds) can induce conformational changes and aggregation of normally conformed α-Syn monomers. Based on this principle, seed amplification technology (SAA), also known as real-time vibration-induced transformation (RT-QuIC), has been developed in recent years and has been used to detect trace amounts of pathological α-Syn aggregates with seeding activity in cerebrospinal fluid (CSF).

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD), affecting more than 10 million people worldwide. Its core pathological feature is the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and the formation of Lewy bodies (LBs) and Lewy neurites (LNs) in the neuronal cytoplasm. The latter are mainly composed of misfolded and abnormally aggregated α-synuclein (α-Syn). Lewy body dementia (DLB) also features Lewy bodies as its pathological feature, forming the "Lewy body disease" lineage together with PD. Furthermore, although multiple system atrophy (MSA) is also a synucleinic disease, its pathological feature is the aggregation of α-Syn (Papp-Lantos bodies) in the glial cytoplasm, which differs from the conformation of Lewy body diseases.

[0004] The prior art patent CN202410034146.1 discloses an Alzheimer's disease auxiliary detection kit and its application based on cerebrospinal fluid sample α-synuclein seed amplification technology. This technology utilizes the SAA principle and monitors the aggregation degree in real time using thioflavin T (ThT) fluorescent dye. It runs under 42℃ shaking / resting cycle conditions for 36~60h and determines whether there are α-Syn pathological seeds in the sample based on the fluorescence intensity. The output is a qualitative (positive / negative) result, which is used to assist in the diagnosis of whether there is α-Syn co-pathology in AD.

[0005] However, the existing qualitative SAA technology mentioned above has the following technical problems: First, qualitative SAAs can only provide a "present / absent" qualitative result, failing to quantify the relative content of pathological seeds in the sample. Clinically, different patients with the same disease exhibit significant differences in pathological burden. Existing SAAs can only determine the presence of α-Syn pathological seeds in a sample, outputting a binary result (positive / negative). However, clinically, different patients with the same disease (such as Parkinson's disease) show vastly different pathological burdens. For example, the concentration of pathological seeds in the cerebrospinal fluid of early-stage PD patients may be only 1 / 10 or even 1 / 100 of that of late-stage patients. Qualitative SAAs cannot distinguish these differences, resulting in limited clinical information. Specifically, qualitative SAAs cannot meet the needs in the following clinical scenarios: differentiating between early and late-stage PD; differentiating between rapidly progressive and slowly progressive PD; and evaluating the dose-response relationship of anti-α-Syn drugs in clinical trials.

[0006] Second, it cannot be correlated with clinical severity. Current technology does not provide any data related to motor function scores (such as MDS-UPDRS III, Hoehn & Yahr staging) or cognitive function scores (such as MoCA). This means that doctors cannot determine whether a patient is in the early or late stage of the disease based on test results, nor can they provide a quantitative basis for individualized treatment. For example, two PD patients who initially test positive may have vastly different disease stages; one may only be H&Y stage 1 (unilateral symptoms), while the other may have progressed to H&Y stage 4 (requiring assisted walking), but current technology cannot distinguish between them.

[0007] Third, it cannot monitor disease progression. Because it only provides positive / negative results, test results for the same patient at different time points (as long as they are all positive) cannot reflect whether the condition is worsening, stabilizing, or improving. This makes qualitative SAA unsuitable for assisting in assessing the progression of a single patient's disease. For clinicians, assessing the changes in a patient's condition over time is a core basis for developing treatment strategies; for patients, understanding their own disease progression trend is an important reference for life planning and psychological adjustment.

[0008] Fourth, it cannot be used to assess drug efficacy. In recent years, several targeted drugs against α-Syn (such as passive immune antibodies, small molecule aggregation inhibitors, and active immunization vaccines) have entered clinical trials. These clinical trials require biomarkers that can quantitatively reflect changes in pathological burden as surrogate endpoints for pharmacodynamics (PD). For example, in clinical trials of antibody drugs, it is necessary to assess whether the drug can reduce the concentration of α-Syn pathological seeds in cerebrospinal fluid. Qualitative SAAs cannot provide this crucial information.

[0009] Fifth, there is a risk of a "hook effect" in extremely high-concentration samples. When the seed concentration in a sample is too high, it may rapidly consume a large amount of substrate in the early stages of the amplification reaction, forming large aggregates, which may prevent the subsequent ThT fluorescence signal from being generated effectively or even decrease it. This phenomenon is called the "hook effect" or "prozone effect," and may lead to high-concentration samples being misjudged as false negatives or unstable results. Existing qualitative SAA techniques have not been optimized to address this phenomenon.

[0010] Sixth, the detection window is relatively short (36-60 hours), which may miss low-concentration seed samples (requiring a longer latency period to generate a detectable signal), and it does not fully utilize the quantitative potential of long-term reactions. The total reaction time of existing technologies is typically 36-60 hours. For low-concentration seed samples, a longer latency period (Lag Phase) may be required to generate a detectable fluorescence signal. The short detection window may lead to some low-concentration samples being misjudged as false negatives. Furthermore, the short-term reaction does not fully utilize the quantitative potential of long-term reactions in SAA.

[0011] Therefore, there is an urgent need in the field for a new method and kit that can quantify α-Syn pathological seed load, correlate with clinical severity, monitor disease progression, and be used for drug efficacy evaluation. Summary of the Invention

[0012] To address the above technical problems, the purpose of this invention is to provide a method for semi-quantitative detection of the pathological seed load of Lewy folded α-synuclein in biological samples. This method includes: initial sample screening, serial dilution, amplification detection, and score calculation. This invention obtains the Lewy folded pathological seed load (LFP score) by performing matrix-matched serial dilutions on initially screened positive samples, combined with long-term seed amplification reactions, and counting the total number of positive wells across all dilutions and replicates. Specifically, this invention achieves the above objective through the synergistic effect of three key methods, jointly solving the technical problem that existing SAA technology cannot quantify the pathological load: First, using a matrix-matched negative control matrix for serial dilution eliminates the influence of sample matrix effects on amplification efficiency; second, obtaining the LFP score by counting the total number of positive wells across multiple dilutions overcomes the hook effect of high-concentration samples in single-concentration detection; third, combining with long-term amplification reactions ensures sufficient amplification of low-concentration seed samples. The synergistic effect of these three methods enables the LFP score to reflect the true pathological load for the first time and is significantly correlated with clinical severity, jointly achieving an upgrade from qualitative to semi-quantitative detection of α-Syn pathological seeds.

[0013] This invention upgrades α-synuclein seed amplification technology from qualitative detection to semi-quantitative detection for the first time, establishing a quantitative correlation between α-synuclein seed amplification signals and clinical disease severity. The LFP score is positively correlated with the Hoehn & Yahr stage and MDS-UPDRS III motor score of Parkinson's disease patients, negatively correlated with the MoCA cognitive score, and correlated with the detection rate of α-Syn co-pathology in AD patients. It can be used for disease auxiliary diagnosis, severity assessment, progression monitoring, and drug efficacy evaluation. The method and kit for semi-quantitative detection of this invention can be used to detect the presence of α-synuclein pathological seeds in biological samples of Alzheimer's disease patients, assisting physicians in determining whether the patient has α-Syn co-pathology.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: A method for semi-quantitative detection of Lewy foldable α-synuclein pathological seed load in biological samples includes the following steps: S1. Initial screening of samples: Take the biological sample to be tested and use the α-synuclein seed amplification technology to test the undiluted sample. Set up no less than 3 replicates. If the number of positive replicates reaches the preset threshold, it is judged as a positive initial screening result. S2. Serial dilution of negative sample matrix: Using negative control biological sample matrix that has been confirmed to be free of α-synuclein pathological seeds, the initially screened positive samples are serially diluted to obtain at least 3 different dilutions. S3. Amplification and detection: α-synuclein seed amplification reaction was performed on the diluted samples at each dilution. At least 3 replicates were set for each dilution. The total reaction time was 120-144 h. S4, LFP score calculation: The total number of positive reactions in all replicates of all dilutions is counted to obtain the Lewis fold pathology score.

[0015] The negative control biological sample matrix that was confirmed to be free of α-synuclein pathological seeds was prepared by mixing equal volumes of at least 10 samples from healthy volunteers who were clinically and biomarker-confirmed to be free of α-Syn pathological seeds, and then used after being confirmed to have no amplification signal by SAA detection using this method.

[0016] Preferably, in S1, the biological sample to be tested includes any one of cerebrospinal fluid, serum, plasma, peripheral blood exosomes, and tissue homogenate. The number of compound holes is set to 4; The preset threshold for the number of positive duplicate holes is 2.

[0017] Interpretation criteria: If the fluorescence intensity of a replicate well reaches or exceeds a preset threshold (e.g., 20% of the average maximum fluorescence value of the positive control), the well is considered positive. Initial screening positive criteria: The number of positive replicate wells reaches a preset threshold (e.g., ≥2 positive out of 4 replicate wells). If the initial screening is positive, proceed to the next step of quantitative evaluation; if negative, report directly as negative without serial dilution. Furthermore, this application utilizes serial dilution to create a peak response range between seed concentration and amplification signal at different dilution levels. The LFP score counts the total number of positive wells across multiple dilutions, not the signal intensity of a single concentration. Therefore, even if extremely high concentration samples exhibit signal decline (hook effect) at certain dilutions (e.g., undiluted), they can still generate positive signals and be counted at higher dilutions, thus avoiding false negatives and achieving accurate quantification of extremely high concentration samples.

[0018] A negative control biological sample matrix, confirmed to be free of α-Syn pathological seeds, was used as the diluent (matrix-matched dilution). This design aimed to eliminate the matrix effect between different dilutions—that is, the influence of differences in matrix composition between the diluent and the original sample on amplification efficiency. Using a matrix homologous to the sample (such as cerebrospinal fluid) for dilution ensures that amplification efficiency depends only on seed concentration, not on changes in matrix composition.

[0019] Preferably, in S2, the dilution ratio of the serial dilution is selected from any one or more combinations of the following, specifically including: 2x, 3x, 5x or 10x serial dilution.

[0020] The above 2x series dilutions include 1:2, 1:4, and 1:8; The above-mentioned 3-fold series dilutions include 1:3, 1:10, 1:30, and 1:100; The above 5-fold series dilutions include 1:5, 1:25, and 1:125; The above 10-fold series dilutions include 1:10, 1:100, and 1:1000.

[0021] The principle for selecting the dilution ratio is to cover the transition range from positive to negative.

[0022] Preferably, in step S3, the α-synuclein seed amplification reaction parameters are: incubation at 35-45°C under alternating cycles of shaking and resting, real-time monitoring using thioflavone T fluorescent dye, excitation wavelength of 440-460 nm, and emission wavelength of 475-490 nm; the total reaction time is 120-144 h.

[0023] Preferably, in step S3, an alternating cycle of 1 minute of oscillation and 1 minute of rest is used; and the incubation is carried out at 42°C.

[0024] Preferably, in step S4, the threshold standard for a positive reaction in the duplicate wells is: the fluorescence intensity of the duplicate wells reaches or exceeds 15-25% of the average maximum fluorescence value of the positive control. Preferably, in S4, the LFP score is classified into three levels of pathological burden as follows: a score of 2-6 corresponds to low pathological burden, a score of 7-14 corresponds to medium pathological burden, and a score of 15-28 corresponds to high pathological burden.

[0025] A detection kit for the method described above, comprising a recombinant α-synuclein substrate, thioflavone T fluorescent dye, reaction buffer, negative matrix diluent, positive control fiber stock solution, and a reaction plate; the recombinant α-synuclein substrate is recombinant α-synuclein with a His6 tag, at a final concentration of 0.05–0.10 mg / mL; the thioflavone T fluorescent dye stock solution has a concentration of 50–200 μM; the reaction buffer is a 4× concentrate containing 400–800 mM sodium chloride, 120–160 mM pH 8.0 phosphate buffer, and detergent; the negative matrix diluent is a mixed negative control sample matrix confirmed to be free of α-synuclein pathological seeds; the positive control fiber stock solution contains pre-prepared α-synuclein fibers incorporated into the negative control sample matrix at a final concentration of 5–10 pM; the reaction plate is a microplate pre-filled with glass beads, the glass beads having a diameter of 0.6–1.0 μm. mm, with 4 to 8 pre-placed per hole.

[0026] Preferably, the final concentration of the recombinant α-synuclein substrate is 0.07 mg / mL; the concentration of the thioflavin T fluorescent dye stock solution is 100 μM; the reaction buffer is a phosphate buffer containing 600 mM sodium chloride and 140 mM pH 8.0; the negative matrix dilution solution is a mixed negative control sample matrix confirmed to be free of α-synuclein pathological seeds; the α-synuclein fibers prepared in advance by the positive control fiber stock solution are incorporated into the negative control sample matrix at a final concentration of 7.5 pM; the reaction plate is a microplate pre-filled with glass beads, the glass beads having a diameter of 0.8 mm and 6 beads pre-filled in each well.

[0027] The use of the method for semi-quantitative detection or the kit as described in the preparation of a product for at least one of the following purposes includes: (1) Kits for assisting in the detection of Parkinson's disease or Lewy body dementia; (2) A reagent to differentiate between Parkinson's disease / Lewy body dementia and multiple system atrophy; (3) Reagents for assisting in the assessment of patients with Parkinson's disease or Lewy body dementia; (4) A reagent to assist in monitoring the progression of Parkinson's disease or Lewy body dementia; (5) Reagents for evaluating the efficacy of anti-α-synuclein targeted drugs in clinical trials.

[0028] Compared with the prior art, the present invention has at least the following technical effects: This invention addresses the technical challenges of existing α-synuclein seed amplification technologies, which, due to the inherent randomness of the amplification reaction, substrate consumption, and the complexity of product aggregation, can only provide qualitative results, and traditional kinetic parameter analysis cannot quantify pathological burden. A breakthrough is achieved through a combination of three core technologies: serial dilutions using a matrix-matched negative control matrix eliminate the interference of sample matrix effects on amplification efficiency; the Lewis fold pathological score is obtained by statistically analyzing the total number of positive wells at multiple dilutions, overcoming the false negative problem caused by the hook effect in high-concentration samples during single-concentration detection; and long-term amplification ensures sufficient detection of low-concentration seed samples. These three methods work synergistically to enable the LFP score to reflect the true pathological burden for the first time and to be significantly correlated with clinical severity (H&Y stage, MDS-UPDRSIII, MoCA), achieving an upgrade from qualitative to semi-quantitative detection of α-Syn pathological seeds.

[0029] This invention provides a method for semi-quantitative detection of Lewy foldable α-synuclein pathological seed load in biological samples, which has the following advantages: (1) This invention achieves semi-quantitative analysis through serial dilution and comprehensive positive counting. Specifically, for samples initially screened as positive, a matrix-matched negative control sample is serially diluted. Then, SAA testing is performed on samples at each dilution, and the total number of positive reactions in all replicates across all dilutions is counted to obtain the LFP score. This method, through serial dilution combined with comprehensive positive counting, obtains an integer score proportional to the concentration of pathological seeds in the sample, achieving relative quantification of the pathological burden. Unlike absolute quantification (such as ELISA), the LFP score does not provide an absolute concentration value, but it allows for repeatable comparisons and grading between different samples and between different time points of the same sample.

[0030] This invention's multi-dilution comprehensive counting design ensures that samples of different concentration ranges fall within the linear detection range by performing detection at multiple dilutions, thus overcoming the false negative problem caused by the hook effect in extremely high-concentration samples using traditional single-concentration SAA. Specifically, by serially diluting high-concentration samples to the linear detection range, the "hook effect" is overcome; by statistically analyzing positive patterns at multiple dilutions, qualitative signals are converted into semi-quantitative scores; and by using a matrix-matched diluent (negative control CSF pool), the influence of sample matrix effects on amplification efficiency is eliminated. The output of SAA technology is upgraded from binary (positive / negative) to integer scores (semi-quantitative scores), achieving for the first time the quantification of α-Syn pathological seed load; establishing the correlation between quantitative indicators and gold standard clinical scales (H&Y staging, UPDRS-III, MoCA) gives the test results clinical interpretability. This allows for semi-quantitative comparison of pathological load between different patients and between different time points within the same patient. Existing technologies cannot provide any quantitative indicators.

[0031] The technical solution of this invention has dual functions of qualitative detection and relative quantitative assessment: Qualitative function: achieved through the initial screening step. The test results of undiluted samples (≥2 / 4 replicates positive / negative) directly provide a qualitative judgment—whether α-Syn pathological seeds are present in the sample.

[0032] (2) The reaction time of the prior art is usually 36~60h, while the present invention extends the reaction time to 120~144h. The method of this application allows low-concentration seed samples to have sufficient amplification time, reducing false negatives; it provides a wider dynamic range for serial dilution detection, enabling low-dilution samples to also generate detectable signals.

[0033] (3) Clinical validation results show that the correlation between LFP score and clinical scales was established for the first time. This invention, through a clinical cohort of 45 patients with PD / DLB, 31 patients with MSA, and 20 controls, demonstrated for the first time that LFP score is significantly correlated with the gold standard clinical scales (H&Y, UPDRS-III, MoCA) (p<0.01); positively correlated with Hoehn & Yahr staging (r=0.39, p=0.0086) and MDS-UPDRS III motor score (r=0.45, p=0.0032), and negatively correlated with MoCA cognitive score (r=-0.56, p=0.00073). This makes LFP score not only a diagnostic indicator but also a quantitative indicator of disease severity. Existing technologies do not provide any such data, and their dynamic parameters (Fmax, TTT, etc.) have been shown to have no robust correlation with clinical severity.

[0034] (4) Applications include longitudinal monitoring and differential diagnosis This invention is the first to demonstrate a longitudinal trend of increasing LFP scores at the individual level over time (LFP scores increased in all seven patients at baseline and during a 12-month follow-up period), and confirms its consistency with cognitive decline. This provides a potential pharmacodynamic biomarker for clinical drug trials. Existing qualitative SAAs cannot achieve individual-level progression monitoring.

[0035] (5) Differentiating between Lewy body disease and MSA: This invention is specific for Lewy folded α-Syn, and the LFP score of PD / DLB patients is significantly higher than that of MSA patients (MSA patients are almost all negative), which helps in clinical differential diagnosis. Although existing technologies also mention the detection of synucleinosis, they have not specifically verified the ability to differentiate MSA.

[0036] This invention can also be used to detect α-Syn copathology in Alzheimer's disease patients. Based on the results of 35 AD patients, this method detected an α-Syn pathological seed positivity rate of 11.4% in AD patients, consistent with the reported incidence of AD copathology in the literature. This provides a new biomarker tool for the accurate subtyping of AD patients. Attached Figure Description

[0037] Figure 1 A schematic diagram illustrating the correlation between LFP score and clinical severity of Parkinson's disease; Figure 2 A schematic diagram of CSF sample testing for PD patients at baseline and follow-up (mean interval 12 months); Figure 3 A comparative diagram of the reaction characteristics of different disease samples in the α-synuclein seed amplification reaction. Figure 4 This is a schematic diagram illustrating the anti-dilution stability and quantitative detection capability analysis of LFP scores. Detailed Implementation

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is 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. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0039] Example 1: LFP scoring method based on α-Syn SAA 1. Sample preparation Cerebrospinal fluid (CSF) was collected from the subjects. Immediately after collection, the samples were centrifuged at 1300 g for 5 minutes at 4°C to remove cell debris. The supernatant was aliquoted into low-adsorption microcentrifuge tubes (0.5 mL per tube) and stored at -80°C for later use. Informed consent was obtained from all subjects for all sample collection, and the collection was approved by the research institution's ethics committee.

[0040] 2. Reagent Preparation (1) Substrate protein: Weigh the lyophilized recombinant His6-α-Syn (purity >95%, endotoxin <1 EU / mg) and dissolve it in substrate dilution buffer (containing 20 mM Tris-HCl, pH 8.0, 50 mM NaCl) to a final concentration of 0.07 mg / mL. After aliquoting, store at -80℃ and avoid repeated freeze-thaw cycles.

[0041] (2) Reaction buffer (4×): containing 600 mM NaCl, 140 mM phosphate buffer (pH 8.0) and 1% detergent (Triton X-100) to promote α-Syn aggregation. It is diluted with deionized water, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4℃.

[0042] (3) ThT solution: Weigh thioflavone T powder, dissolve it in deionized water to a final concentration of 100 μM, filter it through a 0.22 μm filter membrane for sterilization, and store it at 4℃ in the dark. ThT generates fluorescence signals at an excitation wavelength of 448 nm and an emission wavelength of 482 nm, which are used to monitor the aggregation degree of α-Syn in real time.

[0043] (4) Positive control: α-Syn fibers were prepared in advance: Recombinant α-Syn (1 mg / mL) was incubated at 37°C and 1,000 rpm for 72 h to form mature fibrous aggregates. The fibers were diluted to a final concentration of 7.5 pM with negative CSF and aliquoted for storage.

[0044] (5) Negative control: The negative matrix dilution was prepared by mixing equal volumes of CSF samples from at least 10 healthy elderly volunteers who were clinically and biomarker-confirmed to be free of α-Syn pathological seeds to form a mixed negative CSF pool. After confirming the absence of amplification signal by SAA testing, the pool was aliquoted and stored.

[0045] (6) 96-well plate: Use black flat-bottomed 96-well plates (OptiPlate-96, PerkinElmer), with 6 glass beads of 0.8 mm diameter pre-placed in each well. The glass beads are acid-washed, rinsed with deionized water, and sterilized at high temperature before use.

[0046] 3. Initial screening test: Add 85 μL of reaction solution (containing substrate, ThT, and 1× buffer) to each well of a 96-well plate, followed by 15 μL of the CSF sample to be tested, a negative control, and a positive control, with four replicates for each sample. After sealing the plate, place it in a fluorescence plate reader (FLUOstarOmega) and cycle the plate at 42°C with shaking for 1 minute followed by rest for 1 minute. Detect ThT fluorescence (Ex / Em = 448 / 482 nm) every hour for 144 hours.

[0047] Interpretation criteria: Threshold setting: Calculate the average value of the maximum fluorescence value of the positive control wells, and use 20% of this average value as the positive threshold.

[0048] Single-well interpretation: If the maximum fluorescence value of a well reaches or exceeds the threshold within 144 hours, the well is judged as positive; otherwise, it is negative.

[0049] The initial screening positive criteria are as follows: if there are ≥2 positive replicates in the 4 replicates of the sample to be tested, it is considered a positive initial screening result and proceeds to the next step of quantitative assessment; if there are <2 positive replicates, it is considered a negative result and "no α-Syn pathological seeds were detected" is reported directly.

[0050] 4. Serial dilution and LFP score calculation LFP score = Σ i=1 n (P i ) Where: n = the total number of dilutions (in this scheme, n = 7, that is, 7 dilutions: 1:2, 1:4, 1:8, 1:3, 1:10, 1:30, 1:100). i = the i-th dilution; P i = The number of positive wells in 4 replicates at the i-th dilution (values ​​range from 0 to 4); A positive well is defined as: the maximum fluorescence intensity (Fmax) of thioflavone T in that well is ≥ the average Fmax of the positive control × 20%.

[0051] The LFP score is calculated directly from the positive count obtained during the experiment and is closely related to the specific technical conditions such as the dilution scheme, replicate settings, and positive interpretation threshold.

[0052] For samples that initially tested positive, the following dilutions were performed using a negative CSF pool: 1:2, 1:4, 1:8, 1:3, 1:10, 1:30, and 1:100.

[0053]

[0054] Take 15 μL of each dilution and test according to the SAA conditions described above. Each dilution should have 4 replicates. The total number of positive wells across all replicates for all dilutions is the LFP score. In this example, there are 7 dilutions × 4 replicates = 28 wells, with a theoretical score range of 2~28 (since the initial screening confirmed a positive result, at least 2 replicates should be positive).

[0055] LFP score calculation: The total number of positive wells in all replicates across all dilutions is counted (range: 2~28 points). A higher score indicates a higher α-Syn pathological seed load in the sample.

[0056] 5. Interpretation of Results and Clinical Relevance Based on clinical cohort data from 45 patients with PD / DLB, this invention establishes for the first time a grading standard for LFP score and disease severity.

[0057] Based on clinical cohort data, the scoring levels are shown in Table 1 below:

[0058] The above grading criteria were first proposed by the inventors based on clinical cohort data (45 PD / DLB patients) and have been verified to be significantly correlated with the gold standard clinical scale (p<0.01), and can be used as a reference for clinical auxiliary diagnosis and disease monitoring. Currently, there are no universally recognized national or industry standards for α-Syn pathological seed load.

[0059] Example 2: Clinical validation performance test data: 2.1 Subject Cohort: CSF samples were collected from the following groups of subjects, and the sample characteristics are as follows: Table 2. Statistical table of SAA test results for each diagnostic group

[0060] Note: All samples were collected during the patient's first visit. Diagnosis was made independently by two or more neurologists, and a third expert made the final decision in case of disagreement.

[0061] Conclusion: Positive rate in the MSA group: 0% (0 / 31), specificity 100% (distinguishing between Lewy body disease and MSA); Positive rate in the CON group: 0% (0 / 20), specificity 100% (distinguishing between Lewy body disease and healthy controls).

[0062] The above results indicate that this method has extremely high sensitivity (97.8%) for Lewy body disease (PD / DLB), meaning that approximately 98 out of 100 Lewy body disease patients can be correctly detected.

[0063] This method showed 100% specificity for MSA and healthy controls, indicating that it can effectively differentiate between Lewy body disease and MSA. This result demonstrates the clinical diagnostic value of this method, as early-stage PD and MSA can sometimes be difficult to distinguish, although their treatment strategies and prognoses differ significantly.

[0064] In the AD patient group, 4 out of 35 patients clinically diagnosed with AD (11.4%) had positive LFP scores, ranging from 4 to 12.

[0065] This positive rate is consistent with the reported incidence of α-Syn copathology in AD patients in the literature.

[0066] This result indicates that the method can detect α-Syn pathological seeds in AD patients and can be used to help determine whether AD patients have α-Syn co-pathology.

[0067] A small percentage of non-Lewy body disease (PSP, FTD) samples also tested positive, which may reflect the presence of α-Syn copathology in these diseases or be false positives. However, the overall positive rate was low (<30%). This further demonstrates that the method of the present invention has extremely high sensitivity and specificity for Lewy body disease (PD / DLB) and can effectively distinguish between Lewy body disease and MSA.

[0068] Example 3: Correlation between LFP score and clinical severity Correlation analysis was performed between LFP scores and clinical scales in PD patients: Table 3. Correlation between LFP score and clinical scales

[0069] The above results indicate that the LFP score can not only diagnose Lewy body disease, but also quantify the severity of the disease, including motor impairment and cognitive decline.

[0070] like Figure 1 The diagram shows the correlation between LFP score and clinical severity of Parkinson's disease. (a) shows the correlation between LFP score and Hoehn & Yahr stage; (b) shows the correlation between LFP score and MDS-UPDRS III motor score; and (c) shows the correlation between LFP score and MoCA cognitive score.

[0071] Results combined Figure 1It can be seen that: (a) Correlation with disease stage: LFP score is significantly positively correlated with Hoehn & Yahr stage (r=0.39, p=0.0086), and LFP score increases significantly with increasing disease stage. (b) Correlation with motor function: LFP score is significantly positively correlated with MDS-UPDRSIII motor symptom score (r=0.45, p=0.0032), indicating that LFP score can reflect the severity of motor impairment in patients. (c) Correlation with cognitive function: LFP score is significantly negatively correlated with MoCA cognitive score (r=-0.56, p=0.00073), indicating that LFP score can reflect the decline in cognitive function in patients.

[0072] Results combined Figure 1 As can be seen, the LFP score is a quantitative biomarker related to clinical severity and can be used for disease monitoring and prognostic assessment of PD / DLB patients.

[0073] Example 4: Verification of Longitudinal Monitoring Capability CSF samples were collected from 7 patients with Parkinson's disease (PD) at baseline and during follow-up (mean interval 12 months). The results are as follows:

[0074] The results showed that the LFP scores of all 7 patients increased over time, consistent with the decreasing trend of the MoCA cognitive score; the ability to monitor disease progression can reflect pathological progression at the individual level.

[0075] like Figure 2 The diagram shows CSF samples from 7 patients with Parkinson's disease (PD) at baseline and during follow-up (mean interval 12 months). The results showed that the LFP scores of all 7 patients increased over time, consistent with the decreasing trend of the MoCA cognitive score. Figure 2 ab). Figure 2 c further illustrates the changes in LFP scores at the individual level. The baseline and follow-up values ​​for each patient are connected by arrows, all pointing to the upper right (increased LFP score, decreased MoCA score), indicating that LFP scores can reflect pathological progression at the individual level. This demonstrates that the method of this invention can monitor disease progression at the individual level, providing potential pharmacodynamic biomarkers for clinical drug trials.

[0076] Example 5: Method Precision Validation

[0077] 5.6 Anti-interference verification

[0078] In summary, anti-Parkinson's drugs (such as levodopa), antidepressants, and cardiovascular / metabolic drugs do not affect the test results, and there are no false negatives or false positives.

[0079] like Figure 3 The figures show a comparison of the reaction characteristics of different disease samples in the α-synuclein seed amplification reaction. Figure a shows the fluorescence kinetic curves of each group of samples; Figure b shows the area under the curve (AUC) box plots of each group of samples; and Figure c shows the maximum fluorescence intensity (Fmax) box plots of each group of samples.

[0080] Results combined Figure 3 As shown in Figure a, only the PD / DLB group samples exhibited a typical amplification response curve, reaching a peak at approximately 50 hours; healthy controls and other neurodegenerative disease (MSA, PSP, AD, FTD) samples showed no obvious amplification signal.

[0081] Figures b and c show that the AUC and Fmax of the PD / DLB group were significantly higher than those of all other groups (p<0.0001).

[0082] This indicates that the method can specifically identify pathological α-synuclein seeds in PD / DLB samples, and there is no cross-reaction with other disease samples.

[0083] like Figure 4 The diagram shown illustrates the analysis of the dilution stability and quantitative detection capability of the LFP score.

[0084] To verify the sensitivity and specificity of the method of the present invention for semi-quantitative detection, the samples were subjected to a series of gradient dilutions (1:2 to 1:100) before detection.

[0085] Results combined Figure 4 It can be seen that: 1. High sensitivity: In the range of 1:10 and higher concentration dilution factors (such as 1:2, 1:4, 1:8, 1:10), each scoring segment mainly shows red positive signals (3 / 4 or 4 / 4), indicating that this method has excellent detection efficiency when the sample concentration is high.

[0086] 2. High dilution stability: Even under high dilution conditions of 1:30 to 1:100, the test results can be accurately converted into blue negative signals without significant false positive interference.

[0087] 3. Quantitative correlation: The LFP score showed a clear stepwise decreasing trend with increasing dilution factor, indicating that the scoring system can achieve quantitative analysis of sample concentration and accurately reflect the content of pathological α-synuclein seeds in the original sample, which has extremely high clinical application value.

[0088] In this application, alternative sample types, in addition to cerebrospinal fluid, may also be serum, plasma, peripheral blood exosomes, or tissue homogenates. For serum / plasma samples, pretreatment (such as removal of interfering substances like albumin and lipoproteins) or enrichment (such as immunoprecipitation or magnetic nanoparticle capture) may be performed before SAA detection and dilution series.

[0089] Alternative substrate proteins: Recombinant α-Syn substrates are not limited to His6 tags. Wild-type α-Syn without tags or α-Syn with other affinity tags (GST, MBP, etc.) can also be used, as long as their seeding and amplification activities are maintained.

[0090] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for semi-quantitative detection of Lewy foldable α-synuclein pathological seed load in biological samples, characterized in that, Includes the following steps: S1. Initial screening of samples: Take the biological sample to be tested and use the α-synuclein seed amplification technology to test the undiluted sample. Set up no less than 3 replicates. If the number of positive replicates reaches the preset threshold, it is judged as a positive initial screening result. S2. Serial dilution of negative sample matrix: Using negative control biological sample matrix that has been confirmed to be free of α-synuclein pathological seeds, the initially screened positive samples are serially diluted to obtain at least 3 different dilutions. S3. Amplification and detection: α-synuclein seed amplification reaction was performed on the diluted samples at each dilution. At least 3 replicates were set for each dilution. The total reaction time was 120-144 h. S4, LFP score calculation: The total number of positive reactions in all replicates of all dilutions is counted to obtain the Lewis fold pathology score.

2. The method for semi-quantitative detection according to claim 1, characterized in that, In S1, the biological sample to be tested includes any one of cerebrospinal fluid, serum, plasma, peripheral blood exosomes, and tissue homogenate; The number of compound holes is set to 4; The preset threshold for the number of positive duplicate holes is 2.

3. The method for semi-quantitative detection according to claim 1, characterized in that, In S2, the dilution ratio of the serial dilution is selected from any one or more combinations of the following, specifically including: 2x, 3x, 5x or 10x serial dilution.

4. The method for semi-quantitative detection according to claim 1, characterized in that, In S3, the α-synuclein seed amplification reaction parameters are as follows: incubation at 35-45°C under alternating cycles of shaking and resting, real-time monitoring using thioflavin T fluorescent dye, excitation wavelength of 440-460 nm, and emission wavelength of 475-490 nm; the total reaction time is 120-144 h.

5. The method for semi-quantitative detection according to claim 4, characterized in that, In step S3, an alternating cycle of 1 minute of oscillation and 1 minute of rest is used; the incubation is carried out at 42°C.

6. The method for semi-quantitative detection according to claim 1, characterized in that, In S4, the threshold standard for a positive reaction in the duplicate wells is: the fluorescence intensity of the duplicate wells reaches or exceeds 15-25% of the average maximum fluorescence value of the positive control.

7. The method for semi-quantitative detection according to claim 1, characterized in that, In S4, the LFP score is classified into three levels of pathological burden as follows: a score of 2-6 corresponds to low pathological burden, a score of 7-14 corresponds to medium pathological burden, and a score of 15-28 corresponds to high pathological burden.

8. A test kit for use in the method according to any one of claims 1 to 7, characterized in that, The test kit includes recombinant α-synuclein substrate, thioflavin T fluorescent dye, reaction buffer, negative matrix dilution, positive control fiber solution, and reaction plate; The recombinant α-synuclein substrate is a recombinant α-synuclein with a His6 tag, and the final concentration is 0.05~0.10 mg / mL; The concentration of the thioflavin T fluorescent dye mother liquor is 50~200μM; The reaction buffer solution is a 4× concentrate containing 400~800 mM sodium chloride, 120~160 mM pH 8.0 phosphate buffer, and detergent; The negative matrix diluent is a mixed negative control sample matrix that has been confirmed to be free of α-synuclein pathological seeds; The α-synuclein fibers prepared in advance by the positive control fiber stock solution are incorporated into the negative control sample matrix at a final concentration of 5-10 pM. The reaction plate is a microporous plate pre-filled with glass beads, the diameter of which is 0.6~1.0 mm, and 4~8 beads are pre-filled in each well.

9. The detection kit according to claim 8, characterized in that, The final concentration of the recombinant α-synuclein substrate was 0.07 mg / mL; The concentration of the thioflavin T fluorescent dye mother liquor was 100 μM; The reaction buffer is a phosphate buffer containing 600 mM sodium chloride and 140 mM pH 8.

0. The negative matrix diluent is a mixed negative control sample matrix that has been confirmed to be free of α-synuclein pathological seeds; The α-synuclein fibers prepared in advance by the positive control fiber stock solution were incorporated into the negative control sample matrix at a final concentration of 7.5 pM. The reaction plate is a microporous plate pre-filled with glass beads, the glass beads having a diameter of 0.8 mm, with 6 beads pre-filled in each well.

10. The use of the detection kit as described in any one of claims 8-9 in the preparation of a product for detecting Lewis foldable α-synuclein pathological seeds in biological samples, characterized in that, The products include: (1) Kits for assisting in the detection of Parkinson's disease or Lewy body dementia; (2) A reagent to differentiate between Parkinson's disease / Lewy body dementia and multiple system atrophy; (3) Reagents for assisting in the assessment of patients with Parkinson's disease or Lewy body dementia; (4) A reagent to assist in monitoring the progression of Parkinson's disease or Lewy body dementia; (5) Reagents for evaluating the efficacy of anti-α-synuclein targeted drugs in clinical trials.

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