Use of a plasma trail level test in the detection of alzheimer's disease

Abstract

The application discloses application of plasma TRAIL level detection in Alzheimer's disease detection, and relates to the technical field of biomedical detection; application of a substance for detecting the concentration of tumor necrosis factor-related apoptosis-inducing ligand in human plasma in preparation of a reagent for assisting in evaluating the cognitive state of a subject related to Alzheimer's disease; the application comprises: comparing the detection value of the plasma TRAIL concentration of the subject with a preset reference value range or a diagnostic threshold value, wherein when the plasma TRAIL concentration is higher than a first diagnostic threshold value for distinguishing AD from a normal cognitive function state, information is provided for assisting in judging that the subject is more likely to be in an AD state rather than a CU state. The application is based on detection of a plasma sample, only requires routine intravenous blood sampling, and avoids the invasive risk of lumbar puncture and the radioactive exposure and high cost of PET imaging. The used enzyme-linked immunosorbent assay and other technologies are mature and relatively standardized in operation.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to the application of plasma TRAIL level detection in Alzheimer's disease detection. Background Technology

[0002] Alzheimer's disease, an irreversible neurodegenerative disease, has become a major global public health challenge. Its pathophysiological processes include the deposition of β-amyloid protein to form senile plaques and the formation of neurofibrillary tangles by hyperphosphorylated tau protein. Accurate identification in the early stages of the disease, even in the preclinical stage, is of vital importance for timely intervention and slowing the progression of the disease.

[0003] Currently, the gold standard for diagnosing Alzheimer's disease (AD) relies on post-mortem pathological examination of brain tissue. Antemorrhagic diagnosis is primarily based on a thorough clinical evaluation combined with evidence from biomarkers. These biomarkers are mainly divided into two categories: one reflecting Aβ pathology and the other reflecting neuronal damage. However, these existing techniques have the following limitations: ① Cerebrospinal fluid testing requires obtaining samples through lumbar puncture, which is an invasive procedure with low patient acceptance. Furthermore, the sample processing and analysis process is complex, making it difficult to promote in primary healthcare institutions. ② While positron emission tomography (PET) is visually intuitive, the equipment is expensive, the examination costs are high, and it involves radiation exposure, making it unsuitable for large-scale population screening and long-term dynamic monitoring.

[0004] To overcome the aforementioned challenges, developing non-invasive biomarkers based on peripheral blood is an important research direction. Although significant progress has been made in detecting plasma Aβ and Tau proteins, these assays typically require ultrasensitive detection platforms, remain costly, and exhibit varying diagnostic cutoff values ​​across different research cohorts and clinical settings; standardization efforts are still ongoing. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing an application of plasma TRAIL level detection in Alzheimer's disease detection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of a substance for detecting the concentration of tumor necrosis factor-related apoptosis-inducing ligand in human plasma in the preparation of reagents for assisting in the assessment of Alzheimer's disease-related cognitive status in subjects.

[0007] Preferably, the application includes comparing the plasma TRAIL concentration of a subject with a preset reference range or diagnostic threshold, wherein when the plasma TRAIL concentration is higher than a first diagnostic threshold used to distinguish between AD and normal cognitive function, it provides information to help determine that the subject is more likely to be in AD than in CU.

[0008] Preferably, the application is specifically for assisting in the differential diagnosis of Alzheimer's disease and amnesic mild cognitive impairment, wherein when the plasma TRAIL concentration is higher than the second diagnostic threshold used to distinguish between AD and aMCI, it provides information to help determine that the subject is more likely to be in an AD state rather than an aMCI state.

[0009] Preferably, the first diagnostic threshold is 510.81 pg / mL, which corresponds to the diagnostic efficacy in distinguishing AD from CU.

[0010] Preferably, the second diagnostic threshold is 485.67 pg / mL, which corresponds to the diagnostic efficacy in distinguishing AD from aMCI.

[0011] A detection kit for implementing the application, the kit comprising: An immunoassay kit for the specific detection of TRAIL protein concentration in human plasma samples, the kit comprising a solid-phase carrier, a TRAIL-specific capture antibody, a labeled detection antibody, and a substrate for generating a detection signal; The instruction manual contains at least one diagnostic threshold, which is selected from: 510.81 pg / mL for differentiating AD from CU, 485.67 pg / mL for differentiating AD from aMCI, and 503.05 pg / mL for differentiating AD from aMCI and CU combined. The calibration standard contains a known concentration of recombinant human TRAIL protein and is used to establish a standard curve between the detection concentration and the signal value.

[0012] An auxiliary assessment system for Alzheimer's disease-related cognitive states, used to implement the aforementioned application, the system comprising: A data input module is used to receive input data, which includes at least the plasma TRAIL concentration detection value of the subject provided by an external detection device; The data processing and analysis module is communicatively connected to the data input module, and the processing and analysis module is configured as follows: ①Invoke a pre-stored diagnostic threshold database, which contains diagnostic thresholds; ② Compare the input plasma TRAIL concentration detection value with one or more of the diagnostic thresholds; ③ Receive and process subject age information and / or cognitive scale score information from the data input module, and integrate and analyze them with the plasma TRAIL concentration detection value; The results output module is used to generate and output an assessment report, which includes at least information on the probability classification or risk level of the subject belonging to AD, aMCI, or CU status based on the comparative or integrated analysis.

[0013] Preferably, the data processing and analysis module is configured to perform receiver operating characteristic (ROC) curve analysis or logistic regression analysis to calculate the diagnostic efficacy index of the plasma TRAIL concentration detection value for distinguishing different cognitive states. The efficacy index includes one or more of area under the curve, sensitivity, and specificity, and the efficacy index is included in the auxiliary assessment report output.

[0014] The beneficial effects of this invention are as follows: 1. This invention uses plasma samples for testing, requiring only routine venous blood collection, avoiding the invasive risks of lumbar puncture and the radiation exposure and high costs of PET imaging. The enzyme-linked immunosorbent assay (ELISA) and other technologies used are mature and relatively standardized, making them easy to implement in most hospital laboratories, greatly improving clinical accessibility and patient compliance, and suitable for large-scale community screening and long-term follow-up monitoring.

[0015] 2. This invention, through rigorous clinical research, systematically elucidates for the first time that plasma TRAIL levels exhibit specific changes in an AD continuum (cognitively normal CU, aMCI, AD) population after strictly excluding other confounding factors: the level in the AD patient group (501.24±98.38 pg / mL) was significantly higher than that in the aMCI group (417.60±111.16 pg / mL) and the CU group (398.59±134.60 pg / mL), while there was no significant difference between the aMCI and CU groups. This suggests that elevated plasma TRAIL levels may be closely related to the clinical dementia stage of AD, providing new biological evidence for distinguishing AD from aMCI.

[0016] 3. Although this invention shows no difference in TRAIL levels between the aMCI and CU groups, its significantly elevated TRAIL levels in the AD group, combined with its diagnostic efficacy in differentiating AD from aMCI, indicate that this biomarker is valuable in identifying individuals who have progressed to a definitive AD dementia state. Longitudinal monitoring of plasma TRAIL level trends in high-risk individuals provides dynamic information for assessing clinical transformation and progression of the disease. Attached Figure Description

[0017] Figure 1 This is an age-stratified analysis diagram of plasma TRAIL levels according to the present invention; Figure 2This is a comparative analysis of plasma TRAIL levels stratified by diagnostic group (AD, aMCI, and CU) according to the present invention. Figure 3 This is a correlation analysis graph showing the relationship between plasma TRAIL levels and key demographic and clinical indicators (including MMSE score, age, CDR, years of education, and ADL scale score) according to the present invention. Figure 4 This is the ROC curve of plasma TRAIL levels according to the present invention; Figure 1 In the study: (a) Plasma TRAIL levels in pre-segmented age groups of CU participants; (b) Correlation between plasma TRAIL levels and age in the overall cohort; (c) Sex-specific correlations in female participants and (d) male participants; (e) Plasma TRAIL levels in female and male CU participants; and (f) Plasma TRAIL levels in pre-segmented age groups; TRAIL: tumor necrosis factor-associated apoptosis-inducing ligand; CU: normal cognitive function; Figure 2 (a) Differential expression of plasma TRAIL; (b) Differential expression in female and (c) male participants across diagnostic groups; TRAIL: tumor necrosis factor-associated apoptosis-inducing ligand; AD: Alzheimer's disease; aMCI: amnesic mild cognitive impairment; CU: normal cognitive function; Figure 3 (a) All groups; (b) AD diagnosis group, (c) aMCI diagnosis group and (d) CU diagnosis group; TRAIL: tumor necrosis factor-associated apoptosis-inducing ligand; MMSE: Mini-Mental State Examination; CDR: Clinical Dementia Rating Scale; ADL: Activities of Daily Living; Figure 4 (a) ROC curves of plasma TRAIL levels distinguishing AD from CU, AD from aMCI, and aMCI from CU; (b) ROC curves of plasma TRAIL levels distinguishing AD from aMCI+CU, and AD+aMCI from CU; ROC: receiver operating characteristic; TRAIL: tumor necrosis factor-associated apoptosis-inducing ligand; AD: Alzheimer's disease; aMCI: amnesic mild cognitive impairment; CU: normal cognitive function. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] Example 1: A research method for detecting plasma TRAIL levels in Alzheimer's disease detection, as detailed below: 1.1 Research Design Between January 2019 and September 2025, 218 participants aged 59 to 102 years from the Department of Geriatrics at Chongqing People's Hospital were recruited. A separate cohort of 178 cognitively normal (CU) individuals was also included from the hospital's health checkup center for age-related analysis. All participants or their legal representatives provided written informed consent. This study protocol has been approved by the Ethics Committee of Chongqing People's Hospital.

[0020] 1.2 Subject Assessment and Exclusion Criteria All participants underwent standardized assessments, including a comprehensive medical history, physical and neurological examinations, neuropsychological testing, and routine laboratory tests. Cognitive function was assessed using the Chinese version of the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA), while functional status was assessed using the Activities of Daily Living (ADL) scale. Participants with positive cognitive screening results underwent an extended neuropsychological testing suite covering memory, executive function, language, and visuospatial abilities, including an auditory word learning test, a clock drawing test, a line-drawing test, a Boston naming test, and a digit span test. Disease staging and functional impact were quantified using the Clinical Dementia Rating Scale (CDR) and the Pfeffer Outpatient Functional Impairment Scale, and the Hachinski Ischemic Index was used to help rule out vascular causes.

[0021] To rule out secondary cognitive impairment, all patients with abnormal cognitive screening results underwent structural brain imaging (computed tomography or magnetic resonance imaging) and laboratory tests for thyroid function, vitamin B12, folic acid, syphilis, and human immunodeficiency virus (HIV). The final diagnosis was determined collaboratively by experienced neurologists. Amnesic mild cognitive impairment (aMCI) was diagnosed according to the Petersen criteria, and Alzheimer's disease (AD) dementia was diagnosed according to the National Institute on Aging-Alzheimer's Association (NIA-AA) criteria. Cognitively normal (CU) controls were defined as individuals with normal cognitive performance (CDR=0) and no subjective cognitive complaints.

[0022] Individuals meeting any of the following criteria will be excluded from the study: (i) a family history suggestive of autosomal dominant inheritance or early-onset dementia; (ii) a current or past history of significant substance abuse; (iii) a severe mental illness that may confound cognitive assessment, such as schizophrenia, bipolar disorder, or major depressive disorder; (iv) a significant neurological disorder, including a history of stroke, epilepsy, or moderate to severe traumatic brain injury; or (v) an uncontrolled severe systemic disease, such as severe heart, lung, liver, or kidney dysfunction, active autoimmune disease, or any malignancy.

[0023] 1.3 Blood sample collection and plasma TRAIL and APOE genotyping Plasma collection followed the published operating procedures. Fasting venous blood samples were collected in 5mLEDTA tubes and centrifuged at 3000 rpm for 10 minutes at 4°C, followed by plasma separation. After removing blood cell components, the aliquoted blood samples were stored in polypropylene low-adsorption cryovials and kept at -80°C until detection. Plasma TRAIL concentration was detected using an enzyme-linked immunosorbent assay kit

[17] (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer's instructions

[18] . APOE genotyping (ε2, ε3, ε4) was performed using a fluorescent PCR-based kit (Human APOE Genotyping Kit; Xiamen Renrui Biotechnology Co., Ltd., China) according to the manufacturer's operating procedures.

[0024] 1.4 Statistical Analysis Statistical analyses were performed using SPSS software (version 26.0; IBM, Armonk, NY, USA). Normality of the data was assessed using the Shapiro-Wilk test. Continuous variables were expressed as mean ± standard deviation (SD) or median (interquartile range), depending on the circumstances. One-way ANOVA (followed by post-hoc testing) or the Kruskal-Wallis test was used for comparisons between groups. Chi-square tests were used for comparisons between groups of categorical variables. Pearson or Spearman correlation coefficients were used to analyze associations between variables. Age-corrected analysis of covariance (ANCOVA) was used for comparisons of plasma TRAIL levels between groups. Hierarchical multivariate regression analysis was used to further control for potential confounding factors. The diagnostic efficacy of plasma TRAIL was assessed using receiver operating characteristic (ROC) curve analysis, and the area under the curve (AUC) was calculated. Multivariate logistic regression analysis was used to assess independence. A two-sided p-value <0.05 was considered statistically significant.

[0025] 2. Results 2.1 Plasma TRAIL levels in different age groups of cognitively normal subjects To clarify the age-related changes in plasma TRAIL concentration, this study analyzed a cohort of 178 cognitively normal (CU) individuals spanning a wide age range of adults, as detailed below:

[0026] * Use the chi-square test to compare differences in the proportion of women or men between different age groups. SD, standard deviation.

[0027] Plasma TRAIL levels gradually increased with age. The levels in the youngest group (20–29 years) were significantly lower than those in the 40–49 year group (228.93±56.31 pg / mL vs. 285.37±47.48 pg / mL, P=0.001) and the 50–79 year group (228.93±56.31 pg / mL vs. 320.56±48.22 pg / mL, P<0.001). Similarly, the levels in the 30–39 year group were also lower than those in the 50–79 year group (253.20±55.51 pg / mL vs. 320.56±48.22 pg / mL, P<0.001). There were no significant differences in plasma TRAIL levels between adjacent age groups (all P>0.05). Figure 1 a).

[0028] Supporting this trend is the strong positive correlation between plasma TRAIL concentration and age across the entire CU cohort (r=0.579, P<0.001). Figure 1 b). This correlation was observed in women (r=0.542, P<0.001); Figure 1 c) and males (r=0.611, P<0.001); Figure 1 All values ​​remained significant in d). There was no significant difference in plasma TRAIL levels between sexes (271.45±61.14 pg / mL for females and 268.91±64.56 pg / mL for males, P=0.903); Figure 1 e), this result was consistent across all stratified age groups ( Figure 1 f).

[0029] In the subgroup with available genetic data, although the mean age of APOEε4 carriers and non-carriers was comparable (42.25±0.96 years vs. 39.41±11.43 years, P=0.231), there was no significant difference in plasma TRAIL levels between the two groups (P=0.180).

[0030] 2.1 Plasma TRAIL levels in the Alzheimer's disease continuum A total of 218 participants were included, including patients with Alzheimer's disease (AD) (n=78), patients with amnestic mild cognitive impairment (aMCI) (n=70), and sex-matched cognitively normal (CU) controls (n=70). Demographic and clinical characteristics are summarized below:

[0031] AD: Alzheimer's disease; ADL: Activities of daily living; aMCI: Amnesic mild cognitive impairment; CDR: Clinical dementia rating scale; CU: Normal cognitive function; MMSE: Mini-Mental State Examination. As expected, AD patients had the lowest MMSE and ADL scores and the highest CDR scores.

[0032] Age-corrected stratified regression analysis showed significant differences in plasma TRAIL levels among the three groups (P<0.001). Plasma TRAIL levels in the AD group were significantly higher than those in the aMCI group (501.24±98.38 pg / mL vs. 417.60±111.16 pg / mL, P<0.001) and the CU group (501.24±98.38 pg / mL vs. 398.59±134.60 pg / mL, P<0.001). No significant difference was observed between the aMCI group and the CU group. Figure 2 a).

[0033] Stratified by sex confirmed this pattern. In women, plasma TRAIL levels were higher in AD patients than in aMCI patients (489.62±109.16 pg / mL vs. 406.53±113.02 pg / mL, P=0.038) and CU controls (489.62±109.16 pg / mL vs. 404.11±153.45 pg / mL, P=0.027), while there was no difference between the aMCI and CU groups (P>0.999). Figure 2 b). In men, TRAIL levels were also higher in AD patients than in aMCI patients (509.77±89.98 pg / mL vs. 426.40±110.34 pg / mL, P=0.005) and CU controls (509.77±89.98 pg / mL vs. 394.45±120.43 pg / mL, P<0.001), while there was no significant difference between the male aMCI group and the CU group (P=0.491). Figure 2 c).

[0034] 2.3 Correlation between plasma TRAIL and clinical indicators Correlation analysis across the entire cohort showed a significant negative correlation between plasma TRAIL levels and MMSE scores (r=-0.156, P=0.022). Figure 3 a) suggests that higher TRAIL concentrations are associated with poorer cognitive performance. Plasma TRAIL expression levels showed a slight increasing trend with age (r=0.162, P=0.017). Figure 3 a). In contrast, in AD patients ( Figure 3 b) aMCI patients ( Figure 3 c) and the cognitively normal (CU) group ( Figure 3 In d), no clear correlation was observed between TRAIL level and age or MMSE score.

[0035] 2.4 ROC curve analysis of the diagnostic potential of plasma TRAIL levels for AD and aMCI The diagnostic potential of plasma TRAIL was assessed using receiver operating characteristic (ROC) curve analysis. Figure 4 ), as detailed below:

[0036] When differentiating between AD patients and CU controls, the area under the curve (AUC) was 0.652 (95% CI: 0.564–0.740), with a sensitivity of 39.7% and a specificity of 88.6% at the optimal cutoff. This model performed even better in differentiating between AD and aMCI, with an AUC of 0.687 (95% CI: 0.600–0.773), a sensitivity of 56.4%, and a specificity of 78.6%. Figure 4 a).

[0037] The utility of this biomarker in broader clinical differentiation was further evaluated. When differentiating between the combined AD and aMCI group and the CU group, the AUC was 0.630 (95% CI: 0.550–0.710), with a sensitivity of 60.1% and a specificity of 65.7%. Conversely, when differentiating AD patients from the combined aMCI and CU group, the AUC was 0.669 (95% CI: 0.593–0.746), with a sensitivity of 43.6% and a specificity of 74.0%. Figure 4 b).

[0038] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a substance for detecting the concentration of tumor necrosis factor-related apoptosis-inducing ligand in human plasma in the preparation of a reagent for assisting in the assessment of Alzheimer's disease-related cognitive status in subjects.

2. Use according to claim 1, characterized in that, The application includes comparing the plasma TRAIL concentration of a subject with a preset reference range or diagnostic threshold, wherein when the plasma TRAIL concentration is higher than a first diagnostic threshold used to distinguish between AD and normal cognitive function, it provides information to help determine that the subject is more likely to be in AD than in CU.

3. Use according to claim 2, characterized in that, The specific application is to assist in the differential diagnosis of Alzheimer's disease and amnesic mild cognitive impairment. Specifically, when the plasma TRAIL concentration is higher than the second diagnostic threshold used to distinguish between AD and aMCI, it provides information to help determine that the subject is more likely to be in an AD state than an aMCI state.

4. Use according to claim 3, characterized in that, The first diagnostic threshold is 510.81 pg / mL, corresponding to the diagnostic efficacy in distinguishing AD from CU.

5. Use of a plasma TRAIL level test in the detection of Alzheimer's disease according to claim 4, characterized in that, The second diagnostic threshold is 485.67 pg / mL, which corresponds to the diagnostic efficacy in distinguishing AD from aMCI.

6. A test kit for carrying out the use according to any one of claims 1 to 5, characterized in that, The kit contains: An immunoassay kit for the specific detection of TRAIL protein concentration in human plasma samples, the kit comprising a solid-phase carrier, a TRAIL-specific capture antibody, a labeled detection antibody, and a substrate for generating a detection signal.

7. The test kit according to claim 6, characterized in that The kit also includes: instructions describing at least one diagnostic threshold, the diagnostic threshold being selected from: 510.81 pg / mL for differentiating AD from CU, 485.67 pg / mL for differentiating AD from aMCI, and 503.05 pg / mL for differentiating AD from aMCI and CU combined.

8. The detection kit according to claim 7, characterized in that, The kit also includes: a calibration standard containing a known concentration of recombinant human TRAIL protein, used to establish a standard curve between the detection concentration and the signal value.

9. An auxiliary assessment system for Alzheimer's disease-related cognitive states, used to implement the application according to any one of claims 1-5, characterized in that, The system includes: A data input module is used to receive input data, which includes at least the plasma TRAIL concentration detection value of the subject provided by an external detection device; The data processing and analysis module is communicatively connected to the data input module, and the processing and analysis module is configured as follows: ①Invoke a pre-stored diagnostic threshold database, which contains diagnostic thresholds; ② Compare the input plasma TRAIL concentration detection value with one or more of the diagnostic thresholds; ③ Receive and process subject age information and / or cognitive scale score information from the data input module, and integrate and analyze them with the plasma TRAIL concentration detection value; The results output module is used to generate and output an assessment report, which includes at least information on the probability classification or risk level of the subject belonging to AD, aMCI, or CU status based on the comparative or integrated analysis.

10. The auxiliary evaluation system according to claim 9, characterized in that, The data processing and analysis module is configured to perform receiver operating characteristic (ROC) curve analysis or logistic regression analysis to calculate the diagnostic efficacy index of the plasma TRAIL concentration detection value for distinguishing different cognitive states. The efficacy index includes one or more of area under the curve, sensitivity, and specificity, and is included in the output of the auxiliary assessment report.

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