Application of nucleoprotein in urine in noninvasive diagnosis of NIID

Abstract

The present invention proposes an in-urine nuclear protein for NIID diagnosis, the in-urine nuclear protein being a p62 protein, with which it is possible to provide a method, device and kit for non-invasive, simple and low-cost evaluation of NIID. According to the present invention, by comparing the content of the nuclear protein p62 in the urine of the healthy person and the NIID patient, the increase / decrease relationship of the protein between the healthy person and the NIID patient is determined so as to determine the NIID suffering possibility of the subject by using the relationship;

Description

Technical Field

[0001] The invention relates to use of urinary nucleoprotein in non-invasive diagnosis of NIID, belonging to the technical field of biomedical detection. Background Art

[0002] Neuronal intranuclear inclusion disease (NIID) is a rare disease that mainly affects the nervous system. The clinical manifestations of NIID are complex, diverse and highly heterogeneous, and often occur in all age groups. The symptoms of NIID overlap with a variety of neurological lesions, making clinical diagnosis difficult and also posing difficulties for genetic counseling and eugenics. At present, there are three diagnostic methods for NIID: imaging examination, pathological examination, and genetic examination. The imaging features of NIID mainly include curved diffusion-weighted imaging (DWI) high signals in the cortical-medullary junction, DWI high signals in the corpus callosum, symmetrical white matter lesions, cortical swelling and enhancement, extensive brain atrophy, and cerebellar white matter lesions symmetrically distributed along the cerebellar axis. In comparison, the specificity of NIID imaging detection is low, and its classic "ribbon sign" feature can appear in many other neurological diseases, and some sporadic patients do not have this imaging feature. Over-reliance on this imaging feature may lead to missed diagnosis or misdiagnosis of NIID.

[0003] The pathological characteristics of NIID are the detection of eosinophilic transparent inclusion bodies in cells of the central / peripheral nervous system and organs. In recent years, skin biopsy has become a relatively easy and less invasive method for pathological detection of NIID. Skin biopsy mainly involves taking skin tissue from patients to prepare tissue sections, which are then stained with HE. Inclusion bodies can be seen in the nuclei of cells under an optical microscope. However, similar nuclear inclusion bodies are also present in the skin tissue of patients with neurodegenerative diseases with similar clinical phenotypes, such as fragile X-associated tremor / ataxia syndrome (FXTAS). Therefore, the use of HE staining for skin biopsy diagnosis has the disadvantage of poor specificity. Although pathological detection methods are more accurate than imaging, their sampling methods are more invasive. Even if skin biopsy is used, it is not suitable for patients to accept due to its invasiveness and subsequent adverse reactions.

[0004] The genetic characteristic of NIID is the abnormal duplication of GGC in the 5' region of the NOTCH2NLC gene. The results of genetic testing cannot reflect the progression of the disease, and the detection time is long, the efficiency is low, the repeatability is low, the cost is slightly high, and it cannot meet the amplification of large-scale samples, making it difficult to be widely used in the clinical genetic diagnosis of NIID diseases. Although genetic testing of NIID is helpful in diagnosing NIID, it cannot be used as an independent basis for diagnosis, and pathological findings of nuclear inclusions are still required to make a diagnosis of NIID. Moreover, the abnormal duplication of GGC in the 5' region of the NOTCH2NLC gene only occurs frequently in the Asian population. It is extremely important to develop a diagnostic method for NIID that can replace pathological testing.

[0005] With the development of molecular biology and diagnostic technology, liquid biopsy has gradually come to the fore. Liquid biopsy diagnoses diseases by sampling cerebrospinal fluid, saliva, pleural effusion, blood, ascites, urine, etc. Compared with tissue biopsy, liquid biopsy uses specific antibodies to detect pathogenic proteins in patient-derived body fluids. It has the advantages of being non-invasive, highly repeatable, and capable of early diagnosis and dynamic monitoring. However, there are currently no specific antibodies that can be used to conduct relevant liquid biopsies. Therefore, it is necessary to develop a detection method that is specific for NIID. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a use of urinary nucleoprotein in non-invasive diagnosis of NIID.

[0007] The invention provides a urinary nuclear protein for NIID diagnosis, wherein the urinary nuclear protein is p62 protein.

[0008] The present invention relates to an evaluation method and a kit for determining whether a subject suffers from NIID by measuring intranuclear protein in the urine of the subject.

[0009] The technical solution further optimized as the present invention is as follows: The amino acid sequence of the above-mentioned p62 protein is shown in SEQ ID NO.1.

[0010] The present invention also provides a non-invasive diagnostic kit for NIID, comprising the above-mentioned urine nuclear protein for NIID diagnosis.

[0011] Preferably, the above-mentioned kit is a homogeneous digital immunoassay kit for p62, which is used to detect the content of nuclear protein in urine.

[0012] Preferably, the homogeneous digital immunoassay kit comprises at least a p62 protein standard, a p62 detection antibody, and a p62 capture antibody.

[0013] Preferably, the p62 detection antibody and the p62 capture antibody are respectively modified with quantum dots of different colors.

[0014] Preferably, the p62 detection antibody is modified with green fluorescent quantum dots, and the p62 capture antibody is modified with red fluorescent quantum dots.

[0015] The reagent set for detecting the content of nuclear protein in urine of the present invention is used in preparing a kit used in a method for evaluating NIID. The evaluation of NIID is the detection of NIID and the evaluation of the risk prediction of NIID.

[0016] In the above-mentioned use, the evaluation step includes comparing the reference value of the measured value of the nuclear protein in urine selected from the healthy or low-risk group sample with the reference value of the measured value of the nuclear protein in urine selected from the NIID patient sample. The reference value of the measured value of the nuclear protein in urine selected from the healthy or low-risk group sample is the highest value of the measured value, and the reference value of the measured value of the nuclear protein in urine selected from the NIID patient sample is the lowest value of the measured value.

[0017] In the above-mentioned use, if the nuclear protein p62 content in the urine sample to be tested is higher than the baseline value of nuclear protein in the urine of NIID patients, it means that the subject is very likely to suffer from NIID.

[0018] The present invention determines the increase and decrease relationship between healthy people and NIID patients by comparing the content of nuclear protein p62 in urine of healthy people and NIID patients, and then uses this relationship to confirm the possibility of the subject suffering from NIID. The present invention relates to a method and a kit for detecting NIID and predicting the risk of NIID in a subject by measuring the content of nuclear protein p62 in urine of the subject. Specifically, the method includes: a step of measuring the content of nuclear protein p62 in urine sample from the subject; and a step of evaluating the subject's NIID based on the above measurement results.

[0019] The present invention can provide a method, device and kit for evaluating NIID in a non-invasive, simple and low-cost manner. Therefore, the present invention is useful in the field of diagnosis of NIID. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The purification effect diagram of the quantum dots emitting fluorescence modified by the p62 detection antibody in the present invention (size exclusion chromatography). In the figure, A is the purification effect diagram of the quantum dots emitting green fluorescence modified by the p62 detection antibody, and B is the purification effect diagram of the quantum dots emitting red fluorescence modified by the p62 capture antibody.

[0021] Figure 2 The present invention is a flow chart of the homogeneous digital immunoassay for measuring nuclear protein p62 in urine.

[0022] Figure 3 Evaluation of the performance (sensitivity and selectivity) of the homogeneous digital immunoassay for p62 in the present invention Figure 4 This is a schematic diagram for comparing the p62 content in different neurological diseases in the present invention.

[0023] Figure 5 The figure is a receiver operating characteristic curve (ROC) of the urinary nuclear protein p62 content for diagnosing NIID in the present invention. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

[0025] In one embodiment of the present invention, the content of nuclear protein p62 in urine is measured. The measurement method can be a method or means known in the technical field without particular limitation.

[0026] The p62 homogeneous digital immunoassay kit for NIID evaluation of the present invention comprises at least the following units: p62 protein standard, p62 detection antibody modified to emit green fluorescent quantum dots, and p62 capture antibody modified to emit red fluorescent quantum dots. Each antibody is modified with quantum dots of different fluorescent colors. The two antibodies bind to different parts of the antigen and can form a heterochromatic quantum dot-labeled sandwich immune complex with the antigen.

[0027] The p62 homogeneous digital immunoassay kit can distinguish p62-induced immune complexes and free antibodies at the single particle level, so there is no need to separate the immune complexes from the free antibodies. The details are as follows: 1. Preparation, purification and blocking of green fluorescent quantum dots modified with p62 detection antibody 2.5 µL of green fluorescent carboxyl quantum dots (emission wavelength of 585 nm, purchased from Thermo Fisher Scientific Inc) was quickly mixed with 5.0 µL of 5 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (prepared and used immediately, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride, purchased from Thermo Fisher Scientific Inc), and then reacted in the dark at 25 °C for 25 min. 10 µL of the green fluorescent carboxyl quantum dots after reaction was mixed evenly with 20.0 µL of p62 detection antibody (purchased from Abcam Inc), and reacted in the dark at 25 °C for 4 h. The green fluorescent quantum dots were modified on the detection antibody by the reaction of the primary amino group of the antibody with the carboxyl group of the quantum dots. The above reaction solution was centrifuged at 14000 g for 5 min, the supernatant was aspirated, and it was purified by high performance liquid chromatography (size exclusion chromatography). Chromatographic conditions: The column model was SuperoseTM 6 Increase 5 / 150GL (purchased from Cytiva), and the mobile phase was NaHCO 3(50 mM, pH 9.0), flow rate 0.3 mL / min, injection volume 13.0 µL, UV detection wavelength 224.0 nm. Collect the detection antibody fraction modified with green fluorescent quantum dots (see Figure 1 ), stored at 4 ℃ for future use. Before use, mix the purified probe and SuperBlock™ blocking buffer at a volume ratio of 20:1, and react in the dark at 25 ℃ for 30 min. Use a fluorescence spectrophotometer to quantify the concentration of the detection antibody modified with green fluorescent quantum dots. Taking quantum dot 585 as an example, first determine the fluorescence intensity of pure quantum dot 585 with different concentrations, and draw a standard curve of the relationship between fluorescence intensity and concentration. According to the measured fluorescence intensity of the detection antibody modified with green fluorescent quantum dots, substitute it into the standard curve to calculate the concentration value of the detection antibody modified with green fluorescent quantum dots.

[0028] 2. Preparation, purification and blocking of red fluorescent quantum dots modified with p62 capture antibody The preparation, purification and blocking methods of the red fluorescent quantum dots modified with p62 capture antibody were the same as above, except that the detection antibody was replaced by p62 capture antibody (purchased from Abcam Inc) and the green fluorescent quantum dots (emission wavelength of 655 nm, purchased from Thermo Fisher Scientific Inc) were replaced by red fluorescent quantum dots.

[0029] 3. Detection principle like Figure 2 As shown, a transmission grating is added in front of the camera. The grating divides the light collected by the objective lens into two beams, one of which deviates from the original propagation direction and is projected into a strip-shaped light spot on the camera, and the other forms a dot-shaped light spot on the camera along the original propagation direction. The spacing between the two-color quantum dots in the immune complex is smaller than the resolution of the optical microscope, so the dot-shaped light spots of the two heterochromatic quantum dots are completely superimposed. By adjusting the grating constant of the transmission grating and its spacing with the camera, the immune complex labeled with heterochromatic quantum dots will appear as two independent stripes, which is different from the single stripe of the antibody labeled with monochrome quantum dots. Therefore, in the presence of free antibodies, the immune complex can be identified and counted.

[0030] The concentrations of the p62 capture antibody-modified red fluorescent quantum dots and the p62 detection antibody-modified green fluorescent quantum dots in the kit are both 75 pM.

[0031] 4. Evaluation of the p62 homogeneous digital immunoassay kit AImmunoreaction: Take 40 µL of p62 capture antibody-modified red fluorescent quantum dots and p62 detection antibody-modified green fluorescent quantum dots, and mix with 20 µL of p62 protein standards of different concentrations diluted in PBS. Perform the immunoreaction in the dark for 1 h. The concentrations of p62 protein standards are: 0.01, 0.05, 0.24, 1.20, 6.00, 15.00, 30.00, 60.00, 120.00, 240.00 pM.

[0032] B Single particle fluorescence spectral imaging: Single particle spectral imaging of the above immune mixed reaction solution. First, dissolve dextran (DEX) in ultrapure water to prepare a solution with a mass fraction of 0.1%. Mix the 0.1% DEX solution and Tween 20 at a volume ratio of 1:6×10-6 and set aside. Secondly, dilute the immune reaction solution to 1pM with the prepared DEX solution as the detection solution, use a pipette to take 1μL of the detection solution and drop it on the slide, let it stand in the dark for 30 minutes to allow the sample to dry naturally; cover with a coverslip, fix the cover with tape, and place it on Figure 2 The results were observed under an inverted fluorescence microscope.

[0033] C. Data processing: The captured data were processed using Image J to calculate the ratio of red quantum dots to green quantum dots aggregates at different concentrations.

[0034] D Kit sensitivity evaluation: Plot the logarithm of the p62 protein standard concentration as the horizontal axis and the ratio of red quantum dots to green quantum dots aggregates as the vertical axis (see Figure 3 ) Draw a standard curve. In PBS buffer, the linear range of this kit for detecting p62 protein standards is 0.10-15 pM and 5p-240pM, and the detection limit of this kit for detecting p62 protein standards is 0.06 pM.

[0035] E Kit Selectivity Evaluation: Compare the response of the quantum dot-modified antibody to non-target substances, that is, replace the p62 protein standard with non-target substances (see Figure 3 ). Among them, the concentration of non-target protein is 3.3 times that of p62 protein, and the concentration of amino acid mixture and single-stranded DNA mixture is 10 times that of p62 protein. Figure 3 The quantum dot-modified antibody showed little response to non-target substances. It can be seen that this kit has good selectivity for p62 protein standards.

[0036] 5. Clinical sample testing process Extraction of nuclear proteins from urine: Centrifuge 200 mL of urine at 800 g for 5 minutes at 4°C. Rinse the precipitated cells with pre-cooled 1× PBS solution and centrifuge the suspension at 800 g for 5 minutes at 4°C. Repeat the washing of cells twice. Resuspend the cells and count them using a cell counting plate. Add Hypotonic buffer at a ratio of 0.08 mL Hypotonic buffer per 106 cells. After ice bathing for 10 minutes, centrifuge the suspension at 800 g for 5 minutes at 4°C. Discard the supernatant and add 0.4 mL pre-cooled Hypotonic buffer and shake for 30 seconds. Centrifuge the suspension at 2500 g at 4°C for 5 minutes, discard the supernatant and retain the nuclear pellet. Add 0.1 mL Lysis buffer to the pellet, shake and suspend the nuclear pellet, and place it on ice bath for 20 minutes. Centrifuge the extract at 17,000 g for 10 minutes at 4°C and take the supernatant (nuclear protein extract).

[0037] B. Immune reaction: The experimental conditions were the same as those in Example 4A, except that the nuclear protein extract was used instead of the p62 protein standard.

[0038] C Single particle fluorescence spectral imaging: The experimental conditions are the same as those in Example 4 B. D. Data processing: The experimental conditions are the same as those in C of Example 4. The measured aggregation ratios of red and green quantum dots are substituted into the standard curve to determine the concentration of p62 protein in the sample. Example 2 Comparison of p62 content in urine nuclei of patients with different neurological diseases

[0039] The method and kit in Example 1 were used to detect the p62 content in urine of patients with NIID, myasthenia gravis (CI), myasthenia gravis (MG), cerebral infarction (PD) and normal subjects. The results were as follows: Figure 4 shown. Figure 2 The distribution of p62 protein in the above diseases was compared. Figure 4 It can be seen that the p62 content in the urine nuclei of NIID patients is significantly higher than that of other disease groups and healthy people; there is no significant difference in the p62 content in the urine nuclei of patients with other diseases and healthy people. In healthy people, the p62 protein content in the urine nuclei is lower than 1fM or undetectable (lower than the detection limit of this kit), while the p62 protein content in the urine nuclei of NIID patients is at the level of several hundred fM. Therefore, in this embodiment, subjects with p62 protein content in urine nuclei at the level of several hundred fM are NIID patients, and subjects with p62 protein content below 1fM are healthy people or low-risk subjects. Example 3 Receiver operating characteristic curve of p62 in urine nuclei of NIID patients

[0040] The p62 content in urine of NIID patients was detected by the method and kit in Example 1. The results are as follows: Figure 5 The AUC value of p62 in the nuclei of urine cells of NIID patients for diagnosing NIID was 1, indicating that p62 in the nuclei of urine cells of NIID patients has a very good clinical diagnostic value.

[0041] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A urinary nuclear protein for NIID diagnosis, characterized in that: The nuclear protein in the urine is p62 protein.

2. The urinary nuclear protein for NIID diagnosis according to claim 1, characterized in that: The amino acid sequence of the p62 protein is shown in SEQ ID NO.

1.

3. A non-invasive diagnostic kit for NIID, comprising the urinary nuclear protein for NIID diagnosis as described in any one of claims 1-2.

4. A NIID non-invasive diagnostic kit according to claim 3, characterized in that: The kit is a homogeneous digital immunoassay kit for p62.

5. A NIID non-invasive diagnostic kit according to claim 4, characterized in that: The homogeneous digital immunoassay kit at least includes a p62 protein standard, a p62 detection antibody, and a p62 capture antibody.

6. A NIID non-invasive diagnostic kit according to claim 5, characterized in that: The p62 detection antibody and the p62 capture antibody are respectively modified with quantum dots of different colors.

7. A NIID non-invasive diagnostic kit according to claim 6, characterized in that: The p62 detection antibody is modified with green fluorescent quantum dots, and the p62 capture antibody is modified with red fluorescent quantum dots.

8. Use of a reagent set for detecting the content of nuclear protein in urine in a kit for preparing an evaluation method for NIID, characterized in that: The evaluation of NIID is the evaluation of NIID detection and NIID risk prediction.

9. The use according to claim 8, characterized in that The evaluation step includes comparing a reference value among the measured values ​​of the intranuclear protein in urine selected from a sample of a healthy person or a low-risk person with a reference value among the measured values ​​of the intranuclear protein in urine of a NIID patient.

10. The use according to claim 9, characterized in that The content of nuclear protein p62 in the urine sample to be tested is higher than the baseline value of nuclear protein in urine of NIID patients, which indicates that the subject is likely to suffer from NIID.

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