Application of protein marker in preparation of product for early screening, identification and diagnosis of amyotrophic lateral sclerosis
By using the LanCL1 protein biomarker, the problem of insufficient specificity in the early diagnosis of ALS in existing technologies has been solved, achieving highly specific early screening and diagnosis, simplifying the sampling process, reducing the risk of misdiagnosis, shortening the diagnostic delay time, and assisting in disease assessment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-22
- Publication Date
- 2026-03-13
AI Technical Summary
The lack of highly specific biomarkers for the early diagnosis of amyotrophic lateral sclerosis (ALS) in existing technologies leads to diagnostic difficulties and delays. Existing biomarkers such as neurofilament protein and TDP-43 may also be elevated in other neurological diseases, resulting in insufficient specificity.
LanCL1, a protein biomarker of lanethionine synthase C-like protein, was used as a marker in the blood for early screening and diagnosis using a test kit. GSH agarose resin was used for binding and Western blot detection to differentiate ALS from other neurological diseases.
LanCL1 protein is significantly elevated in ALS patients, has high specificity, can accurately diagnose ALS at an early stage, reduces the risk of misdiagnosis, is easy to sample, reduces patient suffering, shortens the diagnostic delay time, and assists in disease assessment.
Smart Images
Figure CN121656569A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of early disease screening, specifically relating to the application of a protein biomarker in the preparation of products for early screening, identification and diagnosis of amyotrophic lateral sclerosis. Background Technology
[0002] Amyotrophic lateral sclerosis (ALS) is a severe, disabling, and fatal neurodegenerative disease that selectively affects the upper and lower motor neurons of the brain and spinal cord. The global incidence of ALS is approximately 1.75 per 100,000 people per year. However, predictive models based on current population size and the number of existing ALS cases in various regions suggest that, due to population aging, the number of global ALS cases will increase by 69% by 2040 compared to 2015, with the largest increase in Africa, followed by Asia. At that time, ALS will place a heavier burden on the social and healthcare systems of all countries, especially developing countries. Because ALS has an insidious onset, lacks specific early clinical manifestations, and currently lacks specific diagnostic biomarkers, early diagnosis is difficult. International reports indicate that the average diagnostic delay for ALS patients is 8-14 months. Therefore, scholars worldwide have been dedicated to finding valuable diagnostic biomarkers to aid in the early diagnosis of ALS patients and to assess disease progression and prognosis.
[0003] Currently, biomarkers for amyotrophic lateral sclerosis (ALS) are mainly classified into the following categories:
[0004] 1. Cerebrospinal fluid biomarkers
[0005] (1-1) Neurofilament protein. Neurofilament protein is one of the most studied cerebrospinal fluid biomarkers. Its concentration in cerebrospinal fluid is correlated with the rate of ALS progression and can be used as an indicator of disease progression. However, the elevation of neurofilament protein is not specific to ALS and can also occur in other neurological diseases, thus its specificity is limited.
[0006] (1-2) TDP-43 protein. The level of TDP-43 in the cerebrospinal fluid of ALS patients is related to the severity and rate of disease progression, but its specificity as a biomarker still needs further verification, and its concentration in cerebrospinal fluid is affected by a variety of factors, such as the method of cerebrospinal fluid collection and processing.
[0007] (1-3) Other proteins. The expression levels of proteins such as CHMP2B and FUS in the cerebrospinal fluid of ALS patients are also altered, but research on these proteins as biomarkers is not yet mature and is still in the exploratory stage.
[0008] 2. Blood biomarkers
[0009] (2-1) Neurofilament protein. Similar to neurofilament protein in cerebrospinal fluid, the level of neurofilament protein light chain (NfL) in blood is also elevated in ALS patients and is associated with disease progression. However, it also suffers from a lack of specificity, as it may also be elevated in other neurodegenerative diseases, which limits its application as a specific diagnostic marker for ALS.
[0010] (2-2) MicroRNAs. Some studies have found that specific microRNAs are abnormally expressed in the blood of ALS patients. They have good stability and are easy to detect, and are expected to become biomarkers for the diagnosis and prognosis of ALS. However, there are still few related studies, and further verification and exploration are needed.
[0011] 3. Genes and epigenetic markers
[0012] (3-1) C9orf72 gene. The hexanucleotide repeat amplification of the C9orf72 gene is one of the important pathogenic genes of ALS. Its related dipeptide repeat proteins, such as polyGR, are being studied as biomarkers of C9-ALS. However, the detection of polyGR is currently mainly focused on the unmethylated form. The detection of its methylated form in CSF and its potential as a biomarker need further research.
[0013] (3-2) DNA methylation. A study published in BMC Med Genomics on January 20, 2025, assessed the potential of biomarkers by analyzing the characteristics of hypomethylated DNA derived from motor neurons. Although motor neuron-derived DNA could not be detected in plasma samples from ALS patients, neuron-derived DNA could be detected in cerebrospinal fluid, providing a new approach for finding motor neuron-specific biomarkers in cerebrospinal fluid.
[0014] 4. Extracellular vesicle biomarkers
[0015] Extracellular vesicles are important mediators of intercellular communication, and the proteins, nucleic acids, and other components they carry may reflect the pathological processes of diseases. Studies have found changes in the number and composition of extracellular vesicles in the cerebrospinal fluid and blood of ALS patients. For example, extracellular vesicles containing specific proteins or miRNAs may serve as biomarkers for ALS. However, research on extracellular vesicle biomarkers is still in its early stages, and their sources, content, and functions need further clarification.
[0016] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention
[0017] This invention provides the application of a protein biomarker in the preparation of products for early screening, identification and diagnosis of amyotrophic lateral sclerosis (ALS), wherein the protein biomarker includes at least LanCL1.
[0018] Preferably, the protein marker is LanCL1.
[0019] To facilitate understanding of this invention, the protein biomarker is described below:
[0020] Lanthionine synthetase C-like protein 1 (LanCL1), also known as P40 or GRP69A, was first isolated from erythrocyte shadows by Mayer Herbert of the University of Vienna in 1998 using affinity chromatography. Due to its molecular weight of 40 kDa, it was initially named P40. Sequence alignment revealed high homology with the prokaryotic LanC (lanthionine synthase C) protein family. These LanC-like (LanCL) proteins share conserved hydrophobic structures, zinc-binding residues, and predicted active residue sites with bacterial LanC. Prokaryotic LanC is a zinc-finger enzyme whose main function is to catalyze the reaction of cysteine residue side chains with dehydrated threonine or serine residues to form thioether crosslinks, ultimately producing macrocyclic thioether (lanthionine) products with strong antibacterial activity.
[0021] Preferably, the expression level of LanCL1 in patients with amyotrophic lateral sclerosis (ALS) is higher than that in healthy individuals.
[0022] Preferably, the expression level of LanCL1 is negatively correlated with the severity of amyotrophic lateral sclerosis (ALS).
[0023] Preferably, the product is a test kit for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS).
[0024] Preferably, the test kit is used to test humans.
[0025] Preferably, the test sample for the test kit is blood.
[0026] The beneficial effects of this invention are as follows:
[0027] (1) High specificity and accurate identification: The serum LanCL1 protein level is significantly elevated only in ALS patients, while there is no significant change in patients with other similar neurological diseases such as Parkinson's disease. It can effectively distinguish ALS from similar diseases, greatly reduce the risk of misdiagnosis and improve diagnostic specificity.
[0028] (2) Sampling is convenient and safe, and the compliance rate is high: The test sample is blood, which can be obtained through routine venous blood collection or finger prick blood collection. The operation is simple and minimally invasive. Compared with cerebrospinal fluid sampling, patients experience less pain and have a higher acceptance rate, which is more conducive to large-scale clinical application.
[0029] (3) Significant value in early diagnosis: In the early stage of ALS, serum LanCL1 levels are significantly higher than those in healthy individuals. This can indicate disease risk when clinical symptoms are atypical and traditional diagnostic methods are difficult to use to make a diagnosis, thus shortening the average diagnostic delay time by 8-14 months and providing a window of opportunity for early intervention.
[0030] (4) It can help assess the condition: The expression level of LanCL1 is negatively correlated with the severity of ALS. By detecting its serum level, it can help determine the stage of the disease (early / mid-late stage) of the patient, and provide an objective reference for treatment plan formulation, efficacy monitoring and prognosis assessment. Attached Figure Description
[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0032] Figure 1 This is a graph showing the results of protein electrophoresis.
[0033] Figure 2 It is a flowchart of the sample collection, processing, and testing process.
[0034] Figure 3 This is a graph showing the protein level expression results of LanCL1 in ALS patients.
[0035] Figure 4 This is a graph showing the protein level expression of LanCL1 in PD patients. Detailed Implementation
[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0037] Example
[0038] This embodiment will elaborate on the discovery process and final results of LanCL1 as a diagnostic marker for ALS:
[0039] (a) LanCL1 protein can be secreted extracellularly.
[0040] This invention has found that overexpression of the myc-LanCL1 plasmid in HeLa cells (commonly used laboratory cells) allows the myc-LanCL1 protein to be detected in the cell culture medium, suggesting that the LanCL1 protein may have the ability to be secreted extracellularly (e.g., Figure 1 (As shown). These findings provide a theoretical basis for detecting LanCL1 in serum.
[0041] (II) Detection protocol for LanCL1 in serum
[0042] In this embodiment, it includes three aspects: sample processing, binding, and detection (e.g. Figure 2 As shown), the details are as follows:
[0043] 1. Serum pretreatment
[0044] After clinical blood collection, serum was separated according to standard operating procedures. After serum separation, 100 μL was transferred to a 1.5 mL centrifuge tube and placed on ice. The serum was then sonicated at 25 kHz and 30% power for 2 seconds. After sonication, the sample was placed on ice to cool. This process was repeated three times.
[0045] After sonication, 900 μL of pre-cooled PBS (phosphate buffered saline) was added to the 100 μL of serum, for a total volume of 1000 μL. Then, an appropriate amount of protease inhibitor was added.
[0046] 2. LanCL1 protein binds to GSH agarose resin
[0047] Glutathione agarose resin (catalog number 16100) from Thermo Scientific was used. 100 μL of glutathione agarose resin was pipetted into a 1.5 mL centrifuge tube, placed on ice, and 900 μL of PBS was added. The centrifuge tube was repeatedly inverted to thoroughly wash the agarose resin. Then, the tube was centrifuged at 3000 rpm for 3 min in a low-temperature centrifuge. The supernatant PBS was carefully discarded, and this process was repeated twice. The pretreated agarose resin was then placed on ice for later use.
[0048] Pretreated serum was added to agarose resin. The centrifuge tubes were then placed on a rotary mixer and incubated at 4°C for 6 hours. The mixture was then centrifuged at 3000 rpm for 3 minutes in a low-temperature centrifuge, and the supernatant was carefully discarded. 900 μL of pre-chilled PBS was added, and the centrifuge tubes were placed on a rotary mixer and incubated at 4°C for 5 minutes. The mixture was then centrifuged at 3000 rpm for 3 minutes in a low-temperature centrifuge, and the supernatant was carefully discarded. The tubes were washed three times with PBS.
[0049] 3. Detection of LanCL1 protein
[0050] This embodiment uses Western blotting (WB) to detect the level of LanCL1 in serum. After the final wash, the PBS was carefully discarded, and 50 μL of protein loading buffer was added. The mixture was vortexed to mix, boiled at 100°C for 3 min, and then centrifuged at 12000 rpm for 2 min. The supernatant was transferred to a new centrifuge tube. LanCL1 protein was detected according to standard Western blotting procedures. The primary antibody used for LanCL1 protein was ABMART product, catalog number PH2742.
[0051] (iii) Serum LanCL1 protein can serve as a marker for ALS.
[0052] This research has been approved by the Biomedical Ethics Review Committee of West China Hospital, Sichuan University (No. 2016-097). All research subjects agreed to participate in this research project and signed informed consent forms. Forty ALS patients who were diagnosed at the Department of Neurology, West China Hospital, Sichuan University between September 2023 and September 2025 were included. Follow-up assessments were conducted at the time of admission and every 6 months thereafter, including basic patient information, clinical symptoms, signs, electromyography, ALS Functional Rating Scale-Revised (ALSFRS-R) score, and survival status. Simultaneously, age- and sex-matched healthy controls (Health Control, HC) and Parkinson's disease (PD) patients were included as controls. HC participants primarily consisted of the patients' spouses and healthy volunteers who underwent physical examinations at West China Hospital of Sichuan University. These participants had no neurological diseases, no positive neurological symptoms or signs, and no family history of neurological diseases. The diagnosis of PD patients was based on the clinical diagnostic criteria established in 2015 by the International Movement Disorders Association and the British Brain Bank. All participating patients met the diagnostic criteria for clinically confirmed PD or clinically probable PD. Whole-exome sequencing was performed on ALS, HC, and PD patients to rule out the possibility of carrying ALS-causing gene mutations. HC and PD patients denied a family history of ALS. Detailed information for each group is as follows:
[0053] (1) Normal healthy controls (Health Control, HC): 30 cases, aged 40-60 years, male-female ratio 1.5:1, no neurological diseases, major organ diseases and autoimmune diseases, physical examination, blood routine and liver and kidney function were normal;
[0054] (2) Early ALS group: 20 cases, aged 40-60, male-female ratio 1.5:1, meeting the revised EI Escorialrevised ALS diagnostic criteria, disease duration ≤1 year, no targeted treatment, and excluded from other neurodegenerative diseases;
[0055] (3) Mid-to-late stage ALS group: 20 cases, aged 40-60 years, male-to-female ratio 15:1, meeting the revised EI Escorialrevised ALS diagnostic criteria, disease course > 1 year, accompanied by limb weakness, muscle atrophy and other obvious motor dysfunction;
[0056] (4) Early PD group: 20 cases, aged 40-80, male-female ratio 1.5:1, meeting the clinical diagnostic criteria set by the International Movement Disorders Association and the British Brain Bank in 2015. Early PD patients must have a disease course of less than 3 years and HY stage ≤2;
[0057] (5) Mid-to-late stage PD group: 20 cases, aged 40-80, male-to-female ratio 1.5:1, meeting the clinical diagnostic criteria set by the International Movement Disorders Association and the British Brain Bank in 2015. Early PD patients need to have a disease course of less than 3 years and HY stage ≥3.
[0058] In this embodiment, LanCL1 was detected in the serum of healthy controls, early-stage ALS patients, and patients with intermediate-to-late-stage ALS. LanCL1 is a GSH-binding protein. We used GSH agarose resin to precipitate LanCL1 from 100 μL of serum, and then performed protein electrophoresis to detect the protein expression of LanCL1. The results showed that compared with healthy controls, the protein level of LanCL1 in the serum of early-stage and intermediate-to-late-stage ALS patients was increased, but compared with early-stage ALS patients, the protein level of LanCL1 in the serum of intermediate-to-late-stage ALS patients was decreased (e.g., ...). Figure 3 (As shown).
[0059] Similarly, in this embodiment, LanCL1 was detected in the serum of healthy controls, early-stage Parkinson's disease (PD) patients, and patients with intermediate-to-late-stage PD. The results showed that compared with healthy controls, the protein levels of LanCL1 in the serum of early-stage and intermediate-to-late-stage PD patients were not significantly different (e.g., ...). Figure 4 (As shown).
[0060] In summary, serum LanCL1 protein levels are closely related to the progression of ALS, while they do not show significant changes in the progression of PD. Therefore, serum LanCL1 protein levels may serve as a specific marker for the early diagnosis and differentiation of ALS, and are not easily affected by other similar diseases.
[0061] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. The application of a protein biomarker in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The protein biomarkers include at least LanCL1.
2. The application of the protein biomarker according to claim 1 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The protein biomarker is LanCL1.
3. The application of the protein biomarker according to claim 1 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The expression level of LanCL1 in patients with amyotrophic lateral sclerosis (ALS) is higher than that in healthy individuals.
4. The application of the protein biomarker according to claim 1 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, LanCL1 expression levels are negatively correlated with the severity of amyotrophic lateral sclerosis (ALS).
5. The application of the protein biomarker according to claim 1 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The product is a test kit for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS).
6. The application of the protein biomarker according to claim 5 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The test kit is used to test humans.
7. The application of the protein biomarker according to claim 6 in the preparation of products for early screening, identification, and diagnosis of amyotrophic lateral sclerosis (ALS), characterized in that, The test sample for the test kit is blood.