Biomarker for detecting diabetic retinopathy, kit and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an application of CDH5 protein and / or SELL protein as a biomarker in preparation of a kit for detecting diabetic retinopathy, the biomarker comprises at least one of CDH5 protein and SELL protein; the amino acid sequence of the CDH5 protein is as shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is as shown in SEQ NO: 2. The CDH5 protein and / or the SELL protein are / is used as the biomarker for detecting diabetic retinopathy, especially for detecting early diabetic retinopathy, good sensitivity and specificity can be obtained, the accuracy of a detection result is ensured, early diagnosis and early treatment are realized, sampling is convenient and non-invasive, and large-scale popularization can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker, reagent kit, and application for detecting diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is one of the most common chronic complications of diabetes, affecting approximately one-third of diabetic patients worldwide. The clinical staging of DR is mainly divided into two stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). NPDR is further classified into mild, moderate, and severe levels. PDR represents the advanced stage of diabetic retinopathy. Due to the current imperfect screening system in primary healthcare, many patients are diagnosed at an advanced stage, significantly increasing the risk of blindness. The risk of DR is significantly positively correlated with the duration of diabetes, but its molecular mechanisms are not fully understood, and existing treatments (such as anti-VEGF drugs and laser surgery) have limited efficacy in only a portion of patients. This situation highlights that insufficient control of risk factors and the lack of early warning indicators often cause clinical intervention to miss the optimal time window. Therefore, revealing the key driving factors of DR and establishing an early diagnostic system have become urgent needs for early intervention and improving patient prognosis.

[0003] Currently, the diagnosis of DR mainly relies on techniques such as fundus photography, fluorescein fundus angiography (FFA), and optical coherence tomography (OCT). However, significant limitations remain: 1) Limitations in technical principles: FFA visualizes vascular leakage through intravenous injection of contrast agents, but it can only capture established structural lesions (such as microaneurysms and hemorrhages), and cannot identify molecular abnormalities before the blood-retinal barrier is damaged. FFA requires puncture to obtain blood, posing a risk of allergic reactions, and the examination process is time-consuming. Although OCT can present the retinal layer structure in high definition, it does not respond to the degradation of early intercellular connection proteins. 2) Deficiencies in clinical applicability (subjective dependence and obstacles to promotion at the grassroots level): Traditional fundus examinations have a high misdiagnosis rate, especially in the non-proliferative stage of DR, where doctors' judgments of small lesions vary significantly. A single OCT device costs over one million yuan and requires professional technicians to operate; FFA requires emergency equipment, making both difficult to popularize in resource-scarce areas and creating significant obstacles to promotion at the grassroots level. 3) Insufficient sensitivity in early diagnosis (delayed time window and molecular blind spot): Current technologies can only detect retinal damage when visible damage is present, by which time patients have usually missed the optimal intervention window. Early biomarkers cannot be detected, and these indicators may appear 3-5 years before imaging abnormalities. 4) Bottlenecks in AI-assisted diagnosis (data dependence and low clinical integration): AI models need to be trained on historical fundus images, but their sensitivity for early cases without imaging features is only 40-50%. FDA-approved AI systems are still recommended for use in conjunction with human interpretation and cannot independently perform graded diagnosis (especially for complex lesions such as macular edema).

[0004] Early diagnosis and intervention of diabetic retinopathy (DR) are crucial for preventing visual impairment. (1) Irreversibility of the time window: Current technology can only diagnose DR in the middle and late stages, at which point the critical window for reversing early microvascular lesions through blood glucose / blood pressure control has been missed; (2) Mismatch of medical resources: Complex examinations are not widely available in primary hospitals, resulting in most patients progressing to moderate ischemia at initial diagnosis, while low-cost and easy-to-operate testing methods can significantly improve screening coverage; (3) Differences in prognostic economics: Early intervention can significantly reduce the 10-year risk of blindness, and the average annual treatment cost is significantly lower than that in the late stage. Therefore, developing a biomarker that can specifically identify DR in the early stages is of decisive significance for achieving hierarchical diagnosis and treatment and improving the global disease burden.

[0005] In recent years, research on the pathogenesis and biomarkers of diabetic retinopathy (DR) has deepened, but highly specific and sensitive early diagnostic biomarkers are still lacking. DR biomarker research mainly faces the following bottlenecks: 1) Insufficient specificity: Inflammatory factors in the blood are also found in other diabetic complications, making it impossible to accurately distinguish between DR and non-DR patients. 2) Lack of dynamic monitoring: For example, glycated hemoglobin (HbA1c) reflects long-term glycemic control, but it cannot assess the risk of retinopathy in real time. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker that can rapidly detect whether diabetic patients have diabetic retinopathy, especially for early-stage diabetic retinopathy.

[0007] The following technical solutions are used to achieve the above objectives.

[0008] The first aspect of the present invention provides the use of CDH5 protein and / or SELL protein as biomarkers in the preparation of a kit for detecting diabetic retinopathy, wherein the biomarkers include CDH5 protein and / or SELL protein; the amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO: 2.

[0009] In some embodiments, the diabetic retinopathy is early diabetic retinopathy.

[0010] A second aspect of the present invention provides the application of a reagent for detecting the expression levels of CDH5 protein and / or SELL protein in the preparation of a kit for detecting diabetic retinopathy, wherein the amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO: 2.

[0011] In some embodiments, the diabetic retinopathy is early diabetic retinopathy.

[0012] In some embodiments, the reagents include those based on ELISA, Western blotting, or protein chip technologies.

[0013] The fourth aspect of the present invention provides the application of a reagent for detecting the expression levels of CDH5 protein and SELL protein in the preparation of a kit for detecting diabetic retinopathy, wherein the amino acid sequence of the CDH5 protein is shown in SEQ NO: 1 and the amino acid sequence of the SELL protein is shown in SEQ NO: 2.

[0014] The fifth aspect of the present invention provides a kit for detecting diabetic retinopathy, comprising reagents for detecting the expression levels of CDH5 protein and / or SELL protein in a sample, wherein the amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO: 2.

[0015] In some embodiments, the test sample is peripheral blood, preferably blood, serum or plasma, more preferably serum.

[0016] The sixth aspect of this invention provides a method for detecting diabetic retinopathy, comprising the following steps:

[0017] Detect the protein expression levels of CDH5 and / or SELL;

[0018] The amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO: 2.

[0019] In some implementations, the detection methods include ELISA, Western blotting, or protein chip technology.

[0020] This invention discovers that both CDH5 and SELL proteins can serve as biomarkers for the detection of diabetic retinopathy, enabling timely, rapid, objective, and accurate detection of diabetic retinopathy, especially in its early stages. Early detection of diabetic retinopathy can be achieved by detecting the expression levels of CDH5 and SELL in serum. Furthermore, the combined use of CDH5 and SELL proteins as biomarkers for the detection of diabetic retinopathy, particularly in its early stages, yields better sensitivity and specificity, ensuring accurate results and enabling early diagnosis and treatment. This can significantly reduce the risk of blindness within 10 years, and the sampling is convenient and non-invasive, allowing for large-scale application. Attached Figure Description

[0021] Figure 1 This is a differential expression map of vitreous protein profiles between the DR and NC groups. Figure 1 A represents the volcano plot analysis of two groups of differentially expressed proteins; Figure 1 B is the GO enrichment analysis of the two groups of differentially expressed proteins; Figure 1 C represents the KEGG enrichment analysis of the two groups of differentially expressed proteins.

[0022] Figure 2 The expression of CDH5 and SELL in each group was detected by Western blotting and ELISA. Figure 2 A represents the CDH5 and SELL protein bands; Figure 2 B- Figure 2 C represents the relative expression levels of CDH5 and SELL; Figure 2 D- Figure 2 E represents the expression levels of CDH5 and SELL as measured by ELISA (Note: compared with the control group). * P < 0.05 ** P < 0.01, *** P < 0.001, **** P < 0.0001).

[0023] Figure 3This is a Western blotting ROC curve of serum CDH5 and SELL in the training set data to verify the diagnosis of DR.

[0024] Figure 4 These are the CDH5 and SELL standard curves; among them, Figure 4 A is the CDH5 standard curve graph. Figure 4 B is the SELL standard curve.

[0025] Figure 5 This is the ROC curve of ELISA validation of CDH5 and SELL in serum for diagnosing DR in the training set data.

[0026] Figure 6 This is the ROC curve of ELISA validation of CDH5 and SELL in serum for diagnosing DR in the validation set data. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0030] In this invention, it was discovered that both CDH5 and SELL proteins can serve as biomarkers for the detection of diabetic retinopathy (DR), enabling timely, rapid, objective, and accurate detection of DR, especially in its early stages. Early detection of DR can be achieved by detecting the expression levels of CDH5 and SELL in serum.

[0031] The inventors have discovered for the first time that the expression levels of CDH5 and SELL proteins are significantly higher in the early stages of diabetic retinopathy than in healthy controls. Therefore, CDH5 and SELL proteins can serve as specific markers for the early stages of diabetic retinopathy. Compared with existing detection methods, the combined use of CDH5 and SELL proteins as markers for diabetic retinopathy, especially early-stage diabetic retinopathy, yields better sensitivity and specificity, ensuring accurate results and enabling early diagnosis and treatment. This can significantly reduce the risk of blindness within 10 years, and the sampling is convenient and non-invasive, allowing for large-scale application.

[0032] In this invention, vitreous fluid from the eyes of diabetic retinopathy (DR) patients and healthy controls was extracted, and 296 differentially expressed proteins were screened using proteomic analysis. Extensive experiments and verification using Western blotting confirmed that CDH5 and SELL were significantly differentially expressed proteins between DR patients and controls. The same differentially expressed proteins, CDH5 and SELL, were then screened from the blood of DR patients and healthy controls, and their expression levels were detected using Western blotting and ELISA, respectively. The results showed that both CDH5 and SELL proteins can serve as specific markers for the early stages of diabetic retinopathy, exhibiting high sensitivity and specificity. Furthermore, the combined use of CDH5 and SELL proteins significantly improved the early diagnosis rate of DR compared to using either CDH5 or SELL alone, demonstrating higher sensitivity and specificity. This provides strong evidence for early intervention and treatment of DR and has significant clinical application value.

[0033] The following are specific examples.

[0034] It should be noted that the amino acid sequences of the CDH5 protein and / or SELL protein involved in the following examples are as follows:

[0035] The amino acid (784) sequence of the CDH5 protein (NCBI Reference Sequence: NP_001786.2; UniProtKB Entry: P33151) is shown in SEQ NO: 1:

[0036] 。

[0037] The amino acid (372) sequence of the SELL protein (NCBI Reference Sequence: NP_000646.3; UniProtKB Entry: P14151) is shown in SEQ. NO: 2 shows: MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDCWTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCD VGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYNPLFIPVAVMVTAFSGLAFIIWLARRLKKGKKSKRSMNDPY.

[0038] Example 1

[0039] 1. Case selection and preparation of vitreous samples

[0040] Patients with indications for vitrectomy at the Department of Ophthalmology, Loudi Central Hospital, from January to December 2022 were selected and divided into the NC group and the DR group. This study was approved by the Medical Ethics Committee of Loudi Central Hospital (2022-Ethics (Research)-25). All patients signed informed consent forms. The NC group consisted of 7 patients with idiopathic macular hole, including 2 males and 5 females, with a mean age of (52.57±17.56) years; the DR group consisted of 7 patients with proliferative diabetic retinopathy (PDR), including 5 males and 3 females, with a mean age of (69.14±11.47) years. This study was approved by the Medical Ethics Committee of Loudi Central Hospital. All patients signed informed consent forms for the surgery. During the vitrectomy, a standard three-channel approach was established for 23G pars plana vitrectomy. The irrigation tubing was first closed, the 23G vitrectomy tip was used to remove the vitreous body and aspirate 0.5 mL of vitreous fluid, and then the irrigation tubing was reopened to begin the surgery. Immediately centrifuge the vitreous sample for 10 minutes (3,000 rpm), collect the supernatant and store it in an EP tube at -80°C for later use.

[0041] 2. Label-free quantitative protein detection in vitreous humor

[0042] This section was commissioned to Shanghai Huaying Biomedical Technology Co., Ltd., and the steps are as follows:

[0043] 1) Protein sample pretreatment

[0044] After thawing the above samples, centrifuge at 13,000 rpm for 15 min at 4°C, and transfer the supernatant to a new centrifuge tube. After determining the protein concentration using BCA, take 1,000 μg of protein solution and transfer it to a new 1.5 mL EP tube. Add acetone solution (acetone to protein solution volume ratio of 6:1) and incubate overnight at -20°C. Centrifuge at 13,000 rpm for 15 min at 4°C, discard the supernatant, and then add 500 μL of pre-cooled ethanol / acetone / acetic acid mixture (50:50:0.1) to the precipitate to wash the precipitate. Repeat this operation once after centrifugation. Then, resuspend the precipitate with guanidine hydrochloride (6M) and TEAB (300 mM), mix well, and determine the protein concentration.

[0045] 2) FASP (Filter Aided Proteome Preparation) enzymatic hydrolysis

[0046] Take 40 μg of the protein sample corresponding to the volume mentioned above, and add 25 mM ammonium bicarbonate to a final volume of 100 μL. After adding DTT (final concentration 20 mM), perform a reduction reaction at 57 °C for 1 h, then add Iodoacetimide (final concentration 90 mM) for alkylation, vortex mix, and incubate at room temperature in the dark for 40 min. Add the solution to a 10 kJ ultrafiltration tube, centrifuge at 12,000 rpm, discard the solution at the bottom of the ultrafiltration tube, and then continue to add the dissociation buffer ammonium bicarbonate. Repeat this operation 4 times after centrifugation. Finally, add trypsin prepared with the dissociation buffer and incubate overnight at 37 °C. Collect the enzymatically digested peptides by centrifugation and concentrate and dry them using a lyophilizer.

[0047] 3) Desalination

[0048] Dissolve the mixed peptides in 0.1% trifluoroacetic acid (TFA) solution, then add the solution to a 100% acetonitrile-activated desalting column that has been pre-equilibrated with 0.1% TFA solution; after centrifugation, continue to wash with 0.1% TFA solution; finally, add 50% acetonitrile solution, centrifuge and collect the eluent; concentrate and dry the eluent using a freeze dryer to remove residual acetonitrile.

[0049] 4) LC-MS analysis

[0050] Lyophilized samples were reconstituted with 0.1% TFA and separated using an EASY-nLC 1000 (Thermo Scientific, USA) with a C18 analytical column (1.9 μm, 75 μm * 20 cm) at a flow rate of 200 nL / min. Tandem mass spectrometry (Orbitrap FusionLumos, Thermo Scientific, USA) was performed using data-dependent scanning at a resolution of 60,000 (FWHM), a mass-to-charge ratio (m / z) range of 350–1600, and HCD fragmentation mode with a collision energy set to 30%.

[0051] 5) Results: Differentially expressed proteins in vitreous samples from the two groups were analyzed. Following the library construction and screening criteria of Shanghai Huaying Biomedical Technology Co., Ltd., the Student's T-test was used to obtain p-values ​​for both groups. The differentially expressed proteins met the following criteria: Fold change ≥ 1.2 or ≤ 0.8333; p-value < 0.05. A total of 164 upregulated proteins and 132 downregulated proteins were identified. Volcano plot analysis of these 296 differentially expressed proteins revealed that the vitreous proteins CDH5 and SELL were significantly elevated in patients with diabetic retinopathy (DR) compared to the non-corporeal membrane oxygenation (NC) group. Figure 1 A). GO enrichment analysis of protein expression in the two groups of samples showed that the most prominent biological functions involved fibrinolysis, complement activation, negative regulation of endopeptidase activity, acute phase response, and classical complement activation pathway; cellular components were mainly concentrated in platelet dense granule lumen, blood microparticles, and platelet α-granule lumen; molecular functions included endopeptidase inhibitor activity, serine-type endopeptidase inhibitor activity, and heparin binding (…). Figure 1 B). KEGG enrichment analysis of differentially expressed proteins in the two groups of samples showed that the differentially expressed proteins mainly occurred during complement and coagulation cascade processes (B). Figure 1 C).

[0052] Example 2

[0053] 1. Serum samples used for Western blot analysis

[0054] This study included patients with type 2 diabetes who visited the Ophthalmology and Endocrinology departments of Loudi Central Hospital between October 2023 and April 2025, as well as healthy individuals who underwent physical examinations during the same period. The study was approved by the Medical Ethics Committee of Loudi Central Hospital (2023-Ethics (Research)-033). All patients signed informed consent forms. 20 mL of fasting venous blood was collected from each participant in the morning. After being left at room temperature for 2 hours, the blood was centrifuged at 1,000g for 15 minutes, and the supernatant was collected and stored at -80℃. A total of 88 patients with type 2 diabetes and 20 healthy individuals who underwent physical examinations during the same period were ultimately included in the study.

[0055] The study subjects were divided into 5 groups: control group (20 healthy individuals), NDR group (n=25 without DR), mild-to-moderate NPDR group (n=23 with mild to moderate non-proliferative DR), severe NPDR group (n=20 with severe non-proliferative DR), and PDR group (n=20 with proliferative DR). Among the 88 patients with type 2 diabetes, there were 42 males (47%) and 46 females (52%), with a mean age of 56.55 ± 8.91 years, ranging from 40 to 71 years. The control group consisted of 20 patients, including 8 males (40%) and 12 females (60%), with a mean age of 58.92 ± 16.01 years, ranging from 41 to 70 years.

[0056] 2. Western blot detection of serum CDH5 and SELL levels

[0057] 1) Plasma protein concentration

[0058] Remove the plasma sample from the -80°C freezer and transfer it to a 4°C freezer to thaw naturally. Add ammonium sulfate from the protein concentration kit (plasma:ammonium sulfate = 9:1) to each plasma sample volume and vortex thoroughly. Incubate the mixture at 4°C for 1 hour. Centrifuge at 15,000g or higher at 4°C for 15 minutes, carefully remove and discard the supernatant, retaining the precipitate. Dry the centrifuge tube by inverting it on absorbent paper and add 1 mL of anhydrous ethanol. Centrifuge at 15,000g at 4°C for 15 minutes. Aspirate the supernatant, retaining the precipitate. Add 200 μL of phosphate buffer from the protein concentration kit to the centrifuge tube. Centrifuge at 15,000g at 4°C for 15 minutes. Aspirate the supernatant, retaining the precipitate. Dry the centrifuge tube by inverting it on absorbent paper. Add 40 μL of RIPA lysis buffer containing protease and phosphatase inhibitors and vortex thoroughly. Centrifuge at 10,000g for 10 min at 4℃, collect the supernatant, and quantify the protein using the BCA method.

[0059] 2) BCA protein quantification

[0060] Add 4 mL of ultrapure water to a tube containing 20 mg BSA and dissolve thoroughly to prepare a 5 mg / mL protein standard solution. Prepare a 96-well plate with standard wells, negative control wells, and test wells. Add 10 μL of ultrapure water to each standard and negative control well, and 8 μL of ultrapure water to each test well. Prepare BSA concentrations of 0, 78.125, 156.25, 312.5, 625, 1250, 2500, and 5000 μg / mL according to Table 1 below. Add 2 μL of the sample to each test well. Add 180 μL of BeyoBCAPlus working solution to each well, mix using a microplate shaker for 30 seconds, and incubate at 37°C for 30 minutes. The absorbance at 570 nm wavelength was measured using an ELISA reader. The absorbance measurements of both the standard and the sample were reduced by the absorbance measurement of the 0 μg / mL blank standard. The absorbance of the standard and the sample after blank correction was then calculated and used to plot the standard curve and calculate the sample concentration.

[0061] Table 1. Preparation of BSA solution

[0062]

[0063] 3) Western Blot detection method

[0064] Add 5X SDS-PAGE protein loading buffer to the dissolved concentrated sample, vortex mix, and place on a metal bath for denaturation at 100℃ for 5 min. Load 15 μg of denatured protein into each well, adjust the voltage to 80V, and stop electrophoresis when bromophenol blue appears. Then place the SDS-PAGE gel in a semi-dry transfer apparatus and transfer at 350 mA for 45–80 min depending on the molecular weight. Block with 5% skim milk powder for 2 h, wash with TBST, and add CDH5 or SELL antibody diluted 1:1000 (both diluted). Incubate overnight at 4℃, wash with TBST, add horseradish peroxidase-labeled secondary antibody (HRP-IgG, 1:2000), and incubate at room temperature for 2 h. Wash with TBST, add ECL developing solution, and finally expose and develop in a GelView 6000Plus intelligent image workstation. Calculate the band grayscale using ImageJ software.

[0065] 3. Western Blot Results

[0066] The expression of related proteins in concentrated serum of each group was detected by Western blot (see...). Figure 2 A- Figure 2C) There were no significant differences between the NDR group and the mild-to-moderate NPDR group and the control group, while the levels in the severe NPDR group and the PDR group were significantly higher than those in the control group (all P < 0.05). The results indicate that the expression of CDH5 and SELL proteins gradually increases with the increase of DR severity.

[0067] 4. Western Blot analysis to validate the diagnostic efficacy of serum CDH5 and SELL in DR.

[0068] The expression levels of CDH5 and SELL in serum samples from each group were detected by Western blotting, and their predictive ability for DR was analyzed using ROC. The results showed that when the grayscale expression level of CDH5 was 0.553, the sensitivity for diagnosing DR was 0.800, the specificity was 0.889, and the AUC was 0.858. When the expression level of SELL was 0.809, the sensitivity was 0.680, the specificity was 0.889, and the AUC was 0.786. Furthermore, the AUC value of the combined CDH5 and SELL predictive model constructed in this embodiment reached 0.920, indicating that the combination of CDH5 and SELL can significantly improve the diagnostic accuracy of DR (see...). Figure 3 ).

[0069] Example 3

[0070] 1. The ELISA method for detecting CDH5 and SELL in serum involved in this embodiment is described below:

[0071] (1) Reagent name and source:

[0072] Human vascular endothelial cadherin (CDH5) ELISA kit, Jiangsu Enzyme Immunoassay Co., Ltd. (Catalog No. MM-64917H1)

[0073] Human L-selectin (SELL) ELISA kit, Jiangsu Enzyme Immunoassay Co., Ltd. (Catalog No. MM-0082H2)

[0074] 1) Preparation of standard curve

[0075] The standard solutions for CDH5 protein were prepared as shown in Table 2 below:

[0076] Table 2. Preparation of Standard Solutions for CDH5 Protein

[0077] Standard concentration serial number dilution 800 pg / mL 1 50μL stock solution 400 pg / mL 2 Mix 50 μL of sample 1 with 50 μL of standard dilution solution. 200 pg / mL 3 Mix 50 μL of sample 2 with 50 μL of standard dilution solution. 100 pg / mL 4 Mix 50 μL of sample 3 with 50 μL of standard dilution solution. 50 pg / mL 5 Mix 50 μL of sample 4 with 50 μL of standard dilution solution. 25 pg / mL 6 Mix 50 μL of sample 5 with 50 μL of standard dilution solution. 0 pg / mL 7 50μL standard diluent

[0078] The standard solutions for SELL protein are prepared as shown in Table 3 below:

[0079] Table 3. Preparation of Standard Solutions for SELL Protein

[0080] Standard concentration serial number dilution 2000 pg / mL 1 50μL stock solution 1000pg / mL 2 Mix 50 μL of sample 1 with 50 μL of standard dilution solution. 500 pg / mL 3 Mix 50 μL of sample 2 with 50 μL of standard dilution solution. 250 pg / mL 4 Mix 50 μL of sample 3 with 50 μL of standard dilution solution. 125 pg / mL 5 Mix 50 μL of sample 4 with 50 μL of standard dilution solution. 62.5 pg / mL 6 Mix 50 μL of sample 5 with 50 μL of standard dilution solution. 31.25 pg / mL 7 Take 50 μL of sample 6 and mix it with 50 μL of standard dilution solution. 0 pg / mL 8 50μL standard diluent

[0081] (2). ELISA detection steps

[0082] Set up blank wells (blank control wells without sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μL of standard to the enzyme-labeled plate, and add 50 μL of serum or vitreous humor to the sample wells. Add the sample to the bottom of the well, avoiding contact with the well walls, and gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 30 min.

[0083] Dilute the 30-fold concentrated washing buffer with distilled water and set aside. Carefully peel off the sealing film, discard the liquid, and shake dry. Fill each well with the washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells. Incubate and wash again. Add 50 μL of chromogenic reagent A to each well, then add 50 μL of chromogenic reagent B, gently vortex to mix, and incubate at 37°C in the dark for 10 minutes.

[0084] Add 50 μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). After blank correction, measure the absorbance (OD value) of each well at a wavelength of 450 nm. Plot a standard curve with concentration on the x-axis and OD value on the y-axis. Then, calculate the concentration of the sample by substituting the OD value into the standard curve.

[0085] CDH5 and SELL standard curves are as follows Figure 4 As shown.

[0086] 2. Serum samples used for ELISA testing

[0087] 1) Serum sample information for ELISA training set

[0088] The training set consisted of patients with type 2 diabetes who visited the ophthalmology and endocrinology departments of Loudi Central Hospital, as well as healthy individuals who underwent physical examinations during the same period. This study was approved by the Medical Ethics Committee of Loudi Central Hospital (2023-Ethics (Research)-033). All patients signed informed consent forms. 20 mL of fasting venous blood was collected from each participant in the morning. After being left at room temperature for 2 hours, the blood was centrifuged at 1000g for 15 minutes, and the supernatant was collected and stored at -80℃. Ultimately, 88 patients with type 2 diabetes and 20 healthy individuals who underwent physical examinations during the same period were collected as the training set.

[0089] The study subjects were divided into 5 groups: control group (20 healthy individuals), NDR group (n=25 without DR), mild-to-moderate NPDR group (n=23 with mild to moderate non-proliferative DR), severe NPDR group (n=20 with severe non-proliferative DR), and PDR group (n=20 with proliferative DR). Among the 88 patients with type 2 diabetes, there were 42 males (47%) and 46 females (52%), with a mean age of 56.55 ± 8.91 years, ranging from 40 to 71 years. The control group consisted of 20 patients, including 8 males (40%) and 12 females (60%), with a mean age of 58.92 ± 16.01 years, ranging from 41 to 70 years.

[0090] 2) Validation set serum samples for ELISA testing

[0091] Patients with type 2 diabetes who visited the Ophthalmology and Endocrinology departments of Loudi Central Hospital, and healthy individuals undergoing physical examinations during the same period, were collected as study subjects, different from those selected in the training set. This study was approved by the Medical Ethics Committee of Loudi Central Hospital (2023-Ethics (Research)-033). All patients signed informed consent forms. 20 mL of fasting venous blood was collected from each subject in the morning, left at room temperature for 2 hours, centrifuged at 1000g for 15 minutes, and the supernatant was collected and stored at -80℃. Finally, 59 patients with type 2 diabetes and 20 healthy individuals undergoing physical examinations during the same period were collected as the validation set.

[0092] The study subjects were divided into 5 groups: control group (20 healthy individuals), NDR group (n=20 without DR), mild-to-moderate NPDR group (24 with mild to moderate non-proliferative DR), severe NPDR group (5 with severe non-proliferative DR), and PDR group (10 with proliferative DR). Among the 59 patients with type 2 diabetes, there were 24 males (41%) and 35 females (59%), with a mean age of 58.46 ± 8.99 years, ranging from 35 to 76 years. The control group consisted of 20 patients, including 7 males (35%) and 13 females (65%), with a mean age of 52.10 ± 13.80 years, ranging from 25 to 70 years.

[0093] 3. ELISA results of CDH5 and SELL levels in serum of each training set group.

[0094] Following the ELISA kit and detection method described above, serum CDH5 levels in each group of the training set were detected.

[0095] SELL expressions (e.g.) Figure 2(As shown in D and E), there was no significant difference between the NDR group and the control group, while the levels in the mild-to-moderate NPDR group, severe NPDR group, and PDR group were significantly higher than those in the control group (all P < 0.05). The results indicate that the expression of CDH5 and SELL proteins gradually increases with the increase of DR severity.

[0096] 4. Correlation analysis results of DR risk factors

[0097] The samples collected through Spearman correlation analysis are shown in Table 4. The analysis revealed a positive correlation between diabetes duration, HbA1c, SELL, CDH5, serum creatinine, fibrinogen, and serum cystatin C and retinopathy of prematurity (T2DM) in patients (all P < 0.05). High-density lipoprotein (HDL) was negatively correlated with retinopathy (DR) (P < 0.05). Other variables such as age, sex, height, weight, BMI, total cholesterol, triglycerides, and low-density lipoprotein (LDL) showed no statistically significant difference in DR incidence (all P > 0.05).

[0098] Table 4. Correlation analysis between clinical data and DR

[0099]

[0100]

[0101] 5. Results of univariate and multivariate logistic regression analyses

[0102] The samples collected through univariate logistic regression analysis were analyzed with CDH5, SELL, high-density lipoprotein, cystatin C, serum creatinine, fibrinogen, HbA1c, and duration of diabetes as independent variables, and the presence or absence of diabetic retinopathy (DR) as the dependent variable. The analysis revealed highly significant correlations between CDH5, SELL, high-density lipoprotein, cystatin C, serum creatinine, fibrinogen, HbA1c, and duration of diabetes and the occurrence of DR (P < 0.01). A multivariate logistic regression model was then established. After controlling for the effects of SELL and cystatin C, the influence of CDH5 and HbA1c on DR remained significant (both P < 0.01), indicating that CDH5 and HbA1c are independent risk factors for DR. The results are shown in Tables 5 and 6.

[0103] Table 5 Univariate Logistic Regression Analysis of DR-Related Variables

[0104]

[0105] Table 6. Multivariate Logistic Regression Analysis of DR-Related Variables

[0106]

[0107]

[0108] 6. ELISA efficacy analysis of serum CDH5 and SELL in diagnosing DR.

[0109] 1) ELISA validation of the diagnostic efficacy of serum CDH5 and SELL in DR using training data.

[0110] Following the ELISA kit and detection method described above, the expression levels of CDH5 and SELL in serum samples from each group of the training set were detected, and their predictive ability for DR was analyzed using ROC. The results showed that when the expression level of CDH5 was 112.455 pg / mL, the sensitivity for diagnosing DR was 0.873, the specificity was 0.978, and the AUC was 0.964. When the expression level of SELL was 93.033 pg / mL, the sensitivity was 0.794, the specificity was 1.000, and the AUC was 0.957. Furthermore, the AUC value of the integrated prediction model of CDH5 and SELL constructed in this study reached 0.971, indicating that CDH5 and SELL can significantly improve the diagnostic accuracy of DR (see...). Figure 5 ).

[0111] 2) ELISA efficacy analysis of serum CDH5 and SELL in diagnosing DR in validation set data.

[0112] Following the ELISA kit and detection method described above, the expression levels of CDH5 and SELL in serum samples from each validation set were detected, and their predictive ability for DR was analyzed using ROC. The results showed that when the expression level of CDH5 was 112.227 pg / mL, the sensitivity for diagnosing DR was 0.949, the specificity was 0.967, and the AUC was 0.958. When the expression level of SELL was 93.033 pg / mL, the sensitivity was 0.821, the specificity was 1.000, and the AUC was 0.937. Furthermore, the AUC value of the integrated prediction model of CDH5 and SELL constructed in this study reached 0.982, indicating that CDH5 and SELL can significantly improve the diagnostic accuracy of DR (see...). Figure 6 ).

[0113] Since the results obtained by the method in this embodiment can only serve as intermediate information and cannot directly determine whether a patient has DR, it is necessary to combine clinical symptoms, fundus photography, fundus fluorescein angiography and optical coherence tomography to ultimately determine the patient's condition.

[0114] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of CDH5 protein and / or SELL protein as biomarkers in the preparation of kits for detecting diabetic retinopathy, wherein, The biomarker includes at least one of CDH5 protein and SELL protein; the amino acid sequence of CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of SELL protein is shown in SEQ NO:

2.

2. The application according to claim 1, wherein the diabetic retinopathy is early diabetic retinopathy.

3. The application of reagents for detecting the expression levels of CDH5 and / or SELL proteins in the preparation of kits for detecting diabetic retinopathy, wherein, The amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO:

2.

4. The application according to claim 3, wherein the diabetic retinopathy is early diabetic retinopathy.

5. The application as described in claim 3 or 4, characterized in that, The reagents include those based on ELISA, Western blotting, or protein chip technology for detection.

6. Application of reagents for detecting CDH5 and SELL protein expression levels in the preparation of kits for detecting diabetic retinopathy, wherein, The amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO:

2.

7. A kit for detecting diabetic retinopathy, characterized in that, The reagent includes reagents for detecting the expression levels of CDH5 protein and / or SELL protein in a sample, wherein the amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO:

2.

8. The kit according to claim 7, characterized in that, The test sample is peripheral blood, preferably blood, serum or plasma, and more preferably serum.

9. A method for detecting diabetic retinopathy, characterized in that, Includes the following steps: Detect the protein expression levels of CDH5 and / or SELL; The amino acid sequence of the CDH5 protein is shown in SEQ NO: 1, and the amino acid sequence of the SELL protein is shown in SEQ NO:

2.

10. The method as described in claim 9, characterized in that, The detection methods include ELISA, Western blotting, or protein chip technology.

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