Biomarker for ankylosing spondylitis and application thereof
By detecting the CD63+CD62LloCD10- low-density neutrophil biomarkers, the specificity and sensitivity issues in the early diagnosis and efficacy assessment of ankylosing spondylitis have been resolved, enabling early diagnosis and dynamic efficacy assessment, and improving the accuracy of diagnosis and treatment and the possibility of individualized treatment.
Patent Information
- Application Number
- CN202610637270.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-09
- Publication Date
- 2026-08-25
AI Technical Summary
Existing technologies lack highly sensitive and specific biomarkers for the early diagnosis and efficacy assessment of ankylosing spondylitis. Traditional diagnostic methods lack specificity and are lagging behind, and efficacy assessment mainly relies on subjective symptoms and non-specific inflammatory markers.
CD63+CD62LloCD10- low-density neutrophils were used as biomarkers. Their number and proportion were detected by flow cytometry and other techniques. Combined with the detection of intracellular reactive oxygen species and proteins related to the formation of extracellular traps of neutrophils, early diagnosis and efficacy assessment were provided.
It enables early, non-invasive auxiliary diagnosis of ankylosing spondylitis, can sensitively reflect treatment effects, provide objective efficacy assessment, and improve the accuracy of diagnosis and treatment and the possibility of individualized treatment.
Smart Images
Figure CN122631898A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of medical testing technology, and in particular relates to a biomarker for the auxiliary diagnosis and efficacy evaluation of ankylosing spondylitis and its application. Background Technology
[0002] Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease that primarily affects the axial joints. Currently, its clinical diagnosis mainly relies on clinical symptom assessment (such as inflammatory back pain), imaging examinations (such as X-ray or MRI), and HLA-B27 genotyping. However, these methods still face significant challenges in early diagnosis: clinical symptoms lack specificity and are highly subjective; imaging changes often appear in the middle and late stages of the disease; and while HLA-B27 positivity is associated with AS, it is not a specific diagnostic indicator and can also be seen in healthy individuals. Furthermore, routine laboratory inflammatory markers used to monitor disease activity, such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), have limited sensitivity and specificity and cannot directly reflect the underlying immunopathological mechanisms and cellular functional changes in AS.
[0003] Currently, there are significant shortcomings in clinical indicators for assessing the efficacy of AS treatment. The judgment of treatment response is mainly based on the improvement of patients' subjective symptoms, physical examination, and changes in the aforementioned non-specific inflammatory markers. These assessment methods are not only highly subjective and easily affected by individual differences, but also have a relatively delayed response, failing to sensitively reflect the biological changes in the immune response in the early stages of treatment.
[0004] Current technologies for the diagnosis and treatment management of ankylosing spondylitis (AS) face two major challenges: first, the lack of highly sensitive and specific early diagnostic biomarkers that reflect the disease's immunopathological mechanisms; and second, the lack of biological monitoring indicators that can objectively, dynamically, and sensitively assess treatment efficacy and potentially predict treatment response. To address these issues, there is an urgent need to develop a novel biomarker-based detection method that can not only assist in the early identification of AS but also provide a reliable basis for efficacy evaluation and dynamic monitoring during treatment. Summary of the Invention
[0005] The purpose of this application is to provide a biomarker for the auxiliary diagnosis and efficacy evaluation of ankylosing spondylitis and its application, aiming to solve the problem that there is currently no highly sensitive and specific biomarker for the auxiliary diagnosis and efficacy evaluation of ankylosing spondylitis in the prior art.
[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides the use of a detection reagent for ankylosing spondylitis-related biomarkers in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, wherein the biomarker is CD63+CD62LloCD10- low-density neutrophils.
[0007] In some embodiments, the product includes a kit for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of treatment for ankylosing spondylitis, wherein the kit includes antibodies for specifically detecting CD63, CD62L, and CD10, and at least one of reagents for detecting intracellular reactive oxygen species levels and antibodies for detecting proteins associated with the formation of extracellular traps of neutrophils.
[0008] In some embodiments, antibodies that specifically detect CD63 include anti-human CD63 antibodies.
[0009] In some embodiments, antibodies that specifically detect CD62L include anti-human CD62L antibodies.
[0010] In some embodiments, antibodies that specifically detect CD10 include anti-human CD10 antibodies.
[0011] In some embodiments, the reagents used to detect intracellular reactive oxygen species levels include 2',7'-dichlorodihydrofluorescein diacetate.
[0012] In some embodiments, antibodies used to detect proteins associated with neutrophil extracellular trap formation include anti-human MPO antibodies, anti-human NE antibodies, anti-human PAD4 antibodies, anti-human H3cit antibodies, and cellular DNA staining agents.
[0013] In some embodiments, flow cytometry is used to detect the number and / or proportion of CD63+CD62LloCD10- low-density neutrophils.
[0014] In some embodiments, antibodies related to proteins associated with neutrophil extracellular trap formation are detected using any one of the following detection methods: immunofluorescence, Western blotting, or flow cytometry.
[0015] In some embodiments, phenotypic characteristics include CD63 positivity, low CD62L expression, and CD10 negativity.
[0016] In some embodiments, the protein associated with the formation of extracellular traps of neutrophils is selected from at least one of citrullinated histone H3, peptidyl arginine deiminase 4, myeloperoxidase, and neutrophil elastase.
[0017] In some embodiments, when used to assist in the diagnosis of ankylosing spondylitis, the level of detected CD63+CD62LloCD10- low-density neutrophils is compared with the level of healthy controls. If the level of CD63+CD62LloCD10- low-density neutrophils in the tested individual is higher than that in healthy controls, it suggests that the tested individual has or is at risk of developing ankylosing spondylitis.
[0018] In some embodiments, when used to evaluate the therapeutic efficacy of ankylosing spondylitis, the levels of CD63+CD62LloCD10- low-density neutrophils in the biological samples of the individual being tested are measured before and after treatment, and the changes in levels before and after treatment are compared; if the level decreases after treatment, it indicates that the treatment is effective.
[0019] In some embodiments, the product includes a system for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of ankylosing spondylitis treatment, wherein the system includes: Data acquisition unit: Acquires biological samples from the individual to be tested; Data analysis unit: Detects the level of CD63+CD62LloCD10- low-density neutrophils in biological samples, including at least one of the following: number, proportion, phenotype, intracellular reactive oxygen species, and antibodies against proteins related to the formation of extracellular traps of neutrophils; Data prediction unit: Based on the data obtained from the data analysis unit, it assists in the diagnosis of ankylosing spondylitis and / or evaluates the efficacy of treatment.
[0020] The application of the detection reagent for biomarkers related to ankylosing spondylitis provided in the first aspect of this application in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, wherein the biomarker is CD63+CD62LloCD10- low-density neutrophils. Among the provided biomarkers, each cell type has a unique phenotype and is closely related to the core pathological mechanism of ankylosing spondylitis—abnormal neutrophil activation. Their number and proportion are significantly higher in ankylosing spondylitis patients than in healthy individuals, thus serving as objective and specific biomarkers. In terms of diagnosis, the biomarker can be quantitatively detected using mature technologies such as flow cytometry, enabling early and non-invasive auxiliary diagnosis and overcoming the shortcomings of traditional indicators in terms of lag and lack of specificity. In terms of efficacy evaluation, the dynamic changes in the biomarker level before and after treatment can sensitively reflect the disease control status: effective treatment is accompanied by a significant decrease in its level, while a sustained failure to decrease or an increase in the level indicates poor or ineffective treatment, which helps to achieve prospective monitoring and individualized treatment adjustments. This method has readily available samples, mature technology, and good potential for clinical translation. It not only improves the accuracy of diagnosis and treatment of ankylosing spondylitis, but also provides a new evaluation tool for related drug development, and has important clinical value and application prospects. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of this application, 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 This is a flow cytometry analysis of the number and proportion of low-density neutrophils provided in Example 1 of this application; Figure 2 This is an immunofluorescence diagram showing the ability of proportional neutrophils and low-density neutrophils to form extracellular traps (NETs) according to Example 1 of this application; Figure 3 This is a Western blot analysis diagram comparing the NET formation capacity of low-density neutrophils and normal-density neutrophils, provided in Example 1 of this application. Figure 4 This is an analysis diagram of the intracellular reactive oxygen species (ROS) levels in CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils measured by the DCFH-DA probe provided in Example 1 of this application. Figure 5 This is a graph showing the changes in the number and proportion of circulating CD63+CD62LloCD10- low-density neutrophils before and after clinical treatment in AS patients, provided in Example 2 of this application. Figure 6 This is a correlation analysis of the ESR and CD63+CD62LloCD10-LDN ratio in AS patients provided in Example 3 of this application; Figure 7 This is a correlation analysis of the CRP and CD63+CD62LloCD10-LDN ratio in AS patients provided in Example 3 of this application. Detailed Implementation
[0023] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.
[0024] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.
[0025] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.
[0026] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.
[0027] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.
[0028] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.
[0029] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.
[0030] The first aspect of this application provides the application of a detection reagent for ankylosing spondylitis-related biomarkers in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, wherein the biomarker is CD63+CD62LloCD10- low-density neutrophils.
[0031] This application presents for the first time the outstanding application value of using CD63+CD62LloCD10- low-density neutrophils as the core biomarker in the early auxiliary diagnosis and dynamic evaluation of the therapeutic efficacy of ankylosing spondylitis.
[0032] Currently, traditional diagnostic markers for ankylosing spondylitis (such as CRP and ESR) lack specificity. The CD63+CD62LloCD10- low-density neutrophil marker provided in this application is closely related to the core of AS's immunopathology—abnormal neutrophil activation and the formation of neutrophil extracellular traps.
[0033] CD63 is an activating granule membrane protein, and its high expression directly indicates neutrophil degranulation and activation status.
[0034] Low expression of CD62L (L-selectin) is a key marker for neutrophils to transition from a resting state to an activated, inflammatory phenotype, and its downregulation is associated with cell migration to inflammatory sites and continued activation.
[0035] CD10 negativity further distinguishes this group of cells from normally differentiated neutrophils, indicating that they are in a special state of differentiation or function.
[0036] The low-density property refers to the co-separation with PBMCs during density gradient centrifugation. This physical property itself is related to the "degranulation" and activation of neutrophils under inflammatory and autoimmune conditions.
[0037] Based on the above phenotypes, CD63+CD62LloCD10- low-density neutrophils represent a special subset of neutrophils that are abnormally activated, hyperfunctional, and have pro-inflammatory potential in patients with ankylosing spondylitis.
[0038] The provided biomarkers exhibit unique phenotypes and are closely related to the core pathological mechanism of ankylosing spondylitis—abnormal neutrophil activation. Their number and proportion are significantly higher in ankylosing spondylitis patients than in healthy individuals, thus serving as objective and specific biomarkers. In diagnosis, these biomarkers can be quantitatively detected using mature techniques such as flow cytometry, enabling early and non-invasive auxiliary diagnosis and overcoming the shortcomings of traditional indicators in terms of lag and lack of specificity. Regarding efficacy evaluation, the dynamic changes in biomarker levels before and after treatment can sensitively reflect disease control: effective treatment is accompanied by a significant decrease in levels, while persistent or rising levels indicate poor or ineffective treatment, facilitating prospective monitoring and individualized treatment adjustments. This method has readily available samples, mature technology, and good clinical translation potential. It not only improves the accuracy of ankylosing spondylitis diagnosis and treatment but also provides a novel evaluation tool for related drug development, demonstrating significant clinical value and application prospects.
[0039] In some embodiments, the product includes a kit for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of treatment for ankylosing spondylitis, wherein the kit includes antibodies for specifically detecting CD63, CD62L, and CD10, and at least one of reagents for detecting intracellular reactive oxygen species levels and antibodies for detecting proteins associated with the formation of extracellular traps of neutrophils.
[0040] The provided kit includes a combination of antibodies specifically targeting CD63, CD62L, and CD10, directly targeting the unique cell phenotypes identified. This ensures high specificity and accuracy, enabling precise identification and quantification of target cell subpopulations from complex blood samples, effectively avoiding false positives or false negatives. Secondly, the kit can be optionally equipped with reagents for detecting intracellular reactive oxygen species (ROS) levels and antibodies for detecting key NET formation proteins (such as H3cit, PAD4, MPO, and NE), achieving a complete assessment from phenotype to function. This not only further validates the pathological activity of this cell subpopulation but also provides in-depth information reflecting the underlying immune status of the disease, enhancing the biological significance and clinical interpretability of the test results.
[0041] In some embodiments, antibodies that specifically detect CD63 include anti-human CD63 antibodies.
[0042] In some embodiments, antibodies that specifically detect CD62L include anti-human CD62L antibodies.
[0043] In some embodiments, antibodies that specifically detect CD10 include anti-human CD10 antibodies.
[0044] In some embodiments, the reagents used to detect intracellular reactive oxygen species levels include 2',7'-dichlorodihydrofluorescein diacetate.
[0045] In some embodiments, antibodies used to detect proteins associated with neutrophil extracellular trap formation include anti-human MPO antibodies, anti-human NE antibodies, anti-human PAD4 antibodies, anti-human H3cit antibodies, and cellular DNA staining agents.
[0046] In some embodiments, flow cytometry is used to detect the number and / or proportion of CD63+CD62LloCD10- low-density neutrophils.
[0047] Flow cytometry can directly provide the absolute number or relative percentage of this cell subset. In clinical applications, the test results for the subject are simply compared with the established healthy reference range. Abnormal values provide strong auxiliary diagnostic evidence that the patient has AS or is in the active phase of the disease. Compared to subjective clinical symptom assessment, delayed imaging changes, and non-specific blood inflammatory markers, this biomarker can reflect abnormal immune cell levels and inflammatory activity in the patient's body earlier and more directly. It is expected to indicate disease risk when clinical symptoms are not obvious or serological indicators are not significantly elevated, thus enabling earlier clinical intervention.
[0048] In some embodiments, antibodies related to proteins associated with neutrophil extracellular trap formation are detected using any one of the following detection methods: immunofluorescence, Western blotting, or flow cytometry.
[0049] This study employs a variety of mature and complementary detection techniques, including immunofluorescence, Western blotting, and flow cytometry, to quantitatively analyze proteins related to neutrophil extracellular net (NET) formation. On one hand, this significantly enhances the clinical applicability and technical flexibility of the biomarker detection system. Given the different technological platforms that various medical institutions or research laboratories may possess, it can be effectively implemented and promoted in various scenarios. On the other hand, multiple methods provide pathways for mutual verification and data validation. For example, flow cytometry allows for rapid, multi-parameter semi-quantitative analysis, while Western blotting provides precise absolute confirmation of molecular weight and protein expression levels. Immunofluorescence allows for direct observation of NET formation at the cellular morphology level. Together, these methods constitute a comprehensive and reliable analytical system, greatly enhancing the scientific rigor and persuasiveness of the detection results.
[0050] In some embodiments, phenotypic characteristics include CD63 positivity, low CD62L expression, and CD10 negativity.
[0051] On the one hand, CD63 positivity indicates that cells are in a degranulation-activated state; low CD62L expression represents the precise capture of the intermediate state of cells activated from circulation and preparing to migrate to the inflammatory site, serving as a sensitive indicator of persistent inflammation; CD10 negativity effectively distinguishes them from terminally differentiated neutrophils performing routine antibacterial functions. The combination of these three factors defines a group of "pathogenic" neutrophils specifically activated in the pathological environment of ankylosing spondylitis, possessing strong pro-inflammatory potential. This precise phenotypic combination makes the detection highly targeted and specific, directly reflecting immune cell abnormalities directly related to disease development and progression, and providing a certain degree of specificity for accurate auxiliary diagnosis or efficacy analysis of ankylosing spondylitis.
[0052] In some embodiments, the protein associated with the formation of extracellular traps of neutrophils is selected from at least one of citrullinated histone H3, peptidyl arginine deiminase 4, myeloperoxidase, and neutrophil elastase.
[0053] Among the proteins associated with neutrophil extracellular net (NET) formation, citrullinated histone H3 is a core marker and key component in NET formation, and its detection directly confirms the occurrence of NET formation. Peptidyl arginine deiminoase 4 is a key rate-limiting enzyme catalyzing histone citrullination and initiating NET formation; its expression level reflects the cell's potential for NET formation. Myeloperoxidase and neutrophil elastase are potent inflammatory mediators and proteases stored in neutrophil granules, released during NET formation and participating in its structural and functional development, directly reflecting cytotoxicity and tissue damage capabilities. Selecting this group of proteins comprehensively covers the key nodes in the NET formation pathway. Detecting these proteins not only confirms that CD63+CD62LloCD10- low-density neutrophils have the "ability" to form NETs, but also assesses the "activity" and "pathogenic potential" of these NETs, greatly enhancing the depth of information and clinical guidance value of the detection results in assessing disease severity and the effectiveness of therapeutic targets.
[0054] In some embodiments, when used to assist in the diagnosis of ankylosing spondylitis, the level of detected CD63+CD62LloCD10- low-density neutrophils is compared with the level of healthy controls. If the level of CD63+CD62LloCD10- low-density neutrophils in the tested individual is higher than that in healthy controls, it suggests that the tested individual has or is at risk of developing ankylosing spondylitis.
[0055] In the early stages of disease or when symptoms are atypical, traditional indicators may not yet be abnormal, but an abnormally elevated level of this biomarker can provide an objective, immunopathologically based early warning signal, prompting clinicians to conduct more in-depth investigations. It can also provide testing instruments with an automatically comparable database and generate risk assessment reports, improving the efficiency and standardization of the diagnostic process.
[0056] In some embodiments, when used to evaluate the therapeutic efficacy of ankylosing spondylitis, the levels of CD63+CD62LloCD10- low-density neutrophils in the biological samples of the individual being tested are measured before and after treatment, and the changes in levels before and after treatment are compared; if the level decreases after treatment, it indicates that the treatment is effective.
[0057] This study provides a real-time, objective, and immunopathologically based biological response indicator for evaluating the treatment efficacy of ankylosing spondylitis, overcoming the limitations of traditional inflammatory markers (such as CRP and ESR) that primarily rely on patient subjective experiences (e.g., pain scores) and delayed responses. This biomarker sensitively reflects the inhibitory effect of therapeutic drugs on the key pathological process of abnormally activated neutrophils in the patient's body. This helps physicians determine the effectiveness of treatment plans, enabling personalized and precise treatment, while also providing real-time monitoring to provide early warning of impending clinical relapse or secondary failure.
[0058] In some embodiments, the product includes a system for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of ankylosing spondylitis treatment, wherein the system includes: Data acquisition unit: Acquires biological samples from the individual to be tested; Data analysis unit: Detects the level of CD63+CD62LloCD10- low-density neutrophils in biological samples, including at least one of the following: number, proportion, phenotype, intracellular reactive oxygen species, and antibodies against proteins related to the formation of extracellular traps of neutrophils; Data prediction unit: Based on the data obtained from the data analysis unit, it assists in the diagnosis of ankylosing spondylitis and / or evaluates the efficacy of treatment.
[0059] The data acquisition unit includes acquiring biological samples from the individual to be tested. Specifically, peripheral blood collected from the individual to be tested is used for detection.
[0060] In some specific embodiments, neutrophils were isolated using Ficoll-Paque density gradient centrifugation. Specifically, whole blood was diluted 1:1 with PBS and carefully plated into 15 mL conical centrifuge tubes containing Ficoll-Paque™ density gradient medium. The sample was centrifuged at 400 × g for 30 minutes at room temperature without using the brake. After centrifugation, low-density PBMCs and a neutrophil-rich pellet were collected. The neutrophil-rich pellet was incubated on ice with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 10 minutes to lyse residual erythrocytes. The lysis reaction was terminated by adding excess PBS, followed by centrifugation at 300 × g for 5 minutes. The purification steps were repeated as needed to obtain highly purified neutrophils.
[0061] The data analysis unit includes detecting the level of CD63+CD62LloCD10- low-density neutrophils in biological samples, including at least one of the following: number, proportion, phenotype, intracellular reactive oxygen species, and antibodies against proteins related to the formation of extracellular traps of neutrophils.
[0062] In some embodiments, flow cytometry is used to determine the number and proportion of low-density neutrophils.
[0063] The specific steps include: obtaining low-density neutrophils, washing PBMCs twice with PBS, resuspending them in 200 μL of PBS, performing cell counting, and adjusting the cell count to 1 × 10⁻⁶. 6 Add antibodies against CD66b, CD15, CD63, CD62L, and CD10, and incubate at °C (4°F) in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 100 μL PBS, and analyze using flow cytometry. CD66b+CD15+ cells were identified as low-density neutrophils, and CD63, CD62L, and CD10 were further identified. 1×10⁻⁶ cells were collected. 4 Cells were analyzed to calculate the number of CD63+CD62LloCD10- low-density neutrophils and their proportion in PBMCs.
[0064] In other embodiments, when used for efficacy assessment, it is necessary to detect changes in the number and proportion of circulating CD63+CD62LloCD10- low-density neutrophils before and after clinical treatment.
[0065] The specific steps include: after obtaining low-density neutrophils, washing PBMCs twice with PBS, resuspending them in 200 μL of PBS, counting the cells, and adjusting the cell count to 1 × 10⁻⁶. 6 Add antibodies against CD66b, CD15, CD63, CD62L, and CD10, and incubate at °C (4°F) in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 100 μL PBS, and analyze using flow cytometry. CD66b+CD15+ cells were identified as low-density neutrophils, and CD63, CD62L, and CD10 were further identified. 1×10⁻⁶ cells were collected. 4 Cells were analyzed to calculate the number of CD63+CD62LloCD10- low-density neutrophils and their proportion in PBMCs.
[0066] In some embodiments, immunofluorescence is used to measure the ratio of neutrophils and the ability of low-density neutrophil extracellular traps (NETs) to form.
[0067] The specific steps include: first, obtaining high-purity neutrophils (PBMCs) and normal-density neutrophils; then, attaching each confocal dish with 285 μL of Corning® Cell-Tak™ cell and tissue adhesive, 5 μL of 1M NaOH, and 10 μL of 1M NaHCO3 for 15 min. After 15 min, discard the adhesive. Wash the PBMCs twice with PBS, resuspend them in 200 μL of cell sorting buffer, and perform cell counting at a ratio of 1 x 102. 6Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C for 30 min in the dark. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils. Count the CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils, adjusting the cell number to 5 × 10⁻⁶ cells per 200 μL. 5 Add the sample to a confocal dish and incubate at 37°C for 12 hours. After 12 hours, discard the supernatant, add Sytox Green for staining for 15 minutes, and wash three times with PBS for 5 minutes each time. Observe under a fluorescence microscope.
[0068] In some embodiments, immunoblotting compares the NET-forming ability of low-density neutrophils and normal-density neutrophils.
[0069] The specific steps include: after obtaining low-density neutrophils and normal-density neutrophils, washing the PBMCs twice with PBS, resuspending them in 200 μL of cell sorting buffer, and counting the cells at a ratio of 1 x 10⁻⁶. 6 Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils.
[0070] Protein samples were lysed using RIPA lysis buffer with phenylmethylsulfonyl fluoride (PMSF) added to a final concentration of 1 mM. Cells or tissues were washed once with pre-chilled PBS, then lysed directly on ice with the lysis buffer added for 30 minutes, gently agitated to promote lysis, and then centrifuged at 12,000 × g for 15 minutes at 4°C. Collect the supernatant and mix it with 5×SDS-PAGE loading buffer at a ratio of 4:1. Then heat at 95°C for 10 minutes to ensure sufficient protein denaturation. Due to limited sample volume and to ensure consistent loading volume across experimental groups, protein quantification was not performed; instead, equal volumes of lysis buffer were directly loaded. Depending on the molecular weight of the target protein, SDS-PAGE electrophoresis was performed using a 7.5% or 10% polyacrylamide gel. Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane at 4°C using a wet transfer method at a constant voltage of 100 V for 90 minutes. After transfer, the membrane was blocked at room temperature for 1 hour with TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) containing 5% skim milk powder. The primary antibody was prepared using TBST buffer containing 5% skim milk powder. After BSA dilution, the membrane was incubated overnight at 4°C. Primary antibodies used included MPO, NE, PAD4, and H3cit. The membrane was washed three times with TBST and then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody: goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L). After a second wash, protein bands were visualized on a chemiluminescence imaging system using an enhanced chemiluminescence reagent.
[0071] In some embodiments, the DCFH-DA probe was used to measure the intracellular reactive oxygen species (ROS) levels in CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils.
[0072] The specific steps include: after obtaining low-density neutrophils and normal-density neutrophils, washing the PBMCs twice with PBS, resuspending them in 200 μL of cell sorting buffer, and counting the cells at a ratio of 1 x 10⁻⁶. 6Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils. Count the CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils, adjusting the cell count to 1 x 10^6 cells per ml. 6 Add 1 μL of DCFH-DA probe, incubate at 37°C for 30 min, wash three times with PBS, and then add 100 μL of PBS. Analyze using flow cytometry, select the PE channel, and compare the mean fluorescence intensity (MFI) between groups.
[0073] The data prediction unit includes the use of data obtained from the data analysis unit to assist in the diagnosis and / or evaluation of the treatment efficacy of ankylosing spondylitis.
[0074] In some embodiments, when used to assist in the diagnosis of ankylosing spondylitis, the level of detected CD63+CD62LloCD10- low-density neutrophils is compared with the level of healthy controls. If the level of CD63+CD62LloCD10- low-density neutrophils in the tested individual is higher than that in healthy controls, it suggests that the tested individual has or is at risk of developing ankylosing spondylitis.
[0075] In some embodiments, when used to evaluate the therapeutic efficacy of ankylosing spondylitis, the levels of CD63+CD62LloCD10- low-density neutrophils in the biological samples of the individual being tested are measured before and after treatment, and the changes in levels before and after treatment are compared; if the level decreases after treatment, it indicates that the treatment is effective.
[0076] The following description is based on specific embodiments.
[0077] The abbreviations in the attached figures indicate: LDNs: Low-density neutrophils; NDNs: Normal density neutrophils: AS: Ankylosing spondylitis patients; HD: Healthy individuals: AS LDN(s): Low-density neutrophils in patients with ankylosing spondylitis; AS NDN(s): Normal density neutrophils in patients with ankylosing spondylitis: HD LDN(s): Low-density neutrophils in healthy individuals; HD NDN(s): Neutrophils of normal density in healthy individuals.
[0078] Example 1 A system for assisting in the diagnosis of ankylosing spondylitis, comprising: (a) Data Acquisition Unit: Acquire biological samples from the individuals to be tested. This includes using peripheral blood collected from the individuals to be tested.
[0079] Specifically, neutrophils were isolated using the Ficoll-Paque density gradient centrifugation method. Whole blood was diluted 1:1 with PBS and carefully plated into 15 mL conical centrifuge tubes containing Ficoll-Paque™ density gradient medium. The samples were centrifuged at 400 × g for 30 minutes at room temperature without using the brakes. After centrifugation, low-density PBMCs and a neutrophil-rich pellet were collected. The neutrophil-rich pellet was incubated on ice with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 10 minutes to lyse residual erythrocytes. Excess PBS was added to terminate the lysis reaction, followed by centrifugation at 300 × g for 5 minutes. The purification steps were repeated as needed to obtain highly purified neutrophils.
[0080] (ii) Data analysis unit: Detects the level of CD63+CD62LloCD10- low-density neutrophils in biological samples. The level includes at least one of the following: number, proportion, phenotype, intracellular reactive oxygen species, and antibodies against proteins related to the formation of extracellular traps of neutrophils.
[0081] First, flow cytometry was used to determine the number and proportion of low-density neutrophils.
[0082] The specific steps include: first, obtaining high-purity neutrophils (PBMCs), washing the PBMCs twice with PBS, resuspending them in 200 μL of PBS, counting the cells, and adjusting the cell count to 1 × 10⁻⁶. 6 Add antibodies against CD66b, CD15, CD63, CD62L, and CD10, and incubate at °C (4°F) in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 100 μL PBS, and analyze using flow cytometry. CD66b+CD15+ cells were identified as low-density neutrophils, and CD63, CD62L, and CD10 were further identified. 1×10⁻⁶ cells were collected. 4Cells were analyzed to calculate the number of CD63+CD62LloCD10- low-density neutrophils and their proportion in PBMCs.
[0083] Second, the ability to form extracellular traps (NETs) of neutrophils and low-density neutrophils was assessed using immunofluorescence.
[0084] The specific steps include: first, obtaining high-purity neutrophils (PBMCs) and normal-density neutrophils; then, attaching each confocal dish with 285 μL of Corning® Cell-Tak™ cell and tissue adhesive, 5 μL of 1M NaOH, and 10 μL of 1M NaHCO3 for 15 min. After 15 min, discard the adhesive. Wash the PBMCs twice with PBS, resuspend them in 200 μL of cell sorting buffer, and perform cell counting at a ratio of 1 x 102. 6 Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C for 30 min in the dark. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils. Count the CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils, adjusting the cell number to 5 × 10⁻⁶ cells per 200 μL. 5 Add the sample to a confocal dish and incubate at 37°C for 12 hours. After 12 hours, discard the supernatant, add Sytox Green for staining for 15 minutes, and wash three times with PBS for 5 minutes each time. Observe under a fluorescence microscope.
[0085] Third, the immunoblotting was used to compare the NET formation capacity of low-density neutrophils and normal-density neutrophils.
[0086] The specific steps include: separating the obtained low-density neutrophils and normal-density neutrophils, washing the PBMCs twice with PBS, resuspending them in 200 μL of cell sorting buffer, and counting the cells at a ratio of 1 x 10⁻⁶. 6Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils.
[0087] Protein samples were lysed using RIPA lysis buffer with phenylmethylsulfonyl fluoride (PMSF) added to a final concentration of 1 mM. Cells or tissues were washed once with pre-chilled PBS, then lysed directly on ice with the lysis buffer added for 30 minutes, gently agitated to promote lysis, and then centrifuged at 12,000 × g for 15 minutes at 4°C. Collect the supernatant and mix it with 5×SDS-PAGE loading buffer at a ratio of 4:1. Then heat at 95°C for 10 minutes to ensure sufficient protein denaturation. Due to limited sample volume and to ensure consistent loading volume across experimental groups, protein quantification was not performed; instead, equal volumes of lysis buffer were directly loaded. Depending on the molecular weight of the target protein, SDS-PAGE electrophoresis was performed using a 7.5% or 10% polyacrylamide gel. Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane at 4°C using a wet transfer method at a constant voltage of 100 V for 90 minutes. After transfer, the membrane was blocked at room temperature for 1 hour with TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) containing 5% skim milk powder. The primary antibody was prepared using TBST buffer containing 5% skim milk powder. After BSA dilution, the membrane was incubated overnight at 4°C. Primary antibodies used included MPO, NE, PAD4, and H3cit. The membrane was washed three times with TBST and then incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody: goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L). After a second wash, protein bands were visualized on a chemiluminescence imaging system using an enhanced chemiluminescence reagent.
[0088] Fourth, the intracellular reactive oxygen species (ROS) levels of CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils were measured using the DCFH-DA probe.
[0089] The specific steps include: after obtaining low-density neutrophils and normal-density neutrophils, washing the PBMCs twice with PBS, resuspending them in 200 μL of cell sorting buffer, and counting the cells at a ratio of 1 x 10⁻⁶. 6 Cells: Add 1 μL of CD66b magnetic beads and incubate at °C for 30 min. Wash twice with buffer, then add 1 ml of buffer. Rinse the magnetic bead column with buffer, then add cell suspension. After all the cell suspension has been added to the centrifuge tube below, add 2 ml of buffer, squeeze out the cells, and centrifuge at 1500 rpm for 5 min to obtain low-density neutrophils. Further add CD63, CD62L, and CD10 antibodies, and incubate at °C in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 500 μL of PBS, and sort using flow cytometry to obtain CD63+CD62LloCD10- low-density neutrophils. Count the CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils, adjusting the cell count to 1 x 10^6 cells per ml. 6 Add 1 μL of DCFH-DA probe, incubate at 37°C for 30 min, wash three times with PBS, and then add 100 μL of PBS. Analyze using flow cytometry, select the PE channel, and compare the mean fluorescence intensity (MFI) between groups.
[0090] (iii) Data prediction unit: Based on the data obtained from the data analysis unit, conduct an assessment for the auxiliary diagnosis of ankylosing spondylitis.
[0091] First, flow cytometry was used to determine the number and proportion of low-density neutrophils.
[0092] Specifically, such as Figure 1 As shown, Figure 1 As shown in Figure A, the absolute number of LDNs in AS patients was significantly higher than that in HD patients. This result directly confirms the presence of an abnormally increased subset of neutrophils in the peripheral blood of patients with ankylosing spondylitis (AS) or in the pathological state studied. This subset has a low physical density and is distributed in a mononuclear cell layer after density gradient centrifugation. This significant increase suggests that the expansion of low-density neutrophils may be related to disease-associated systemic inflammation or abnormal bone marrow release, making it a potentially pathologically significant cell population. Figure 1Figure B shows the statistical results of the proportion of low-density neutrophils in a specific cell population (possibly total neutrophils or nucleated cells in the peripheral blood mononuclear cell layer), compared between ankylosing spondylitis (AS) patients and healthy donors (HD). Data showed that a total of 24 volunteers were provided, including 8 AS patients and 16 healthy individuals; the proportion of low-density neutrophils was significantly higher in the AS patient group than in the healthy donor group. This finding is crucial, indicating that the increase in low-density neutrophils is not merely a general increase in the total number of neutrophils, but a specific change in their relative proportion. This specific increase in proportion further supports low-density neutrophils as a specific biomarker of ankylosing spondylitis-related immune abnormalities, better distinguishing between disease and health states, and possessing potential for auxiliary diagnosis.
[0093] Second, the ability of normal neutrophils and low-density neutrophils to form extracellular trapping networks (NETs) was analyzed.
[0094] Specifically, such as Figure 2 As shown, according to Figure 2 Figure A shows a qualitative comparison of NET formation between low-density neutrophils (LDNs) from AS patients and healthy HD donors. The figure clearly indicates that the NET structures formed by low-density neutrophils (ASLDNs) from AS patients are the most abundant and prominent. The order of NET formation capacity can be inferred as: AS LDNs > HD LDNs > AS NDNs ≈ HD NDNs. This directly demonstrates that low-density neutrophils, especially those derived from AS patients, have a stronger tendency towards NETosis (NET formation). Figure 2 As shown in Figure B, the MFI value of the AS-low-density neutrophil group was the highest, significantly higher than the other three groups, indicating that the AS-low-density neutrophil group had the strongest NETs-forming ability. In healthy donors, the MFI of HD-low-density neutrophils was also higher than that of HD-NDNs, suggesting that even in the absence of obvious disease, low-density neutrophils naturally possess a stronger NETs-forming potential than NDNs. However, the number of these cells is extremely low in HD donors, while the number and proportion of LDNs are significantly increased in AS patients, and the AS disease state greatly amplifies this difference.
[0095] It can be seen that AS patients have an increased number and proportion of low-density neutrophils, and these cells produce NETs more strongly than NDNs. This may be a key cellular mechanism driving chronic inflammation and tissue destruction in AS joints and entheses. High levels of NETs can release large amounts of citrullinated proteins, proteases, and antimicrobial peptides, directly damaging tissues and acting as autoantigens to induce and maintain autoimmune responses.
[0096] Third, we analyzed the immunoblotting to compare the NET formation capabilities of low-density neutrophils and normal-density neutrophils.
[0097] Specifically, such as Figure 3 As shown, Figure 3 As shown in Figure A, myeloperoxidase (MPO) expression was highest in low-density neutrophils (AS-LDN) from AS patients, significantly higher than in NDNs from AS patients (AS-NDN) and healthy donors (HD-NDN). This indicates that low-density neutrophils from AS patients possess a richer reserve of granular contents and a stronger potential for oxidative bursts and tissue damage. Figure 3 As shown in Figure B, the relative expression level of neutrophil elastase (NE) was significantly upregulated in AS-low-density neutrophils, also higher than in AS-NDN and HD-NDN. High levels of NE expression are directly associated with stronger proteolytic activity and cytotoxicity of NETs. Figure 3 As shown in Figure C, the expression level of PAD4 (peptidyl arginine deiminase 4) directly determines the potential for NETosis in cells. Data shows that the expression level of PAD4 in AS-low-density neutrophils is significantly higher than in the AS-NDN and HD-NDN groups. This mechanistically explains why low-density neutrophils in AS patients exhibit the strongest tendency for NET formation—because they express the highest levels of this "starter switch." Figure 3 As shown in Figure D, citrullinated histone (H3cit) expression was most prominent in AS-low-density neutrophils. This not only confirms the functional activity of PAD4 in low-density neutrophils, but also directly demonstrates that the low-density neutrophils of AS patients are in an active NETosis state or have a very high NETosis readiness state.
[0098] Immunoblot analysis provided decisive evidence at the molecular mechanism level: CD63+CD62LloCD10- low-density neutrophils, especially in patients with ankylosing spondylitis, acquired abnormally enhanced NETs formation capacity by specifically upregulating the expression of PAD4, MPO, and NE and producing H3cit.
[0099] Fourth, the DCFH-DA probe was used to measure the intracellular reactive oxygen species (ROS) levels in CD63+CD62LloCD10- low-density neutrophils and normal-density neutrophils.
[0100] Specifically, such as Figure 4As shown, compared to AS-NDNs and HD-NDNs, AS-low-density neutrophils exhibited the highest fluorescence intensity, indicating the highest level of intracellular reactive oxygen species (ROS). This suggests that this cell subpopulation, originating from patients with ankylosing spondylitis, is in an abnormal, high-level state of oxidative stress. This is not only a key metabolic characteristic of cell activation and hyperfunction (such as enhanced NETosis), but also directly related to its tissue damage potential. This finding further demonstrates the scientific basis for low-density neutrophils as a biomarker for the diagnosis and treatment assessment of AS from a cellular metabolic perspective, and provides a basis for its application in monitoring the efficacy of targeted therapies for oxidative stress.
[0101] In this embodiment, when assisting in the diagnosis of ankylosing spondylitis, the level of detected CD63+CD62LloCD10- low-density neutrophils is compared with that of healthy controls. If the number of CD63+CD62LloCD10- low-density neutrophils and their proportion of PBMCs in the tested individual are higher than those in healthy controls, it suggests that the tested individual has or is at risk of developing ankylosing spondylitis.
[0102] Example 2 The system for evaluating the therapeutic efficacy of ankylosing spondylitis, compared with Example 1, differs in the following in the data analysis unit (II): it requires detecting changes in the number and proportion of circulating CD63+CD62LloCD10- low-density neutrophils before and after clinical treatment. The specific steps include: after obtaining PBMCs, washing them twice with PBS, resuspending them in 200 μL of PBS, performing cell counting, and adjusting the cell number to 1 × 10⁻⁶. 6 Add antibodies against CD66b, CD15, CD63, CD62L, and CD10, and incubate at °C (4°F) in the dark for 30 min. After incubation, wash twice with PBS, resuspend in 100 μL PBS, and analyze using flow cytometry. CD66b+CD15+ cells were identified as low-density neutrophils, and CD63, CD62L, and CD10 were further identified. 1×10⁻⁶ cells were collected. 4 Cells were analyzed to calculate the number of CD63+CD62LloCD10- low-density neutrophils and their proportion in PBMCs.
[0103] Furthermore, in the (iii) data prediction unit: based on the data obtained from the data analysis unit, an assessment is conducted for the auxiliary diagnosis of ankylosing spondylitis.
[0104] according to Figure 5 The study analyzed the changes in the number and proportion of circulating CD63+CD62LloCD10- low-density neutrophils before and after clinical treatment in patients.
[0105] It can be seen that, Figure 5 A shows the absolute number of low-density neutrophils before and after treatment (unit: ×10). 5 The comparison clearly shows that the absolute number of low-density neutrophils after treatment was significantly reduced compared to before treatment. When assessing the efficacy of treatment for ankylosing spondylitis, the levels of CD63+CD62LloCD10- low-density neutrophils in the biological samples of the tested individuals were measured before and after treatment, and the changes in levels before and after treatment were compared; a decrease in levels after treatment indicated effective treatment. The decrease in absolute numbers means that successful treatment not only alleviated clinical symptoms but also directly led to a substantial reduction in this abnormally activated, hyperfunctional pathogenic neutrophil subset in the peripheral blood. This reflects the correction of the patient's immune disorder by treatment at the cellular level. Figure 5 Figure B shows a comparison of the percentage of low-density neutrophils before and after treatment. The results also show a highly significant decrease in the relative proportion of low-density neutrophils after treatment compared to before treatment. This decrease in proportion is more telling than the change in absolute number. It indicates that after treatment, not only did the number of low-density neutrophils decrease, but their composition in the cell population also returned to a normal trend. This suggests that the treatment may more specifically target or influence the pathological pathways leading to abnormal proliferation and activation of low-density neutrophils, rather than simply reducing neutrophils generally.
[0106] Example 3 Correlation analysis of CD63+CD62LloCD10-LDN ratio with ankylosing spondylitis (AS) Specific steps: Step 1: Erythrocyte sedimentation rate (ESR) detection Peripheral blood was collected from patients with ankylosing spondylitis (AS). 0.4 ml of erythrocyte sedimentation rate (ESR) diluent was added to a test tube, followed by venous blood collection. 1.6 ml of blood was added to the test tube and mixed thoroughly immediately. The liquid was aspirated to the "0" mark using a Westergren ESR tube. Any blood adhering to the tube tip was wiped away, and the tube was placed upright on an ESR rack. The tube was incubated at room temperature for 1 hour, and the erythrocyte sedimentation rate (ESR) in mm was observed. ESR data were recorded for both AS and hepatic degeneration (HD) patients.
[0107] Step 2: Serum C-Reactive Protein (CRP) Detection Peripheral blood was collected from AS patients. Whole blood was left at room temperature for 30 minutes to 2 hours until natural coagulation and serum separation. The blood was then centrifuged at approximately 1000g for 10 minutes at 4°C, and the yellow supernatant was collected as serum. The prepared serum was kept on ice until use. Samples or standards of different concentrations were added at 100 μl / well to the corresponding wells of a 96-well plate. The wells were sealed with a clear sealing film and incubated at room temperature for 120 minutes. The plate was washed 5 times, and the last wash was patted dry on thick absorbent paper. 100 μl of biotinylated antibody was added to each well. The wells were sealed with a clear sealing film and incubated at room temperature for 60 minutes. The plate was washed 5 times, and the last wash was patted dry on thick absorbent paper. 100 μl of horseradish peroxidase-labeled streptavidin was added to each well. The wells were sealed with a white sealing film and incubated at room temperature in the dark for 20 minutes. The plate was washed 5 times, and the last wash was patted dry on thick absorbent paper. Add 100 μl of TMB solution as a colorimetric reagent per well, seal the wells with white sealing film, and incubate at room temperature in the dark for 15-20 minutes. Add 50 μl of stop solution per well, mix well, and immediately measure A. 450 Values. Record CRP data for patients with AS and HD.
[0108] Step 3: Determination of the proportion of CD63+CD62LloCD10-LDNs in peripheral blood PBMCs CD63+CD62LloCD10-LDNs were obtained as described above. These were then added to PBMCs and thoroughly mixed. The proportion of CD63+CD62LloCD10-LDNs in the PBMCs was determined by flow cytometry. The proportion of CD63+CD62LloCD10-LDNs in AS and HD patients was recorded. ESR, CRP, and other parameters were analyzed using a GraphPad Prism 10. The data on the proportion of CD63+CD62LloCD10-LDNs were integrated to obtain the correlation analysis between ESR and the proportion of CD63+CD62LloCD10-LDNs in AS patients. Figure 6 Correlation analysis of CRP and the proportion of CD63+CD62LloCD10-LDN in AS patients ( Figure 7 It can be seen that ESR and CRP in AS patients are highly correlated with the proportion of CD63+CD62LloCD10-LDN.
[0109] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. The application of a detection reagent for biomarkers associated with ankylosing spondylitis in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The biomarker is CD63+CD62LloCD10- low-density neutrophils.
2. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 1 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The product includes a kit for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of ankylosing spondylitis treatment, wherein the kit includes antibodies for specifically detecting CD63, CD62L and CD10, and at least one of the following: reagents for detecting intracellular reactive oxygen species levels and antibodies for detecting proteins related to the formation of extracellular traps of neutrophils.
3. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 2 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, Antibodies that specifically detect CD63 include anti-human CD63 antibodies; and / or, Antibodies that specifically detect CD62L include anti-human CD62L antibodies; and / or, Antibodies that specifically detect CD10 include anti-human CD10 antibodies; and / or, Reagents used to detect intracellular reactive oxygen species levels include 2',7'-dichlorodihydrofluorescein diacetate; and / or, Antibodies used to detect proteins associated with neutrophil extracellular trap formation include anti-human MPO antibodies, anti-human NE antibodies, anti-human PAD4 antibodies, anti-human H3cit antibodies, and cellular DNA staining agents.
4. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 3 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The number and / or proportion of CD63+CD62LloCD10- low-density neutrophils were detected by flow cytometry.
5. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 3 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The antibodies associated with the formation of extracellular traps of neutrophils were detected using any one of the following methods: immunofluorescence, Western blotting, or flow cytometry.
6. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 2 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The phenotypic characteristics include CD63 positivity, low CD62L expression, and CD10 negativity.
7. The use of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 2 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The protein associated with the formation of the extracellular trap of neutrophils is selected from at least one of citrullinated histone H3, peptidyl arginine deiminase 4, myeloperoxidase, and neutrophil elastase.
8. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 2 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, When used to assist in the diagnosis of ankylosing spondylitis, the level of detected CD63+CD62LloCD10- low-density neutrophils is compared with the level of healthy controls. If the level of CD63+CD62LloCD10- low-density neutrophils in the tested individual is higher than that in the healthy controls, it suggests that the tested individual has or is at risk of developing ankylosing spondylitis.
9. The application of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 2 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, When used to evaluate the efficacy of treatment for ankylosing spondylitis, the levels of CD63+CD62LloCD10- low-density neutrophils in the biological samples of the tested individuals were measured before and after treatment, and the changes in levels before and after treatment were compared; if the levels decreased after treatment, it indicated that the treatment was effective.
10. The use of the detection reagent for biomarkers related to ankylosing spondylitis according to claim 1 in the preparation of products for early diagnosis and / or efficacy evaluation of ankylosing spondylitis, characterized in that, The product includes a system for assisting in the diagnosis of ankylosing spondylitis and / or for evaluating the efficacy of ankylosing spondylitis treatment, wherein the system includes: Data acquisition unit: Acquires biological samples from the individual to be tested; Data analysis unit: Detects the level of CD63+CD62LloCD10- low-density neutrophils in the biological sample, the level including at least one of the following: number, proportion, phenotype, intracellular reactive oxygen species, and antibodies against proteins related to the formation of extracellular traps of neutrophils; Data prediction unit: Based on the data obtained from the data analysis unit, it assists in the diagnosis of ankylosing spondylitis and / or evaluates the efficacy of treatment.