Application of biomarker in preparation of sALS related diagnosis product and sALS diagnosis product

Abstract

The invention discloses an application of a biomarker in preparation of sALS related diagnostic products and a product for diagnosing sALS, and belongs to the technical field of biomedical treatment, FCN1 and / or PSAP are / is taken as the biomarker, the biomarker is realized by detecting the level of FCN1 and / or PSAP in a biological sample on the protein level, the amino acid sequence of FCN1 is as shown in SEQ ID: 1, and the amino acid sequence of PSAP is as shown in SEQ ID: 2 to SEQ ID: 5. According to the invention, FCN1 and / or PSAP are / is used as biomarkers for research on sALS diagnosis, so that improvement of sALS diagnosis level is facilitated.

Description

Technical Field

[0001] The present invention relates to the application of biomarkers in the preparation of sALS-related diagnostic products and products for diagnosing sALS, and specifically to the application of FCN1 and / or PSAP as biomarkers in the preparation of early diagnostic products for sALS, and products for early diagnosis of sALS, and belongs to the field of biomedical technology. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease and the most common type of motor neuron disease (MND). The main clinical features of ALS are progressive muscle atrophy, weakness, fasciculations, bulbar palsy, and pyramidal tract signs caused by the involvement of upper and lower motor neurons. Some patients may have varying degrees of cognitive and / or behavioral disorders and other manifestations of frontotemporal lobe involvement. Among ALS patients, about 10% of patients have familial ALS (fALS). Currently, multiple genes have been found to be associated with fALS. The remaining nearly 90% of cases are sporadic ALS (sALS). However, the cause of ALS has not yet been clearly determined. Most ALS patients die from complications caused by respiratory tract infections, dysphagia, respiratory muscle paralysis, etc. Therefore, early diagnosis is one of the key points of ALS diagnosis and treatment, which helps to implement drug treatment, nutritional management, and respiratory support as soon as possible, thereby improving the quality of life of patients and prolonging their survival.

[0003] In the general clinical diagnosis of ALS, determining the extent of upper and lower motor neuron involvement is a key step in diagnosis. At present, the clinical diagnosis of this disease mainly relies on clinical examination of patients (detailed medical history and meticulous physical examination), neuroelectrophysiological examination (such as nerve conduction examination, concentric needle electromyography examination, magnetic stimulation motor evoked potential, etc.) and exclusion of other diseases. In addition, other examinations such as neuroimaging, biochemical tests (such as serum creatine kinase, cerebrospinal fluid protein, C-reactive protein, homocysteine, etc.), cerebrospinal fluid or serum neurofilament light chain detection, pulmonary function tests and blood gas analysis are helpful to differentiate ALS from other diseases, exclude structural damage of patients, and evaluate neuronal damage or respiratory function. The early diagnosis of ALS is difficult due to the hidden and diverse early clinical manifestations of ALS patients and the lack of specific biological diagnostic indicators. The average delay in diagnosis from onset to diagnosis is about 9-15 months. In addition, the early diagnosis of the disease requires effective differentiation from multiple system atrophy (MSA), cervical spondylosis, lumbar spondylosis, multifocal motor neuropathy, late-onset spinal muscular atrophy and other diseases, making the diagnosis of ALS relatively difficult.

[0004] There are not many studies on ALS-related biomarkers. The reported markers mainly include motor neuron-related markers (such as phosphorylated neurofilament heavy chain, neurofilament light chain, cystatin C, thyroxine transporter), inflammatory state-related markers (such as monocyte chemoattractant protein 1, miR-451), skeletal muscle-related markers (such as miR-338-3p, miR-206) and metabolism-related markers (such as homocysteine, glutamate, cholesterol, etc.). The related studies of these markers are mostly focused on the body state, nerve damage, inflammation or prognosis of ALS patients. So far, there is still a lack of effective biomarkers for the diagnosis of ALS.

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

[0006] Ficolin-1 (FCN1) is a subtype of ficolin, a glycoprotein with collagen-like domains and fibrinogen-like domains that exists in different human tissues. Ficolin is a soluble oligomeric defense protein with lectin-like activity and is therefore considered an important innate immune molecule. However, there are no reports on the use of FCN1 as a marker in the diagnosis of sALS. Summary of the invention

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

[0008] The present invention is achieved through the following technical scheme: application of biomarkers in the preparation of sALS-related diagnostic products, using FCN1 and / or PSAP as biomarkers, and 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 sequence of PSAP is shown in SEQ ID: 2 to SEQ ID: 5.

[0009] The biological sample is plasma.

[0010] The diagnostic product detects the protein level of FCN1 and / or PSAP in a biological sample by ELISA.

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

[0012] The diagnostic products include test 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 level of FCN1 and / or PSAP in a biological sample by ELISA as described above, or a diagnostic product determined by detecting the up-regulated protein level of FCN1 and / or PSAP 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: (1) The present invention is based on highly sensitive deep proteomics technology and enzyme-linked immunosorbent assay (ELISA), which can provide a convenient and reliable examination index in peripheral blood, and can achieve early diagnosis of sALS through the detection of plasma protein markers, providing important clinical value for the diagnosis and treatment of sALS.

[0016] (2) The novel diagnostic markers for sALS provided by the present invention: FCN1 and PSAP were significantly upregulated in sALS, with a consistent trend of change between the sALS disease group and different disease control groups. The diagnostic performance evaluation in different cohorts ranged from medium to high levels, demonstrating that these two protein markers are reliable and universal in the diagnosis of sALS.

[0017] (3) The present invention uses a commercial ELISA kit to quantitatively detect the content of FCN1 and PSAP in the plasma of subjects. The results show that the FCN1 concentration in the plasma of sALS patients is 0.583 ± 0.219 ng / mL, and the PSAP concentration is 0.613 ± 0.183 ng / mL. In the control group (including patients with various types of nervous system diseases such as cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, etc.), the FCN1 concentration in the plasma of patients is 0.379 ± 0.200 ng / mL, and the PSAP concentration is 0.444 ± 0.166 ng / mL. The expression levels of FCN1 and PSAP in the plasma of sALS patients are significantly upregulated compared with those in the control group, and the differences are statistically significant ( p < 0.05), indicating that these two plasma protein markers are stable and universal for the diagnosis of sALS.

[0018] (4) Based on the current status of clinical diagnosis of ALS, the disease needs to be differentiated from a variety of diseases with similar symptoms in the general clinical diagnosis process, including cervical spondylosis, lumbar spondylosis, multifocal motor neuropathy, late-onset spinal muscular atrophy, Hirayama disease, Kennedy disease, hereditary spastic paraplegia, ALS superimposed syndrome, etc. Therefore, the present invention uses different disease controls in the research process to identify molecular markers with reliable diagnostic performance and wide application for sALS to meet the clinical needs in this regard. Experiments have shown that the plasma protein markers FCN1 and PSAP provided by the present invention can still show good diagnostic ability in the differentiation of sALS from different types of disease control groups.

[0019] In summary, the present invention demonstrates that FCN1 and PSAP proteins in plasma can assist in the diagnosis of sALS under relatively low-invasive and convenient conditions. These two protein markers have stable and reliable performance for the diagnosis of sALS and its differential diagnosis from various types of nervous system diseases including MSA, cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, spinocerebellar ataxia, etc., and have a wide range of applications, which can provide important clinical value for the diagnosis process of sALS. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The differentially expressed proteins between the ALS group and the control group; Figure 2 It is the enrichment analysis diagram of biological process; Figure 3 The results of enrichment analysis of differentially expressed proteins; Figure 4 Comparison of FCN1 and PSAP plasma levels in the two cohorts and ROC curve characteristics. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0022] Example 1: Determination of biomarkers Pre-treatment 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 People's Liberation Army from November 2020 to January 2022, and the patient data were completed through hospitalization medical records.

[0023] Inclusion criteria for sALS patients: 1) first diagnosis, ALS diagnosis meets the revised El Escorial World Federation of Neurology criteria; 2) no family history of ALS.

[0024] Exclusion criteria: 1) combined with other neurodegenerative diseases; 2) combined with brain trauma, tumors, and paraneoplastic syndrome; 3) combined with serious medical diseases.

[0025] The included subjects were divided into a discovery cohort and a validation cohort. The discovery cohort consisted of 10 sALS patients and 23 non-ALS (MSA) patients, and the validation cohort consisted of 28 sALS patients and 22 non-ALS (cervical spondylotic myelopathy, peripheral neuropathy, Guillain-Barré syndrome, spinocerebellar ataxia and other neurological diseases) patients. In the discovery cohort and validation cohort, there were no statistical differences between the ALS group and the control group in terms of age, gender, diabetes, hypertension, BMI, and history of smoking and drinking.

[0026] Test samples: plasma samples from enrolled subjects.

[0027] The biomarkers were determined as follows: (1) Plasma sample collection: Use commercial EDTA-anticoagulated blood collection tubes to collect fasting venous whole blood from patients on the morning before hospitalization and on the day of outpatient consultation. Centrifuge at 4000 × g for 10 min at room temperature. Collect 500 μL of plasma, freeze it in 1.5 mL EP tubes, and store it in a -80°C refrigerator for later use.

[0028] (2) Pretreatment: A plasma low-abundance protein enrichment magnetic bead kit (DMB kit, Beijing Qinglian Biotech Co., Ltd.) was used to enrich the low-abundance proteins of the discovery cohort plasma samples and perform pretreatment for mass spectrometry analysis according to the DMB kit. The peptides after enzyme cleavage were frozen at -80°C for subsequent LC-MS / MS analysis.

[0029] (3) Sample data acquisition: The cleaved 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 reversed-phase capillary column (150 µm × 250 mm) (ReproSil-Pur C18-AQ 1.9 µm, Dr. Maisch, Germany). The eluted peptides were analyzed using a Q-Exactive HF-X (ThermoFisher Scientific, USA) and library data were acquired using data-dependent acquisition (DDA) mode. Sample data were then acquired using a full mass scan from 350 to 1500 m / z using data-independent acquisition (DIA) mode, and a total of 2164 proteins were identified.

[0030] (4) Sample result analysis: The original DIA files collected by Q-Exactive HF-X were processed using DIA-NN (version 1.81) according to the following settings: UniProt human database (2022.03.17), carbamidomethyl (C) as fixed modification, oxidation (M) and acetyl (protein N-terminus) 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 parameters were set to normalization dependent on retention time between samples. Clinical sample analysis was performed by DIA-NN (version 1.81) search against the self-built database to obtain the relative expression abundance data of sample proteins.

[0031] (5) Data processing: Data were processed using bioinformatics methods to compare and identify differentially expressed proteins between the ALS group and the control group, and to analyze the molecular functions and pathways associated with the differentially expressed proteins.

[0032] Specifically, the relative protein expression abundance was normalized, logarithmically transformed, and missing values ​​were interpolated using Perseus software. Proteins with missing values ​​> 80% were filtered out. All data were logarithmically transformed and Student's t The difference between the two groups was compared by the test. The difference was > 1.5 or < 0.67, and pThe screening rule of p < 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 draw volcano maps, heat maps, and principal component analysis (PCA). Bioinformatics analysis included gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis.

[0033] Among them, the differential protein results were visualized by volcano plots and heat maps. According to the screening criteria, a total of 320 DEPs were obtained between the ALS group and the control group. Compared with the control group, there were 188 up-regulated differential proteins in the ALS group and 132 down-regulated differential proteins. The results of principal component analysis showed that DEPs could significantly distinguish the ALS group from the control group. Figure 1 shown.

[0034] Depend on Figure 1 As can be seen, Figure A is a volcano plot, which shows the difference in -log10 ( p Figure 3 (value) and log2 (fold change) show the distribution of differentially expressed proteins identified by non-targeted proteomics in the discovery cohort, where blue represents proteins that are down-regulated in the ALS group compared with the control group, and red represents proteins that are up-regulated in the ALS group; Figure B is a PCA diagram of differentially expressed proteins, and the differentially expressed proteins displayed by PCA can clearly distinguish ALS and control groups; Figure C is a heat map display of differentially expressed proteins.

[0035] The results of bioinformatics analysis showed that the upregulated DEPs in the ALS group were mainly involved in biological processes such as complement activation and lipoprotein metabolism, and the related pathways included cholesterol metabolism and cGMP-PKG signaling pathway. The downregulated DEPs in the ALS group were mainly involved in biological processes such as negative regulation of the endogenous apoptosis signaling pathway induced by oxidative stress, positive regulation of dendritic spine morphogenesis, and axon extension. The related pathways involved the ErbB signaling pathway and the regulation pathway of the actin cytoskeleton, see Figure 2 and Figure 3 Therefore, based on the biological functions involved in DEPs and the changes in the levels of related signal pathways, the present invention can also provide a certain theoretical basis and potential direction for the research related to the pathogenesis of sALS.

[0036] like Figure 2As shown in the figure, A to C are the GSEA results based on the overall proteomics data. It can be seen from the figure that compared with the control group, the expression levels of phospholipid biosynthesis, lipid biosynthesis and negative regulation of cell projection tissue in the ALS group were significantly upregulated (Nominal p < 0.01). Figure 3 As shown in Figure 1, A and B are the GO and KEGG enrichment analysis results of differentially expressed proteins, respectively.

[0037] Through differential gene expression analysis, it was found that compared with the MSA patients in the control group, the protein expression levels of both in the plasma of sALS patients were significantly upregulated, with the difference multiples of 3.89 and 3.57, respectively, ranking in the top 15 in the differential protein list. Furthermore, through literature research and the applicability of commercial ELISA kits, it was determined that the protein markers for sALS diagnosis were FCN1 and PSAP.

[0038] The amino acid sequence of FCN1 is shown in SEQ ID: 1: MELSGATMARGLAVLLVLFLHIKNLPAQAADTCPEVKVVGLEGSDKLTILRGCPGLPGAPGPKGEAGVIGERGERGLPGAPGKAGPVGPKGDRGEKGMRGEKGDAGQSQSCATGPRNCKDLLDRGYFLSGWHTIYLPDCRPLTVLCDMDTDGGGWTVFQRRMD GSVDFYRDWAAYKQGFGSQLGEFWLGNDNIHALTAQGSSELRVDLVDFEGNHQFAKYKSFKVADEAEKYKLVLGAFVGGSAGNSLTGHNNNFFSTKDQDNDVSSSNCAEKFQGAWWYADCHASNLNGLYLMGPHESYANGINWSAAKGYKYSYKVSEMKVRPA.

[0039] The amino acid sequences of PSAP are shown in SEQ ID: 2 to SEQ ID: 5: Isoform 1 (SEQ ID: 2): MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0040] Isoform 2(SEQ ID:3): MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0041] Isoform 3(SEQ ID:4): MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN。

[0042] Variant 1(SEQ ID:5): .

[0043] Example 2: Detection of biomarkers The protein markers determined in Example 1 above: FCN1 and PSAP were verified by quantitative detection of enzyme-linked immunosorbent assay (ELISA) in the validation cohort, and the ELISA kit was a commercial kit. PSAP kit: Human Prosaposin (PSAP) ELISA Kit of Wuhan Huamei Bioengineering Co., Ltd., catalog number: CSB-E12837h, sensitivity of 0.041 ng / mL, linear range of 0.041-30 ng / mL; FCN1 kit: Human FCN1 (Ficolin-1) ELISA Kit of Wuhan Feien Biotechnology Co., Ltd., catalog number: EH0135, sensitivity of 0.094 ng / mL, linear range of 0.156-10 ng / mL.

[0044] The concentration of candidate markers in plasma was detected by ELISA according to the standard procedure provided in the kit instructions, and the standard curve was prepared using Curve Expert to calculate the concentration of the corresponding markers in the samples.

[0045] (I) The specific steps of ELISA detection of FCN1 are as follows: (a) Equilibrate the reagents and well plates at room temperature for 30 min and prepare the standards by serial dilution.

[0046] (b) After washing the plate twice, add 100 µL of the standard or sample to be tested to each well, cover with a plate sticker and incubate at 37°C for 90 min.

[0047] (c) Discard the liquid in the wells and wash the plate twice; add 100 μL of biotinylated antibody working solution, cover with a new plate sticker, and incubate in a 37°C incubator for 1 h. Discard the liquid and wash the plate three times.

[0048] (d) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well and incubate at 37°C for 30 min.

[0049] (e) Discard the liquid and wash the plate 5 times.

[0050] (f) Add 90 µL of substrate solution to each well and incubate at 37°C in the dark for 15 min. 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.

[0051] (g) The standard curve was prepared using Curve Expert 1.4 software and the FCN1 concentration in the samples was calculated.

[0052] (II) The specific steps of ELISA detection of PSAP are as follows: (a) Equilibrate the reagents and well plates at room temperature for 30 min and prepare the standards by serial dilution.

[0053] (b) Add 100 µL of standard or test sample to each well, cover with a plate sticker and incubate at 37°C for 2 h.

[0054] (c) Discard the liquid in the wells, add 100 µL of biotin-labeled antibody working solution, cover with a new plate sticker, and incubate at 37°C for 1 h. Discard the liquid and wash the plate 3 times.

[0055] (d) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well and incubate at 37°C for 1 h.

[0056] (e) Discard the liquid and wash the plate 5 times.

[0057] (f) Add 90 µL of substrate solution to each well and incubate at 37°C in the dark for 15 min. 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.

[0058] (g) The standard curve was prepared using Curve Expert 1.4 software, and the PSAP concentration in the samples was converted.

[0059] Statistical methods: Graphpad Prism v9.0, IBM SPSS Statistics v26.0, and Medcalc v20.010 were used for statistical analysis. Categorical variables were expressed as the number of cases (proportion %), and the chi-square or Fisher's exact test was used for comparison between the two groups. Continuous variables were judged to be normally distributed. Normally distributed data were described by mean ± standard deviation, and skewed distribution data were described by median and interquartile range. The variance between the two groups was determined to be equal, and Student's t Inspection, Welch's t Test or Mann-Whitney U The test was performed to test the difference between the two groups. The receiver operating characteristic (ROC) curve was 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 markers. AUC is an important indicator of test accuracy. The larger the AUC, the higher the diagnostic value of the test. The Youden Index is also called the correct diagnosis index. Its value range is between (0-1). The closer it is to 1, the better the accuracy. In this application, the diagnostic standard is set at the maximum correct diagnosis index. p < 0.05 was considered statistically significant.

[0060] Commercial ELISA kits were used to quantitatively detect the differences in the plasma levels of FCN1 and PSAP between the ALS group and the control group in the validation cohort. Figure 4 shown.

[0061] Figure 4 A and B are the comparison of FCN1 and PSAP levels between the two groups in the discovery cohort, Figure 4Comparison of FCN1 and PSAP levels between the two groups in the validation cohorts in C and D. The results showed that the FCN1 concentration in the plasma of 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 patients with various types of nervous system diseases such as cervical spondylotic myelopathy, peripheral neuropathy, and Guillain-Barré syndrome), the FCN1 concentration in the plasma was 0.379 ± 0.200 ng / mL, and the PSAP concentration was 0.444 ± 0.166 ng / mL. The expression of FCN1 and PSAP in the plasma of sALS patients was significantly upregulated compared with that in the control group, and the differences were statistically significant ( p < 0.05), and this trend was consistent with the results in the discovery cohort (e.g. Figure 4 The results show that these two plasma protein markers are stable and universal for the diagnosis of sALS.

[0062] In order to understand the performance evaluation of FCN1 and PSAP in sALS diagnosis, the present invention draws the ROC curves of two independent cohorts based on the relative expression and quantitative verification results of proteomics, see Figure 4 As shown in E to H, the ROC curve characteristics are shown in Table 1 ROC curve characteristics of FCN1 and PSAP.

[0063] Table 1 ROC curve characteristics of FCN1 and PSAP

[0064] The analysis results showed that FCN1 and PSAP can effectively distinguish sALS patients from MSA patients in the control group (FCN1: AUC = 0.870, sensitivity 0.700, specificity 0.957; PSAP: AUC = 0.939, sensitivity 0.900, specificity 0.826). FCN1 (cut-off value = 0.414 ng / mL) and PSAP (cut-off value = 0.473 ng / mL) also showed good diagnostic performance (AUC = 0.735, sensitivity 0.714, specificity 0.682) for the differentiation of sALS from various types of neurological diseases including cervical spondylotic myelopathy, peripheral neuropathy, and Guillain-Barré syndrome.

[0065] Example 3: ELISA kit The ELISA kit provided in this example can detect the protein levels of protein markers FCN1 and PSAP in the plasma samples of the subjects, and the detection method is as described in Example 2.

[0066] Specifically, the ELISA kit at least includes substances for detecting the level of FCN1 or PSAP protein, and other required buffer solutions or related standard reagents.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. The use of biomarkers in the preparation of sALS-related diagnostic products, characterized in that: FCN1 and / or PSAP are used as biomarkers 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 sequence of PSAP is shown in SEQ ID: 2 to SEQ ID:

5.

2. The use according to claim 1, characterized in that: The biological sample is plasma.

3. The use according to claim 2, characterized in that: The diagnostic product detects the protein level of FCN1 and / or PSAP in a biological sample by ELISA.

4. The use according to claim 2, characterized in that: The diagnostic product is determined by detecting upregulated protein levels of FCN1 and / or PSAP in a biological sample.

5. The use according to claim 2, characterized in that: The diagnostic products include test kits, test strips or other medically feasible products.

6. A product for diagnosing sALS, characterized in that: Comprising a diagnostic product as claimed in claim 3 or 4.

7. The product according to claim 6, characterized in that: Includes test kits, test strips or other medically feasible products.

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