Antibody detection kit for central nervous system idiopathic inflammatory demyelination disease and preparation method thereof

Abstract

The invention relates to a central nervous system idiopathic inflammatory demyelination disease antibody detection kit and a preparation method thereof. The central nervous system idiopathic inflammatory demyelination disease antibody detection kit comprises an antibody detection strip, a working solution, a control group and an auxiliary agent, wherein MOG, AQP4, 293T cells of empty carrier protein, GFAP, MBP, and hela cells of empty carrier protein are spotted on the antibody detection strip. The kit disclosed by the invention can be used for simultaneously detecting four autoantibodies, namely MOG, AQP4, GFAP and MBP, so that the comprehensiveness and accuracy of diagnosis are remarkably improved, and the diagnosis accuracy of the idiopathic inflammatory demyelination disease of the central nervous system is favorably improved. The immune state of the patient can be reflected more comprehensively, and the risk of misdiagnosis and missed diagnosis is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of cell detection, and in particular to an antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system and a preparation method thereof. Background Art

[0002] Idiopathic inflammatory demyelinating diseases of the central nervous system, including multiple sclerosis (MS) and neuromyelitis optica (NMO), are serious diseases caused by autoimmune reactions, leading to neurological impairment. Early diagnosis is crucial to improving patient prognosis, and currently commonly used diagnostic methods mainly rely on clinical symptoms and imaging examinations. Although these methods are important, there may be a risk of misdiagnosis or missed diagnosis in specific cases. Therefore, it is particularly important to develop rapid and sensitive biomarker detection technologies.

[0003] Existing detection technologies mainly focus on the detection of antibodies in serum or cerebrospinal fluid. Although traditional detection methods such as ELISA and Western blot have high specificity, they are cumbersome, time-consuming, and require high laboratory conditions. In addition, these methods usually target a single antigen, making it difficult to detect multiple related antibodies at the same time and unable to provide comprehensive diagnostic information.

[0004] In terms of the development of antibody detection kits, there is currently a lack of products that can quickly and easily detect multiple related antibodies at the same time. Existing kits usually do not have multiple detection functions, resulting in the need for multiple tests during the diagnosis process, increasing the burden and waiting time for patients. At the same time, the sensitivity and specificity of the test need to be improved to ensure accurate identification of specific antibodies in complex clinical samples.

[0005] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0006] Based on this, the kit of the present invention detects four autoantibodies, namely MOG, AQP4, GFAP and MBP. This product provides a more comprehensive detection method by comprehensively detecting four autoantibodies, namely MOG, AQP4, GFAP and MBP. This technical solution not only improves the early diagnosis ability of idiopathic inflammatory demyelinating diseases, but also provides a clearer basis for the differential diagnosis of diseases. Through the joint detection of multiple key antibodies, it can better reflect the immune status of patients, provide support for clinical decision-making, promote the implementation of individualized treatment, and thus improve the prognosis of patients.

[0007] One object of the present invention is to provide an antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system, wherein the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system comprises: an antibody detection strip, a working solution, a control group, and an auxiliary agent;

[0008] The antibody detection strip is spotted with MOG, AQP4, 293T cells with empty carrier protein and GFAP, MBP, Hela cells with empty carrier protein.

[0009] Specifically, idiopathic inflammatory demyelinating diseases (IIDDs) of the central nervous system are a group of diseases related to autoimmunity, with CNS demyelination and inflammation as the main pathological features. Clinically, multiple sclerosis, neuromyelitis optica spectrum disorders, and acute disseminated encephalomyelitis are common. The onset of IIDDs is associated with several autoantibodies, including anti-myelin oligodendrocyte glycoprotein (MOG) antibodies, anti-aquaporin 4 (AQP4) antibodies, glial fibrillary acidic protein (GFAP) antibodies, anti-myelin basic protein (MBP) antibodies, and anti-myelin associated glycoprotein antibodies, among which MOG antibodies and AQP4 antibodies are the most common.

[0010] Furthermore, the working solution is bovine serum albumin (BSA).

[0011] Furthermore, the auxiliary agent is selected from one or more of a sealing agent, a fluorescent secondary antibody, and a PBS buffer.

[0012] Furthermore, the fluorescent secondary antibody is selected from one or more of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and tetraethylrhodamine.

[0013] Furthermore, the material of the sealing agent is glycerol containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane, and thymol.

[0014] Furthermore, the control group includes a positive control and a negative control.

[0015] Furthermore, the negative control is a protein protectant.

[0016] Furthermore, the positive control is positive goat serum and protein protective agent containing recombinant antigens of MOG, AQP4, GFAP and MBP proteins.

[0017] The present invention also provides a method for preparing the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system, comprising the following steps:

[0018] The 293T cells with MOG, AQP4 and empty vector protein and the Hela cells with GFAP, MBP and empty vector protein are spotted on glass slides respectively, and then cut to form antibody detection strips to obtain the antibody detection kit for chronic inflammatory demyelinating polyradiculoneuropathy;

[0019] The antibody detection strip and other components are packaged to obtain the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system.

[0020] The present invention has the following beneficial effects:

[0021] The kit of the present invention can simultaneously detect four autoantibodies, namely, MOG, AQP4, GFAP and MBP, which significantly improves the comprehensiveness and accuracy of the diagnosis, and helps to improve the diagnostic accuracy of idiopathic inflammatory demyelinating diseases of the central nervous system. It can more comprehensively reflect the immune status of the patient, reduce the risk of misdiagnosis and missed diagnosis, and can identify relevant antibodies in the early stage of the disease, thereby achieving early diagnosis, significantly improving disease progression, and reducing the complexity and economic burden of subsequent treatment for patients. The kit of the present invention has the characteristics of being easy and fast to operate, and can obtain results in a shorter time, reducing the waiting time and diagnosis cycle of the patient, thereby achieving more timely therapeutic intervention, and providing a basis for the auxiliary diagnosis of clinical idiopathic inflammatory demyelinating diseases of the central nervous system and the differential diagnosis with other diseases. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0023] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0024] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified in all instances by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary depending on the desired properties to be obtained by the present invention.

[0025] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. These techniques have been fully described in existing literature.

[0026] The antigenic proteins of MOG, AQP4, GFAP, and MBP described herein are known in the art, and their amino acid sequences are known in the art and can be found in professional databases such as NCBI.

[0027] Protein protective agent, ABS01-0250, was purchased from Zhengzhou Sairun Biotechnology Co., Ltd.

[0028] MOG, AQP4, and 293T cells expressing empty vector protein, and GFAP, MBP, and HeLa cells expressing empty vector protein were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0029] Example 1

[0030] A central nervous system idiopathic inflammatory demyelinating disease antibody detection kit, the central nervous system idiopathic inflammatory demyelinating disease antibody detection kit comprising the following components: 3 antibody detection strips, 1 working solution (BSA), 1 sealing agent (containing sodium chloride, sodium dihydrogen phosphate, sodium hydrogen phosphate, 1,4-diazobicyclo [2.2.2] octane, thymol glycerol, the mass ratio is 9:1:0.5:0.1:0.1:9), 1 buffer (PBS buffer), 1 positive control (containing MOG, AQP4, GFAP, MBP protein recombinant antigen positive goat serum, protein protective agent, the mass ratio is 1:0.1), 1 negative control (protein protective agent), 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0031] The preparation method of the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system comprises the following steps:

[0032] MOG, AQP4, 293T cells with empty vector protein and GFAP, MBP, Hela cells with empty vector protein are spotted on glass slides at 25°C and 40% humidity, and then cut to form antibody detection strips to obtain the antibody detection kit for chronic inflammatory demyelinating polyradiculoneuropathy;

[0033] The antibody detection strip and other components are packaged according to the above mass fractions and frozen in liquid nitrogen to obtain the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system.

[0034] Example 2

[0035] A kit for detecting antibodies for idiopathic inflammatory demyelinating diseases of the central nervous system, comprising the following components: 3 antibody test strips, 1 working solution (BSA), 1 sealing agent (containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane, thymol glycerol, with a mass ratio of 10:1:0.5:0.1:0.1:9), 1 buffer (PBS buffer), 1 positive control (containing positive goat serum of recombinant antigens of MOG, AQP4, GFAP, and MBP proteins, and a protein protective agent, with a mass ratio of 1:0.1), 1 negative control (protein protective agent), and 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0036] The preparation method of the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system comprises the following steps:

[0037] MOG, AQP4, 293T cells with empty vector protein and GFAP, MBP, Hela cells with empty vector protein are spotted on glass slides at 30°C and 35% humidity, and then cut to form antibody detection strips to obtain the antibody detection kit for chronic inflammatory demyelinating polyradiculoneuropathy;

[0038] The antibody detection strip and other components are packaged according to the above mass fractions and frozen in liquid nitrogen to obtain the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system.

[0039] Example 3

[0040] A kit for detecting antibodies for idiopathic inflammatory demyelinating diseases of the central nervous system, comprising the following components: 3 antibody test strips, 1 working solution (BSA), 1 sealing agent (containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane, thymol glycerol, with a mass ratio of 9:1:0.5:0.1:0.1:10), 1 buffer (PBS buffer), 1 positive control (containing positive goat serum of recombinant antigens of MOG, AQP4, GFAP, and MBP proteins, and a protein protective agent, with a mass ratio of 1:0.2), 1 negative control (protein protective agent), and 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC));

[0041] The preparation method of the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system comprises the following steps:

[0042] MOG, AQP4, 293T cells with empty vector protein and GFAP, MBP, Hela cells with empty vector protein are spotted on glass slides at 25°C and 35% humidity, and then cut to form antibody detection strips to obtain the antibody detection kit for chronic inflammatory demyelinating polyradiculoneuropathy;

[0043] The antibody detection strip and other components are packaged according to the above mass fractions and frozen in liquid nitrogen to obtain the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system.

[0044] Test Case

[0045] The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system prepared in Example 1 was used as a kit for performance testing.

[0046] The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system prepared in Example 1 was used as a kit to detect 20 MOG, AQP4, GFAP, MBP negative sera and 20 MOG, AQP4, GFAP, MBP positive sera respectively;

[0047] Test method:

[0048] A1. Equilibrate room temperature: Take out the antibody test strip and place it at room temperature for 30 minutes;

[0049] A2. Sample incubation: Add 100 μl of the diluted serum sample to be tested to each well of the reaction plate in a fume hood, place the antibody test strip upside down on the reaction plate, and incubate at room temperature for 1 hour;

[0050] A3. Cleaning: Immerse the antibody test strip in a washing tank filled with PBS for 5 minutes, take out the antibody test strip, and wipe off the excess liquid on the edge and back with absorbent paper;

[0051] A4. Secondary antibody incubation: Add 100 μl of fluorescent secondary antibody to each well of the dry and clean reaction plate, quickly turn the antibody detection strip upside down onto the reaction plate, and incubate at room temperature for 1 hour;

[0052] A5. Washing: Immerse the antibody test strip in a washing tank filled with PBS for 5 minutes in a dark environment. Take out the antibody test strip and wipe off the excess liquid on its edge and back with absorbent paper;

[0053] A6. Sealing: Add 40 μl of sealing agent to each reaction area and cover with a coverslip;

[0054] A7. Observe the fluorescence signal: read the green fluorescence signal under the microscope in the green fluorescence band (490-520nm), observe with a 20x objective lens, and analyze the results.

[0055] Table 1 Kit performance test results of Example 1

[0056]

[0057] It can be concluded from Table 1 that the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system of Example 1 of the present invention can effectively detect four different antibodies, namely MOG, AQP4, GFAP and MBP.

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0059] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. An antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system, characterized in that: The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system comprises: an antibody detection strip, a working solution, a control group, and an auxiliary agent; The antibody detection strip is spotted with MOG, AQP4, 293T cells with empty carrier protein and GFAP, MBP, Hela cells with empty carrier protein.

2. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 1, characterized in that: The working solution is bovine serum albumin.

3. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 1, characterized in that: The auxiliary agent is selected from one or more of a sealing agent, a fluorescent secondary antibody, and a PBS buffer.

4. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 3, characterized in that: The fluorescent secondary antibody is selected from one or more of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and tetraethylrhodamine.

5. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 3, characterized in that: The material of the sealing agent is glycerol containing sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1,4-diazobicyclo[2.2.2]octane and thymol.

6. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 1, characterized in that: The control group includes a positive control and a negative control.

7. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 7, characterized in that: The negative control is a protein protectant.

8. The antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to claim 7, characterized in that: The positive control is positive goat serum and protein protective agent containing recombinant antigens of MOG, AQP4, GFAP and MBP proteins.

9. A method for preparing an antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system according to any one of claims 1 to 8, characterized in that: The steps include: The 293T cells with MOG, AQP4 and empty vector protein and the Hela cells with GFAP, MBP and empty vector protein are spotted on glass slides respectively, and then cut to form antibody detection strips to obtain the antibody detection kit for chronic inflammatory demyelinating polyradiculoneuropathy; The antibody detection strip and other components are packaged to obtain the antibody detection kit for idiopathic inflammatory demyelinating diseases of the central nervous system.

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