Application of FSTL1 protein as marker for idiopathic normal pressure hydrocephalus and detection method thereof

Abstract

This invention provides the application, detection method, and kit of human follicle-staphylin-like protein (FSTL1) as a marker for idiopathic normal pressure hydrocephalus. The NCBI Gene ID of FSTL1 is 11167, the RefSeq mRNA number is NM_007085.5, the RefSeq protein code is NP_009016.1, and the UniProt number is Q12841. This invention solves the technical problems of existing detection technologies, such as the inability to recognize molecules early, insufficient marker specificity, and insufficient sensitivity for detecting low-abundance proteins.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection, specifically to the application and detection method of FSTL1 protein as a marker for idiopathic normal pressure hydrocephalus. Background Technology

[0002] Idiopathic normal pressure hydrocephalus is a chronic neurological disorder that commonly affects the elderly. Clinically, it presents as a triad of gait disturbance, cognitive decline, and urinary incontinence, with incidence increasing with age. Its core pathological mechanism involves abnormal cerebrospinal fluid circulation dynamics, leading to ventricular enlargement while maintaining normal intracranial pressure. Without timely intervention, it can lead to progressive deterioration of neurological function, severely impacting patients' quality of life and increasing the burden of care on families. Current clinical diagnosis and treatment primarily rely on "imaging diagnosis + shunt surgery." Diagnosis depends on cranial CT / MRI to observe ventricular enlargement, and treatment mainly involves ventriculoperitoneal shunt surgery. However, postoperative outcomes vary greatly among individuals, and some patients miss the optimal intervention window due to delayed diagnosis or misdiagnosis.

[0003] Current iNPH detection technologies have the following limitations: A core deficiency in imaging diagnostic techniques: While head CT / MRI is a commonly used diagnostic tool, it can only identify morphological changes in mid-to-late-stage ventricular enlargement, failing to capture early molecular pathological changes, leading to difficulties in early diagnosis; furthermore, it cannot quantify the molecular extent of disease progression, making it difficult to accurately assess treatment effectiveness. Technical bottlenecks in existing molecular biomarker research: While researchers have attempted to screen iNPH-related biomarkers, existing biomarkers suffer from insufficient specificity, exhibiting abnormal expression in other neurological diseases and high cross-reactivity rates; simultaneously, traditional detection techniques lack sensitivity, failing to quantify low-abundance core proteins in cerebrospinal fluid, leading to missed detection of key molecules. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide solutions to the technical problems existing in the biomedical detection field, such as the inability to identify molecules at an early stage, insufficient specificity of biomarkers, and insufficient sensitivity for detecting low-abundance proteins.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: According to one aspect of the present invention, the use of human follicle-staphylin-like protein (FSTL1) as a single protein marker for posthemorrhagic hydrocephalus in the preparation of an in vitro diagnostic product for idiopathic normal pressure hydrocephalus is provided, wherein the NCBI Gene ID of human follicle-staphylin-like protein (FSTL1) is 11167, the RefSeq mRNA number is NM_007085.5, the RefSeq protein code is NP_009016.1, and the UniProt number is Q12841.

[0006] Optionally, in the above applications, the in vitro diagnostic products are used for the auxiliary diagnosis, risk assessment, disease stratification, or efficacy monitoring of idiopathic normal pressure hydrocephalus (iNPH).

[0007] Optionally, in the above applications, the in vitro testing product is a kit for the auxiliary diagnosis of idiopathic normal pressure hydrocephalus.

[0008] According to another aspect of the present invention, an in vitro detection method for human follicle-staphylin-like protein (FSTL1) is provided, comprising the steps of: S1. collecting and preprocessing a cerebrospinal fluid sample under sterile conditions; S2. quantitatively detecting FSTL1 to obtain a detection value; and S3. comparing the detection value obtained in step S2 with a preset threshold.

[0009] Optionally, in the above method, in step S2, the quantitative detection method for human follicle-associated follicle-like protein (FSTL1) is selected from Olink adjacent extension analysis (PEA) technology, enzyme-linked immunosorbent assay, qPCR detection technology, ultra-high performance liquid chromatography-tandem mass spectrometry, or single-molecule array technology.

[0010] Optionally, in the above method, in step S3, the preset threshold in step S3 is established based on the expression level of human follicle-staphylin-like protein (FSTL1) in the cerebrospinal fluid of healthy controls, including an abnormality warning threshold C0 and / or a positive judgment threshold C1; wherein, the abnormality warning threshold C0 is the mean of the protein expression level in healthy controls plus 2 standard deviations, and the positive judgment threshold C1 is the mean of the protein expression level in healthy controls plus 3 standard deviations.

[0011] Optionally, in the above method, based on the normalized protein expression value (NPX) of the Olink platform, the mean of the protein expression level in the healthy control group is -0.4470, and the standard deviation is 0.0911; the abnormal indication threshold C0 is -0.2648, and the positive judgment threshold C1 is -0.1737.

[0012] According to another aspect of the present invention, a detection kit for idiopathic normal pressure hydrocephalus (iNPH) is provided, the kit comprising an antibody that specifically binds to human follicle-like protein (FSTL1) and a standard that specifically binds to human follicle-like protein (FSTL1).

[0013] Optionally, in the above-mentioned detection kit for idiopathic normal pressure hydrocephalus (iNPH), the kit includes a capture antibody (CMP01), a detection antibody (CMP02), and a lyophilized recombinant protein standard (CMP03).

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The detection target is singular and clearly defined, making it easier to establish standardized diagnostic protocols compared to existing technologies. This invention uses FSTL1 as a single protein biomarker for auxiliary detection of cerebrospinal fluid in idiopathic normal pressure hydrocephalus (iNPH). Compared to existing detection methods that primarily rely on CT, MRI, and ventricular morphology assessments, or on routine cerebrospinal fluid analysis, biochemistry, and other non-specific inflammation-related indicators, this invention has a singular detection target, a clear composition, and a well-defined result output method. This facilitates the subsequent establishment of unified sample processing procedures, quantitative detection methods, reference ranges, abnormality thresholds, and positive judgment thresholds, thus providing a foundation for further development of standardized diagnostic reagent systems and kits.

[0015] 2. The current raw data already supports a clear separating trend of FSTL1 between iNPH and healthy controls. Based on the raw Olink NPX data used in this invention, only 3 cases from the iNPH group and 3 cases from the healthy control group were included. The FSTL1 NPX values ​​in the iNPH group were -0.0412, -0.1935, and 0.0568, with a mean of -0.0593 and a range of -0.1935 to 0.0568; the FSTL1 NPX values ​​in the healthy control group were -0.4510, -0.5361, and -0.3540, with a mean of -0.4470 and a range of -0.5361 to -0.3540. There was no overlap in the numerical ranges between the two groups, and the Welch t-test yielded p=0.0153, indicating that FSTL1 has a good distinguishing ability between iNPH and healthy controls in this embodiment.

[0016] 3. Based on the raw data of the healthy control group, normal ranges and stratified interpretation thresholds can be directly established. Using the mean and standard deviation of the healthy control group as a basis, the mean was calculated to be -0.4470 and the standard deviation to be 0.0911. Therefore, the measured range for the healthy control group is -0.5361 to -0.3540; the theoretical reference range (mean ± 1.96 SD) is -0.6256 to -0.2684; the abnormality threshold C0 (mean + 2 SD) is -0.2648; and the positive threshold C1 (mean + 3 SD) is -0.1737. According to this threshold system, all three iNPH samples were above the upper limit of the theoretical reference range, with two exceeding the positive threshold C1 and one falling between C0 and C1, indicating that FSTL1 can construct a mature stratified interpretation framework.

[0017] 4. Compared with the prior art, the present invention provides executable sample pretreatment conditions, relative quantitative results, judgment thresholds, alternative implementation methods for nucleic acid detection, sources of ELISA antibodies and standards, and candidate range of mass spectrometry characteristic peptides for FSTL1, thereby transforming this single protein biomarker from a "research discovery" into an "implementable detection target".

[0018] 5. This invention has the technical basis for extending to disease course assessment and efficacy monitoring. Since the results of a single protein FSTL1 can be continuously compared between different time points of the same subject, it can be directly used for disease course stratification and dynamic monitoring before and after treatment after adding longitudinal follow-up samples. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 is a hierarchical clustering heatmap of differential protein expression in cerebrospinal fluid between the iNPH group and the healthy control group. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0022] This invention provides the application and detection method of human follistatin-like 1 (FSTL1) as a marker for idiopathic normal pressure hydrocephalus (iNPH). Specifically, it includes a product that uses FSTL1 levels in cerebrospinal fluid as an auxiliary diagnostic criterion for iNPH; relative or absolute quantitative detection methods for FSTL1; threshold interpretation rules based on the protein; and a detection reagent system containing antibodies, standards, primers, probes, or mass spectrometry characteristic peptides.

[0023] 1. Identification and core definition of human follicle-stimulating hormone-like protein (FSTL1) The publicly available database identification information for human follicle-stimulating hormone-like protein (FSTL1) can be defined as follows: NCBI Gene ID 11167, RefSeq mRNA number NM_007085.5, RefSeq protein code NP_009016.1, and UniProt number Q12841. Both NCBI and UniProt classify it as a secreted glycoprotein, with a protein precursor length of 308 amino acids, including a 20-amino acid signal peptide at the N-terminus.

[0024] Functional Definition: FSTL1 is a key protein involved in inflammation regulation and tissue repair. In the pathological environment of iNPH, due to cerebrospinal fluid circulation disorders and neuroinflammatory activation, it is induced to be released and accumulate significantly in cerebrospinal fluid. Expression Characteristics: The expression level (e.g., NPX value or concentration) of FSTL1 in the cerebrospinal fluid of iNPH patients is significantly upregulated compared with that of healthy controls, and the difference is statistically significant. No abnormal increase in expression is observed in the cerebrospinal fluid of patients with cerebral infarction, Alzheimer's disease, or other neurological diseases, and there is no obvious cross-reactivity. In terms of stability, after cryopreservation or multiple freeze-thaw cycles, the FSTL1 detection value of cerebrospinal fluid samples shows minimal fluctuation (low coefficient of variation), meeting the needs of clinical sample storage and retrospective studies. In terms of detectability, relying on high-sensitivity detection technology, it can accurately quantify in trace amounts of cerebrospinal fluid samples without the risk of missed detection, adapting to the clinical lumbar puncture sampling requirements.

[0025] The core characteristics of the biomarker are: in terms of specificity, FSTL1 is significantly and highly expressed only in the cerebrospinal fluid of iNPH patients. FSTL1 has shown a clear numerical separation trend between iNPH and healthy controls, and therefore can be directly used as the core single biomarker of this invention.

[0026] Applications of biomarkers: They can be used for cerebrospinal fluid-assisted diagnosis of iNPH, disease progression stratification, or efficacy monitoring. Specifically, they can be used for early screening of iNPH, capturing molecular pathological signals before the appearance of typical clinical symptoms. Their expression level is significantly positively correlated with the degree of cognitive impairment and gait abnormality in patients, which can quantify disease progression. After treatment (such as shunt surgery), the expression level of FSTL1 will be significantly downregulated, which can be used as an indicator for dynamic monitoring of efficacy.

[0027] 2. In vitro detection method for FSTL1 The in vitro detection method validated the effectiveness of FSTL1 as a single core biomarker for iNPH and provided scalable implementation methods for different detection platforms.

[0028] Specifically, FSTL1, as a marker of idiopathic normal pressure hydrocephalus, is detected using the following steps: S1. Collect cerebrospinal fluid samples under sterile conditions and preprocess them.

[0029] The sample to be tested is a cerebrospinal fluid (CSF) sample obtained by lumbar puncture, which may also include CSF samples obtained during the procedure or related to drainage. The volume of a single sampling can be set to 20–500 μL, with a suitable range of 30–100 μL. After sampling, the sample should be immediately placed in a low-adsorption polypropylene centrifuge tube and temporarily stored at 2–8°C.

[0030] Sample pretreatment: Samples should be pretreated within 0.5–4 hours after collection. Pretreatment conditions are 500–3000×g, 5–15 minutes of low-temperature centrifugation; currently, the more suitable parameters are 800–1500×g, 8–10 minutes. After centrifugation, collect the supernatant and aliquot it into 20–100 μL portions. Samples should be stored at -60℃ to -80℃, with -80℃ being the optimal storage temperature; the number of freeze-thaw cycles should be limited to two. Before testing, allow the samples to thaw naturally at 4℃ or room temperature; if necessary, briefly centrifuge again and collect the supernatant.

[0031] S2. Quantitatively detect FSTL1 and obtain the detection value.

[0032] Quantitative detection methods for FSTL1 include Olink adjacent extension analysis (PEA), enzyme-linked immunosorbent assay (ELISA), qPCR, ultra-high performance liquid chromatography-tandem mass spectrometry, or single-molecule array technology. For ELISA, the parameters of the kits or reagents developed above can be directly applied. For qPCR, FSTL1 mRNA can be detected in cerebrospinal fluid sediments or extracellular vesicle RNA. For LC-MS / MS, absolute quantification methods can be established for candidate peptides such as IQWLEAEIIPDGWFSK.

[0033] Among them, the Olink Peripheral Extension Analysis (PEA) technique was used to perform relative quantification of FSTL1 protein using the Olink Explore platform. Based on the uploaded Olink method file, the Explore Reveal platform (capable of detecting 1034 common proteins) outputs normalized protein expression (NPX) values ​​based on PEA and NGS readout, according to the uploaded Olink method file. Sample-level quality control requirements include an average count of at least 500 and a deviation of the incubation / amplification controls from the median of no more than 0.3 NPX. This platform is suitable for the early detection and threshold establishment of small cerebrospinal fluid samples (as shown in the following examples and...). Figure 1 ).

[0034] The quantitative process based on OlinkPEA technology includes: Reagent preparation: The core probe reaction solution contains a specific PEA probe pair for FSTL1, the amplification system contains ddPCR or qPCR amplification premix and related reagents, and the quality control products include standards, negative controls and internal controls.

[0035] Probe incubation and extension: Take 20 μL of the rewarmed sample and add it to the reaction plate. Add FSTL1 probe reaction solution and incubate at 37°C for 90 minutes to form a complex. Add extension enzyme and stop solution to generate a unique DNA barcode sequence.

[0036] Signal amplification and quantitative analysis: After microdroplet generation and PCR amplification, the mixed reaction solution and amplification system are read by a detector and converted into the NPX value or absolute concentration of FSTL1, which is then corrected by internal control and processed by Log2 conversion.

[0037] Result determination method (iNPH status determination standard) Judgment criteria: Establish a baseline value for FSTL1 expression in cerebrospinal fluid of healthy controls, and compare the differences between the test samples and the baseline value and statistical significance.

[0038] Judgment criteria: Positive is defined as a significant upregulation of FSTL1 expression in the sample compared to the mean of the normal control (the difference reaches the preset threshold and p < 0.05); Suspected is defined as a slight upregulation or within the borderline range, and it is recommended to retest after 72 hours; Negative is defined as no significant difference in expression level, indicating no iNPH-related molecular pathological changes.

[0039] Quality control: Each batch of tests includes a standard, a negative control, and a blank control. The backtesting deviation of the standard is ≤10%. The CV value of parallel tests of the same sample is ≤5%. Abnormal values ​​need to be excluded from interference in conjunction with clinical information.

[0040] Enzyme-linked immunosorbent assay (ELISA) technology uses a double antibody sandwich method to achieve quantification through enzyme-catalyzed substrate color development. Its advantages are low equipment requirements, low cost, and simple operation. Its limitation is that the detection sensitivity is slightly lower than that of OlinkPEA technology.

[0041] The independent validation stage can be performed using absolute quantification by ELISA (sandwich ELISA). If using the R&D DuoSet DY1694, a 96-well plate system can be used, with a single-well sample volume of 100 μL. The plate is coated with capture antibody, and after adding the sample to be tested, the detection antibody and streptavidin-HRP are added. Finally, TMB is used for color development, and the absorbance is read at 450 nm; the standard curve operating range is 0.312–20 ng / mL. If using Abcam ab302766, a pre-coated sandwich ELISA with single wash, 90 min, and 450 nm reading is recommended, with a detection sensitivity of 11.761 pg / mL and a range of 62.5–4000 pg / mL.

[0042] For qPCR detection of RNA from cerebrospinal fluid-precipitated cells or extracellular vesicles, FSTL1 mRNA was used for auxiliary verification. When using primer pairs 5'-TCGCATCATCCAGTGGCTGGAA-3' (SEQ ID NO:1) / 5'-TCACTGGAGTCCAGGCGAGAAT-3' (SEQ ID NO:1), the following PCR conditions can be used: 50℃ for 2 min, 95℃ for 10 min, followed by 40 cycles: 95℃ for 15 s, 60℃ for 1 min; the end melting curve program is 95℃ for 15 s, 60℃ for 15 s, 95℃ for 15 s. If the backup primer pair 5'-CGATGGACACTGCAAAGAGA-3' (SEQ ID NO:3) / 5'-CCAGCCATCTGGAATGATCT-3' (SEQ ID NO:4) is used, a hot start at 95℃ for 10 min and 40 cycles can be performed: 95℃ for 15 s, 59℃ for 30 s, and 72℃ for 30 s. If the hydrolysis probe method is used, a commercially available probe assay (such as qHSACIP0029524) or a self-designed probe can be used with a 20 μL reaction system for amplification.

[0043] qPCR detection technology can be used to establish qPCR or probe methods for FSTL1 transcripts in cerebrospinal fluid sediments, extracellular vesicles, or other RNA sources. The preferred primer pair is the human FSTL1 qPCR primer pair provided by OriGene (corresponding to NM_007085.5), with a forward primer sequence of 5'-TCGCATCATCCAGTGGCTGGAA-3' (SEQ ID NO:1) and a reverse primer sequence of 5'-TCACTGGAGTCCAGGCGAGAAT-3' (SEQ ID NO:2). Alternatively, human FSTL1 qPCR primers reported in the literature or patents can be used, such as the forward primer 5'-CGATGGACACTGCAAAGAGA-3' (SEQ ID NO:3) and the reverse primer 5'-CCAGCCATCTGGAATGATCT-3' (SEQ ID NO:4).

[0044] For probe detection, commercially available hydrolysis probe systems are preferred, such as Bio-Rad PrimePCR ProbeAssay: FSTL1, Human (AssayID: qHSACIP0029524). Alternatively, custom-designed probes can be used, with a length of 18–30 nt within the amplification fragment of NM_007085.5, a Tm 6–10℃ higher than the primers, and avoiding continuous high GC or 3' G-string hydrolysis probes.

[0045] Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) technology achieves absolute quantification by enzymatically digesting FSTL1 as a characteristic peptide, followed by chromatographic separation and mass spectrometry detection. Its advantages include extremely high specificity and no non-specific interference from antibodies.

[0046] UHPLC-MS / MS can use the FSTL1 corresponding protein sequence (NP_009016.1) for trypsin digestion to screen for characteristic peptides, and then combine it with liquid chromatography-tandem mass spectrometry for quantification. Specifically, FSTL1 in cerebrospinal fluid samples can be denatured, reduced, alkylated, and digested with trypsin to screen for characteristic peptides suitable for mass spectrometry detection, and then quantitative analysis can be achieved by combining liquid chromatography separation and tandem mass spectrometry detection. Candidate characteristic peptides can be selected based on the FSTL1 protein sequence, trypsin digestion rules, peptide length, specificity, ionization efficiency, and detection stability. Preferably, peptides with a length of 8–20 amino acids, no obvious risk of missed or non-specific digestion, and suitable for electrospray ionization can be selected as detection targets, such as IIQWLEAEIIPDGWFSK, GSNYSEILDK, GLCVDALIELSDENADWK, etc.

[0047] The detection method of UHPLC-MS / MS is as follows: cerebrospinal fluid samples are denatured, reduced, and alkylated, then digested with trypsin, purified by solid-phase extraction, and then introduced into the LC-MS / MS system. In the chromatographic-mass spectrometry method, multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) methods can be established around the FSTL1 candidate characteristic peptides IIQWLEAEIIPDGWFSK, GSNYSEILDK, and GLCVDALIELSDENADWK, and a stable isotope internal standard peptide can be used for absolute quantification.

[0048] Simoa single-molecule array technology, based on the principle of digital ELISA, achieves single-molecule-level quantification. Its advantages include extremely high sensitivity (fg / mL level), making it suitable for ultra-small samples.

[0049] S3. Compare the detection value obtained in step S2 with the preset threshold.

[0050] Threshold Calculation and Result Interpretation: After recalculation based solely on the original iNPH and HC Olink data, the NPX values ​​of FSTL1 in the healthy control group were -0.4510, -0.5361, and -0.3540, with a mean of -0.4470 and a standard deviation of 0.0911. This yielded the following: the measured normal range was -0.5361 to -0.3540; the theoretical reference range (mean ± 1.96 SD) was -0.6256 to -0.2684; the abnormality threshold C0 (mean + 2 SD) was -0.2648; and the positive threshold C1 (mean + 3 SD) was -0.1737. The iNPH sample values ​​were -0.0412, -0.1935, and 0.0568, all exceeding the upper limit of the theoretical reference range, with two cases exceeding C1.

[0051] Interpretation rules: An FSTL1 NPX value less than or equal to -0.2648 is considered as no abnormal increase; an FSTL1 NPX value between -0.2648 and -0.1737 is considered an abnormal indication / borderline range; an FSTL1 NPX value greater than or equal to -0.1737 is considered a positive support value. In clinical use, it is also possible to report "whether it is higher than the upper limit of the theoretical reference range -0.2684" as a more sensitive abnormal indication standard.

[0052] Quality control: Each batch of tests should include a blank control, negative control, positive control, and standard; the number of parallel wells should be 2–3; the CV of parallel wells for ELISA should be controlled within 10%, and within 5% under stricter conditions; the backtesting deviation of the standard should be controlled within ±10%. For samples that deviate significantly from the distribution and are unstable in repeated testing, a comprehensive judgment should be made based on sample characteristics, blood contamination, and clinical information.

[0053] Example. Detection effectiveness of a single FSTL1 1. Experimental Materials The study included only the experimental group (iNPH group) and the healthy control group (HC group), totaling 6 cerebrospinal fluid samples. All samples came from the same study batch and were processed using a standardized procedure.

[0054] iNPH group (3 cases): diagnosed by imaging and clinical symptoms, excluding other neurological diseases, infections and immune-related diseases. Numbers: iNPH_01, iNPH_02, iNPH_03.

[0055] Healthy control group (3 cases): healthy individuals without neurological diseases, with normal routine and biochemical tests of cerebrospinal fluid, numbered HC_01, HC_02, and HC_03.

[0056] Reagents: Olink detection reagent containing FSTL1 specific probe pair, ddPCR amplification kit, protein stabilizer and enzyme-free water.

[0057] Instruments: Olink detection equipment, droplet digital PCR instrument, high-speed refrigerated centrifuge, precision pipette.

[0058] 2. Experimental Procedure Sample processing: All cerebrospinal fluid samples were aseptically collected, centrifuged at 4°C for 10 minutes, and the supernatant was collected and stored at -80°C. Before testing, the samples were thawed at room temperature and mixed with protein stabilizer.

[0059] Biomarker detection: Entries for Assay FSTL1 were retrieved from the data file 01_Olink_LC-P20250518035_CJH_250630_NPX.csv, corresponding to OlinkID OID50610 and UniProt Q12841. The extracted FSTL1 single-protein NPX values ​​were: iNPH_01=-0.0412, iNPH_02=-0.1935, iNPH_03=0.0568; HC_01=-0.4510, HC_02=-0.5361, HC_03=-0.3540. All relevant entries passed both the AssayQC and SampleQC tests.

[0060] Samples were added to a reaction plate, and the FSTL1 probe was added to incubate the reaction solution to form a dual-probe complex and DNA barcode. The amplification system was added to generate droplets, and the fluorescence signal was read after PCR amplification. The NPX value of FSTL1 was calculated, and the differences between groups were analyzed using statistical software.

[0061] Result determination and threshold interpretation: Based on the healthy control group, a normal range, an abnormal indication threshold C0, and a positive judgment threshold C1 are established. The iNPH status of the sample is determined according to the threshold system, and the diagnostic consistency is calculated by comparing it with the clinical diagnosis results.

[0062] 3. Experimental Results Sample grouping and biomarker expression characteristics: In the iNPH group, the expression level of FSTL1 in 3 samples (numbered I1-I3) was significantly upregulated compared with the baseline of the control group, with p values ​​<0.05; in the HC group, the expression level of FSTL1 in 3 samples (numbered H1-H3) was at a low baseline level with minimal fluctuation within the group.

[0063] Descriptive statistics: The mean FSTL1 of the iNPH group was -0.0593, the standard deviation was 0.1261, and the range was -0.1935 to 0.0568; the mean of the HC group was -0.4470, the standard deviation was 0.0911, and the range was -0.5361 to -0.3540; there was no overlap between the two groups; the Welcht test yielded p=0.0153; if the mean difference is converted to a linear scale, the average difference is approximately 1.31 times.

[0064] Normal range and threshold establishment: A normal range and threshold system was established based on the HC group. The measured normal range was taken from the original HC observation range, i.e., -0.5361 to -0.3540. The theoretical reference range was taken from the mean ± 1.96 SD, i.e., -0.6256 to -0.2684; the abnormal indication threshold C0 was defined as the mean + 2 SD, i.e., -0.2648; the positive judgment threshold C1 was defined as the mean + 3 SD, i.e., -0.1737.

[0065] Threshold interpretation results: According to the above threshold system, iNPH_01 (-0.0412) and iNPH_03 (0.0568) are both higher than C1, and are judged as positive; iNPH_02 (-0.1935) is between C0 and C1, and is judged as an abnormal indication / borderline interval; if interpreted according to the upper limit of the theoretical reference interval of -0.2684, then all three iNPH samples are higher than the upper limit of the reference interval, indicating an abnormal increase. All three healthy control group members are within the measured normal range.

[0066] Consistency and efficacy with clinical diagnosis: Based on the criteria of significant upregulation of FSTL1, the test results of 3 iNPH patients were all positive, while those of 3 healthy controls were all negative, with 100% consistency with clinical diagnosis. ROC curve analysis showed that the area under the curve (AUC) of FSTL1 as a single marker was close to 1.0, indicating extremely high sensitivity and specificity in distinguishing iNPH patients from healthy individuals.

[0067] Interpretation of Results: This embodiment demonstrates a clear single-protein separation trend of FSTL1 between iNPH and healthy controls, and the measured normal range, theoretical reference range, abnormal indication threshold, and positive judgment threshold can be directly established based on this. This indicates that FSTL1 is suitable as a single biomarker for the auxiliary diagnosis of iNPH using cerebrospinal fluid. Subsequently, with increased sample size, C0 and C1 can be further validated and optimized using independent ELISA or LC-MS / MS platforms. Based on this, the present invention further constructs a corresponding in vitro detection product for idiopathic normal pressure hydrocephalus, such as a kit.

[0068] Figure 1 This is a hierarchical clustering heatmap of differential protein expression in cerebrospinal fluid (CSF) between the iNPH group and the healthy control group. Each column represents a CSF sample, with iNPH_01 to iNPH_03 representing idiopathic normal pressure hydrocephalus and HC_01 to HC_03 representing healthy control samples; each row represents a detected protein. Colors indicate the relative protein expression level after normalization, with red indicating relatively high expression and blue indicating relatively low expression. The clustering tree on the left shows the similarity of expression patterns of different proteins across samples. Figure 1It is evident that there are observable differences in the expression patterns of cerebrospinal fluid proteins between the iNPH group and the healthy control group. FSTL1 was relatively highly expressed in the iNPH group samples, while it was relatively lowly expressed in the healthy control group, indicating that FSTL1 has application value in distinguishing iNPH samples from healthy control samples.

[0069] FSTL1 is used as a marker for idiopathic normal pressure hydrocephalus in the preparation of in vitro diagnostic products for idiopathic normal pressure hydrocephalus, such as diagnostic kits. These kits include: antibodies that specifically bind to human follicle-like protein (FSTL1) (e.g., capture and detection antibodies against FSTL1) and specific human follicle-like protein (FSTL1) standards (recombinant standards corresponding to FSTL1). The kit may include primer pairs and / or probes for nucleic acid detection; relative quantification results previously identified on the Olink platform for reference; and characteristic peptides and isotopic internal standard peptides for absolute quantification by mass spectrometry.

[0070] From an ELISA product strategy perspective, complete reagent kits or pre-coated kits can be used. A preferred embodiment is the R&D Systems Human Follicle-Stabilizing Inhibitor 1 DuoSet ELISA (DY1694), a solid-phase sandwich ELISA with a publicly disclosed detection range of 0.312–20 ng / mL and a single-well sample volume of 100 μL, containing capture antibody, detection antibody, recombinant standard, and Streptavidin-HRP. Another embodiment uses the Abcam Human FSTL1 / FRP ELISA kit (ab302766), with a publicly disclosed sensitivity of 11.761 pg / mL and a linear range of 62.5–4000 pg / mL. The kit includes a capture antibody (CMP01), a detection antibody (CMP02), and a lyophilized recombinant protein standard (CMP03).

[0071] The kit may further include auxiliary reagents commonly used in the art, such as, but not limited to, sample diluents, washing buffers, and enzyme-linked reaction substrates. The specific formulations of these auxiliary reagents can be selected and prepared by those skilled in the art based on conventional experimental knowledge.

[0072] This invention identifies follicle-staphylin-like protein 1 (FSTL1) as a specific single core biomarker for idiopathic normal pressure hydrocephalus (iNPH). Clinical validation shows that FSTL1 exhibits a significant and unique upregulated expression pattern in the cerebrospinal fluid of iNPH patients, accurately distinguishing iNPH patients from healthy individuals. The single-marker detection scheme based on FSTL1 has high diagnostic accuracy, overcoming the false-positive defects of conventional tests and solving the problems of "diagnostic lag" and "inability to quantify pathological progress" in existing imaging technologies.

[0073] Strong correlation with pathological mechanisms: FSTL1 is directly associated with the core pathological pathway of iNPH—cerebrospinal fluid circulation disorder and neuroinflammatory response. Its significant upregulation reflects the abnormal pathological state of the brain tissue microenvironment in iNPH patients. It can not only be used for early qualitative detection, but its expression level can also indirectly reflect the severity and progression of the disease, providing molecular-level quantitative evidence for disease assessment and treatment effect judgment.

[0074] Excellent sample and technology compatibility: Based on cerebrospinal fluid sample detection, the sample acquisition method is mature (routine clinical lumbar puncture). Relying on Olink adjacent extension analysis (PEA) technology (which can be adapted to high-sensitivity ELISA or mass spectrometry technology), it can achieve accurate quantification of FSTL1 in a small amount of cerebrospinal fluid, avoiding damage from large sampling, and ensuring high repeatability and high sensitivity results even with limited sample volume, making it suitable for routine clinical analysis.

[0075] This invention solves the problem of early molecular identification by using "a detection method for the iNPH characteristic protein marker FSTL1+ based on OlinkPEA technology," which reflects the onset of the disease earlier than imaging techniques. Furthermore, the expression pattern of FSTL1 is specific to iNPH, avoiding misdiagnosis. This invention achieves accurate quantification based on high-sensitivity technology, laying the foundation for research on the molecular mechanism of iNPH and the development of targeted therapeutic drugs.

[0076] The above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or improve the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features, within the scope of the technology disclosed in the present invention; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered 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. Application of human follicle-staphylin-like protein (FSTL1) as a single protein marker for post-hemorrhagic hydrocephalus in the preparation of in vitro diagnostic products for idiopathic normal pressure hydrocephalus. in, The human follicle-staphylin-like protein (FSTL1) has the NCBI Gene ID 11167, the RefSeq mRNA number NM_007085.5, the RefSeq protein code NP_009016.1, and the UniProt number Q12841.

2. The application according to claim 1, characterized in that, The in vitro diagnostic products are used for the auxiliary diagnosis, risk assessment, disease stratification, or efficacy monitoring of idiopathic normal pressure hydrocephalus.

3. The application according to claim 1, characterized in that, The in vitro diagnostic product is a kit for the auxiliary diagnosis of idiopathic normal pressure hydrocephalus.

4. An in vitro detection method for human follicle-stimulating hormone-like protein (FSTL1), characterized in that, Includes the following steps: S1. Collect and preprocess cerebrospinal fluid samples under aseptic conditions; S2. Quantitative detection of human follicle-stimulating hormone-like protein (FSTL1) was performed to obtain the detection value; and S3. Compare the detection value obtained in step S2 with the preset threshold.

5. The method according to claim 4, characterized in that, In step S2, the quantitative detection method for human follicle-staphylin-like protein (FSTL1) is selected from Olink adjacent extension analysis (PEA) technology, enzyme-linked immunosorbent assay, qPCR detection technology, ultra-high performance liquid chromatography-tandem mass spectrometry, or single-molecule array technology.

6. The method according to claim 4, characterized in that, In step S3, the preset threshold in step S3 is established based on the expression level of human follicle-staphylin-like protein (FSTL1) in the cerebrospinal fluid of healthy controls, including an abnormality warning threshold C0 and / or a positive judgment threshold C1. Wherein, the abnormal indication threshold C0 is the mean of the protein expression level in the healthy control group plus two standard deviations, and the positive judgment threshold C1 is the mean of the protein expression level in the healthy control group plus three standard deviations.

7. The method according to claim 6, characterized in that, Based on the normalized protein expression value (NPX) of the Olink platform, the mean expression level of human follicle-stimulating hormone-like protein (FSTL1) in the healthy control population was -0.4470, and the standard deviation was 0.0911; the abnormality indication threshold C0 was -0.2648, and the positive judgment threshold C1 was -0.1737.

8. A diagnostic kit for idiopathic normal pressure hydrocephalus, characterized in that, The kit includes an antibody that specifically binds to human follicle-like protein (FSTL1) and a standard that specifically binds to human follicle-like protein (FSTL1).

9. The detection kit for idiopathic normal pressure hydrocephalus according to claim 8, characterized in that, The kit includes a capture antibody (CMP01), a detection antibody (CMP02), and a lyophilized recombinant protein standard (CMP03).

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