Kit for detecting antibody of myasthenia gravis and preparation method of kit

Abstract

The invention relates to a kit for detecting a myasthenia gravis antibody and a preparation method of the kit. The kit for detecting the myasthenia gravis antibody comprises the following components: a myasthenia gravis antibody detection plate, a working solution, PBS (Phosphate Buffer Solution), a fluorescent secondary antibody and a mounting medium, wherein the myasthenia gravis antibody detection plate is prepared by mixing hela cells containing MUSK, LRP4, Titin and RYR1 proteins with modified polystyrene and then spotting the mixture on a glass slide; the modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine. According to the present invention, the existence of the four antibodies such as MUSK, LRP4, Titin and RYR1 can be simultaneously detected in the same system. The multiple detection mode can comprehensively reflect the antibody spectrum of the myasthenia gravis patient, reduce the sample size and the detection time, reduce the detection cost, and improve the detection efficiency and the clinical application value.

Description

Technical Field

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

[0002] Myasthenia gravis (MG) is an autoimmune disease characterized by skeletal muscle weakness and fatigability. Its pathogenesis usually involves the attack of autoantibodies produced by the body's immune system on receptors or related proteins at the neuromuscular junction. Common pathogenic antibodies include antibodies against acetylcholine receptors (AChR), muscle-specific kinases (MUSK), low-density lipoprotein receptor-related protein 4 (LRP4), titin, and ryanodine receptor (RYR1).

[0003] Traditional myasthenia gravis antibody detection methods mainly rely on radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Although these methods have certain application value, they have problems such as low sensitivity and specificity, complex operation, and long detection time. Moreover, most of them are single antibody detection and cannot meet the clinical demand for multi-antibody combined detection. In addition, these methods usually require the use of radioactive isotopes or strong acid-base reagents, which poses a safety hazard and increases the difficulty and cost of operation.

[0004] The emergence of multiplex detection technology makes it possible to detect multiple targets in a single sample at the same time, significantly improving detection efficiency and reducing sample consumption. However, in the process of multiplex detection, how to improve the adsorption capacity of detection carriers for cells and proteins, enhance hydrophilicity, and ensure the specific binding of antibodies and the stability of fluorescent signals is still a technical problem that needs to be solved.

[0005] Some of the myasthenia gravis antibody detection kits currently on the market mostly use polystyrene materials as the substrate of the detection plate. Polystyrene materials themselves have weak adsorption capacity for cells and poor hydrophilicity, which is not conducive to the stable binding of cells and may lead to instability and reduced sensitivity of the test results. In addition, the existing detection methods can usually only detect a certain antibody and cannot achieve simultaneous detection of multiple antibodies. It is difficult to fully reflect the antibody spectrum of myasthenia gravis patients, which limits its extensiveness in clinical applications.

[0006] Therefore, it is necessary to develop a new technical solution to solve the deficiencies in the prior art. Summary of the invention

[0007] Based on this, the present invention proposes to use modified polystyrene material as the substrate of the antibody detection plate. By modifying polystyrene, the adsorption capacity of its surface to cells and proteins is significantly improved, and its hydrophilicity is enhanced, thereby improving the specific binding of antibodies and ensuring the sensitivity and stability of detection. At the same time, this scheme designs a multiple antibody detection plate, which can simultaneously detect four antibodies, MUSK, LRP4, Titin and RYR1, in the same detection system, so as to have a more comprehensive understanding of the patient's antibody spectrum and provide stronger support for clinical diagnosis and treatment.

[0008] One object of the present invention is to provide a kit for detecting antibodies of myasthenia gravis, wherein the kit comprises the following components: a myasthenia gravis antibody detection plate, a working solution, PBS, a fluorescent secondary antibody, and a sealing agent;

[0009] Wherein, the myasthenia gravis antibody detection plate is a mixture of Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins and modified polystyrene and then spotted on a glass slide;

[0010] The modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine.

[0011] Specifically, myasthenia gravis (MG) is a disorder of neuromuscular transmission at the neuromuscular junction caused by an autoimmune reaction, and its main clinical manifestations are partial or systemic skeletal muscle weakness and fatigue. Antibody detection is of great significance for the diagnosis of myasthenia gravis. Acetylcholine receptor (AChR) antibodies are the most common pathogenic antibodies; in addition, antibodies against other components of the postsynaptic membrane, including muscle-specific receptor tyrosine kinase (MuSK), low-density lipoprotein receptor-related protein 4 (LRP4) and ryanodine receptor (RyR) antigens, have been found to be involved in the pathogenesis of MG. These antibodies can interfere with AChR aggregation, affect AChR function and neuromuscular junction (NMJ) signal transmission. Among patients with myasthenia gravis, 10-20% of patients cannot detect AchR antibodies in their serum, which is called serum AchR antibody negative MG (Seronegative MG, SNMG). However, about 80% of patients with myasthenia gravis can detect thymic lesions, of which about 10% will develop thymic epithelial cell tumors or thymic carcinomas. Titin antibodies are highly correlated with myasthenia gravis accompanied by thymic lesions. Detection of Titin antibodies is of great significance for the diagnosis of thymoma myasthenia gravis. MuSK antibodies are another important marker for myasthenia gravis. MuSK antibodies can be detected in about 50% of SNMG. The detection of MuSK antibodies can assist in the diagnosis of myasthenia gravis more comprehensively. Anti-LRP4 antibodies are also detected in some SNMG patients, and about 10% of SNMG patients have anti-LRP4 antibodies. Anti-RyR1 antibodies can also be detected in SNMG patients, which are more common in elderly MG patients. They have a high directivity for thymic tumors, indicating a poor prognosis, and their clinical manifestations are also mainly systemic MG. Therefore, detection of the above four antibodies is particularly important in the diagnosis of MG.

[0012] Furthermore, positive controls and negative controls are included.

[0013] Furthermore, the chloromethyl polystyrene resin is cross-linked with 1-2% DVB.

[0014] Furthermore, the working fluid is goat serum or bovine serum.

[0015] Furthermore, the fluorescent secondary antibody is stained with fluorescein isothiocyanate.

[0016] Furthermore, the sealing medium is glycerol.

[0017] Furthermore, the negative control is 8-12 wt % goat serum.

[0018] Furthermore, the positive control is a recombinant antigen containing MUSK, LRP4, Titin, and RYR1 proteins.

[0019] The present invention also provides a method for preparing the kit for detecting antibodies of myasthenia gravis, comprising the following steps:

[0020] S1. Adding chloromethyl polystyrene resin to a solvent, adding dopamine, mixing and heating the reaction, filtering and washing to obtain the modified polystyrene;

[0021] S2. After mixing Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins with a modified polystyrene solution, the mixture is spotted onto a glass slide, the glass slide is sub-assembled into the reaction plate, and the test strip is formed after cutting, thereby obtaining a kit for antibody detection of myasthenia gravis.

[0022] Furthermore, in step S1, the heating temperature is 75-85°C.

[0023] The present invention also provides application of the myasthenia gravis antibody detection kit in detecting myasthenia gravis antibodies.

[0024] The present invention has the following beneficial effects:

[0025] The present invention first modifies polystyrene by reacting chloromethyl polystyrene resin with dopamine. The modified polystyrene material provides a larger surface area, which significantly enhances the adsorption capacity of cells and proteins, thereby effectively improving the fixation effect of cells on the test plate and reducing cell shedding, thereby improving the sensitivity and stability of the detection. At the same time, this modified material has better hydrophilicity, so that the protein has higher activity and stability on its surface, ensuring the specific binding of antibodies during the detection process, reducing the occurrence of non-specific binding, and further improving the accuracy and reliability of the detection; in addition, the present invention can simultaneously detect the presence of four antibodies against MUSK, LRP4, Titin and RYR1 in the same system. This multiple detection method can fully reflect the antibody spectrum of patients with myasthenia gravis, reduce the sample volume and detection time, reduce the detection cost, and improve the detection efficiency and clinical application value. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] HeLa cells containing MUSK, LRP4, Titin, and RYR1 proteins were purchased from Beijing Jingyi Medical Testing Laboratory Co., Ltd.

[0030] Chloromethyl polystyrene resin, 1% DVB cross-linked, was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd.

[0031] Polystyrene, P107086, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0032] Example 1

[0033] A kit for detecting antibodies of myasthenia gravis, comprising the following components: 2 parts of myasthenia gravis antibody detection plates, 2 parts of working solution (10 g / 100 ml goat serum), 20 parts of PBS (0.01 mol / L), 0.5 parts of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 parts of mounting medium (glycerol), 2 parts of positive control (containing recombinant antigens of MUSK, LRP4, Titin, RYR1 proteins) and 2 parts of negative control (10 wt% goat serum);

[0034] Wherein, the myasthenia gravis antibody detection plate is a hela cell containing MUSK, LRP4, Titin, and RYR1 proteins spotted on a glass slide;

[0035] The modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine.

[0036] The preparation method of the above-mentioned myasthenia gravis antibody detection kit comprises the following steps:

[0037] S1. 10 parts of chloromethyl polystyrene resin were added to 100 parts of toluene, swollen for 3 hours, and then 7 parts of dopamine were added, mixed and heated to 80 ° C for 24 hours, washed with anhydrous ethanol, 0.1 mol / LNaOH, and deionized water to neutrality, filtered, washed, and dried to obtain the modified polystyrene;

[0038] S2. Add 5 mg / ml PBS solution of HeLa cells containing MUSK, LRP4, Titin, and RYR1 proteins to 2wt% modified polystyrene boric acid buffer (ensuring that the HeLa cells containing MUSK, LRP4, Titin, and RYR1 proteins are 2wt%), mix, and spot on a glass slide at a temperature of 24°C and a humidity of 40%, sub-assemble the glass slide into the reaction plate, cut to form the test strip, and then package it together with other components according to the above mass fraction, and freeze it in liquid nitrogen to obtain the kit for antibody detection of myasthenia gravis.

[0039] Example 2

[0040] A kit for detecting antibodies of myasthenia gravis, comprising the following components: 2 parts of myasthenia gravis antibody detection plates, 2 parts of working solution (10 g / 100 ml goat serum), 20 parts of PBS (0.01 mol / L), 0.5 parts of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 parts of mounting medium (glycerol), 2 parts of positive control (containing recombinant antigens of MUSK, LRP4, Titin, RYR1 proteins) and 2 parts of negative control (10 wt% goat serum);

[0041] Wherein, the myasthenia gravis antibody detection plate is a hela cell containing MUSK, LRP4, Titin, and RYR1 proteins spotted on a glass slide;

[0042] The modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine.

[0043] The preparation method of the above-mentioned myasthenia gravis antibody detection kit comprises the following steps:

[0044] S1. 10 parts of chloromethyl polystyrene resin were added to 100 parts of toluene, swollen for 3 hours, and then 7 parts of dopamine were added, mixed and heated to 80 ° C for 24 hours, washed with anhydrous ethanol, 0.1 mol / LNaOH, and deionized water to neutrality, filtered, washed, and dried to obtain the modified polystyrene;

[0045] S2. Add 5 mg / ml PBS solution of Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins to 2wt% modified polystyrene boric acid buffer (ensuring that the Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins are 3wt%), mix, and spot on a glass slide at a temperature of 24°C and a humidity of 40%, sub-assemble the glass slide into the reaction plate, cut to form the test strip, and then package it together with other components according to the above mass fraction, and freeze it in liquid nitrogen to obtain the kit for antibody detection of myasthenia gravis.

[0046] Example 3

[0047] A kit for detecting antibodies of myasthenia gravis, comprising the following components: 2 parts of myasthenia gravis antibody detection plates, 2 parts of working solution (10 g / 100 ml goat serum), 20 parts of PBS (0.01 mol / L), 0.5 parts of fluorescent secondary antibody (with fluorescein isothiocyanate (FITC)), 5 parts of mounting medium (glycerol), 2 parts of positive control (containing recombinant antigens of MUSK, LRP4, Titin, RYR1 proteins) and 2 parts of negative control (10 wt% goat serum);

[0048] Wherein, the myasthenia gravis antibody detection plate is a hela cell containing MUSK, LRP4, Titin, and RYR1 proteins spotted on a glass slide;

[0049] The modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine.

[0050] The preparation method of the above-mentioned myasthenia gravis antibody detection kit comprises the following steps:

[0051] S1. 10 parts of chloromethyl polystyrene resin were added to 100 parts of toluene, swollen for 3 hours, and then 7 parts of dopamine were added, mixed and heated to 80 ° C for 24 hours, washed with anhydrous ethanol, 0.1 mol / LNaOH, and deionized water to neutrality, filtered, washed, and dried to obtain the modified polystyrene;

[0052] S2. Add 5 mg / ml PBS solution of HeLa cells containing MUSK, LRP4, Titin, and RYR1 proteins to 2wt% modified polystyrene boric acid buffer (ensuring that the HeLa cells containing MUSK, LRP4, Titin, and RYR1 proteins are 2wt%), mix, and spot on a glass slide at a temperature of 24°C and a humidity of 35%, sub-assemble the glass slide into the reaction plate, cut to form the test strip, and then package it together with other components according to the above mass fraction, and freeze it in liquid nitrogen to obtain the kit for antibody detection of myasthenia gravis.

[0053] Comparative Example 1

[0054] A kit for detecting antibodies for myasthenia gravis. The difference between this comparative example and Example 1 is that step S1 is removed in this comparative example, that is, modified polystyrene is removed, and other components and preparation methods are the same.

[0055] Comparative Example 2

[0056] A kit for detecting antibodies of myasthenia gravis, wherein polystyrene latex of equal mass is used to replace modified polystyrene, and other materials and preparation methods are the same as those in Example 1.

[0057] Test Case

[0058] The kits for detecting antibodies for myasthenia gravis prepared in Example 1 and Comparative Examples 1-2 were used as kits for performance testing.

[0059] The myasthenia gravis antibody detection kits prepared in Example 1 and Comparative Examples 1-2 were used as the kits to detect 20 MUSK, LRP4, Titin, RYR1 negative sera and 20 MUSK, LRP4, Titin, RYR1 positive sera;

[0060] Test method:

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

[0062] 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 cell matrix slide upside down on the reaction plate, and incubate at room temperature for 1 hour;

[0063] A3. Washing: Immerse the cell matrix slide in a washing tank filled with PBS for 5 minutes, take out the cell matrix slide, and wipe off the excess liquid on the edge and back with absorbent paper;

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

[0065] A5. Washing: Immerse the cell matrix slide in a washing tank filled with PBS for 5 minutes in a dark environment. Take out the cell matrix slide and wipe off the excess liquid on the edge and back with absorbent paper.

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

[0067] A7. Observe the fluorescence signal: Read the green fluorescence result under the microscope, observe with a 20x objective lens, and analyze the result.

[0068] Table 1 Performance test results of the kit for detecting myasthenia gravis antibodies of Example 1 and Comparative Examples 1-2

[0069]

[0070] It can be concluded from Table 1 that the kit for detecting antibodies for myasthenia gravis of Example 1 of the present invention can improve the sensitivity and accuracy of detection, can detect multiple antibodies at the same time, and provides a more efficient, sensitive and reliable detection method for the diagnosis of myasthenia gravis.

[0071] 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.

[0072] 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. A kit for detecting antibodies for myasthenia gravis, characterized in that: The kit for detecting antibodies of myasthenia gravis comprises the following components: a myasthenia gravis antibody detection plate, a working solution, PBS, a fluorescent secondary antibody, and a sealing agent; Wherein, the myasthenia gravis antibody detection plate is a mixture of Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins and modified polystyrene and then spotted on a glass slide; The modified polystyrene is obtained by reacting chloromethyl polystyrene resin with dopamine.

2. The kit for detecting antibodies for myasthenia gravis according to claim 1, characterized in that: Positive and negative controls were also included.

3. The kit for detecting antibodies of myasthenia gravis according to claim 1, characterized in that: The chloromethyl polystyrene resin is 1-2% cross-linked.

4. The kit for detecting antibodies for myasthenia gravis according to claim 1, characterized in that: The working solution is goat serum or bovine serum.

5. The kit for detecting antibodies for myasthenia gravis according to claim 1, characterized in that: The fluorescent secondary antibody is stained with fluorescein isothiocyanate.

6. The kit for detecting antibodies of myasthenia gravis according to claim 1, characterized in that: The mounting medium is glycerol.

7. The kit for detecting antibodies of myasthenia gravis according to claim 2, characterized in that: The negative control is 8-12 wt % goat serum.

8. The kit for detecting antibodies of myasthenia gravis according to claim 2, characterized in that: The positive control is a recombinant antigen containing MUSK, LRP4, Titin, and RYR1 proteins.

9. A method for preparing a kit for detecting antibodies against myasthenia gravis according to any one of claims 1 to 8, characterized in that: The steps include: S1. Adding chloromethyl polystyrene resin to a solvent, adding dopamine, mixing and heating the reaction, filtering and washing to obtain the modified polystyrene; S2. After mixing Hela cells containing MUSK, LRP4, Titin, and RYR1 proteins with a modified polystyrene solution, the mixture is spotted onto a glass slide, the glass slide is sub-assembled into the reaction plate, and the test strip is formed after cutting to obtain the kit for antibody detection of myasthenia gravis.

10. The method for preparing a kit for detecting antibodies of myasthenia gravis according to claim 9, characterized in that: In step S1, the heating temperature is 75-85°C.

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