Material for detecting autoimmune encephalitis antibody as well as preparation method and application thereof

Abstract

The invention relates to a material for detecting an autoimmune encephalitis antibody and a preparation method of the material. The material for detecting the autoimmune encephalitis antibody comprises the following components: an autoimmune encephalitis antibody detection plate, a working solution, PBS (Phosphate Buffer Solution), a fluorescent secondary antibody and a mounting medium, by using the modified hydroxyapatite, the binding force between the antigen protein and the detection plate is remarkably enhanced, and the adsorption stability of cells and protein on the detection plate is improved. The enhanced adsorption capacity is helpful for keeping the native conformation and activity of the antigen protein, so that the sensitivity and specificity of antibody detection are improved. The aminated hydroxyapatite has higher hydrophilicity, so that the surface of the aminated hydroxyapatite can be combined with antigen protein more easily. The improvement of hydrophilicity enhances the stability and consistency of the detection material, is helpful for improving the repeatability and reliability of the detection result, and ensures that the consistent result can be obtained under different detection conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of cell detection, in particular to a material for detecting autoimmune encephalitis antibodies and a preparation method and application thereof. Background Art

[0002] Autoimmune encephalitis is a type of inflammatory disease of the central nervous system caused by an autoimmune response, of which antibody-mediated autoimmune encephalitis is the most common type. Recent studies have found that various autoantibodies (such as those against NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX) are closely associated with different types of autoimmune encephalitis. Therefore, rapid and accurate detection of these antibodies is of great clinical significance for early diagnosis, treatment planning, and prognosis assessment.

[0003] Currently, commonly used antibody detection methods in clinical practice include enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence (IF). These methods have limitations, such as low sensitivity and specificity, cumbersome procedures, time-consuming procedures, and the need for specialized equipment. Furthermore, these methods often only detect one or a few antibodies, making it difficult to simultaneously detect multiple autoimmune encephalitis-associated antibodies, thus limiting their application in clinical diagnosis.

[0004] In existing antibody detection methods, antigen proteins are typically immobilized on solid surfaces such as glass slides or microplates. However, this immobilization method often causes structural changes in the antigen protein or partial loss of its natural activity, thereby affecting the efficiency of antibody binding and resulting in decreased detection sensitivity. In addition, commonly used solid surface materials (such as glass and plastic) have poor adsorption properties for cells and proteins, making it difficult to maintain the stability and activity of the antigen protein.

[0005] Hydroxyapatite, an inorganic material commonly used in biomaterials and detection platforms, is widely used in medical diagnostics due to its excellent biocompatibility and ability to bind to a variety of biomolecules (such as proteins and nucleic acids). However, the low hydrophilicity of unmodified hydroxyapatite can easily lead to inefficient adsorption of proteins or cells, thus affecting detection results and stability.

[0006] Existing detection methods typically utilize a single antigen target design and are unable to simultaneously detect multiple antibodies associated with autoimmune encephalitis. Multiplexed detection requires the spatial distribution and identification of different antigens. Traditional methods struggle to effectively coexist and fully expose multiple antigenic proteins, limiting the sensitivity and accuracy of multiplexed antibody detection. Summary of the Invention

[0007] Based on this, the present technical solution aims to provide an improved autoimmune encephalitis antibody detection material and method to improve the sensitivity and specificity of detection and realize the simultaneous detection of multiple related antibodies. By using modified hydroxyapatite (amino-hydroxyapatite), the binding force between the antigen protein and the detection plate is enhanced, thereby improving the adsorption stability of cells and proteins on the detection plate, maintaining the natural structure and activity of the antigen protein, and enhancing the sensitivity of antibody detection. By amino-modifying hydroxyapatite, its surface hydrophilicity is improved, making it easier to bind to the antigen protein, thereby improving the stability and consistency of the detection material, ensuring the repeatability and reliability during the detection process. At the same time, by spotting multiple autoimmune encephalitis-related antigen proteins (NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, DPPX) on a single detection plate at the same time, combined with high-efficiency fluorescent secondary antibody labeling technology, it is possible to simultaneously identify and quantify multiple autoimmune encephalitis antibodies in a single detection process, improve detection efficiency, reduce costs, and be suitable for large-scale clinical detection needs.

[0008] One object of the present invention is to provide a material for detecting autoimmune encephalitis antibodies, wherein the material for detecting autoimmune encephalitis antibodies comprises the following components: an autoimmune encephalitis antibody detection plate, a working solution, PBS, a fluorescent secondary antibody, and a mounting medium;

[0009] The autoimmune encephalitis antibody detection panel is a mixture of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins and modified hydroxyapatite, which are then spotted on a glass slide.

[0010] The modified hydroxyapatite is amino-modified hydroxyapatite.

[0011] Furthermore, positive controls and negative controls are also included.

[0012] Furthermore, the working solution is PBS buffer of goat serum.

[0013] Furthermore, the fluorescent secondary antibody is stained with fluorescein isothiocyanate.

[0014] Furthermore, the mounting medium is glycerol.

[0015] Furthermore, the negative control is 8-12 wt% goat serum.

[0016] Furthermore, the positive control is a recombinant antigen containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins.

[0017] Specifically, autoimmune encephalitis is associated with antibodies against neuronal cell surface or synaptic proteins. In recent years, the spectrum of chronic inflammatory brain diseases characterized by the presence of antigen-specific antibodies in serum and cerebrospinal fluid has expanded significantly. Because the clinical, imaging, and laboratory manifestations of autoimmune encephalitis and infectious encephalitis share many similarities, their clinical diagnosis is somewhat difficult. It has been reported that positive autoantibodies to neural antigens such as NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX are highly suggestive of autoimmune encephalitis. This product comprehensively detects autoantibodies to NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX, which can assist in the diagnosis of limbic encephalitis and encephalitis of unknown cause, and provide a basis for the clinical diagnosis of autoimmune encephalitis and its differential diagnosis from other diseases.

[0018] The present invention also provides a method for preparing the material for detecting autoimmune encephalitis antibodies, comprising the following steps:

[0019] S1. Adding hydroxyapatite nanoparticles to a solvent, then adding a hydrolysis coupling agent solution, stirring evenly, and heating the reaction to obtain modified hydroxyapatite;

[0020] S2. HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins are mixed with a modified hydroxyapatite dispersion, spotted onto a glass slide, and cut to form the test strip to obtain the material for detecting autoimmune encephalitis antibodies.

[0021] Furthermore, in step S1, the heating temperature is 50-65°C.

[0022] The present invention also provides a kit comprising the material for detecting autoimmune encephalitis antibodies.

[0023] The present invention has the following beneficial effects:

[0024] The present invention significantly enhances the binding force between the antigen protein and the test plate by using modified hydroxyapatite (aminated hydroxyapatite), and improves the adsorption stability of cells and proteins on the test plate. This enhanced adsorption capacity helps to maintain the natural conformation and activity of the antigen protein, thereby improving the sensitivity and specificity of antibody detection. Aminated hydroxyapatite has higher hydrophilicity, making its surface more easily bound to the antigen protein. This improvement in hydrophilicity enhances the stability and consistency of the detection material, helps to improve the repeatability and reliability of the test results, and ensures that consistent results can be obtained under different test conditions. On a single test plate, multiple autoimmune encephalitis-related antigen proteins (such as NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, DPPX) are spotted simultaneously, and combined with fluorescent secondary antibody labeling technology, simultaneous detection of multiple related antibodies is achieved. This can significantly improve detection efficiency, reduce the cost of single detection, and meet the clinical demand for multiple antibody detection, which is helpful for early diagnosis and accurate typing of autoimmune encephalitis. By utilizing modified hydroxyapatite and designing a system for the coexistence of multiple antigens, the team addresses challenges inherent in traditional detection methods, such as structural changes in antigenic proteins, sensitivity, and the difficulty in detecting multiple antibodies simultaneously. This approach provides a simple, sensitive, and highly specific multiplex antibody detection platform. This approach enables rapid and accurate detection of multiple autoimmune encephalitis antibodies in complex clinical settings, providing a reliable basis for disease diagnosis and treatment. DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0026] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0027] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0028] Hydroxyapatite, HANPs, Institute of Biomaterials, East China University of Science and Technology, particle size 20 nm.

[0029] Aminopropyltriethoxysilane (APTS), a silane coupling agent, was purchased from Sigma Chemical Company.

[0030] HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0031] Example 1

[0032] A material for detecting autoimmune encephalitis antibodies, comprising the following components: 2 autoimmune encephalitis antibody detection plates, 2 working solutions (10 g / 100 ml goat serum in PBS buffer), 20 PBS (0.01 mol / L), 0.5 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 mounting media (glycerol), 2 positive controls (containing recombinant antigens of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins), and 2 negative controls (10 wt% goat serum).

[0033] The autoimmune encephalitis antibody detection panel is a mixture of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins and modified hydroxyapatite, which are then spotted on a glass slide.

[0034] The modified hydroxyapatite is amino-modified hydroxyapatite.

[0035] The method for preparing the material for detecting autoimmune encephalitis antibodies comprises the following steps:

[0036] S1-1. 10 parts of a silane coupling agent solution (silane coupling agent: anhydrous ethanol = 1:1, v / v) were added to 90 parts of anhydrous ethanol, the pH value was adjusted to 8.5, and the mixture was stirred and hydrolyzed at room temperature for 1 h to obtain a hydrolyzed coupling agent solution;

[0037] S1-2. Immerse 1 part of hydroxyapatite nanoparticles in anhydrous ethanol solution, ultrasonically shake for 30 minutes, wash and filter, then add to the hydrolysis coupling agent solution, stir in a 60 ° C water bath for 6 hours, filter, wash, and dry to obtain amino hydroxyapatite nanoparticles;

[0038] S2. A 5 mg / ml PBS solution of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins is added to a 2 wt% modified hydroxyapatite borate buffer (ensuring that the HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins are 3 wt%). After mixing, the mixture is spotted on a glass slide at a temperature of 25°C and a humidity of 35% to obtain an autoimmune encephalitis antibody detection plate. The test strips are cut into pieces and packaged together with other components according to the above-mentioned mass fractions. The strips are frozen in liquid nitrogen to obtain the material for detecting autoimmune encephalitis antibodies.

[0039] Example 2

[0040] A material for detecting autoimmune encephalitis antibodies, comprising the following components: 3 autoimmune encephalitis antibody detection plates, 2 working solutions (10 g / 100 ml goat serum in PBS buffer), 15 PBS (0.01 mol / L), 1 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 mounting media (glycerol), 2 positive controls (containing recombinant antigens of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins), and 2 negative controls (10 wt% goat serum).

[0041] The autoimmune encephalitis antibody detection panel is a mixture of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins and modified hydroxyapatite, which are then spotted on a glass slide.

[0042] The modified hydroxyapatite is amino-modified hydroxyapatite.

[0043] The method for preparing the material for detecting autoimmune encephalitis antibodies comprises the following steps:

[0044] S1-1. 10 parts of a silane coupling agent solution (silane coupling agent: anhydrous ethanol = 1:1, v / v) were added to 90 parts of anhydrous ethanol, the pH value was adjusted to 8.5, and the mixture was stirred and hydrolyzed at room temperature for 1 h to obtain a hydrolyzed coupling agent solution;

[0045] S1-2. Immerse 1 part of hydroxyapatite nanoparticles in anhydrous ethanol solution, ultrasonically shake for 30 minutes, wash and filter, then add to the hydrolysis coupling agent solution, stir in a 60 ° C water bath for 6 hours, filter, wash, and dry to obtain amino hydroxyapatite nanoparticles;

[0046] S2. A 5 mg / ml PBS solution of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins is added to a 2 wt% modified hydroxyapatite borate buffer (ensuring that the HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins are 3 wt%). After mixing, the mixture is spotted on a glass slide at a temperature of 25°C and a humidity of 35% to obtain an autoimmune encephalitis antibody detection plate. The test strips are cut into pieces and packaged together with other components according to the above-mentioned mass fractions. The strips are frozen in liquid nitrogen to obtain the material for detecting autoimmune encephalitis antibodies.

[0047] Example 3

[0048] A material for detecting autoimmune encephalitis antibodies, comprising the following components: 2 autoimmune encephalitis antibody detection plates, 2 working solutions (10 g / 100 ml goat serum in PBS buffer), 20 PBS (0.01 mol / L), 0.5 fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 mounting media (glycerol), 2 positive controls (containing recombinant antigens of NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins), and 2 negative controls (10 wt% goat serum).

[0049] The autoimmune encephalitis antibody detection panel is a mixture of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins and modified hydroxyapatite, which are then spotted on a glass slide.

[0050] The modified hydroxyapatite is amino-modified hydroxyapatite.

[0051] The method for preparing the material for detecting autoimmune encephalitis antibodies comprises the following steps:

[0052] S1-1. 10 parts of a silane coupling agent solution (silane coupling agent: anhydrous ethanol = 1:1, v / v) were added to 90 parts of anhydrous ethanol, the pH value was adjusted to 8.5, and the mixture was stirred and hydrolyzed at room temperature for 1 h to obtain a hydrolyzed coupling agent solution;

[0053] S1-2. Immerse 1 part of hydroxyapatite nanoparticles in anhydrous ethanol solution, ultrasonically shake for 30 minutes, wash and filter, then add to the hydrolysis coupling agent solution, stir in a 60 ° C water bath for 6 hours, filter, wash, and dry to obtain amino hydroxyapatite nanoparticles;

[0054] S2. A 5 mg / ml PBS solution of HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins is added to a 2 wt% modified hydroxyapatite borate buffer (ensuring that the HeLa cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins are 2 wt%), mixed, and spotted on a glass slide at a temperature of 30°C and a humidity of 30% to obtain an autoimmune encephalitis antibody detection plate, which is then cut into test strips, packaged together with other components according to the above-mentioned mass fractions, and frozen in liquid nitrogen to obtain the material for detecting autoimmune encephalitis antibodies.

[0055] Comparative Example 1

[0056] A material for detecting autoimmune encephalitis antibodies. The difference between this comparative example and Example 1 is that: this comparative example removes step S1, and uses unmodified hydroxyapatite of equal mass to replace the modified hydroxyapatite. Other components and preparation methods are the same.

[0057] Test Case

[0058] The materials for detecting autoimmune encephalitis antibodies prepared in Example 1 and Comparative Example 1 were used as kits for performance testing.

[0059] The materials for detecting autoimmune encephalitis antibodies prepared in Example 1 and Comparative Example 1 were used as kits to detect 30 NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, DPPX negative sera and 30 NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, DPPX positive sera;

[0060] Test method:

[0061] A1. Equilibrate to room temperature: Remove the cell matrix slide and place it at room temperature for 30 minutes.

[0062] A2. Sample Incubation: In a fume hood, add 100 μl of the diluted serum sample to each well of a reaction plate. Place the cell matrix slide upside down on the plate and incubate at room temperature for 1 hour.

[0063] A3. Washing: Immerse the cell matrix slide in a washing tub filled with PBS for 5 minutes. Remove the cell matrix slide and wipe the edges and back of the slide clean of excess liquid with absorbent paper.

[0064] A4. Secondary Antibody Incubation: Add 100 μl of fluorescent secondary antibody to each well of a clean, dry reaction plate. Quickly invert the cell matrix slide onto the plate and incubate at room temperature for 1 hour.

[0065] A5. Washing: Immerse the cell matrix slide in a washing tub filled with PBS for 5 minutes in the dark. Remove the cell matrix slide and wipe the edges and back of the slide clean of excess liquid with absorbent paper.

[0066] A6. Mounting: Add 100 μl of mounting medium to each reaction area and cover with a coverslip;

[0067] A7. Observe the fluorescence signal: Read the green fluorescence signal under a microscope using a 20x objective lens and analyze the results.

[0068] Table 1 Test results of material properties for detecting autoimmune encephalitis antibodies in Example 1 and Comparative Example 1

[0069]

[0070]

[0071] According to Table 1, it can be concluded that the kit of Example 1 of the present invention helps to improve the repeatability and reliability of the test results, and can simultaneously identify and quantify multiple autoimmune encephalitis antibodies in a single test process, thereby improving the detection efficiency.

[0072] 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 invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0073] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method 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 can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A material for detecting autoimmune encephalitis antibodies, characterized in that: The materials for detecting autoimmune encephalitis antibodies include the following components: an autoimmune encephalitis antibody detection plate, a working solution, PBS, a fluorescent secondary antibody, and a sealing agent; Wherein, the autoimmune encephalitis antibody detection plate is a mixture of Hela cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX antigen proteins and modified hydroxyapatite and then spotted on a glass slide; The modified hydroxyapatite is aminated hydroxyapatite.

2. The material for detecting autoimmune encephalitis antibodies according to claim 1, characterized in that: Positive and negative controls were also included.

3. The material for detecting autoimmune encephalitis antibodies according to claim 1, characterized in that: The working solution is goat serum PBS buffer.

4. The material for detecting autoimmune encephalitis antibodies according to claim 1, characterized in that: The fluorescent secondary antibody is stained with fluorescein isothiocyanate.

5. The material for detecting autoimmune encephalitis antibodies according to claim 1, characterized in that: The mounting medium is glycerol.

6. The material for detecting autoimmune encephalitis antibodies according to claim 2, characterized in that: The negative control is 8-12 wt % goat serum.

7. The material for detecting autoimmune encephalitis antibodies according to claim 2, characterized in that: The positive control is a recombinant antigen containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins.

8. The method for preparing a material for detecting autoimmune encephalitis antibodies according to any one of claims 1 to 7, characterized in that: The steps include: S1. adding hydroxyapatite nanoparticles to a solvent, then adding a hydrolysis coupling agent solution, stirring evenly, and heating to react to obtain modified hydroxyapatite; S2. Hela cells containing NMDAR, GABABR, AMPAR1, AMPAR2, CASPR2, IgLON5, LGI1, and DPPX proteins are mixed with a modified hydroxyapatite dispersion, spotted on a glass slide, and cut to form the test strip to obtain the material for detecting autoimmune encephalitis antibodies.

9. The method for preparing a material for detecting autoimmune encephalitis antibodies according to claim 8, characterized in that: In step S1, the heating temperature is 50-65°C.

10. A kit, characterized in that: The invention comprises the material for detecting autoimmune encephalitis antibodies as described in any one of claims 1 to 7.

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