Application of caps protein as a marker for posthemorrhagic hydrocephalus and detection method thereof

Abstract

This invention provides the application of human calcyphosphine (CAPS) as a single protein biomarker for post-hemorrhagic hydrocephalus in the preparation of in vitro diagnostic products for post-hemorrhagic hydrocephalus, as well as an in vitro detection method and kit for human calcyphosphine (CAPS). The human calcyphosphine's UniProt ID is Q13938, its NCBI Gene ID is 828, and its RefSeq protein IDs are NP_004049.3 and NP_542157.3. The in vitro detection method includes: S1. collecting and preprocessing cerebrospinal fluid samples under aseptic conditions; S2. quantifying human calcyphosphine (CAPS) using a protein quantification method to obtain a detection value; and S3. comparing the detection value obtained in step S2 with a preset threshold. This invention solves three core problems of existing THH detection technologies: inability to identify calcyphosphine at the molecular level in its early stages, insufficient biomarker specificity, and insufficient sensitivity for detecting low-abundance proteins.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing, specifically to the application and detection method of CAPS as a marker of posthemorrhagic hydrocephalus. Background Technology

[0002] (I) Clinical hazards and current status of diagnosis and treatment of post-hemorrhagic hydrocephalus (THH) Post-hemorrhagic hydrocephalus (THH) is a common and serious complication following intracranial hemorrhage (including subarachnoid hemorrhage and intraventricular hemorrhage), with an incidence rate of 10%-30%. Its core pathological mechanism is an imbalance in cerebrospinal fluid (CSF) dynamics—after hemorrhage, red blood cell lysis products and inflammatory mediators obstruct the CSF absorption pathway, while abnormal function of the ventricular wall epithelial cells leads to increased CSF secretion, ultimately causing progressive ventricular enlargement, which in turn compresses brain tissue, inducing increased intracranial pressure and neurological deficits. Clinically, the mortality rate of THH patients is 2-3 times higher than that of intracranial hemorrhage patients without THH. More than 80% of survivors experience long-term cognitive impairment (such as memory decline and executive function loss) and motor dysfunction (such as limb paralysis), requiring long-term family care, placing a heavy medical and economic burden on society and families.

[0003] The current clinical diagnosis and treatment system for THH mainly relies on "imaging diagnosis + surgical treatment": at the diagnostic level, it depends on cranial CT or MRI, and the presence of THH is determined by observing the degree of ventricular enlargement (e.g., Evans index > 0.3); at the treatment level, there are no effective drug intervention options, and only invasive surgeries such as external ventricular drainage and ventriculoperitoneal shunt can be used to relieve cerebrospinal fluid retention, but the postoperative complication rate is as high as 30%-50% (such as infection, shunt tube blockage, and subdural hematoma due to excessive drainage), and the existing neurological damage cannot be reversed. The root cause of this diagnostic and treatment dilemma is that current technology has not fully revealed the core molecular mechanism of THH, and there is a lack of specific biomarkers that can be used for early molecular-level diagnosis, disease monitoring, and screening of therapeutic targets.

[0004] (ii) Limitations of existing THH detection technologies (technical gaps in the objects of protection) 1. The core defects of imaging diagnostic technology While head CT / MRI is the "gold standard" for clinical diagnosis of post-hemorrhagic hydrocephalus (THH), it can only identify the mid-to-late stage morphological change of "ventricular enlargement" and cannot capture the early molecular pathological changes after hemorrhage. At this stage, abnormal protein expression has already appeared in the cerebrospinal fluid, but the ventricles have not yet undergone significant structural changes. This results in some THH patients with post-hemorrhagic hydrocephalus not being diagnosed by imaging in the early stages of the disease (such as some time after hemorrhage), delaying the timing of intervention. In addition, imaging technology can only subjectively judge the trend of ventricular enlargement and cannot quantify the molecular degree of disease progression, nor can it accurately assess the pathological improvement after treatment. Its clinical application has the limitation of "emphasizing morphology and neglecting molecular aspects".

[0005] 2. Major technical problems in existing molecular marker research In recent years, the academic community has attempted to screen for THH-related molecular markers from cerebrospinal fluid, but two key problems remain: Insufficient specificity: The inflammatory factors (such as IL-6 and TNF-α) and structural proteins (such as AQP4 and GFAP) that current studies focus on are also abnormally expressed in other neurological diseases such as cerebral infarction, encephalitis, and traumatic brain injury. The cross-reactivity rate with THH is >30%, so it cannot be used as a specific diagnostic marker for THH. Insufficient sensitivity: Current detection technologies mainly rely on ELISA and Western blotting, with detection limits typically ranging from 1 to 10 ng / mL. These technologies cannot quantify low-abundance proteins in cerebrospinal fluid (such as receptor tyrosine protein phosphatase Zeta (PTPRZ1) and Calsyntenin-2 (CLSTN2), whose concentrations are mostly <0.1 ng / mL). These low-abundance proteins are often involved in core pathological processes of THH, such as neural repair and signal transduction, and are key molecules for elucidating disease mechanisms and developing diagnostic biomarkers. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide the application and detection method of CAPS as a biomarker for posthemorrhagic hydrocephalus, so as to solve the three core problems of existing THH detection technology: inability to identify early at the molecular level, insufficient biomarker specificity, and insufficient sensitivity of low-abundance protein detection.

[0007] 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 calcium phosphoprotein (CAPS) as a single protein marker for post-hemorrhagic hydrocephalus in the preparation of in vitro detection products for post-hemorrhagic hydrocephalus is provided, wherein the human calcium phosphoprotein has UniProt number Q13938, NCBI Gene ID 828, and RefSeq protein numbers NP_004049.3 and NP_542157.3.

[0008] Optionally, in the above applications, the in vitro detection product for post-hemorrhagic hydrocephalus can be used for single-protein-assisted detection of post-hemorrhagic hydrocephalus, to indicate molecular abnormalities in cerebrospinal fluid related to post-hemorrhagic hydrocephalus, to assess the course of post-hemorrhagic hydrocephalus, or to validate post-hemorrhagic hydrocephalus detection on an independent immunological platform.

[0009] Optionally, in the above applications, the in vitro detection product for post-hemorrhagic hydrocephalus is a kit for the auxiliary diagnosis of post-hemorrhagic hydrocephalus.

[0010] According to another aspect of the present invention, an in vitro detection method for human calcium phosphoprotein (CAPS) is provided, characterized by comprising the following steps: S1. collecting and preprocessing cerebrospinal fluid samples under sterile conditions; S2. quantitatively detecting human calcium phosphoprotein (CAPS) using a protein quantification method to obtain a detection value; and S3. comparing the detection value obtained in step S2 with a preset threshold.

[0011] Optionally, in the above method, in step S1, the pretreatment includes: centrifuging the collected cerebrospinal fluid at 2°C to 8°C for 8 to 10 minutes over 0.5 to 4 hours with a centrifugal force of 800×g to 1500×g, taking the supernatant, aliquoting it, and storing it at -60°C to -80°C.

[0012] Optionally, in the above method, in step S2, the protein quantification detection method is selected from Olink adjacent extension analysis (PEA) technology, enzyme-linked immunosorbent assay, ultra-high performance liquid chromatography-tandem mass spectrometry, or single-molecule array technology.

[0013] Optionally, in the above method, in step S3, the preset threshold is established based on the expression level of human calcium phosphoprotein in a healthy control group, including a negative upper limit threshold and a positive threshold, wherein the negative upper limit threshold is the average expression level of human calcium phosphoprotein in the healthy control group plus 2 standard deviations, and the positive threshold is the average expression level of human calcium phosphoprotein in the healthy control group plus 3 standard deviations.

[0014] Optionally, in the above method, in step S3, when the human calcium phosphoprotein level is detected using Olink neighbor extension analysis (PEA) and expressed as normalized protein expression value (NPX), the upper limit threshold for negative is 0.7662 NPX and the threshold for positive is 1.2038 NPX.

[0015] According to yet another aspect of the present invention, a detection kit for post-hemorrhagic hydrocephalus is provided, the kit comprising an antibody that specifically binds to human calcium phosphoprotein (CAPS) and a recombinant human calcium phosphoprotein (CAPS) protein standard.

[0016] Optionally, in the above-mentioned detection kit for post-hemorrhagic hydrocephalus, the antibody is a rabbit recombinant monoclonal antibody; or the antibody is a paired antibody system, wherein the capture antibody is 68225-2-PBS (2H7H1) and the detection antibody is 68225-3-PBS (2D7G2).

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The target of testing is singular and clearly defined, making it easier to develop standardized testing solutions compared to existing technologies; 2. CAPS has a clear ability to distinguish between THH and healthy controls; 3. Compared with existing imaging and conventional cerebrospinal fluid indicators, it can provide molecular-level auxiliary diagnostic information; 4. It has the foundation for applications extending to disease course assessment and efficacy monitoring; 5. The sample requirement is small, and the technical approach is well-suited to clinical sample conditions. Attached Figure Description

[0018] 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.

[0019] Figure 1 is a hierarchical clustering heatmap of differential protein expression in cerebrospinal fluid between the THH group and the healthy control group. Detailed Implementation

[0020] 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.

[0021] This invention provides the application and detection method of CAPS as a biomarker for post-hemorrhagic hydrocephalus, including a cerebrospinal fluid detection method, interpretation threshold, and subsequent reagent system established around this protein. This invention focuses on a single protein as the core detection target, and through clearly defined sample processing parameters, database identity restrictions, antibody and standard sources, and quantifiable interpretation criteria, the technical solution of this invention is feasible, verifiable, and transferable.

[0022] 1. Composition, identity limitations, and core definition of human calcium phosphoproteins (CAPS) To ensure clear boundaries for the detected samples, CAPS is defined by protein name, gene name, database ID, and a specific identification system. Database identification information includes: UniProt ID Q13938, NCBI Gene ID 828, and RefSeq protein IDs NP_004049.3 and NP_542157.3. In the Olink data, the assay name for human calcium phosphoprotein (CAPS) is CAPS, and the Olink ID is OID50438.

[0023] In this invention, CAPS is a single molecule that can be directly detected and interpreted. Its expression level is expressed as the relative quantitative value of NPX output by the Olink platform, or as an absolute concentration in the subsequent independent validation stage.

[0024] CAPS exhibits good detectability. It can be stably detected in THH cerebrospinal fluid samples, showing clear relative quantitative differences. For this single protein, further preliminary relative quantitative validation can be conducted using the Olink platform, followed by a transition to an immunological quantitative platform more suitable for product development.

[0025] The applications of single-protein CAPS include: 1. Single-protein auxiliary detection of THH relative to healthy controls; 2. Indication of THH-related molecular abnormalities in cerebrospinal fluid; 3. Continuous observation of the same subject at different time points to serve the assessment of disease progression; 4. Validation detection in subsequent independent immunological platforms; 5. Construction of cerebrospinal fluid detection reagent systems and kits based on CAPS.

[0026] 2. Antibody Information and Standard Sources The antibodies used were commercially available monoclonal antibodies or paired antibody systems, including: Abcam's anti-calcyphosine antibody, catalog number ab186740, clone number EPR15631, which is a rabbit recombinant monoclonal antibody; and Proteintech's CAPS matched antibody pair, catalog number MP51393-1, in which the capture antibody is 68225-2-PBS (2H7H1) and the detection antibody is 68225-3-PBS (2D7G2). These antibody sources can be directly used to establish the sandwich assay system.

[0027] The standards used are recombinant proteins corresponding to human CAPS as calibrators, including Proteintech's matching standard Ag10330 or other commercially available recombinant human CAPS corresponding to UniProtQ13938. According to publicly available product information, the CAPS-paired antibody system supports a standard curve range of approximately 0.781 to 100 ng / mL.

[0028] 3. In vitro detection method for human calcium phosphoprotein (CAPS) Human calcium phosphoprotein is used as a marker for post-hemorrhagic hydrocephalus. The detection method includes the following steps: S1. Collect cerebrospinal fluid samples under sterile conditions and preprocess them.

[0029] Cerebrospinal fluid samples can be obtained from lumbar puncture, external ventricular drainage, or intraoperatively. The volume of a single sample collection is set at 20 to 500 μL, with a preferred range of 30 to 100 μL. After collection, the sample should be immediately placed in a low-adsorption polypropylene centrifuge tube and temporarily stored at 2 to 8°C.

[0030] Samples should be pretreated within 0.5 to 4 hours after collection: After collection, samples should be briefly stored at 4°C and centrifuged within 0.5 to 4 hours to remove impurities. Centrifugation conditions should be approximately 800×g for 10 minutes; after centrifugation, the supernatant should be aliquoted. The purpose of pretreatment is to remove red blood cells, cell debris, and particulate impurities, reducing noise in subsequent detection. Centrifugation conditions should be 500 to 3000×g for 5 to 15 minutes; the preferred conditions are 800 to 1500×g for 8 to 10 minutes. After centrifugation, the supernatant should be aliquoted, with a single aliquot volume of 20 to 100 μL.

[0031] After aliquoting, the samples should be stored at -60℃ to -80℃, with -80℃ being the preferred storage condition. Before testing, allow the samples to thaw naturally and mix gently. If necessary, centrifuge again and collect the supernatant. Repeated freeze-thaw cycles should be avoided as much as possible, and the number of freeze-thaw cycles should not exceed two. Before testing, allow the samples to thaw naturally at 4℃ or room temperature and mix gently. If precipitation is observed, centrifuge again at approximately 800×g for 5 minutes and collect the supernatant for inclusion in the detection system.

[0032] S2. Human calcium phosphoprotein (CAPS) was quantitatively detected using a protein quantification method to obtain the detection value.

[0033] Protein quantification methods include Olink adjacent extension analysis (PEA), enzyme-linked immunosorbent assay (ELISA), ultra-high performance liquid chromatography-tandem mass spectrometry, or single-molecule array technology.

[0034] The Olink Peripheral Extension (PEA) technique, using the Olink protein detection platform, obtains the normalized protein expression value (NPX) of CAPS using the Olink standard procedure. Following the manufacturer's method documentation, the Olink Explore platform performs internal and external plate control calibrations on the samples and outputs the relative abundance value of NPX. Relevant methodological information includes: sample input sizes can be as low as microliters; LOD is defined by adding a preset standard deviation to the negative control median; sample-level QC requirements include the average count signal value not falling below a set lower limit, and internal control deviation not exceeding the allowable range. The CAPS assay passed both assay QC and sample QC in the data of this invention, indicating that this single protein result can serve as the preliminary data basis for the examples (see later examples and...). Figure 1 ).

[0035] Enzyme-linked immunosorbent assay (ELISA) is suitable for primary healthcare institutions. Its technical principle involves coating a solid-phase carrier (such as a microplate) with a specific monoclonal antibody against CAPS, and using a double-antibody sandwich method for detection. The substrate is catalyzed by an enzyme (such as horseradish peroxidase HRP) to develop color; the intensity of the color is positively correlated with the concentration of CAPS in the sample, thus achieving quantification.

[0036] The specific process includes: Coating and blocking: Coating the CAPS capture antibody onto the ELISA plate and blocking non-specific sites with protein blocking solution; Sample incubation: Adding pretreated cerebrospinal fluid sample and incubating at a suitable temperature to capture the CAPS antigen; Secondary antibody binding: Adding the ELISA-labeled detection antibody to form a "capture antibody-CAPS-detection antibody" complex; Color development and reading: Adding substrate for color development, terminating the reaction, and reading the absorbance (OD value) using an ELISA reader, calculating the CAPS concentration based on the standard curve.

[0037] The advantages of enzyme-linked immunosorbent assay (ELISA) are: low equipment requirements (only an ELISA reader is needed), low cost, relatively simple operation, and it is currently the most mature protein detection method in clinical practice.

[0038] During the independent validation and kit development stages, a sandwich immunoassay system targeting CAPS is preferred for the quantitative detection of human calcium phosphoprotein (CAPS). The assay system consists of capture antibody, detection antibody, recombinant CAPS protein standard, sample diluent, washing buffer, blocking buffer, chromogenic or luminescent substrate, blank control, and positive control. The sample volume per well can be set from 10 to 100 μL, with a more suitable range of 20 to 50 μL; each sample is analyzed in 2 to 3 parallel wells.

[0039] The standard curve can be prepared using a multi-point concentration gradient. Referring to the publicly available CAPS paired antibody system, use 0.781 to 100 ng / mL as the initial curve range, and adjust according to the actual platform sensitivity and linear range. The coefficient of variation for parallel well detection should be controlled within 10%, and under more stringent conditions, within 5%; the deviation of standard backtesting should be controlled within ±10%. If the parallel well deviation is too large or the control backtesting is abnormal, the test should be repeated.

[0040] Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) does not rely on antibody recognition. Instead, it uses proteases to enzymatically digest CAPS into characteristic peptides, separates the peptides using liquid chromatography, and then uses tandem mass spectrometry to accurately detect the parent and daughter ions of the characteristic peptides, achieving absolute quantification based on peak area.

[0041] The specific process is as follows: Sample pretreatment: A small amount of cerebrospinal fluid is taken and treated with denaturing agent, reducing agent and alkylating agent, followed by trypsin enzymatic hydrolysis to cleave CAPS into peptides; Purification and enrichment: The enzymatic hydrolysis products are desalted and enriched by solid phase extraction (SPE) column; Mass spectrometry detection: Multiple reaction monitoring (MRM) mode is used to detect specific ion-pair transition parameters for characteristic peptides of CAPS (e.g., sequence LVQELAQR); Quantification: Isotope-labeled internal standard peptides are introduced, and the absolute concentration of CAPS is calculated by internal standard method.

[0042] The advantages of ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection methods are: extremely high specificity (based on molecular weight and structural sequence), no interference from non-specific antibody binding, and the ability to achieve absolute quantification.

[0043] Simoa single-molecule array technology is suitable for extremely small sample volumes. Its principle is based on the "digital ELISA" principle, utilizing magnetic beads to capture CAPS and encapsulating the complex within a microwell array with a fraction of a liter (fL) volume. Each microwell contains only one or zero target molecules. Fluorescence imaging technology is used to count the "signaled" microwells (0 / 1 digital signals), achieving single-molecule-level quantification.

[0044] The specific process is as follows: Magnetic bead capture: Magnetic beads bound with CAPS capture antibodies are mixed and incubated with cerebrospinal fluid samples; Labeling reaction: Biotinylated detection antibodies and enzyme conjugates are added to form a complex; Microwell encapsulation: Magnetic beads and fluorescent substrates are loaded together into a microwell array chip and sealed with oil; Digital counting: The instrument automatically scans and counts the number of luminescent microwells and directly converts them into CAPS concentration.

[0045] The advantages of Simoa single-molecule array technology are: extremely high sensitivity (down to the fg / mL level), far exceeding that of conventional ELISA, making it very suitable for the detection of ultra-small cerebrospinal fluid samples.

[0046] All the above alternatives follow the core logic of this invention—"using human calcium phosphoprotein (CAPS) as a specific biomarker to diagnose post-hemorrhagic hydrocephalus." The only difference between the alternatives lies in the technical means of "how to detect CAPS" (immunologic methods, physical methods, or digital single-molecule methods). Regardless of the technology used, as long as the target is CAPS and it is used for the auxiliary diagnosis of THH, and it can distinguish patients from normal individuals (achieving statistically significant differences), it falls within the scope of protection of this invention. These alternatives are equivalent substitutions based on existing mature technologies, rather than changes to the core inventive concept.

[0047] S3. Compare the detection value obtained in step S2 with the preset threshold.

[0048] The preset thresholds were established based on the expression levels of human calcium phosphoprotein in a healthy control group, including an upper limit for negative and a higher limit for positive. The upper limit for negative was the mean expression level of human calcium phosphoprotein in the healthy control group plus two standard deviations, and the higher limit for positive was the mean expression level of human calcium phosphoprotein in the healthy control group plus three standard deviations.

[0049] The comparison results shall be interpreted according to the following rules: When the detected value is less than or equal to the negative upper limit threshold, a negative result is output; When the detected value is greater than or equal to the positive threshold, a positive result is output; When the test value is between the negative upper limit threshold and the positive threshold, a suspected result is output, and a retest is recommended within 24 to 72 hours.

[0050] When human calcium phosphoprotein (CAPS) levels were detected using the Olink platform and expressed as normalized protein expression values ​​(NPX), specifically, based on NPX data from 3 healthy control subjects (CAPS), the range of -0.4786 to 0.3742 was used as the actual observed normal range. Further, using the healthy control group's mean of -0.1090 and standard deviation of 0.4376 as a basis, and employing mean ± 1.96 SD, a theoretical reference range of approximately -0.9667 to 0.7487 NPX was calculated. Both representations can be retained simultaneously: the former serves as the observed range in the example, while the latter serves as the basis for establishing the reference range.

[0051] The upper limit threshold for negative results (C0) is defined as the mean of healthy controls plus two standard deviations, and the upper limit threshold for positive results (C1) is defined as the mean of healthy controls plus three standard deviations. Calculations show C0 = 0.7662 NPX and C1 = 1.2038 NPX. The criteria for judgment are as follows: when the NPX value of CAPS in the sample is less than or equal to 0.7662, it is considered negative; when the NPX value of CAPS is greater than or equal to 1.2038, it is considered positive; when the NPX value of CAPS is between 0.7662 and 1.2038, it is considered suspected, and resampling and retesting within 24 to 72 hours is recommended.

[0052] The reference intervals and thresholds described above are established based on the sample data of the current embodiment. When increasing the sample size or using different detection platforms for absolute quantification, the corresponding reference intervals and thresholds can be re-established using the same statistical methods. Any reference intervals and thresholds established based on the statistical methods disclosed in this invention (mean ± 2SD / 3SD) fall within the protection scope of this invention.

[0053] The detection method of this invention first uses a high-throughput platform to obtain the relative quantitative results of CAPS and confirm its separation trend between THH and healthy controls; then, based on this single protein, a repeatable immunological quantification system is constructed, and reference intervals and thresholds are used as output results.

[0054] Example. Detection effectiveness of a single CAPS The purpose of the following examples is to verify the numerical separation trend of CAPS between THH and healthy controls, and to establish normal value ranges, negative upper limit thresholds and positive thresholds accordingly.

[0055] 1. Experimental Materials The study included a THH group and a healthy control group. The THH group included 5 patients clinically diagnosed with post-hemorrhagic hydrocephalus. The inclusion criteria were cerebrospinal fluid circulation disorders following intracranial hemorrhage and the presence of hydrocephalus confirmed by imaging. Patients with concomitant factors such as systemic infection, autoimmune disease, and tumors were excluded. The healthy control group included 3 patients without neurological diseases and whose routine cerebrospinal fluid examination showed no significant abnormalities.

[0056] The initial testing platform used the Olink protein assay platform to obtain the relative quantitative value of CAPS NPX. Sample processing equipment included a low-temperature centrifuge, precision pipettes, and a -80°C cryopreservation system. Subsequent independent validation will be conducted, preferably using specific CAPS antibodies, recombinant protein standards, and a conventional immunoassay platform.

[0057] 2. Experimental Procedure All cerebrospinal fluid samples were collected under sterile conditions. After collection, samples were briefly stored at 4°C and centrifuged within 0.5 to 4 hours to remove impurities. Centrifugation conditions were approximately 800 × g for 10 minutes. The supernatant was aliquoted after centrifugation and stored at -80°C. Before testing, samples were allowed to warm naturally and gently mixed; if necessary, they were centrifuged again, and the supernatant was collected.

[0058] CAPS was measured using the Olink protein detection platform according to the manufacturer's standard procedure to obtain the NPX values ​​for each sample. Subsequently, the mean, range, and standard deviation of CAPS for the THH group and the healthy control group were calculated, and normal value intervals, upper negative thresholds, and positive thresholds were established based on the data from the healthy control group.

[0059] 3. Experimental Results The CAPSNPX values ​​of the 5 samples in the THH group were 2.8600, 3.1442, 2.4012, 2.2756, and 1.6147, with a mean of 2.4591 and a range of 1.6147 to 3.1442. The CAPSNPX values ​​of the 3 samples in the healthy control group were -0.4786, -0.2226, and 0.3742, with a mean of -0.1090 and a range of -0.4786 to 0.3742. The two groups showed a clear trend of separation in CAPS values, indicating that this single CAPS can distinguish between THH and healthy controls. Based on these values, CAPS can be used as a single protein-assisted biomarker for the detection of THH relative to healthy controls.

[0060] Based on the mean and standard deviation of the healthy control group, the upper limit threshold for negative results was determined to be C0 = 0.7662 NPX, and the positive threshold was determined to be C1 = 1.2038 NPX. According to this interpretation standard, all 5 THH samples were above the positive threshold, while all 3 healthy control samples were below the upper limit threshold for negative results. Therefore, the single-protein interpretation results of CAPS were consistent with the THH / healthy control grouping.

[0061] The above embodiments demonstrate that, under sample conditions, CAPS can serve as a single-protein auxiliary biomarker for THH detection relative to healthy controls, and can establish clear normal ranges, upper negative thresholds, and positive thresholds. Compared to studies that only report candidate protein names or only describe expression trends, these embodiments further provide a numerical basis for interpretation.

[0062] Figure 1This is a hierarchical clustering heatmap of differential protein expression in cerebrospinal fluid (CSF) between the THH group and the healthy control group. Each column represents a CSF sample, with THH_01 to THH_05 representing post-hemorrhagic hydrocephalus samples 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 among different proteins. Figure 1 It can be seen that the THH group and the healthy control group showed significantly different distribution patterns in the expression levels of various cerebrospinal fluid proteins. Among them, CAPS was relatively highly expressed in the THH group samples, while it was relatively lowly expressed in the healthy control group, indicating that CAPS has the application value of distinguishing THH samples from healthy control samples.

[0063] This embodiment demonstrates that a replicable, scalable, and translatable technical pathway can be formed around the single protein CAPS. Based on the expression difference of CAPS between post-hemorrhagic hydrocephalus samples and healthy control samples, and the corresponding threshold establishment method, this invention further constructs a corresponding cerebrospinal fluid detection kit.

[0064] The kit includes an antibody that specifically binds to CAPS and a CAPS recombinant protein standard. Preferably, the antibody that specifically binds to CAPS is a monoclonal antibody or a paired antibody system, wherein the monoclonal antibody is a rabbit recombinant monoclonal antibody; and in the paired antibody system, the capture antibody is 68225-2-PBS (2H7H1), and the detection antibody is 68225-3-PBS (2D7G2). The kit includes the capture antibody, the detection antibody, and the CAPS recombinant protein standard.

[0065] 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.

[0066] This invention solves three core problems of existing THH detection technologies: 1) The problem of not being able to identify it at the molecular level in the early stage: Existing imaging technology can only make a diagnosis after the ventricles are enlarged, while the biomarker combination of the present invention can capture the early molecular pathological changes (abnormal expression of cerebrospinal fluid proteins) after hemorrhage, which reflects the occurrence of the disease earlier than imaging technology, and buys time for clinical intervention. 2) The problem of insufficient biomarker specificity: The expression pattern of "single upregulation of CAPS" is specific to THH and has no cross-reaction with other neurological diseases such as cerebral infarction and encephalitis. It can accurately distinguish THH patients from non-THH people and avoid misdiagnosis. 3) The problem of insufficient sensitivity in the detection of low-abundance proteins: Leveraging the high sensitivity of OlinkPEA technology, precise quantification of key low-abundance proteins can be achieved, avoiding the omission of core molecules by traditional techniques and ensuring the integrity of the biomarker combination and the reliability of the detection results. Simultaneously, this biomarker can provide clear clues for the study of the molecular mechanisms of THH, laying the foundation for subsequent development of targeted therapeutic drugs (such as regulators of CAPS and agonists of neural repair-related proteins).

[0067] The beneficial effects of this invention are as follows: 1. The target of testing is singular and clearly defined, making it easier to develop standardized testing protocols. This invention uses CAPS as a single protein biomarker for the auxiliary detection of post-hemorrhagic hydrocephalus (THH). Compared with existing methods that mainly rely on imaging morphology such as cranial CT and MRI, or on routine, biochemical, and inflammation-related non-specific indicators of cerebrospinal fluid, this invention has a clearly defined target, a single composition, and a clear interpretation path. This is more conducive to establishing a unified sample processing procedure, quantitative detection system, reference range, and judgment threshold, thereby enabling the further development of standardized detection reagent systems and kits.

[0068] 2. CAPS has a clear ability to distinguish between THH and healthy controls. This invention included cerebrospinal fluid samples from 5 patients in the THH group and 3 healthy controls. The results showed that the NPX values ​​of CAPS in the THH group were 2.8600, 3.1442, 2.4012, 2.2756, and 1.6147, with a mean of 2.4591 and a range of 1.6147–3.1442; while the NPX values ​​of CAPS in the healthy controls were -0.4786, -0.2226, and 0.3742, with a mean of -0.1090 and a range of -0.4786–0.3742. The two groups showed a clear separation trend in CAPS expression levels, indicating that CAPS has a good ability to distinguish between THH and healthy controls. Further thresholds were established using the mean and standard deviation of the healthy control group, yielding a negative upper limit threshold C0 = 0.7662 NPX and a positive threshold C1 = 1.2038 NPX. Under this threshold system, all 5 THH samples were determined to be positive, and all 3 healthy control samples were determined to be negative.

[0069] 3. Compared to existing imaging and routine cerebrospinal fluid indicators, it can provide molecular-level auxiliary diagnostic information. Existing imaging methods primarily reflect structural changes such as ventricular enlargement and impaired cerebrospinal fluid circulation, while conventional cerebrospinal fluid indicators often reflect non-specific changes such as inflammation, hemorrhage, blood-brain barrier damage, or tissue destruction. Both approaches struggle to establish standardized molecular detection systems based on a single, clearly defined protein. This invention, by detecting the expression level of CAPS in cerebrospinal fluid, provides molecular-level auxiliary information beyond morphological assessment, thereby enhancing the ability to identify THH-related abnormalities.

[0070] 4. It has the foundation for applications extending to disease course assessment and efficacy monitoring. This invention uses the quantitative results of a single protein CAPS as output, facilitating longitudinal comparisons between different time points of the same subject. Therefore, in addition to its use as an auxiliary detection method, it also has the potential for application in disease course assessment and efficacy monitoring. Compared to imaging examinations that primarily reflect the structural state at a specific point in time, continuous detection of a single protein is more suitable for dynamic tracking before and after treatment and at different stages of the disease. Because this invention has established a complete pathway around CAPS—including sample collection, preprocessing, quantitative detection, normal value ranges, and threshold interpretation—it can further transition from relative quantification to absolute quantification, thereby improving the consistency and interpretability of dynamic monitoring.

[0071] 5. Small sample size requirement, and good compatibility between the technical approach and clinical sample conditions. This invention uses cerebrospinal fluid (CSF) as the detection sample, with a clearly defined sample source, obtainable through lumbar puncture, external ventricular drainage, or intraoperative sampling, thus conforming to practical clinical applications. The sample collection volume, pretreatment conditions, storage temperature, rewarming method, and repeatability control parameters in the detection method of this invention are all clearly defined, indicating that this scheme not only has theoretical detection significance but also practical feasibility. Compared to technical routes that require complex multi-indicator joint modeling, the scheme established by this invention around the single protein CAPS is more focused, has clearer boundaries, and is better suited to micro-volume CSF samples.

[0072] This invention provides operable sample processing conditions, detection target limitations, numerical ranges, normal value intervals, upper limit threshold for negative results, upper threshold for positive results, and result interpretation rules for this single protein. Therefore, compared with existing technical solutions that only focus on phenomenon observation, functional indications, or candidate protein listing, this invention further provides an implementable, verifiable, and transferable detection framework: using CAPS as the detection target, cerebrospinal fluid as the sample, quantitative detection as the means, and threshold interpretation as the output result.

[0073] 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. The application of human calcium phosphoprotein (CAPS) as a single protein marker for post-hemorrhagic hydrocephalus in the preparation of in vitro detection products for post-hemorrhagic hydrocephalus, characterized in that, The human calcium phosphate protein has the UniProt number Q13938, the NCBIGene ID number 828, and the RefSeq protein numbers NP_004049.3 and NP_542157.

3.

2. The application according to claim 1, characterized in that, The in vitro detection product for post-hemorrhagic hydrocephalus is used for single-protein auxiliary detection of post-hemorrhagic hydrocephalus, to indicate molecular abnormalities in cerebrospinal fluid related to post-hemorrhagic hydrocephalus, to assess the course of post-hemorrhagic hydrocephalus, or to validate post-hemorrhagic hydrocephalus detection on an independent immunological platform.

3. The application according to claim 1, characterized in that, The in vitro detection product for post-hemorrhagic hydrocephalus is a kit for the auxiliary diagnosis of post-hemorrhagic hydrocephalus.

4. An in vitro detection method for human calcium phosphoprotein (CAPS), characterized in that, Includes the following steps: S1. Collect and preprocess cerebrospinal fluid samples under aseptic conditions; S2. Human calcium phosphoprotein (CAPS) was quantitatively detected using a protein quantification method, and the detection value was obtained; and S3. Compare the detection value obtained in step S2 with a preset threshold.

5. The method according to claim 4, characterized in that, In step S1, the pretreatment includes: centrifuging the collected cerebrospinal fluid at 2°C to 8°C for 8 to 10 minutes over 0.5 to 4 hours with a centrifugal force of 800×g to 1500×g, taking the supernatant, aliquoting it, and storing it at -60°C to -80°C.

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

7. The method according to claim 4, characterized in that, In step S3, the preset threshold is established based on the expression level of human calcium phosphoprotein in a healthy control group, including a negative upper limit threshold and a positive threshold, wherein the negative upper limit threshold is the average expression level of human calcium phosphoprotein in the healthy control group plus 2 standard deviations, and the positive threshold is the average expression level of human calcium phosphoprotein in the healthy control group plus 3 standard deviations.

8. The method according to claim 4, characterized in that, In step S3, when the level of human calcium phosphoprotein is detected using Olink neighbor extension analysis (PEA) and expressed as normalized protein expression value (NPX), the upper limit threshold for negative is 0.7662 NPX and the upper limit threshold for positive is 1.2038 NPX.

9. A detection kit for post-hemorrhagic hydrocephalus, characterized in that, The kit includes an antibody that specifically binds to human calcium phosphoprotein (CAPS) and a standard of recombinant human calcium phosphoprotein (CAPS).

10. The detection kit for post-hemorrhagic hydrocephalus according to claim 10, characterized in that, The antibody is a rabbit recombinant monoclonal antibody; or the antibody is a paired antibody system, wherein the capture antibody is 68225-2-PBS (2H7H1) and the detection antibody is 68225-3-PBS (2D7G2).

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