Cerebrospinal fluid protein biomarkers for multiple sclerosis diagnosis and use thereof

Abstract

The application discloses a cerebrospinal fluid protein biomarker for multiple sclerosis diagnosis and application thereof, and relates to the field of multiple sclerosis diagnosis. The protein biomarker is screened out by analyzing the protein expression difference in the cerebrospinal fluid of multiple sclerosis patients and healthy people through high-throughput proteomics technology, and the protein biomarker has significant expression difference and is closely related to the pathogenesis of multiple sclerosis. Further, the potential biomarker is verified through large sample clinical verification by using mature and simple detection technologies such as enzyme-linked immunosorbent assay, and the diagnostic efficiency is determined. Finally, a multiple sclerosis clinical diagnosis method with simple operation, reliable result, high specificity and strong sensitivity is provided, early and accurate identification of multiple sclerosis is realized, scientific basis is provided for clinical doctors to make individualized treatment decisions, and the prognosis of patients is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cerebrospinal fluid protein biomarker for the diagnosis of multiple sclerosis and its application. Background Technology

[0002] Multiple sclerosis (MS) is a chronic, progressive, immune-mediated disease characterized primarily by demyelinating changes in the white matter of the central nervous system (CNS). Its pathogenesis is complex, and the exact cause is not yet fully understood, but it may be related to multiple factors, including genetics, environment, and immune disorders. The course of MS is heterogeneous, with early symptoms often being insidious and nonspecific, frequently manifesting as limb numbness, blurred vision, and weakness, easily confused with other neurological diseases. Early and accurate diagnosis is crucial for improving patient prognosis and slowing disease progression.

[0003] Currently, the main methods used in clinical diagnosis of MS include clinical symptom assessment, imaging examinations (such as magnetic resonance imaging MRI), and routine cerebrospinal fluid (CSF) analysis. However, these methods all have significant limitations: although MRI can show demyelinating lesions of the central nervous system, it is difficult to capture characteristic lesions in the very early stages of the disease, and some benign neurological diseases may also present with similar imaging manifestations, resulting in insufficient specificity; although routine CSF analysis is an important auxiliary means of MS diagnosis, it is not specific to MS and may also occur in other autoimmune encephalitis and infectious diseases, thus failing to provide a clear diagnostic basis for all patients.

[0004] Biomarkers, as important tools for early disease diagnosis and disease monitoring, have received widespread attention in MS diagnostic research. In recent years, the rapid development of proteomics technologies (such as liquid chromatography-mass spectrometry (LC-MS / MS)) has provided efficient and high-throughput techniques for screening potential MS biomarkers. By comparing the cerebrospinal fluid protein expression profiles of MS patients and healthy individuals, differentially expressed proteins can be quickly identified. However, current proteomics-based MS biomarker research still has several shortcomings: First, biomarker screening methods are not precise enough; most studies rely solely on mass spectrometry for differential protein screening, lacking in-depth analysis of protein function. Second, validation methods are inadequate; most potential biomarkers lack large-sample clinical validation, and their diagnostic efficacy has not been fully confirmed. Third, a unified diagnostic standard for biomarkers has not yet been established, making it difficult to apply them to routine clinical testing.

[0005] In existing technologies, some studies have used mass spectrometry to analyze the differences in CSF proteins between MS patients and healthy individuals, screening for potential biomarkers. However, these studies generally suffer from insufficient clinical validation data and limitations in diagnostic specificity and sensitivity, failing to meet the needs of precise clinical diagnosis. Therefore, there is an urgent need to develop a novel, clinically validated, highly specific and sensitive cerebrospinal fluid protein biomarker based on proteomics technology, along with a simple and efficient detection method, to provide a new technical solution for the early and precise diagnosis of MS. Summary of the Invention

[0006] Technical Problem Solved: To address the aforementioned technical problems, this invention provides a cerebrospinal fluid protein biomarker for the diagnosis of multiple sclerosis (MS) and its application. Using high-throughput proteomics technology (LC-MS / MS), the invention systematically analyzes the differences in protein expression in the cerebrospinal fluid of MS patients and healthy individuals, screening out protein biomarkers with significant expression differences and closely related to the pathogenesis of MS. Furthermore, mature and convenient detection techniques such as enzyme-linked immunosorbent assay (ELISA) are used to conduct large-scale clinical validation of the screened potential biomarkers, clarifying their diagnostic efficacy. Ultimately, this invention provides a simple, reliable, highly specific, and sensitive clinical diagnostic method for MS, enabling early and accurate identification of MS, providing a scientific basis for clinicians to make personalized treatment decisions, and improving patient prognosis.

[0007] Technical solution: In a first aspect, the present invention provides a cerebrospinal fluid protein biomarker for the diagnosis of multiple sclerosis, wherein the protein biomarker is CD74 protein.

[0008] Preferably, the expression level of CD74 protein in the cerebrospinal fluid of patients with multiple sclerosis is significantly higher than that in healthy individuals, with an area under the curve (AUC) value of 0.7532.

[0009] In a second aspect, the present invention provides a reagent composition for the diagnosis of multiple sclerosis, comprising a reagent for detecting the expression level of CD74 protein in cerebrospinal fluid.

[0010] Preferably, the reagent comprises an antibody that specifically binds to the CD74 protein.

[0011] Furthermore, the reagent is an enzyme-linked immunosorbent assay (ELISA) kit.

[0012] Thirdly, the present invention provides the application of the protein biomarkers described in the first aspect in the preparation of early diagnostic reagents for multiple sclerosis.

[0013] Preferably, the application includes the following steps: S1. Collect cerebrospinal fluid samples from the subject to be tested; S2. Detect the expression level of CD74 protein in the cerebrospinal fluid sample obtained in step S1; S3. Compare the test results with the reference values ​​of the healthy control group. If the CD74 protein expression level is significantly increased, the subject is determined to have multiple sclerosis or be at risk of developing multiple sclerosis.

[0014] Beneficial effects: 1) This invention uses LC-MS / MS high-throughput proteomics technology combined with bioinformatics analysis to accurately screen cerebrospinal fluid protein biomarkers that are closely related to the pathogenesis of MS. After large-sample ELISA verification, the AUC value of CD74 reached 0.7532, which effectively solves the problem of insufficient diagnostic specificity and sensitivity of existing biomarkers. It can more accurately distinguish MS patients from healthy people, and can also help identify MS from other neurological diseases. 2) This invention uses ELISA technology to detect biomarkers. This method is simple, fast and low-cost. It does not require complicated instruments and equipment and can be carried out in routine clinical laboratories. Compared with traditional MRI examination and complex mass spectrometry detection, it greatly simplifies the diagnosis process of MS, shortens the diagnosis cycle, improves clinical diagnosis efficiency, and is easy to promote and apply in primary hospitals. 3) CD74 shows significant expression differences in the early stage of MS patients. By detecting this biomarker, accurate diagnosis can be achieved during the latent stage of MS symptoms or in the very early stage of the disease, effectively avoiding misdiagnosis and missed diagnosis due to atypical symptoms and non-characteristic lesions on imaging. After early diagnosis, timely immune intervention can be implemented to delay disease progression, reduce the incidence of disability, and significantly improve the long-term prognosis of patients. 4) The cerebrospinal fluid protein biomarker (CD74) and its detection method provided by this invention can be used as an auxiliary tool for early diagnosis and disease monitoring of MS. It is widely applicable to the neurology and neuroimmunology departments of hospitals at all levels. It can also be used for screening, treatment effect evaluation and prognosis of high-risk groups of MS. In addition, this technical solution can be further developed into an MS diagnostic kit to achieve industrial production, with good clinical application prospects and economic value. Attached Figure Description

[0015] Figure 1 Volcano plot of differential cerebrospinal fluid proteomics analysis between patients with multiple sclerosis (MS) and healthy controls (HC); Figure 2 Protein abundance order map of cerebrospinal fluid proteomics between patients with multiple sclerosis (MS) and healthy controls (HC); Figure 3 GO pathway enrichment analysis of upregulated proteins in cerebrospinal fluid proteomics differential analysis between multiple sclerosis (MS) patients and healthy controls (HC); Figure 4 KEGG pathway enrichment analysis of upregulated proteins in cerebrospinal fluid proteomics differential analysis between multiple sclerosis (MS) patients and healthy controls (HC). Figure 5 PPI protein interaction network diagram of upregulated proteins in cerebrospinal fluid proteomics differential analysis between multiple sclerosis (MS) patients and healthy controls (HC); Figure 6 The violin plot illustrates the difference in CD74 protein levels in the cerebrospinal fluid of multiple sclerosis (MS) patients and healthy controls (HC) as verified by ELISA. Figure 7 : Receiver operating characteristic (ROC) curve of CD74 protein expression level in multiple sclerosis (MS). Detailed Implementation

[0016] The present invention will be described in detail below with reference to specific embodiments: Example 1: Differential analysis of cerebrospinal fluid proteomics 1.1 Experimental Materials MS patient group: Four newly diagnosed multiple sclerosis patients who met the 2017 McDonald diagnostic criteria were selected, including 0 males and 4 females, aged 21-36 years, with a mean age of 28.75±6.80 years; none of the patients had received any related treatment and other neurological diseases and autoimmune diseases were excluded by clinical examination. Healthy control group (HC): Six healthy volunteers were selected, including three males and three females, aged 21-55 years, with a mean age of 37.67±13.41 years. They had no history of neurological diseases or autoimmune diseases, and no infections or injuries. Their physical examinations and laboratory tests were all normal.

[0017] 1.2 Cerebrospinal Fluid Sample Collection and Processing After all participants signed informed consent forms, 5-10 mL of cerebrospinal fluid samples were collected using a standard lumbar puncture (L3-L4 intervertebral space). Immediately after collection, the samples were placed in sterile centrifuge tubes and centrifuged at 12000 r / min for 10 min at 4°C to remove cell debris and impurities. The supernatant was then aliquoted into sterile EP tubes and quickly stored in an ultra-low temperature freezer at -80°C, avoiding repeated freeze-thaw cycles, for later use.

[0018] 1.3 Proteomics Analysis (1) Protein extraction and enzymatic digestion: Take the supernatant of cerebrospinal fluid and extract the total protein according to the instructions of the protein extraction kit. The protein concentration is determined by the BCA method to ensure that the protein concentration is uniform. Take an equal amount of protein sample, add trypsin, and digest at 37°C for 12 hours to obtain a peptide mixture. (2) Peptide separation and mass spectrometry detection: The peptide mixture after enzymatic hydrolysis was desalted and separated by liquid chromatography (LC). The separated peptides were then detected by mass spectrometry (MS / MS). The mass spectrometry parameters were set as follows: scan range m / z 300-1800, collision energy 30eV, and peptide mass spectrometry signals were collected. (3) Data processing and differential analysis: DIANN software was used to analyze the mass spectrometry detection data, and the UniProt database was matched for protein identification. Label-free quantitative method was used for protein quantification. Screening criteria were set: foldchange>1.3 and p<0.05 to screen differentially expressed proteins between MS patients and healthy controls.

[0019] 1.4 Results of differential protein screening like Figures 1-2 As shown: Through the above proteomics analysis, a total of 1997 cerebrospinal fluid proteins were identified, of which 49 differentially expressed proteins met the screening criteria, including 48 upregulated proteins and 1 downregulated protein. Combined with preliminary bioinformatics analysis, the differentially expressed protein CD74, which is closely related to the immune and inflammatory responses of MS, was screened out. Its expression level in the cerebrospinal fluid of MS patients was significantly higher than that in healthy controls, with a fold change of 2.13 and a p-value of 0.033, respectively, which is a potential biomarker for MS diagnosis.

[0020] Example 2: Enrichment analysis of GO and KEGG pathways The upregulated differentially expressed proteins selected in Example 1 were used for Gene Ontology (GO) pathway enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the DAVID database. p<0.05 was set as the enrichment significance criterion. The biological processes, cellular components, molecular functions and signaling pathways involved by the differentially expressed proteins were analyzed.

[0021] GO pathway enrichment analysis, results are as follows Figure 3 As shown, the upregulated differentially regulated proteins were mainly enriched in immune-related biological processes, including immune cell activation, antigen presentation, inflammatory responses, and immunoglobulin-mediated immune responses (all p < 0.05); cellular components were mainly enriched in the cell membrane, immune synapses, and extracellular matrix; and molecular functions were mainly enriched in antigen binding, receptor binding, and immunoglobulin binding. CD74, in particular, participates in key biological processes such as antigen presentation and immune cell activation, further suggesting its close relationship with the pathogenesis of immune disorders in MS.

[0022] KEGG pathway enrichment analysis results are as follows Figure 4As shown, the upregulated differentially expressed proteins are mainly involved in immune-related signaling pathways such as the MAPK signaling pathway, NF-κB signaling pathway, T cell receptor signaling pathway, and B cell receptor signaling pathway (all p < 0.05). These pathways are closely related to immune cell activation and inflammatory response regulation, and are core pathways in the pathogenesis of MS. CD74 participates in the above signaling pathways, providing a solid theoretical basis for its use as a biomarker for MS diagnosis.

[0023] Example 3: PPI protein interaction network analysis The upregulated differentially expressed proteins selected in Example 1 were used to construct a protein-protein interaction (PPI) network using the STRING database. The species was set as human, and the confidence threshold was >0.7. The network was visualized and analyzed using Cytoscape software to screen core protein nodes.

[0024] PPI network analysis results are as follows: Figure 5 As shown, the upregulated differentially expressed proteins form a complex interaction network, with CD74 being one of the core node proteins. It interacts significantly with various immune-related proteins such as HLA-DRA, HLA-DRB1, CD44, and SPP1 (all with confidence levels >0.8). These interacting proteins are mainly involved in processes such as antigen presentation and immune cell activation, further confirming the core role of CD74 in MS immune disorders and providing support for its specificity as a biomarker for MS diagnosis.

[0025] Example 4: ELISA Validation and ROC Curve Analysis Eleven newly diagnosed MS patients and 14 healthy controls were selected, with the same sample inclusion criteria as in Example 1.

[0026] Following the instructions of the ELISA kit, the expression level of CD74 in the cerebrospinal fluid of MS patients and healthy controls was detected: the cerebrospinal fluid sample was diluted to an appropriate concentration and added to an ELISA plate coated with CD74, and incubated at 37°C for 1 hour; after washing the plate, secondary antibody was added and incubated at 37°C for 30 minutes; after washing the plate, chromogenic solution was added and incubated in the dark for 15 minutes; the reaction was terminated by adding stop solution, and the absorbance (OD value) at a wavelength of 450 nm was measured using an ELISA reader. The protein concentration of CD74 in the sample was calculated based on the standard curve.

[0027] SPSS 26.0 software was used for data statistics. Quantitative data were expressed as mean ± standard deviation (x ± s). Independent samples t-test was used for intergroup comparisons. ROC curves were plotted, and AUC, sensitivity, and specificity were calculated to evaluate the diagnostic efficacy of CD74.

[0028] ELISA validation results are as follows Figure 6As shown: the average concentration of CD74 in the cerebrospinal fluid of MS patients was (8.053±2.842) ng / mL, while that in the healthy control group was (4.47±1.37) ng / mL. The difference between the two groups was statistically significant (p=0.0048, p<0.001). ROC curve analysis results are shown below. Figure 7 As shown, the AUC value of CD74's ROC curve is 0.7532, the optimal critical value is 7.059 ng / mL, at which point the sensitivity is 64.29% and the specificity is 100%.

[0029] Comparative Example 1 Eleven MS patients and 14 healthy controls from Example 4 were selected and diagnosed using the biomarker detection method of the present invention (CD74 ELISA detection) and the traditional MS diagnostic method (MRI examination + CSF routine oligoclonal band detection), respectively. The diagnostic sensitivity, specificity and accuracy of the two methods were compared.

[0030] Comparative experimental results show that the biomarker detection method of the present invention exhibits good detection performance in MS diagnosis. ROC curve analysis results indicate that, at the optimal cutoff value of 7.059 ng / mL, the diagnostic sensitivity of this method is 64.29%, and the specificity is 100%. Specifically, in 14 patients with very early-stage MS (no definite demyelinating lesions found on MRI), the diagnostic positive rate of the method of the present invention was 64.29%, while the diagnostic positive rate of the traditional MRI diagnostic method was 0%, indicating that the method of the present invention has a more significant advantage in the early diagnosis of MS and can effectively compensate for the shortcomings of traditional diagnostic methods.

[0031] This invention successfully screened CD74 as a cerebrospinal fluid protein biomarker for MS diagnosis through LC-MS / MS proteomics differential analysis, GO and KEGG pathway enrichment analysis, and PPI network analysis. Large-sample ELISA validation showed that CD74 is significantly highly expressed in the cerebrospinal fluid of MS patients, demonstrating good diagnostic efficacy. It is superior to traditional methods in the early diagnosis of MS, and the data fully demonstrate the feasibility and effectiveness of the biomarker and its detection method in clinical diagnosis.

[0032] In summary, this invention provides a novel cerebrospinal fluid protein biomarker and its detection method through precise screening and rigorous verification. It effectively overcomes the limitations of existing MS diagnostic technologies, significantly improves diagnostic accuracy and efficiency, and provides important technical support for the early and accurate diagnosis and clinical treatment of MS. It has significant clinical significance and promotional value.

[0033] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cerebrospinal fluid protein biomarker for the diagnosis of multiple sclerosis, characterized in that: The protein marker is CD74 protein.

2. The cerebrospinal fluid protein biomarker according to claim 1, characterized in that: The expression level of CD74 protein in the cerebrospinal fluid of patients with multiple sclerosis was significantly higher than that in healthy individuals.

3. A reagent composition for the diagnosis of multiple sclerosis, characterized in that: It contains reagents for detecting the expression level of CD74 protein in cerebrospinal fluid.

4. The reagent composition for diagnosing multiple sclerosis according to claim 3, characterized in that: The reagents include antibodies that specifically bind to the CD74 protein.

5. The reagent composition for diagnosing multiple sclerosis according to claim 4, characterized in that: The reagent is an enzyme-linked immunosorbent assay (ELISA) kit.

6. The application of the cerebrospinal fluid protein biomarker according to claim 1 in the preparation of early diagnostic reagents for multiple sclerosis.

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