Use of substances that specifically bind to the S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis.

Abstract

This invention relates to the pharmaceutical field, and particularly to the use of substances that specifically bind to S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis (ARN). By detecting the level of S-100B protein in the aqueous humor, the extent of optic nerve damage caused by ARN can be objectively and accurately observed. This can aid in the early clinical diagnosis of ARN, enabling timely and appropriate treatment, effectively reducing the risk of retinal detachment and blindness, and helping clinicians predict disease progression and visual prognosis, achieving early intervention, precision treatment, and reducing drug abuse and waste of medical resources.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of substances that specifically bind to the S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis. Background Technology

[0002] Acute retinal necrosis (ARN) is a serious, blinding eye disease that can occur at any age, primarily manifested as necrotizing retinitis, occlusive retinal vasculitis, and vitreous inflammation. The disease progresses rapidly, and if not diagnosed and treated promptly and correctly, the treatment outcome is poor, with a high incidence of retinal detachment, poor visual prognosis, and a high risk of blindness. In some acute cases, patients develop light perception as little as a week after the onset of symptoms.

[0003] Currently, the diagnosis of acute retinitis retinopathy (ARN) mainly relies on clinical ophthalmologists' experience and judgment based on the patient's clinical presentation. However, due to difficulties in observation caused by vitreous opacity and the subjectivity of physical examination, the early accurate diagnosis rate is low, and misdiagnosis is common. This is especially true because early symptoms of ARN lack specificity and are very similar to those of diseases such as uveitis, further complicating diagnosis. Current treatment challenges include insufficient timeliness, lack of targeted antiviral therapy, and inadequate control of disease progression. Early and accurate diagnosis and timely treatment, aiming to control disease progression before retinal detachment occurs, are crucial for improving the treatment outcomes and prognosis of ARN patients. Clinically, there is an urgent need for objective and accurate biomarkers to aid in the early accurate diagnosis of ARN, assess disease severity, and predict prognosis.

[0004] S-100B protein is a type of nerve tissue protein and a marker protein of glial cells. Its level can initially reflect the severity of nerve damage. The higher the S-100B protein content, the more severe the nerve damage and the worse the prognosis. However, there is no public information on the correlation between S-100B protein in the aqueous humor and ARN. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide the use of a substance that specifically binds to the S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis, thereby solving the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides the use of a substance that specifically binds to the S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis.

[0007] The S-100B protein is either wild-type S-100B protein or mutant S-100B protein.

[0008] The S-100B protein is either a full-length sequence or a protein fragment, meaning that the acute retinal necrosis diagnostic or prognostic product includes a substance capable of specifically binding to the full-length sequence or protein fragment of the S-100B protein.

[0009] The acute retinal necrosis diagnostic product is designed to distinguish patients with acute retinal necrosis from healthy individuals or patients with other diseases, such as uveitis.

[0010] The acute retinal necrosis diagnostic product can also be a product used to determine the severity of acute retinal necrosis.

[0011] As described above, the use of the substance of the present invention that specifically binds to S-100B protein in the preparation of diagnostic or prognostic products for acute retinal necrosis (ARN) has the following beneficial effects: By detecting the level of S-100B protein in the aqueous humor, the extent of optic nerve damage caused by ARN can be objectively and accurately observed. This can help in the early clinical diagnosis of ARN, timely adoption of appropriate treatment methods, effectively reducing the risk of retinal detachment and blindness, and helping in the clinical prediction of disease progression and visual prognosis. It enables early intervention and precise treatment, reducing drug abuse and waste of medical resources, resulting in significant economic benefits for patients and society. Using aqueous humor S-100B protein as a biomarker for optic nerve damage can also be extended to other ophthalmic diseases that can lead to optic nerve damage, including glaucoma, optic neuritis, and infectious endophthalmitis. Attached Figure Description

[0012] Figure 1 The difference in S-100B protein levels in the aqueous humor between ARN patients and uveitis (UV) patients, as shown in this invention, is illustrated.

[0013] Figure 2 The ROC curves are shown as plotted based on the expression levels of S-100B protein in aqueous humor samples from ARN patients and control patients.

[0014] Figure 3 The results show the correlation between S-100B levels in the aqueous humor and the percentage of clinically observed retinal necrosis area.

[0015] Figure 4 The results show the correlation analysis between aqueous humor S-100B levels and retinal detachment survival rate. Detailed Implementation

[0016] Current reports on S-100B detection primarily focus on its application in blood and cerebrospinal fluid-related disease detection, with very few reports on its detection in aqueous humor. Studies on diagnosing ARN by detecting the expression of secreted proteins in the aqueous humor of patients are scarce, and no reports have yet established a link between S-100B and ARN diagnosis. This invention collects aqueous humor from ARN patients in the disease group and a control group of patients with uveitis, a disease with early symptoms similar to ARN and easily misdiagnosed. The expression level of S-100B protein in the test samples is measured. If the expression level of S-100B protein is higher than the diagnostic value of 9095 pg / mL, ARN can be diagnosed. The diagnostic value obtained through comparative analysis of the disease group and the control group has very high clinical application value. Furthermore, correlation analysis between the level of S-100B protein in aqueous humor and the percentage of retinal necrosis area yielded the correlation coefficient and linear formula, which can more objectively help clinicians understand the severity of the disease. Patients with aqueous humor S-100B protein concentrations >40,000 pg / mL have a significantly increased risk of developing retinal detachment and can therefore serve as a prognostic marker for ARN patients.

[0017] Based on the above, the present invention provides the use of substances that specifically bind to the S-100B protein or its encoding gene in the preparation of diagnostic or prognostic products for acute retinal necrosis (ARN).

[0018] The S-100B protein of this invention is a calcium-binding protein belonging to the S-100 family.

[0019] In some embodiments of the present invention, the S-100B protein is a wild-type S-100B protein or a mutant S-100B protein.

[0020] The S-100B protein is either a full-length sequence or a protein fragment, meaning that the acute retinal necrosis diagnostic or prognostic product includes a substance capable of specifically binding to the full-length sequence or protein fragment of the S-100B protein.

[0021] The S-100B protein or its encoding gene is derived from an animal, preferably a mammal; the mammal is preferably a rodent, even-toed ungulate, perissodactyl, lagomorph, primate, etc. The mammal includes, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammal vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammal invertebrates.

[0022] In a preferred embodiment, the S-100B protein or its encoding gene is derived from humans. The encoding gene for the human S-100B protein is located in the 2q33-35 region of human chromosomes.

[0023] The substance that specifically binds to the S-100B protein or its encoding gene is not specifically limited, as long as it can specifically bind to the S-100B protein or its encoding gene. For example, it can be an antibody, a functional fragment of an antibody, a conjugated antibody, a nucleic acid molecule, a lipid, a small chemical molecule, a polypeptide, a protein, a lentivirus, an adeno-associated virus, a nanoparticle, a liposome, an extracellular vesicle, or a cell.

[0024] The acute retinal necrosis diagnostic product is designed to distinguish patients with acute retinal necrosis from healthy individuals or patients with other diseases, such as uveitis.

[0025] The acute retinal necrosis diagnostic product can also be used to determine the severity of acute retinal necrosis. Higher expression levels of the S-100B protein or its encoding gene detected by the acute retinal necrosis diagnostic product indicate more severe acute retinal necrosis. Clinically, the severity of acute retinal necrosis can be measured as the percentage of retinal necrosis area.

[0026] The acute retinal necrosis prognostic product refers to a product used to predict disease progression, such as a product that predicts the probability of retinal detachment (RD) leading to vision loss.

[0027] In this invention, "prognosis" refers to the prediction of the potential consequences of trauma or disease (e.g., ARN). Prognostic indicators include short-term and long-term efficacy indicators, including but not limited to HR, ORR, DCR, OS, PFS, DFS, and RFS. HR refers to the hazard ratio, used to compare the risk of a specific event (e.g., disease recurrence, death) occurring in two groups (e.g., the observation group and the control group) within a specific timeframe. HR calculations are typically based on survival analysis models, such as the Cox proportional hazards model. In practice, statistical software (e.g., R, SAS, SPSS) can be used to construct the Cox model and calculate the HR. If HR > 1, the risk in the observation group is higher than that in the control group. If HR < 1, the risk in the observation group is lower than that in the control group. If HR = 1, the risks in both groups are equal.

[0028] The effective ingredient in the diagnostic or prognostic product may be solely a substance that specifically binds to the S-100B protein or its encoding gene, or it may contain other substances. That is, the substance used to detect the S-100B protein or its encoding gene is the sole or one of the effective ingredients of the product. The product can be used in combination with other products or alone.

[0029] The product can be a single-component substance or a multi-component substance.

[0030] The product may be a commercially available reagent or kit, or a self-made reagent. A commercially available kit, for example, is an ELISA kit. The components of the ELISA kit include a biotin-labeled antibody or functional fragment of an antibody that specifically binds to the S-100B protein, as well as sample diluent, horseradish peroxidase-labeled avidin, standards, chromogenic solution, and stop solution.

[0031] There are no special restrictions on the form of the product; it can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0032] The product is primarily designed for samples containing aqueous humor.

[0033] Acute retinal necrosis (ARN) is a serious inflammatory eye disease, usually caused by a viral infection, that can lead to rapid necrosis of the retina and vision loss.

[0034] This invention does not specifically limit the etiology, clinical manifestations, severity, or presence of complications of acute retinal necrosis. Any acute retinal necrosis can be diagnosed using the diagnostic product of this invention.

[0035] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0037] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. 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. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0038] Example 1

[0039] This invention collected aqueous humor samples from 36 patients with ARN and 38 patients with uveitis (UV), and used an ELI SA kit to detect the expression level of S-100B in the aqueous humor samples. Statistical analysis was then performed on the differences in S-100B expression in the aqueous humor of ARN and uveitis patients. Furthermore, scatter plots of S-100B detection concentrations and Kaplan-Meier survival curves for the two differential diagnoses were plotted using GraphPad Prism 9.5.1, and single ROC curves were plotted using MedCalc 19.6.4 to analyze the specificity and sensitivity of S-100B protein for the diagnosis of ARN. Details are as follows:

[0040] I. Experimental Materials

[0041] The 96-well microplate (8 rows × 12 columns) pre-coated with anti-S-100B protein antibody used in this invention was purchased from Cusabio Technology LLC., and the S-100B (human soluble protein-100B) ELISA kit used in this invention was purchased from Cusabio Technology LLC., product number CSB-E08065h. The reagent components are shown below:

[0042] 1. Standard (Recombinant S-100B protein)

[0043] 2. Biotin-labeled detection antibody (anti-S-100B protein, 1×100)

[0044] 3. Horseradish peroxidase-labeled avidin (1×100)

[0045] 4. Biotin-labeled antibody dilution buffer (buffer solution containing Proclin 300 (1:3000))

[0046] 5. Horseradish peroxidase-labeled avidin dilution solution (buffer solution containing Proclin 300 (1:3000))

[0047] 6. Sample dilution buffer (containing Proclin 300 (1:3000))

[0048] 7. Concentrated washing solution (25×Tris-HCl buffer solution)

[0049] 8. Substrate solution (3,3',5,5'-tetramethylbenzidine diamine salt)

[0050] 9. Stop solution (1 mol / L sulfuric acid)

[0051] II. Experimental Procedure

[0052] 1. Sample collection

[0053] Aqueous humor was collected from 36 patients with aeroid retinitis (ARN) and 38 patients with uveitis (UV) as a control group. Because other inflammatory markers differ significantly between ARN and healthy individuals, and because extracting aqueous humor from healthy individuals is ethically unacceptable, using healthy individuals as a control group cannot accurately reflect diagnostic accuracy. Furthermore, UV is difficult to differentiate from ARN in its early stages, leading to misdiagnosis. Therefore, UV patients were selected as the control group. The aqueous humor from the 36 ARN patients was obtained from patients diagnosed according to clinical criteria; the aqueous humor from the 36 uveitis patients was obtained from control patients without fundus necrosis. The collected aqueous humor was placed in 1.5 mL EP tubes, centrifuged at 4000×g for 10 minutes, and the supernatant was collected and frozen at -80°C. Before use, the aqueous humor was thawed at 4°C and aliquoted, avoiding repeated freeze-thaw cycles.

[0054] 2. ELISA detection of S-100B protein content in aqueous humor

[0055] A) Set up standard wells and sample wells, and add 100 μL of standard at different concentrations to each standard well.

[0056] B) Add 98 μL of sample diluent to the sample well, then add 2 μL of the sample to be tested, mix well, seal with film, and incubate at 37°C for 120 min.

[0057] C) Discard the liquid, add 100 μL of biotin-labeled antibody, affix a new sealing film, and incubate at 37°C for 60 min.

[0058] D) Discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 2 minutes, shake off the washing liquid, pat dry with absorbent paper, and repeat this washing process 3 times.

[0059] E) Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well, seal with sealing film, and incubate at 37°C for 60 min.

[0060] F) Discard the liquid, pat dry with absorbent paper, fill each well with washing solution, let stand for 2 minutes, shake off the washing solution, pat dry with absorbent paper, and repeat this washing process 5 times.

[0061] G) Add 90 μL of substrate solution to each well and incubate at 37°C in the dark for 15 min.

[0062] H) Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm wavelength using an ELISA reader within 5 min.

[0063] 3. t-test analysis

[0064] Plotting a standard curve: In an Excel worksheet, plot the standard concentration on the x-axis and the corresponding OD value on the y-axis to create a linear regression curve for the standard. Calculate the S-100B concentration for each sample using the curve equation. Analyze whether there is a significant difference in S-100B protein levels between the ARN group and the control group (UV group) using a t-test. The test results are as follows: Figure 1 As shown, the results indicate a significant difference in S-100B protein levels in the aqueous humor between patients with ARN and those with uveitis (UV). Compared to UV patients, ARN patients showed significantly elevated S-100B protein levels in their aqueous humor, suggesting a close relationship between S-100B expression and ARN, and therefore, it could serve as a biomarker for ARN diagnosis.

[0065] Example 2: ROC curve analysis, sensitivity, and specificity

[0066] ROC curves were plotted using GraphPad Prism 9.5.1 to analyze the diagnostic value of S-100B protein in ARN. Based on the expression levels of S-100B protein in aqueous humor samples from 36 ARN patients and 38 control patients, ROC curves were plotted to evaluate the diagnostic ability of S-100B in differentiating between ARN and UV patients.

[0067] Based on the ROC curve of S-100B protein in differentiating ARN and UV patients, the S-100B concentration with the highest Youden index was used as the cutoff value (i.e., the diagnostic value). Simultaneously, the corresponding AUC, 95% confidence interval, sensitivity, and specificity were calculated. The formulas for calculating sensitivity and specificity are as follows:

[0068]

[0069] Where TPR stands for sensitivity.

[0070] TP stands for True Positives, which is the number of samples that are correctly predicted as positive.

[0071] FN stands for False Negatives, which is the number of positive samples that are incorrectly predicted as negative.

[0072]

[0073] Where FPR stands for specificity.

[0074] FP stands for False Positives, which is the number of negative samples that are incorrectly predicted as positive.

[0075] TN stands for True Negatives, which is the number of samples that are correctly predicted as negative.

[0076] Test results as follows Figure 2 As shown, the diagnostic value was 9095 pg / mL, the AUC value of its ROC curve was 0.904, the sensitivity was 75%, the specificity was 100%, and the Youden index was 0.75. The results indicate that the expression level of S-100B in human aqueous humor has very high specificity and sensitivity for the diagnosis of ARN.

[0077] Example 3: Correlation Analysis of Biomarkers and Percentage of Retinal Necrosis Area

[0078] Patients diagnosed with ARN underwent ultra-wide-angle fundus imaging (UWF) using an Optos 200Tx laser scanning ophthalmoscopy retinal imaging system. The acquired images were assessed and agreed upon by two experienced physicians regarding the percentage of active retinal necrosis. A linear regression analysis of aqueous humor S-100B protein concentration versus the percentage of active retinal necrosis was plotted using GraphPad Prism 9.5.1, and the results are as follows: Figure 3 As shown in the figure. The results indicate that the level of S-100B in the aqueous humor is correlated with the percentage of clinically observed retinal necrosis area, with a correlation coefficient r of 0.689, suggesting that the expression level of S-100B can reflect the severity of the disease.

[0079] Example 4: Biomarkers and Prognosis of ARN

[0080] To analyze the relationship between aqueous humor S-100B protein concentration and retinal detachment (retinal detachment) survival rate, the aqueous humor S-100B protein concentrations of all ARN patients were ranked. Using the median value of 40,000 pg / mL as the cutoff, patients were divided into two groups: >40,000 pg / mL and <40,000 pg / mL. Patients were followed up for a maximum of one year, and follow-up was terminated upon the occurrence of retinal detachment. Kaplan-Meier curves were plotted using GraphPad Prism 9.5.1. The results showed a significant correlation between aqueous humor S-100B levels and the risk of retinal detachment. Figure 4 (P = 0.0248). Patients with S-100B protein concentrations >40,000 pg / mL had a significantly increased risk of developing retinal detachment, with a hazard ratio (HR) of 3.207 (95% confidence interval, 1.159–8.877). This suggests that aqueous humor S-100B protein may serve as a prognostic biomarker for patients with ARN.

[0081] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of a substance specifically binding to aqueous S-100B protein for the manufacture of a product for the prognosis of acute retinal necrosis.

2. Use according to claim 1, characterized in that, The S-100B protein is a wild type S-100B protein.

3. Use according to claim 1, characterized in that, The S-100B protein is a full length sequence.

4. Use according to claim 1, characterized in that, The S-100B protein is derived from an animal.

5. Use according to claim 4, characterized in that, The S-100B protein is derived from a mammal.

6. Use according to claim 5, characterized in that, The S-100B protein is derived from a human.

7. Use according to claim 1, characterized in that, The substance specifically binding to aqueous S-100B protein is selected from the group consisting of an antibody, an antibody functional fragment.

8. Use according to claim 1, characterized in that, The product for the prognosis of acute retinal necrosis is an ELISA kit.

9. Use according to claim 8, characterized in that, The components of the ELISA kit further comprise any one or more of the following: sample diluent, biotin-labeled detection antibody, horseradish peroxidase-labeled avidin, standard, chromogenic solution, or stop solution.

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