Exosome protein as molecular marker and kit for diagnosing uveitis
A kit prepared using IDH3a protein and specific antibodies from exosomes has solved the problems of specificity and sensitivity in the diagnosis of uveitis, enabling non-invasive early screening and improving diagnostic accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-03
AI Technical Summary
Current technologies lack specific biomarkers, making the diagnosis of uveitis reliant on imaging methods. This approach is highly subjective, operationally dependent, and has a high rate of early missed diagnoses, making non-invasive early screening impossible.
Using the IDH3a protein in exosomes as a molecular marker, and combined with an antibody that specifically recognizes the IDH3a protein, a kit for the diagnosis of uveitis was prepared, including components such as lysis buffer and blocking buffer, and detected through peripheral plasma samples.
It enables non-invasive, highly sensitive, easy-to-operate, and low-cost early screening, improving the diagnostic accuracy of uveitis.
Smart Images
Figure CN121784293A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular diagnostics, specifically relating to exosomal proteins as molecular markers and kits for diagnosing uveitis. Background Technology
[0002] Uveitis is a common blinding inflammatory eye disease with a complex pathogenesis involving multiple causes, including infection, autoimmune reactions, and genetic factors. Epidemiological data indicates a global incidence of approximately 38 per 100,000, severely impacting patients' quality of life and vision. Currently, clinical diagnosis primarily relies on imaging methods such as funduscopy, fluorescein angiography (FFA), and optical coherence tomography (OCT). These methods suffer from high subjectivity, operational dependence, and a high rate of early missed diagnoses. The lack of specific biomarkers is a major bottleneck in uveitis diagnosis; existing inflammatory markers such as IL-6 and TNF-α lack specificity, hindering non-invasive early screening.
[0003] Exosomes, as nanoscale (30-150 nm) extracellular vesicles, can carry biological information such as proteins and nucleic acids, and have significant potential in disease diagnosis. Exosomes can be non-invasively extracted from bodily fluids such as plasma and reflect changes in the tissue microenvironment. However, current research on exosomes mainly focuses on oncology and neurodegenerative diseases, and their application in the diagnosis of uveitis remains unexplored. Summary of the Invention
[0004] To address the above deficiencies, this invention provides an exosomal protein as a molecular marker for diagnosing uveitis. The molecular marker is the IDH3a protein in exosomal tissues, and the sequence of the IDH3a protein is shown in SEQ ID NO:1.
[0005] Furthermore, the exosomes are plasma exosomes.
[0006] The present invention also discloses a kit for diagnosing uveitis, the kit comprising an antibody that specifically recognizes the IDH3a protein.
[0007] Furthermore, the kit also includes one or more of the following: lysis buffer, blocking buffer, washing buffer, secondary antibody, chromogenic substrate, protein loading buffer, electrophoresis buffer, transfer buffer, membrane blocking buffer, and PVDF membrane.
[0008] Furthermore, the antibody is a mouse anti-human IDH3a monoclonal antibody or a rabbit anti-human IDH3a polyclonal antibody.
[0009] Compared with the prior art, the present invention has the following advantages:
[0010] 1. Highly non-invasive, requiring only a peripheral blood plasma sample;
[0011] 2. High specificity and sensitivity; experiments showed that IDH3a expression was significantly downregulated in the patient group.
[0012] 3. Simple to operate, standardized reagent kit components, suitable for clinical laboratories;
[0013] 4. Low cost and easy to promote and apply. Compared with existing imaging diagnostics, this invention can achieve early screening and improve diagnostic accuracy. Attached Figure Description
[0014] Figure 1 Electron micrographs of exosomes in the uveitis group and the normal control group in this invention.
[0015] Figure 2 The results of NTA particle size analysis of exosomes in the uveitis group and the normal control group in this invention are shown.
[0016] Figure 3 This is a schematic diagram illustrating the expression of marker proteins on the surface of exosomes in this invention.
[0017] Figure 4 This is a comparison of the detection results of the uveitis group and the normal control group during the Western blotting detection of IDH3a protein in this invention.
[0018] Figure 5 This is the quantitative analysis result of the IDH3a protein Western Blot diagram in this invention. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0020] Example 1
[0021] This embodiment provides exosomal proteins as molecular markers for diagnosing uveitis. The molecular marker is a protein in plasma exosomals: isocitrate dehydrogenase 3a, or IDH3a. The sequence of the IDH3a protein is shown in SEQ ID NO:1.
[0022] Example 2
[0023] 1) Test reagents
[0024] The plasma samples from the healthy control group and uveitis patients in this invention were obtained from the First Affiliated Hospital of Zhengzhou University. Other...
[0025] The reagents or consumables used are commercially available or can be obtained by those skilled in the art through public channels.
[0026] This embodiment included 30 patients diagnosed with uveitis and 30 age- and sex-matched normal volunteers.
[0027] Inclusion criteria: (1) Uveitis patients with an age of onset of less than 16 years; (2) All patients had the same medication regimen (cyclosporine combined with glucocorticoids); (3) Affected eye: bilateral; (4) Duration of disease: chronic inflammation (duration of disease lasting 3 months or more); (5) Uveitis patients who underwent detailed medical history collection, eye examination and related laboratory tests; (6) The control group and the disease group were not related by blood.
[0028] Exclusion criteria: (1) Exclude patients with other eye diseases and other autoimmune diseases such as systemic lupus erythematosus and inflammatory bowel disease; (2) Incomplete clinical case data.
[0029] Sample collection: Fasting peripheral venous blood was collected, and plasma was separated. Due to the low yield of exosomes, plasma samples from individuals of similar age and sex were pooled (10 individuals per sample, with a disease group:control group ratio of 3:3). Exosomes were then extracted from the pooled samples for subsequent testing.
[0030] II) Test Methods
[0031] 1) Peripheral blood collection and extraction and identification of plasma exosomes
[0032] Peripheral blood was collected using EDTA anticoagulant blood collection tubes. The tubes were centrifuged at 2500g for 15 minutes, and the supernatant plasma was collected into a 2ml sterile tube.
[0033] Store in a -80°C freezer.
[0034] 2) Extraction of plasma exosomes
[0035] Exosomes were extracted using the Exoquick reagent kit (System Biosciences). Plasma was thawed in a 25°C water bath after being removed from the freezer, then transferred to centrifuge tubes and centrifuged at 3000g for 10 min at 4°C to remove cell debris. The supernatant was transferred to a new centrifuge tube and centrifuged at 10000g for 20 min at 4°C to remove impurities. 0.4 ml of Exoquick reagent (System Biosciences) was added to every 1.6 ml of supernatant and incubated at 4°C for 30 min. After centrifugation (3000g × 30 min), the supernatant was discarded, followed by another centrifugation (3000g × 5 min) and then discarding the supernatant.
[0036] 3) Identification of exosomes
[0037] The identification process includes three steps: transmission electron microscopy observation of exosome morphology, exosome diameter particle analysis, and identification of characteristic exosome proteins, as detailed below:
[0038] a. Observation of exosome morphology by transmission electron microscopy: 5 μl of the obtained exosome suspension was added to a Formvar-carbon copper grid. After cleaning the copper grid with PBS, it was placed on 50 μl of 1% glutaraldehyde solution for 5 min, then washed with 100 μl of ddH2O for 2 min. It was stained with uranyl oxalate and methylcellulose solution. After absorbing excess liquid on filter paper, it was dried in air for 5 min. The morphology of the obtained exosomes was identified using a HITACHI-HT7700 transmission electron microscope.
[0039] The results are as follows Figure 1 As shown, both groups of plasma exosomes exhibited complete and uniformly shaped round and oval cup-shaped vesicle structures under transmission electron microscopy (TEM), thus the precipitate obtained was identified as exosomes.
[0040] b. Exosome diameter analysis using the ZetaView PMX 110 particle tracker: First, the ZetaView PMX 110 detector is tested for performance using standards. Once the test is passed, exosome samples can be loaded. Note that the exosome samples need to be diluted to an appropriate concentration with PBS solution to avoid clogging the injection needle and affecting the detection results due to excessively high sample concentration.
[0041] like Figure 2 As shown, the results indicated that the average particle size of exosomes in the uveitis group was 106.01 nm, and the average concentration was 8.10 × 10⁻⁶. 7 Particles / ml; the average particle size of the healthy control group was 110.10 nm, and the average concentration was 7.12 × 10⁻⁶. 7 Particles / ml, particle size quality assessment criteria are: particle size: 30~150 nm; particle concentration: 10 7 ~10 11 Therefore, the detection results of both groups of samples are consistent with the calipericyloid characteristics of exosomes;
[0042] c. Western blotting analysis of exosome surface-specific markers: The expression of exosome surface characteristic proteins CD81, TSG101, HSP70 and exosome-negative protein Calnexin (cell group as control) in uveitis group (exosome 1 and exosome 2) and normal control group (exosome 3 and exosome 4) samples was detected by WB assay. Figure 3As shown, the results indicated that CD81, TSG101, and HSP70 antibodies were expressed positively in both the uveitis group and the normal control group, while the exosome-negative protein Calnexin was not expressed, proving that the extracted samples were exosomes.
[0043] 4) Extraction of exosomal proteins
[0044] Add 50 μL of RIPA lysis buffer (containing 1 mM PMSF) to the exosome precipitate extracted by the Exoquick kit (System Biosciences) and lyse on ice for 30 min.
[0045] Centrifuge at 4℃ and 12000rpm for 15min, collect the supernatant, and store at -80℃ for later use.
[0046] 5) Western Blot detection of IDH3a protein
[0047] 1. Protein concentration determination:
[0048] Protein concentration was determined using the BCA method, and the loading amount was adjusted to 20 μg.
[0049] 2. SDS-PAGE electrophoresis:
[0050] Prepare a 10% separating gel and a 5% stacking gel;
[0051] Mix the protein sample with 5× loading buffer at a ratio of 4:1 and boil in a water bath for 5 minutes.
[0052] Load 20 μg of protein per well, concentrate electrophoresis at 80V to the separating gel, then switch to 120V and continue electrophoresis to the bottom of the bromophenol blue gel.
[0053] 3. Transfer:
[0054] Wet transfer method was used, with constant current at 200mA for 60 min;
[0055] The transfer buffer composition is: 25 mM Tris, 190 mM glycine, and 20% methanol.
[0056] The PVDF membrane is activated with methanol before use.
[0057] 4. Blocking and antibody incubation:
[0058] After transfer, seal with 5% skim milk powder (prepared by TBST) at room temperature for 2 hours;
[0059] Add mouse anti-human IDH3a monoclonal antibody (1:1000 dilution) and incubate overnight at 4°C;
[0060] Wash the membrane with TBST 3 times, 10 minutes each time;
[0061] Add HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution) and incubate at room temperature for 1 hour;
[0062] Wash the membrane with TBST 3 times, 10 minutes each time.
[0063] 5. Color development and imaging:
[0064] Use ECL chemiluminescent reagent to uniformly cover the membrane surface;
[0065] Exposure and image acquisition in a chemiluminescence imaging system.
[0066] 6. Results Analysis:
[0067] ImageJ software was used to analyze the grayscale values of the bands, with the total protein amount in each lane as an internal reference, to calculate the relative expression level of IDH3a (e.g., ...). Figure 4 and Figure 5 As shown, Figure 4 (C1, C2, C3: healthy control group samples; U1, U2, U3: uveitis patient samples). The results showed that IDH3a expression in the patient group was significantly lower than that in the control group.
[0068] Statistical analysis and graphing were performed using Graphpad Prism 10 software. Continuous variables conforming to a normal distribution were analyzed using...
[0069] Mean ± standard deviation (mean ± SD) indicates that the data comparison between the two groups was performed using an independent samples t-test.
[0070] Example 3
[0071] This example is a kit prepared based on the IDH3a protein as a molecular marker for diagnosing uveitis, as described in Example 1. The kit includes mouse anti-human IDH3a monoclonal antibody or rabbit anti-human IDH3a polyclonal antibody (1 mL, 1 mg / mL); RIPA lysis buffer (10 mL); 5% skim milk blocking buffer (50 mL); TBST washing buffer (100 mL); HRP goat anti-mouse secondary antibody (1 mL); ECL chromogenic substrate (10 mL); 5× protein loading buffer (5 mL); electrophoresis buffer (500 mL); transfer buffer (500 mL); and PVDF membranes (10 sheets).
[0072] It should be noted that the structure described in this invention can be implemented in many different forms and is not limited to the embodiments described. Any equivalent transformations made by those skilled in the art based on the description and drawings of this invention, or direct or indirect applications in other related technical fields, such as the loading and unloading of other items, are included within the protection scope of this invention.
Claims
1. Exosomal proteins as molecular markers for diagnosing uveitis, characterized by: The molecular marker is the IDH3a protein in exosomes, and the sequence of the IDH3a protein is shown in SEQ ID NO:
1.
2. The exosomal protein as described in claim 1 as a molecular marker for diagnosing uveitis, characterized in that: The exosomes are plasma exosomes.
3. A reagent kit for diagnosing uveitis, characterized in that: The kit includes an antibody that specifically recognizes the IDH3a protein as described in claim 1.
4. The kit as described in claim 3, characterized in that: The kit also includes lysis buffer, blocking buffer, washing buffer, secondary antibody, chromogenic substrate, protein loading buffer, electrophoresis buffer, transfer buffer, membrane blocking buffer, and PVDF membrane.
5. The kit according to claim 3, characterized in that: The antibody is either a mouse anti-human IDH3a monoclonal antibody or a rabbit anti-human IDH3a polyclonal antibody.