Application of biomarkers in the preparation of sALS-related diagnostic products and products for diagnosing sALS

Abstract

This invention discloses the application of biomarkers in the preparation of sALS-related diagnostic products and the products themselves, belonging to the field of biomedical technology. It utilizes FCN1 and / or PSAP as biomarkers, achieved by detecting the levels of FCN1 and / or PSAP in biological samples at the protein level. The amino acid sequence of FCN1 is shown in SEQ ID: 1, and the amino acid sequences of PSAP are shown in SEQ ID: 2 to SEQ ID: 5. This invention uses FCN1 and / or PSAP as biomarkers in sALS diagnostic research, contributing to the improvement of sALS diagnostic accuracy.

Description

Technical Field

[0001] This invention relates to the application of biomarkers in the preparation of sALS-related diagnostic products and products for diagnosing sALS. Specifically, it involves the application of FCN1 and / or PSAP as biomarkers in the preparation of early diagnostic products for sALS, as well as products for the early diagnosis of sALS, belonging to the field of biomedical technology. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease, the most common type of motor neuron disease (MND). ALS is characterized by progressive muscle atrophy, weakness, fasciculations, bulbar palsy, and pyramidal tract signs, resulting from involvement of both upper and lower motor neurons. Some patients may also experience varying degrees of cognitive and / or behavioral impairments related to frontotemporal lobe involvement. Approximately 10% of ALS patients have familial ALS (fALS), and several genes have been identified as associated with fALS. The remaining nearly 90% of cases are sporadic ALS (sALS). However, the etiology of ALS remains unclear. Most ALS patients die from complications such as respiratory infections, dysphagia, and respiratory muscle paralysis. Therefore, early diagnosis is crucial for ALS treatment, facilitating the timely implementation of drug therapy, nutritional management, and respiratory support, thereby improving patients' quality of life and prolonging their survival.

[0003] In the general clinical diagnosis of ALS, determining the extent of upper and lower motor neuron involvement is a crucial step. Currently, the clinical diagnosis of this disease mainly relies on the patient's clinical examination (detailed medical history and meticulous physical examination), neurophysiological examinations (such as nerve conduction studies, core needle electromyography, and magnetic stimulation motor evoked potentials), and the exclusion of other diseases. In addition, other examinations such as neuroimaging, biochemical tests (such as serum creatine kinase, cerebrospinal fluid proteins, C-reactive protein, and homocysteine), cerebrospinal fluid or serum neurofilament light chain tests, pulmonary function tests, and blood gas analysis are helpful in differentiating ALS from other diseases, ruling out structural damage in patients, and assessing neuronal damage or respiratory function. Early diagnosis of ALS is challenging due to its insidious and diverse early clinical manifestations and lack of specific biological diagnostic indicators. The average delay from onset to diagnosis is approximately 9-15 months. Furthermore, early diagnosis requires effective differentiation from multiple diseases such as multiple system atrophy (MSA), cervical spondylosis, lumbar spondylosis, multifocal motor neuropathy, and late-onset spinal muscular atrophy, making ALS diagnosis particularly difficult.

[0004] Research on ALS-related biomarkers is currently limited. The reported biomarkers mainly include motor neuron-related biomarkers (such as phosphorylated neurofilament heavy chains, neurofilament light chains, cystatin C, and thyroxine transporter), inflammatory state-related biomarkers (such as monocyte chemotactic protein 1 and miR-451), skeletal muscle-related biomarkers (such as miR-338-3p and miR-206), and metabolic-related biomarkers (such as homocysteine, glutamate, and cholesterol). Research on these biomarkers has mostly focused on the physical condition, nerve damage, inflammation, or prognosis of ALS patients. To date, there is still a lack of effective biomarkers for the diagnosis of ALS.

[0005] Prosaposin (PSAP) is the precursor form of the non-enzymatic glycoproteins sphingolipid activator proteins A, B, C, and D, and participates in the lysosomal degradation of sphingolipids. Studies have shown that PSAP plays a protective role in neurons and glial cells by activating G protein-mediated pathways. PSAP is widely expressed in the nervous system and muscles, and has neurotrophic effects. PSAP is abundant in the central and peripheral nervous systems, including autonomic, motor, and sensory nerves, and is mainly located in the lysosomal granules of neurons. Existing patent CN111060693A discloses the role of PSAP protein in the pathogenesis of ALS and its verification method, revealing the influence of PSAP on neurodegeneration and its role in the pathogenesis of ALS. The results show that PSAP gene knockout mice can delay the onset of ALS, thus PSAP can be used as a new therapeutic target for ALS.

[0006] Ficolin-1 (FCN1) is a subtype of fibrin, a glycoprotein containing collagen-like and fibrinogen-like domains found in various human tissues. Because fibrin is a soluble oligomeric defense protein with lectin-like activity, it is considered an important innate immune molecule. However, there are currently no reports on the use of FCN1 as a biomarker in the diagnosis of sALS. Summary of the Invention

[0007] This invention aims to provide the application of biomarkers in the preparation of sALS-related diagnostic products and products for diagnosing sALS. Using FCN1 and / or PSAP as biomarkers in sALS diagnostic research helps to improve the level of sALS diagnosis.

[0008] The present invention is achieved through the following technical solution: the application of biomarkers in the preparation of sALS-related diagnostic products, using FCN1 and / or PSAP as biomarkers, and achieving this by detecting the levels of FCN1 and / or PSAP in biological samples at the protein level, wherein the amino acid sequence of FCN1 is shown in SEQ ID: 1, and the amino acid sequences of PSAP are shown in SEQ ID: 2 to SEQ ID: 5.

[0009] The biological sample was plasma.

[0010] The diagnostic product detects the protein levels of FCN1 and / or PSAP in biological samples using an ELISA method.

[0011] The diagnostic product is identified by detecting upregulated protein levels of FCN1 and / or PSAP in biological samples.

[0012] The diagnostic products include reagent kits, test strips, or other medically feasible products.

[0013] The present invention also provides a product for diagnosing sALS, including a diagnostic product for detecting the protein levels of FCN1 and / or PSAP in a biological sample by means of an ELISA method as described above, or a diagnostic product for determining the level by detecting upregulation of FCN1 and / or PSAP protein levels in a biological sample.

[0014] Furthermore, products for diagnosing sALS include kits, test strips, or other medically feasible products.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] (1) Based on highly sensitive deep proteomics technology and enzyme-linked immunosorbent assay (ELISA), this invention can provide a convenient and reliable test indicator in peripheral blood, enabling early diagnosis of sALS through the detection of plasma protein markers, and providing important clinical value for the diagnosis and treatment of sALS.

[0017] (2) The novel diagnostic biomarkers for sALS provided by this invention: FCN1 and PSAP are significantly upregulated in sALS. The sALS disease group and different disease control groups show consistent trends. Their diagnostic performance is assessed from moderate to high in different cohorts, which reflects the reliable and universal characteristics of these two protein biomarkers in the diagnosis of sALS.

[0018] (3) This invention uses a commercially available ELISA kit to quantitatively detect the levels of FCN1 and PSAP in the plasma of subjects. The results showed that the concentration of FCN1 in the plasma of sALS patients was 0.583 ± 0.219 ng / mL, and the concentration of PSAP was 0.613 ± 0.183 ng / mL. In the control group (including various types of neurological diseases such as cervical spondylosis, peripheral neuropathy, and Guillain-Barré syndrome), the concentration of FCN1 in the plasma was 0.379 ± 0.200 ng / mL, and the concentration of PSAP was 0.444 ± 0.166 ng / mL. The expression levels of FCN1 and PSAP in the plasma of sALS patients were significantly upregulated compared with those in the control group, and the differences were statistically significant. p The values ​​of < 0.05 indicate that these two plasma protein markers are stable and universally applicable for the diagnosis of sALS.

[0019] (4) Based on the current status of clinical diagnosis of ALS, this disease needs to be differentiated from various diseases with some similar symptoms during general clinical diagnosis, including cervical spondylosis, lumbar spondylosis, multifocal motor neuropathy, late-onset spinal muscular atrophy, Hirayama disease, Kennedy disease, hereditary spastic paraplegia, and ALS plus syndrome. Therefore, this invention used different disease controls in the research process to identify reliable and widely applicable molecular markers for sALS diagnosis to meet clinical needs in this regard. Experiments have shown that the plasma protein markers FCN1 and PSAP provided by this invention still demonstrate good diagnostic ability in differentiating sALS from control groups of different types of diseases.

[0020] In summary, this invention demonstrates that plasma FCN1 and PSAP proteins can be used for the auxiliary diagnosis of sALS under relatively low-invasive and convenient conditions. These two protein markers have stable and reliable performance in the diagnosis of sALS and in the differential diagnosis of various types of neurological diseases, including MSA, cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, and spinocerebellar ataxia. They also have a wide range of applications and can provide important clinical value for the diagnosis of sALS. Attached Figure Description

[0021] Figure 1 Differentially expressed proteins between the ALS group and the control group;

[0022] Figure 2 A graph for enrichment analysis of biological processes;

[0023] Figure 3 Results of differentially expressed protein enrichment analysis;

[0024] Figure 4Comparison of FCN1 and PSAP plasma levels and ROC curve characteristics between two cohorts. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0026] Example 1: Determination of Biomarkers

[0027] Plasma samples were collected from 38 patients with sALS and 45 patients with non-ALS (including multiple system atrophy (MSA), cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, spinocerebellar ataxia, and other neurological diseases with some similar symptoms to sALS) who were initially diagnosed at the First Medical Center of the General Hospital of the Chinese People's Liberation Army between November 2020 and January 2022, before treatment. Patient information was also improved through inpatient medical records.

[0028] Inclusion criteria for sALS patients: 1) Initial diagnosis of ALS that meets the revised El Escorial criteria for the World Federation of Neurological Societies; 2) No family history of ALS.

[0029] Exclusion criteria: 1) Comorbid neurodegenerative diseases; 2) Comorbid brain injury, tumor, paraneoplastic syndrome; 3) Comorbid serious internal medical diseases.

[0030] The included study subjects were divided into a discovery cohort and a validation cohort. The discovery cohort consisted of 10 patients with severe acute atrial spondylitis (sALS) and 23 patients with non-ALS (MSA), while the validation cohort consisted of 28 patients with sALS and 22 patients with non-ALS (neurological diseases such as cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, and spinocerebellar ataxia). There were no statistically significant differences between the ALS group and the control group in terms of age, sex, diabetes, hypertension, BMI, and history of smoking and alcohol consumption.

[0031] Test sample: Plasma sample from enrolled subjects.

[0032] The methods for determining biomarkers are as follows:

[0033] (1) Plasma sample collection: Fasting venous whole blood was collected from enrolled patients on the morning of the day before treatment after hospitalization and from outpatients on the day of their visit using commercially available EDTA anticoagulant blood collection tubes. The plasma was centrifuged at 4000×g for 10 minutes at room temperature, and 500 μL of plasma was collected, aliquoted and frozen into 1.5 mL EP tubes and stored at -80℃ for later use.

[0034] (2) Pretreatment: The plasma low abundance protein enrichment magnetic bead kit (DMB kit, Beijing Qinglian Biotech Co., Ltd.) was used to perform low abundance protein enrichment and mass spectrometry analysis pretreatment of plasma samples from the discovery cohort according to the DMB kit. The enzyme-digested peptides were frozen at -80℃ for subsequent LC-MS / MS analysis.

[0035] (3) Sample Data Acquisition: The enzyme-digested peptides were loaded onto a C18 pre-column (ReproSil Pur C18-AQ 3-µm, Dr. Maisch, Germany) and desalted using an EASY nLC 1200 system (Thermo Fisher Scientific, USA) with 12 µL of 100% solution A (0.1% formic acid). Gradient separation was then performed for 60 min using a C18 reverse-phase capillary column (150 µm × 250 mm) (ReproSil-Pur C18-AQ 1.9 µm, Dr. Maisch, Germany). The eluted peptides were analyzed using Q-Exactive HF-X (Thermo Fisher Scientific, USA), and library data were acquired using data-dependent acquisition (DDA) mode. Subsequently, full-mass scans at 350-1500 m / z were acquired using data-independent acquisition (DIA) mode to obtain sample data, identifying a total of 2164 proteins.

[0036] (4) Sample Result Analysis: The raw DIA files acquired by Q-Exactive HF-X were processed using DIA-NN (version 1.81) with the following settings: UniProt human database (March 17, 2022), carbamoyl methyl (C) as a fixed modification, and oxidation (M) and acetyl (N-terminus of proteins) as variable modifications. Trypsin specificity was set to allow two missed cleavage sites, and the FDR of proteins and peptides was less than 1%. The data filtering Q-value cutoff was 0.01, and the normalization parameter was set to retention time-dependent normalization between samples. Clinical sample analysis was performed using a self-built database to search the DIA-NN (version 1.81) database and obtain the relative expression abundance data of sample proteins.

[0037] (5) Data processing: Data processing was carried out using bioinformatics methods to identify differentially expressed proteins between the cohort ALS group and the control group, and to analyze the molecular functions and pathways of the differentially expressed proteins.

[0038] Specifically, Perseus software was used to perform analyses such as normalization, logarithmic transformation, and imputation of missing values ​​according to a normal distribution for protein relative expression abundance. Proteins with missing values ​​> 80% were filtered out. All data underwent logarithmic transformation and were analyzed using Student's algorithm. tThe test was used to compare differences between groups. The difference was considered to be greater than 1.5 or less than 0.67. p A screening rule of < 0.05 was used to screen differentially expressed proteins (DEPs) between groups. After z-score normalization, the data were exported for subsequent visualization analysis. Online tools such as Hiplot (https: / / hiplot.com.cn) were used to create volcano plots and heatmaps, and principal component analysis (PCA) was performed. Bioinformatics analysis included geneontology (GO) analysis and analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0039] The differentially expressed protein (DEP) results were visualized using volcano plots and heatmaps. A total of 320 DEPs were obtained between the ALS group and the control group according to the screening criteria. Compared with the control group, 188 differentially expressed proteins were upregulated and 132 were downregulated in the ALS group. Principal component analysis results showed that the DEPs could clearly distinguish the ALS group from the control group. (See [link to relevant documentation]). Figure 1 As shown.

[0040] Depend on Figure 1 As can be seen in the figure, A is a volcano plot, which shows the difference in protein levels between the ALS group and the control group by -log10 ( p The values ​​(value) and log2 (Fold Change) show the distribution of differentially expressed proteins identified by non-targeted proteomics in the discovery cohort. Blue represents proteins downregulated in the ALS group compared to the control group, and red represents proteins upregulated in the ALS group. Figure B is the PCA plot of differentially expressed proteins, and the PCA plot in the figure clearly distinguishes the differentially expressed proteins between ALS and the control group. Figure C is a heatmap display of differentially expressed proteins.

[0041] Bioinformatics analysis revealed that upregulated DEPs in the ALS group primarily involved biological processes such as complement activation and lipoprotein metabolism, with related pathways including cholesterol metabolism and the cGMP-PKG signaling pathway. Downregulated DEPs in the ALS group primarily involved biological processes such as negative regulation of oxidative stress-induced endogenous apoptosis signaling pathways, positive regulation of dendritic spine morphogenesis, and axonal elongation, with related pathways including the ErbB signaling pathway and the actin cytoskeleton regulatory pathway. (See [link to relevant documentation]). Figure 2 and Figure 3 As shown. Therefore, based on the changes in the biological functions and related signaling pathways involved in DEPs, this invention can also provide a certain theoretical basis and potential direction for research on the pathogenesis of sALS.

[0042] like Figure 2 As shown in the figure, A to C represent the GSEA results based on overall proteomics data. As can be seen from the figure, compared with the control group, the ALS group showed significantly upregulated expression levels in phospholipid biosynthesis, lipid biosynthesis, and negative regulation by cell projection tissues. p < 0.01). For example... Figure 3 As shown in the figure, A and B are the GO and KEGG enrichment analysis results of differentially expressed proteins, respectively.

[0043] Differential gene expression analysis revealed that, compared with the control group of MSA patients, the expression levels of both proteins in the plasma of sALS patients were significantly upregulated, with fold differences of 3.89 and 3.57, respectively, ranking among the top 15 differentially expressed proteins. Furthermore, through literature review and assessment of the applicability of commercially available ELISA kits, FCN1 and PSAP were identified as the protein biomarkers for the diagnosis of sALS.

[0044] The amino acid sequence of FCN1 is shown in SEQ ID: 1:

[0045] MELSGATMARGLAVLLVLFLHIKNLPAQAADTCPEVKVVGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDTDGGGWTVFQRRMD GSVDFYRDWAAYKQGFGSQLGEFWLGNDNIHALTAQGSSELRVDLVDFEGNHQFAKYKSFKVADEAEKYKLVLGAFVGGSAGNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPHESYANGINWSAAKGYKYSYKVSEMKVRPA.

[0046] The amino acid sequences of PSAP are shown in SEQ ID: 2 to SEQ ID: 5:

[0047] Isoform 1 (SEQ ID: 2):

[0048] MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0049] Isoform 2(SEQ ID:3):

[0050] MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0051] Isoform 3(SEQ ID:4):

[0052] MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0053] Variant 1(SEQ ID:5):

[0054] .

[0055] Example 2: Detection of biomarkers

[0056] The protein biomarkers FCN1 and PSAP identified in Example 1 were validated in a validation cohort using enzyme-linked immunosorbent assay (ELISA). The ELISA kits were commercially available. The PSAP kit was a Human Prosaposin (PSAP) ELISA Kit from Wuhan Huamei Biotechnology Co., Ltd., catalog number CSB-E12837h, with a sensitivity of 0.041 ng / mL and a linear range of 0.041-30 ng / mL. The FCN1 kit was a Human FCN1 (Ficolin-1) ELISA Kit from Wuhan Feien Biotechnology Co., Ltd., catalog number EH0135, with a sensitivity of 0.094 ng / mL and a linear range of 0.156-10 ng / mL.

[0057] The concentration of candidate biomarkers in plasma was detected using ELISA. The detection was performed according to the standard procedure provided in the kit instructions. A standard curve was generated using Curve Expert, and the concentration of the corresponding biomarker in the sample was calculated.

[0058] (I) The specific operating steps for ELISA detection of FCN1 are as follows:

[0059] (a) Equilibrate the reagents and well plates at room temperature for 30 min, and prepare the standard by serial dilution.

[0060] (b) After washing the plate twice, add 100 µL of standard or test sample to each well, cover with the plate and incubate at 37°C for 90 min.

[0061] (c) Discard the liquid in the wells and wash the plate twice; add 100 µL of biotin-labeled antibody working solution, cover with a new plate, and incubate at 37°C for 1 h. Discard the liquid and wash the plate three times.

[0062] (d) Add 100 µL of horseradish peroxidase-labeled avidin working solution to each well and incubate at 37°C for 30 min.

[0063] (e) Discard the liquid and wash the plate 5 times.

[0064] (f) Add 90 µL of substrate solution to each well, incubate at 37°C in the dark for 15 minutes, add 50 µL of stop solution to terminate the reaction, and measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0065] (g) A standard curve was generated using Curve Expert 1.4 software, and the concentration of FCN1 in the sample was calculated.

[0066] (II) The specific operating steps for ELISA detection of PSAP are as follows:

[0067] (a) Equilibrate the reagents and well plates at room temperature for 30 min, and prepare the standard by serial dilution.

[0068] (b) Add 100 µL of standard or test sample to each well, cover with a plate and incubate at 37°C for 2 h.

[0069] (c) Discard the liquid in the wells, add 100 µL of biotin-labeled antibody working solution, cover with a new plate, and incubate at 37°C for 1 h. Discard the liquid and wash the plate 3 times.

[0070] (d) Add 100 µL of horseradish peroxidase-labeled avidin working solution to each well and incubate at 37°C for 1 h.

[0071] (e) Discard the liquid and wash the plate 5 times.

[0072] (f) Add 90 µL of substrate solution to each well, incubate at 37°C in the dark for 15 minutes, add 50 µL of stop solution to terminate the reaction, and measure the optical density (OD value) of each well at a wavelength of 450 nm.

[0073] (g) A standard curve was generated using Curve Expert 1.4 software, and the PSAP concentration in the sample was calculated.

[0074] Statistical methods: Statistical analysis was performed using Graphpad Prism v9.0, IBM SPSS Statistics v26.0, and Medcalc v20.010. Categorical variables were expressed as examples (proportions %), and chi-square or Fisher's exact test was used for comparisons between two groups. Continuous variables were used to determine whether they were normally distributed. Normally distributed data were described using mean ± standard deviation, and skewed distributed data were described using median and interquartile range. The equality of variance between two groups was also assessed using Student's... t Inspection, Welch's t Test or Mann-Whitney U The test performed a difference test between the two groups. Receiver operating characteristic (ROC) curves were used to reflect the balance between sensitivity and specificity, and the area under the ROC curve (AUC), sensitivity, and specificity were calculated to evaluate the diagnostic performance of the candidate biomarker. AUC is an important indicator of test accuracy; the higher the AUC, the higher the diagnostic value of the test. The Youden Index, also known as the correct diagnostic index, ranges from 0 to 1, with values ​​closer to 1 indicating better accuracy. In this application, the diagnostic criterion is set at the maximum correct diagnostic index. p A value < 0.05 is considered statistically significant.

[0075] The differences in FCN1 and PSAP levels in the plasma of subjects in the validation cohort between the ALS group and the control group were quantitatively detected using a commercially available ELISA kit. The results are as follows: Figure 4 As shown.

[0076] Figure 4 In the table, A and B represent the comparison of FCN1 and PSAP levels between two groups in the discovery queue, respectively. Figure 4The levels of FCN1 and PSAP were compared between the two groups in the C and D validation cohorts. The results showed that the plasma FCN1 concentration in sALS patients was 0.583 ± 0.219 ng / mL, and the PSAP concentration was 0.613 ± 0.183 ng / mL. In the control group (including various types of neurological diseases such as cervical spondylosis, peripheral neuropathy, and Guillain-Barré syndrome), the plasma FCN1 concentration was 0.379 ± 0.200 ng / mL, and the PSAP concentration was 0.444 ± 0.166 ng / mL. The expression levels of FCN1 and PSAP in the plasma of sALS patients were significantly upregulated compared to the control group, and the differences were statistically significant. p < 0.05), and this trend is consistent with its results in the discovery queue (e.g. Figure 4 As shown in Figures A to D), these two plasma protein markers are stable and universally applicable for the diagnosis of sALS.

[0077] To assess the performance of FCN1 and PSAP in the diagnosis of sALS, this invention plotted ROC curves for FCN1 and PSAP in two independent cohorts based on proteomics-based relative expression and quantitative validation results. (See attached text.) Figure 4 As shown in E to H, the ROC curve characteristics are shown in Table 1 for FCN1 and PSAP.

[0078] Table 1. ROC curve characteristics of FCN1 and PSAP

[0079]

[0080] The analysis results showed that FCN1 and PSAP could effectively differentiate between sALS patients and control group MSA patients (FCN1: AUC = 0.870, sensitivity 0.700, specificity 0.957; PSAP: AUC = 0.939, sensitivity 0.900, specificity 0.826). Furthermore, FCN1 (cut-off value = 0.414 ng / mL) and PSAP (cut-off value = 0.473 ng / mL) also demonstrated good diagnostic performance in differentiating sALS from various other neurological diseases, including cervical spondylosis, peripheral neuropathy, and Guillain-Barré syndrome (AUC = 0.735, sensitivity 0.714, specificity 0.682).

[0081] Example 3: ELISA kit

[0082] The ELISA kit provided in this embodiment can detect the protein levels of the protein markers FCN1 and PSAP in the plasma samples of the subjects, and the detection method is as described in Example 2.

[0083] Specifically, the ELISA kit includes at least the substance for detecting FCN1 or PSAP protein levels, as well as other required buffer solutions or related standard reagents.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The application of biomarker FCN1 in the preparation of sALS diagnostic kits, characterized in that: The protein level of FCN1 in plasma was detected by ELISA. An FCN1 concentration >0.414 ng / mL indicated saline symptomatic ALS. The amino acid sequence of FCN1 is as shown in SEQ ID:

1. The diagnosis of sALS refers to the differentiation of sALS from other neurological diseases, such as MSA, cervical spondylotic myelopathy, peripheral neuropathy, and Guillain-Barré syndrome.

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