Application of reagents for detecting P-selectin in the preparation of products for the auxiliary diagnosis of glaucoma

Abstract

This invention discloses the application of reagents for detecting P-selectin in the preparation of products for auxiliary diagnosis of glaucoma, relating to the field of auxiliary diagnostic technology for glaucoma. Compared with healthy controls, glaucoma patients show significantly elevated plasma P-selectin levels, and similarly, patients with primary open-angle and primary angle-closure glaucoma also exhibit significantly elevated P-selectin levels. The plasma P-selectin levels of patients with different disease severities are significantly higher than those of healthy controls. P-selectin concentration demonstrates good differential diagnostic efficacy between healthy controls and glaucoma patients, providing a new option for auxiliary diagnosis of glaucoma.

Description

Technical Field

[0001] This invention relates to the field of glaucoma auxiliary diagnostic technology, and more specifically, to the application of reagents for detecting P-selectin in the preparation of products for auxiliary diagnosis of glaucoma. Background Technology

[0002] Glaucoma is a complex, multifactorial neurodegenerative disease characterized by progressive degeneration of retinal ganglion cells, and is one of the leading causes of irreversible blindness worldwide. Research data shows that approximately 80 million people worldwide were affected by this disease in 2021, and this number is projected to exceed 100 million by 2040. Although elevated intraocular pressure (IOP) is widely recognized as the most important risk factor, a portion of patients still have IOP within the normal range. This indicates that other pathogenic mechanisms exist besides intraocular pressure. The retina has traditionally been considered an immune-immune organ, but this view has been increasingly challenged in recent years. Activation of the peripheral immune system has been proven to be a crucial factor in the pathogenesis of glaucoma, with both innate and adaptive immune components involved in this process.

[0003] P-selectin is an adhesion molecule stored in Weibel-Palade bodies of vascular endothelial cells. Upon inflammatory stimulation, it rapidly translocates to the cell surface, triggering leukocyte rolling and adhesion onto endothelial cells, constituting a crucial first step in transendothelial migration (TEM). Previous studies have shown that during acute cerebral ischemia / reperfusion, P-selectin is rapidly upregulated on the surface of brain microvascular endothelial cells, mediating neutrophil rolling, firm adhesion, and subsequent trans-BBB migration. In Alzheimer's disease (AD), β-amyloid (Aβ) peptide activates endothelial cells, promoting rapid surface expression of P-selectin and facilitating the entry of peripheral leukocytes into the brain parenchyma. These infiltrating cells can activate microglia, forming a positive feedback loop of inflammation and accelerating the pathological progression of AD.

[0004] Currently, no studies have explored whether P-selectin plays a role in the pathogenesis of glaucoma.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of reagents for detecting P-selectin in the preparation of products for the auxiliary diagnosis of glaucoma, so as to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides the application of a reagent for detecting P-selectin in the preparation of products for the auxiliary diagnosis of glaucoma.

[0008] Secondly, the present invention provides the application of a reagent for detecting P-selectin in the preparation of products for the auxiliary diagnosis of patients with intermediate or severe glaucoma.

[0009] Thirdly, the present invention also provides an apparatus for auxiliary diagnosis of glaucoma, comprising: an input module, a control module, and an output module, wherein the input module is configured to: input the content of P-selectin in a subject sample; the control module is configured to: obtain the risk or probability of the tested sample having glaucoma based on the data from the input module; and the output module is configured to: output the risk or probability of the tested sample having glaucoma.

[0010] The present invention has the following beneficial effects: This invention discovers P-selectin, an auxiliary diagnostic biomarker for glaucoma. Compared with healthy controls, glaucoma patients show significantly elevated plasma P-selectin levels, and this elevation is also observed in patients with primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG). Patients with different disease severities (early, moderate, severe, and terminal stages) exhibit significantly elevated P-selectin levels compared to healthy controls. Testing shows that plasma P-selectin concentration has moderate to good differential diagnostic efficacy in distinguishing healthy controls from glaucoma patients; particularly for patients with intermediate and severe glaucoma damage, it exhibits higher differential diagnostic efficacy.

[0011] Therefore, this invention provides a new option for the auxiliary diagnosis of glaucoma. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Plasma P-selectin levels in glaucoma patients. (A) Elevated plasma P-selectin levels in glaucoma patients; (B) Comparison of plasma P-selectin levels in healthy controls (HC), primary angle-closure glaucoma (PACG), and primary open-angle glaucoma (POAG) subgroups. The Mann-Whitney U test was used for intergroup comparisons, and the Kruskal-Wallis test was used to determine differences between PACG, POAG, and healthy controls. p<0.001; Figure 2 To investigate the correlation between plasma P-selectin levels and glaucoma severity and optic nerve damage. (A) Plasma P-selectin levels in different glaucoma patient groups, stratified by disease severity using the Advanced Glaucoma Intervention Study (AGIS) stage. (B) Plasma P-selectin levels stratified by retinal nerve fiber layer (RNFL) thickness. (C) Receiver operating characteristic (ROC) curve analysis was used to evaluate the ability of plasma P-selectin to distinguish patients with different disease severity in the AGIS stage and to differentiate between healthy and glaucoma patients. Differences in all parameters were assessed using an unpaired Wilcoxon rank-sum test. Significance is expressed as **p<0.01, ***p<0.001; Figure 3 The results of the validation study show the ability of plasma P-selectin levels to differentiate between healthy individuals and glaucoma patients. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0015] The term “subject” as used in this article can be understood as anyone involved in glaucoma-assisted diagnosis.

[0016] In a first aspect, the present invention provides the application of a reagent for detecting P-selectin in the preparation of products for the auxiliary diagnosis of glaucoma.

[0017] This invention has found that there are significant differences in plasma P-selectin levels between healthy controls and glaucoma patients, and that the levels of plasma P-selectin also differ significantly between healthy controls and glaucoma patients of different severities.

[0018] P-selectin exhibits extremely high diagnostic efficacy in distinguishing healthy samples from glaucoma patients, with an AUC value exceeding 0.697 on the ROC curve. Plasma P-selectin also demonstrates good discriminative ability for glaucoma of varying severity. Therefore, this invention provides a novel detection method for the auxiliary diagnosis of glaucoma.

[0019] In a preferred embodiment of the present invention, the reagent for detecting P-selectin is selected from reagents for detecting P-selectin content.

[0020] In a preferred embodiment of the present invention, the reagent for detecting P-selectin is selected from reagents used for detecting the content of P-selectin in plasma.

[0021] In a preferred embodiment of the present invention, the reagent used to detect the content of P-selectin in plasma is selected from antibodies against P-selectin carrying detectable markers.

[0022] In a preferred embodiment of the present invention, the detectable marker is selected from biotin, chemiluminescent reagents, or radioisotopes.

[0023] In a preferred embodiment of the present invention, the product used to assist in the diagnosis of glaucoma is selected from reagent kits.

[0024] In a preferred embodiment of the present invention, the kit further includes a reagent that can bind to biotin; the biotin-binding reagent is selected from conjugates of enzymes that can catalyze substrate color development and avidin.

[0025] In a preferred embodiment of the present invention, the kit further includes at least one of the following substances: a solid support, a positive control, a negative control, a standard, a washing solution, and a substrate.

[0026] The solid support may be selected from, for example, porous plates, but is not limited thereto.

[0027] Anti-P-selectin antibodies containing biotin can specifically bind to P-selectin in the sample to form antibody-antigen conjugates. Utilizing the property that enzymes that catalyze substrate color development and avidin conjugates can bind to biotin on the antibody, an enzyme-avidin conjugate-antibody-antigen complex is obtained. Then, the substrate corresponding to the enzyme is added, and after a colorimetric reaction, it is detected on equipment such as an ELISA reader. The content of P-selectin in the sample can be obtained based on the relationship between fluorescence intensity and P-selectin content (standard curve).

[0028] In a preferred embodiment of the present invention, the enzyme capable of catalyzing the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, or glucose-6-phosphate dehydrogenase.

[0029] The reagent used to detect the P-selectin content in plasma is selected from horseradish peroxidase and streptavidin conjugate.

[0030] Secondly, the present invention provides the application of a reagent for detecting P-selectin in the preparation of products for the auxiliary diagnosis of patients with severe glaucoma.

[0031] After testing, P-selectin showed higher diagnostic efficacy for patients with intermediate and severe glaucoma damage.

[0032] Patients with intermediate and severe glaucoma damage are classified according to the AGIS disease staging system, which divides glaucoma into four subgroups (early, intermediate, severe, and terminal).

[0033] Thirdly, the present invention also provides an apparatus for auxiliary diagnosis of glaucoma, comprising: an input module, a control module, and an output module, wherein the input module is configured to: input the content of P-selectin in a subject sample; the control module is configured to: obtain the risk or probability of the tested sample having glaucoma based on the data from the input module; and the output module is configured to: output the risk or probability of the tested sample having glaucoma.

[0034] In a preferred embodiment of the present invention, the control module is configured to compare the content of P-selectin in the subject sample with a preset threshold. If the content is greater than the preset threshold, the risk of the tested sample having glaucoma is assessed; if the content is less than the preset threshold, the risk of the tested sample not having glaucoma is assessed. For example, the preset threshold is 20.65 ng / ml.

[0035] The preset threshold refers to the threshold used when diagnosing disease risk. It is used to directly compare the content of the marker in the subject's sample with the preset threshold, and output the subject's disease risk or disease course based on the comparison result.

[0036] Specifically, the preset thresholds include, but are not limited to, positive judgment values, predicted probability thresholds, and other critical values ​​that can be used to divide results and clarify judgment boundaries. The setting of these thresholds should be based on the performance requirements of the target detection scenario (such as sensitivity, specificity, accuracy, etc.) and determined through clinical data validation, statistical model analysis (such as ROC curve analysis, Youden index calculation, etc.) or industry standard calibration, so as to ensure their validity and reliability in the detection method or diagnostic model.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 This embodiment tests the plasma P-selectin concentration levels in patients and healthy controls.

[0039] (a) Sample collection This embodiment recruits glaucoma patients and matched healthy controls, and collects blood samples, as detailed below.

[0040] This study included 219 participants, comprising 125 patients diagnosed with glaucoma and 94 healthy volunteers. The study received ethical approval from the institution's institutional review committee (approval number: 201968), and all procedures were conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from each participant regarding the use of clinical information. Glaucoma diagnosis was performed by experienced ophthalmologists through a comprehensive eye assessment, taking into account patient age, family history, and typical clinical presentations. Participants underwent multiple ophthalmic examinations: intraocular pressure measurement, retinal nerve fiber layer (RNFL) thickness measurement, and visual field (VF) analysis. Intraocular pressure was recorded using a Goldmann applanation tonometer, RNFL thickness was measured using optical coherence tomography (OCT), and VF was located using a standard automated perimeter. Inclusion criteria required a confirmed diagnosis of primary glaucoma. Exclusion criteria included: secondary glaucoma, a history of ophthalmic surgery within the past 6 months, or coexisting autoimmune diseases, inflammatory diseases, or neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Disease severity was graded using the Advanced Glaucoma Intervention Study (AGIS) scale. Ninety-four age- and sex-matched healthy volunteers served as the control group; participants (or their first-degree relatives) with glaucoma, eye discomfort, IOP > 21 mmHg, recent eye surgery, any autoimmune / inflammatory disease or neurological disorder, or currently taking immunomodulatory drugs were excluded.

[0041]

[0042] Table 1 lists a total of 123 patients with mild / moderate / severe / terminal glaucoma. Two patients did not provide visual field test reports and were therefore not included in the mild / moderate / severe / terminal grouping.

[0043] Age is expressed as mean ± SD. Abbreviations: AGIS: Advanced Glaucoma Intervention Study Score; POAG (Primary Open-Angle Glaucoma); PACG (Primary Angle-Closure Glaucoma). Age differences between glaucoma patients and healthy controls were analyzed using a two-tailed unpaired Student's t-test, and gender differences were analyzed using a chi-square test. P < 0.05 was considered statistically significant.

[0044] After fasting for 8 hours in the morning, venous blood was collected from each subject in an EDTA anticoagulant vacuum container. The blood collection tubes were centrifuged at 3000 rpm, and the supernatant was immediately frozen in an ultra-low temperature freezer at -80°C until analysis.

[0045] (ii) Plasma P-selectin was measured using a P-selectin ELISA kit.

[0046] P-selectin ELISA kit (Catalog No.: EH3818, Wuhan Feien).

[0047] The detection method is as follows: 1. Preparation of washing solution Take 720ml of ultrapure water into a 1000ml narrow-mouth bottle, pour 30ml of concentrated washing solution (25 times volume dilution) into the narrow-mouth bottle, mix thoroughly to obtain the 1× washing solution required for the experiment, and let it stand for later use.

[0048] 2. Preparation of Standard Products 2.1 Place the lyophilized standard tubes (kit catalog number: EH3818, Wuhan Feien) into a pre-chilled centrifuge and centrifuge at 10000 rpm, 4℃, for 1 min. Add 1 ml of the standard & sample diluent from the kit to the standard tubes, let stand at room temperature for 2 min, and mix thoroughly.

[0049] 2.2 Take seven sterile EP tubes and label them 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, and blank, respectively. Add 300 μl of standard solution and sample diluent to each EP tube. Pipette 300 μl of standard solution into the 1 / 2 EP tube and mix thoroughly. Then, pipette 300 μl of standard solution from the already mixed 1 / 2 EP tube and add it to the 1 / 4 EP tube, mixing thoroughly. Continue this process. The EP tube labeled blank contains only standard solution and sample diluent.

[0050] 3. Preparation of biotinylated antibody working solution 3.1 Prepare and use immediately. Calculate the required working fluid volume based on the number of samples to be tested. The calculation method is (number of samples tested + 3) × 100 μl.

[0051] 3.2 Dilute the antibody with antibody diluent at a ratio of 1 / 100 and mix thoroughly. (For example, add 10 μl of biotinylated antibody to 990 μl of biotinylated antibody diluent).

[0052] 4. Preparation of HRP-streptavidin conjugate (SABC) Prepare and use immediately. Calculate the required volume of working solution for this experiment based on the sample size. Refer to step 3 above for the preparation method of biotin antibody working solution.

[0053] 5. Measurement Procedure 5.1 Remove the samples and kits 20 minutes before the experiment and allow them to equilibrate to room temperature (18-25℃). Remove the ELISA plates according to the number of samples to be tested. If there are any remaining ELISA plates, seal them and store them at -20℃.

[0054] 5.2 Sample Addition: Add 100 μl of appropriately diluted sample and 100 μl of standard to the corresponding wells, and add 100 μl of sample diluent to the blank wells. Cover with a membrane and incubate at 37°C for 90 min. Avoid blowing air bubbles and prevent the pipette tip from touching the well wall during sample addition.

[0055] 5.3 Washing and Adding Biotinylated Antibody: Prepare the biotinylated antibody working solution 10 minutes in advance. After the time is up, remove the membrane, remove the liquid from the wells, and add 350 μL of the prepared washing buffer to each well using a multipipe. Do not soak the plate; shake off the washing buffer and gently tap the plate on absorbent paper 2-3 times. Repeat the washing once. Then, add 100 μL of the prepared biotinylated antibody working solution to each well, reattach the membrane, and incubate at 37°C for 60 minutes.

[0056] 5.4 Washing and Adding SABC: Prepare the HRP-Streptavidin Conjugate (SABC) working solution 10 minutes in advance. After the time is up, remove the membrane, remove the liquid from the wells, tap the wells 2-3 times with medium pressure on absorbent paper, add 350 μL of washing buffer to the wells using a multipipeline, let stand for 1 minute, shake off the washing buffer, and tap the wells 2-3 times with medium pressure on absorbent paper. Add another 350 μL of washing buffer, repeating 3 times. Finally, add 100 μL of HRP-Streptavidin Conjugate (SABC) working solution to the wells, cover with a membrane, and incubate at 37°C for 30 minutes.

[0057] 5.5 Washing and Adding TMB: Remove the membrane and wash the plate 5 times following the procedure in 5.4. Then add 90 μl of TMB substrate to each well, reattach the membrane, and incubate in a 37°C incubator in the dark for approximately 15 minutes. The reaction time can be shortened or extended depending on the actual color development, but should not exceed 30 minutes. Terminate the reaction when a good blue gradient is observed in the standard.

[0058] 5.6 Add reaction termination solution: Add 50 μl of reaction termination solution. The color will change from blue to yellow. Note that the order of adding the termination solution should be the same as the order of adding the TMB substrate solution.

[0059] 5.7 OD value determination: Preheat the microplate reader 15 minutes in advance. After termination, immediately measure the absorbance at 450 nm using the microplate reader and read the OD value.

[0060] 5.8 Concentration Calculation: Based on the concentration and OD value of the standard, the standard curve is calculated using Curve Expert software, and then the OD450 value of the sample is substituted into the concentration calculation.

[0061] The plasma P-selectin concentration levels in patients and healthy controls can be measured by following the steps described above.

[0062] Plasma P-selectin levels were measured using a commercially available P-selectin ELISA kit (catalog number: EH3818, Wuhan Feien). Statistical analysis was performed using R language (version 4.1.3). Differences in P-selectin expression between glaucoma patients and healthy controls were compared. Compared with healthy controls, glaucoma patients showed a statistically significant increase in plasma P-selectin levels (…). Figure 1 (See Figure A). Among the subtypes of glaucoma, primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG) are the most common.

[0063] Therefore, plasma P-selectin levels in the PACG and POAG groups were compared with those in the healthy control group. Compared with the control group, the median plasma P-selectin levels in both the PACG and POAG groups were significantly increased. However, there was no significant difference in plasma P-selectin levels between the POAG and PACG groups. Figure 1 (B)

[0064] Example 2 This embodiment tests the relationship between plasma P-selectin levels and glaucoma nerve damage and glaucoma severity.

[0065] To assess the relationship between P-selectin levels and the staging of glaucoma neurodegenerative disease, we simultaneously measured structural and functional parameters. Plasma P-selectin levels in glaucoma patients with different disease severities were compared using the AGIS disease staging system. Patients were divided into four subgroups (early, moderate, severe, and terminal stages) according to the AGIS staging system. Results showed that glaucoma severity was associated with elevated P-selectin levels, and P-selectin levels in patients with different disease severities were significantly different from those in healthy controls. Figure 2 (A)

[0066] At the same time, patients were grouped according to the thickness of their RNFL (retinal nerve fiber layer) to examine the correlation between plasma P-selectin levels and optic nerve damage.

[0067] Optical coherence tomography (OCT): OCT is a non-invasive imaging technique that uses low-coherence light to perform high-resolution cross-sectional imaging of the retina, directly measuring RNFL thickness. Due to its objective, quantitative, and repeatable characteristics, OCT has become a core tool for diagnosing and monitoring optic nerve diseases such as glaucoma.

[0068] It was found that thinning of RNFL was associated with increased P-selectin levels. Figure 2 (B). However, P-selectin levels are not affected by IOP (intraocular pressure), age, or sex.

[0069] The above results confirm that plasma P-selectin is associated with optic nerve damage in glaucoma and the severity of glaucoma.

[0070] Furthermore, the area under the receiver operating characteristic (ROC) curve was used to evaluate the ability of P-selectin to distinguish between healthy controls and glaucoma patients in the sample set of Example 1. Figure 2 The threshold for distinguishing between healthy individuals and glaucoma patients was 20.65 ng / ml, and the AUC value of the ROC curve for distinguishing between healthy individuals and glaucoma patients was 0.697. Plasma P-selectin concentration had moderate to good differential efficacy in distinguishing between healthy controls and patients at different stages of glaucoma injury. After stratification according to AGIS classification, the area under the curve (AUC) values ​​were: early stage 0.694, intermediate stage 0.711, severe stage 0.773, and terminal stage 0.693. Figure 2 (C)

[0071] Example 3 This embodiment tests the diagnostic efficacy of plasma P-selectin levels in the validation set.

[0072] The validation study included a healthy control group (27 cases) and glaucoma patients (45 cases). The AUC value of the ROC curve was 0.731. (See details below.) Figure 3 .

[0073] In summary, this invention provides the first evidence of differential expression of plasma P-selectin in healthy controls, glaucoma, and glaucoma of varying severities. Plasma P-selectin demonstrates good discriminative ability for different severities of glaucoma. This suggests that plasma P-selectin can be used as an adjunct to the diagnosis of glaucoma and its severity, providing a novel detection method for this purpose.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting P-selectin in the preparation of products for the auxiliary diagnosis of glaucoma.

2. The application according to claim 1, characterized in that, The reagent for detecting P-selectin is selected from reagents for detecting P-selectin content.

3. The application according to claim 2, characterized in that, The reagent for detecting P-selectin is selected from reagents for detecting the content of P-selectin in plasma or whole blood, and the product for assisting in the diagnosis of glaucoma is selected from kits.

4. The application according to claim 3, characterized in that, The reagent used to detect the P-selectin content in plasma is selected from antibodies against P-selectin carrying detectable markers.

5. The application according to claim 4, characterized in that, The detectable marker is selected from biotin, chemiluminescent reagents, or radioisotopes.

6. The application according to claim 3, characterized in that, The kit also includes a biotin-binding reagent; the biotin-binding reagent is selected from conjugates of enzymes that catalyze substrate color development and avidin.

7. The application according to claim 6, characterized in that, The enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, or glucose-6-phosphate dehydrogenase.

8. The use of a reagent for detecting P-selectin in the preparation of products for the auxiliary diagnosis of patients with intermediate or severe glaucoma.

9. A device for auxiliary diagnosis of glaucoma, characterized in that, It includes: The system includes an input module, a control module, and an output module. The input module is configured to input the content of P-selectin in the subject sample. The control module is configured to obtain the risk or probability of glaucoma in the tested sample based on the data from the input module. The output module is configured to output the risk or probability of glaucoma in the tested sample.

10. The device for auxiliary diagnosis of glaucoma according to claim 9, characterized in that, The control module is configured to compare the content of P-selectin in the subject sample with a preset threshold. If the content is greater than the preset threshold, the risk of the tested sample having glaucoma is assessed. If the content is less than the preset threshold, the risk of the tested sample not having glaucoma is assessed.

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