Application of substance specifically binding to S-100B protein in preparation of diagnosis product or prognosis product for acute retinal necrosis

Abstract

The invention relates to the field of medicine, in particular to application of a substance specifically binding to S-100B protein to preparation of an acute retinal necrosis diagnosis product or prognosis product. By detecting the level of S-100B protein in aqueous humor, the condition of optic nerve injury caused by acute retinal necrosis can be objectively and accurately observed, clinical early diagnosis of ARN can be helped, a correct treatment method can be adopted in time, the risk of retinal detachment and blindness can be effectively reduced, clinical prediction of disease development and vision prognosis can be helped, early intervention can be realized, and clinical diagnosis of acute retinal necrosis can be realized. Accurate treatment is achieved, and medicine abuse and waste of medical resources are reduced.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to use of a substance that specifically binds to an S-100B protein in the preparation of an acute retinal necrosis diagnosis product or a prognosis product. Background Art

[0002] Acute retinal necrosis (ARN) is a serious blinding eye disease that can occur at any age, with necrotizing retinitis, occlusive retinal vasculitis, and vitritis as the main manifestations. The disease progresses rapidly. If it is not diagnosed and treated promptly and correctly, the treatment effect is poor, the incidence of retinal detachment is high, the visual prognosis is poor, and blindness is likely to occur. In some acute cases, patients develop vision with only light perception within a week from the onset of symptoms.

[0003] At present, the diagnosis of acute retinopathy is mainly determined by clinical ophthalmologists based on their experience of the patient's clinical manifestations. However, due to the difficulty of observation caused by vitreous opacity and the subjectivity of physical examination, the early correct diagnosis rate of the disease is low and misdiagnosis is prone to occur. In particular, the lack of specificity of the early symptoms of ARN and the great similarity to diseases such as uveitis make the diagnosis more difficult. At present, the problems in treatment include untimely treatment, lack of targeted antiviral treatment, and insufficient control of disease progression. Early correct diagnosis and timely treatment, and striving to control the progression of the disease before retinal detachment occurs, are of great significance for improving the efficacy and prognosis of ARN patients. Clinically, there is an urgent need for objective and accurate biomarkers to help with the early correct diagnosis of ARN and to assess the severity and prognosis of the disease.

[0004] S-100B protein is a type of neural tissue protein and a marker protein of glial cells. Its level can preliminarily 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 aqueous humor and ARN. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of a substance that specifically binds to the S-100B protein in the preparation of an acute retinal necrosis diagnostic product or a prognostic product, so as to solve the problems in the prior art.

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

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

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

[0009] The acute retinal necrosis diagnostic product is a product that distinguishes patients with acute retinal necrosis from healthy people or patients with other diseases such as uveitis.

[0010] The acute retinal necrosis diagnostic product may also be a product for determining the severity of acute retinal necrosis.

[0011] As described above, the use of the substance that specifically binds to the S-100B protein of the present invention in the preparation of an acute retinal necrosis diagnostic product or a prognostic product has the following beneficial effects: by detecting the level of the S-100B protein in the aqueous humor, it is possible to objectively and accurately observe the optic nerve damage caused by acute retinal necrosis, which can help the clinical early diagnosis of ARN, timely adopt the correct treatment method, effectively reduce the risk of retinal detachment and blindness, and help the clinic predict the development of the disease and the visual prognosis, achieve early intervention, precise treatment, reduce drug abuse and waste of medical resources, and have high economic benefits for patients and society. Using the aqueous humor S-100B protein as a biomarker for optic nerve damage can also be expanded to other ophthalmic diseases that can cause optic nerve damage, including glaucoma, optic neuritis, infectious endophthalmitis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0013] Figure 2 Shown is a ROC curve based on the expression level of S-100B protein in aqueous humor samples of ARN patients and control patients.

[0014] Figure 3 Results are shown as the correlation between the level of S-100B in aqueous humor and the percentage of retinal necrosis area observed clinically.

[0015] Figure 4 Shown are the results of correlation analysis between aqueous humor S-100B levels and retinal detachment survival rate. DETAILED DESCRIPTION

[0016] The detection of S-100B is currently reported to be mainly used for the detection of related diseases in blood and cerebrospinal fluid, and there are very few reports on the detection of S-100B protein in human aqueous humor. There are few studies on the diagnosis of ARN by detecting the expression of secretory proteins in the aqueous humor of patients, and there is currently no report on the connection between S-100B and ARN diagnosis. The present invention collects the aqueous humor of patients with ARN in the disease group, and the control group is a uveitis with early symptoms similar to ARN and easy to misdiagnose, and detects the expression of S-100B protein in the sample to be tested. If the expression of S-100B protein is higher than the diagnostic value of 9095pg / mL, it can be diagnosed as ARN, and the diagnostic value obtained by the comparative analysis of the disease group control disease has a very high clinical application value. In addition, the correlation analysis of the level of S-100B protein in aqueous humor and the percentage of retinal necrosis area obtained the correlation coefficient and linear formula of the two, which can help the clinic understand the severity of the disease more objectively. Patients with aqueous humor S-100B protein concentration >40,000 pg / mL have a significantly increased risk of developing retinal detachment and can therefore also serve as a prognostic marker for ARN patients.

[0017] Based on the above content, the present invention provides the use of a substance that specifically binds to S-100B protein or its encoding gene in the preparation of an acute retinal necrosis (ARN) diagnostic product or prognostic product.

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

[0019] In certain 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 a full-length sequence or a protein fragment, that is, the acute retinal necrosis diagnostic product or prognostic product includes a substance that can specifically bind 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, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The mammal includes, for example, humans, non-human primates (such as monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep or other non-human mammals; non-mammals include, for example, non-mammalian vertebrates, such as birds (such as chickens or ducks) or fish, and non-mammalian invertebrates.

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

[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, an antibody functional fragment, 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 a product that distinguishes patients with acute retinal necrosis from healthy people or patients with other diseases such as uveitis.

[0025] The acute retinal necrosis diagnostic product can also be a product for judging the severity of acute retinal necrosis. When the expression of S-100B protein or its encoding gene detected by the acute retinal necrosis diagnostic product is higher, the acute retinal necrosis is more severe. Clinically, the severity of acute retinal necrosis can be measured by the percentage of retinal necrosis area.

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

[0027] In the present invention, the "prognosis" refers to the prediction of the possible consequences of trauma or disease (such as ARN), and the prognostic indicators include short-term efficacy indicators and long-term efficacy indicators, including but not limited to HR, ORR, DCR, OS, PFS, DFS, and RFS. Among them, HR refers to the hazard ratio (HR), which is used to compare the risk of an event (such as disease recurrence, death, etc.) occurring in two groups (such as the observation group and the control group) within a specific time. The calculation method of HR is usually based on a survival analysis model, such as the Cox proportional hazard model. In practical applications, statistical software (such as R, SAS, SPSS, etc.) can be used to construct a Cox model and calculate HR. If HR>1, it means that the risk of the observation group is higher than that of the control group. If HR<1, it means that the risk of the observation group is lower than that of the control group. If HR=1, it means that the risks of the two groups are the same.

[0028] In the diagnostic product or prognostic product, the active ingredient that exerts its function may be only a substance that specifically binds to the S-100B protein or its encoding gene, and may also contain other substances. That is, the substance used to detect the S-100B protein or its encoding gene is the only active ingredient or one of the active 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 can be a commercial reagent or a kit or a homemade reagent. The commercial kit is, for example, an ELISA kit. The components of the ELISA kit include a biotin-labeled antibody or antibody functional fragment that can specifically bind to the S-100B protein, a sample diluent, a horseradish peroxidase-labeled avidin, a standard, a color developing solution, and a stop solution.

[0031] The form of the product is not particularly limited and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0032] The sample type mainly targeted by the product is aqueous humor.

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

[0034] The present invention does not specifically limit the etiology, clinical manifestations, severity, and presence or absence of complications of the acute retinal necrosis. As long as it is acute retinal necrosis, the diagnostic product of the present invention can be used for diagnosis.

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

[0036] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0037] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0038] Example 1

[0039] The present invention collected aqueous humor samples from 36 ARN patients and 38 uveitis (UV) patients, and used an ELISA kit to detect the expression level of S-100B in the aqueous humor samples. The expression difference of S-100B in the aqueous humor of ARN patients and uveitis patients was statistically analyzed. In addition, GraphPad Prism 9.5.1 was used to draw the S-100B detection concentration scatter plot and Kaplan-Meier survival curve of two differential diagnosis diseases, and MedCalc 19.6.4 was used to draw a single ROC curve to analyze the specificity and sensitivity of S-100B protein for ARN diagnosis. The details are as follows:

[0040] 1. Experimental Materials

[0041] The 96-well ELISA plate (8 rows × 12 columns) pre-coated with anti-S-100B protein antibody used in the present invention was purchased from Wuhan Huamei Biotechnology Co., Ltd. (Cusabio Technology LLC.), and the S-100B (human soluble protein-100B) ELISA kit used in the present invention was purchased from Wuhan Huamei Biotechnology Co., Ltd. (Cusabio Technology LLC.), product number CSB-E08065h, and the reagent components are as follows:

[0042] 1. Standard product (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 diluent (buffered solution containing Proclin 300 (1:3000))

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

[0047] 6. Sample diluent (buffered solution containing Proclin 300 (1:3000))

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

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

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

[0051] 2. Experimental steps

[0052] 1. Collect samples

[0053] The aqueous humor of 36 patients with ARN was collected, and the aqueous humor of 38 patients with uveitis (UV) was collected as a control group. Since other inflammatory indicators will have obvious differences between ARN and healthy people, and it is ethically unacceptable to extract aqueous humor from healthy people, healthy people as controls cannot accurately reflect the accuracy of diagnosis; and UV is difficult to distinguish from ARN in early symptoms and is easy to misdiagnose, so the present invention uses UV patients as controls. Among them, the aqueous humor of 36 patients with ARN comes from patients diagnosed by clinical diagnostic criteria; the aqueous humor of 36 patients with uveitis comes from control disease patients without fundus necrotic lesions. The collected aqueous humor is placed in a 1.5mL EP tube, and the supernatant is aspirated after centrifugation at 4000×g for 10 minutes, and placed in a -80℃ refrigerator for frozen storage. Before use, take out the aqueous humor and thaw it in a 4℃ refrigerator and aliquot it to avoid repeated freezing and thawing.

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

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

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

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

[0058] D) 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 3 times.

[0059] E) Add 100 μL of detection antibody labeled with horseradish peroxidase (HRP) to each well, seal the plate with sealing film, and incubate in a 37°C incubator 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 a wavelength of 450 nm using an ELISA reader within 5 minutes.

[0063] 3. Analysis by t-test

[0064] Draw the standard curve: In the Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis to draw the standard linear regression curve, and calculate the concentration value of S-100B in each sample according to the curve equation. The t-test method was used to analyze whether the S-100B protein levels in the ARN group and the control group (UV group) were significantly different. The test results are as follows Figure 1 As shown in the results, there was a significant difference in the level of S-100B protein in the aqueous humor between ARN patients and uveitis (UV) patients. Compared with UV patients, the level of S-100B protein in the aqueous humor of ARN patients was significantly increased, indicating that the expression level of S-100B is closely related to ARN and can be used as a biomarker for the diagnosis of ARN.

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

[0066] GraphPad Prism 9.5.1 was used to draw ROC curves and analyze the value of S-100B protein in the diagnosis of ARN. Based on the expression level of S-100B protein in aqueous humor samples of 36 ARN patients and 38 control patients, ROC curves were drawn to evaluate the ability of S-100B to distinguish ARN from UV patients.

[0067] The ROC curve for distinguishing ARN and UV patients based on S-100B protein diagnosis was used. The S-100B concentration value with the largest Youden index was used as the cutoff value (i.e., the diagnostic value). The corresponding AUC and 95% confidence interval, sensitivity, and specificity were calculated at the same time. The sensitivity and specificity calculation formulas are as follows:

[0068]

[0069] Where TPR is the sensitivity,

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

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

[0072]

[0073] Among them, FPR is specificity,

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

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

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

[0077] Example 3 Correlation analysis between markers and retinal necrosis area percentage

[0078] Patients with confirmed ARN underwent ultra-wide-angle fundus imaging (UWF) examinations using the 200Tx laser scanning ophthalmoscope retinal imaging system from the UK Optos company. The collected images were evaluated by two experienced physicians and a consensus was reached on the percentage of active retinal necrosis area. GraphPad Prism 9.5.1 was used to draw a linear regression analysis of the aqueous humor S-100B protein concentration and the percentage of active retinal necrosis area. The results are shown in Figure 2. Figure 3 The results showed that the level of S-100B in aqueous humor was correlated with the percentage of retinal necrosis area observed clinically, with a correlation coefficient r value of 0.689, indicating that the expression level of S-100B can reflect the severity of the disease.

[0079] Example 4 Markers 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 concentration of all ARN patients was ranked, and the median value of 40000pg / mL was taken as the cutoff value. The patients were divided into two groups: >40000pg / mL and <40000pg / mL. The patients were followed up for a maximum of one year, and the follow-up was terminated when retinal detachment occurred. GraphPad Prism 9.5.1 was used to draw the Kaplan-Meier curve, and the results showed that there was a significant correlation between the aqueous humor S-100B level and the risk of retinal detachment ( Figure 4 ; P = 0.0248). Patients with S-100B protein concentration > 40000 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 can be used as a prognostic marker for ARN patients.

[0081] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present 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 the present invention.

Claims

1. Use of a substance that specifically binds to S-100B protein or its encoding gene in the preparation of a diagnostic product or a prognostic product for acute retinal necrosis.

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

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

4. The use according to claim 1, characterized in that The S-100B protein or its encoding gene is derived from animals, preferably from mammals; more preferably from humans.

5. The use according to claim 1, characterized in that: The substance that specifically binds to the S-100B protein or its encoding gene is selected from antibodies, functional fragments of antibodies, nucleic acid molecules, lipids, small chemical molecules, polypeptides, liposomes or cells.

6. The use according to claim 1, characterized in that: The acute retinal necrosis diagnostic product is a product for distinguishing patients with acute retinal necrosis from healthy people or patients with other eye diseases.

7. The use according to claim 6, characterized in that The acute retinal necrosis diagnostic product is a product for distinguishing patients with acute retinal necrosis from patients with uveitis.

8. The use according to claim 1, characterized in that The acute retinal necrosis diagnostic product is a product used to determine the severity of acute retinal necrosis.

9. The use according to claim 1, characterized in that The acute retinal necrosis diagnostic product or prognostic product is an ELISA kit; preferably, the components of the ELISA kit also include any one or more of the following: sample diluent, biotin-labeled detection antibody, horseradish peroxidase-labeled avidin, standard, color developing solution or stop solution.

10. The use according to claim 1, characterized in that The sample type of the acute retinal necrosis diagnostic product or prognostic product is aqueous humor.

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