A cell fixation post-antigen repair composition for a myasthenia gravis antibody profile (IgG) detection kit (cellular immunofluorescence method), and products and applications thereof

Abstract

This invention provides a cell-fixed antigen retrieval composition, its product, and its application for a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay), relating to the field of in vitro detection technology. The cell-fixed antigen retrieval composition of this invention comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and buffer solution. It effectively breaks the cross-linking state of antigen molecules during cell fixation, fully exposes antigen epitopes, protects antigen immunogenicity, and does not damage cell morphology or interfere with subsequent antibody binding. Simultaneously, it can accurately achieve simultaneous detection of multiple specific antibodies, including AChR, Titin, RyR1, Musk, and LRP4. Used in the preparation of a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay), it effectively overcomes the shortcomings of existing antigen retrieval methods and has significant clinical application value and promising industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, specifically relating to a cell-fixed antigen retrieval composition for a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay), its products, and applications. Background Technology

[0002] Cell slides are commonly used cell experimental tools in the field of biotechnology. Their core principle involves seeding and culturing cells on specially designed glass slides (usually tissue culture-treated glass or plastic slides), allowing the cells to adhere and grow on the slide surface. Once the cells reach a suitable density, they undergo subsequent processing such as fixation and staining for various cytological detections and analyses. As an important carrier for cell morphology observation and molecular expression detection, cell slides can achieve the localization, qualitative, and semi-quantitative analysis of intracellular proteins, nucleic acids, and other biomolecules while preserving the natural morphology and physiological characteristics of cells. They are widely used in basic research fields such as cell biology, immunology, and pathology, and play an irreplaceable role in clinical diagnosis and treatment monitoring of tumors, infectious diseases, and immune diseases, as well as in drug development, candidate drug efficacy evaluation, and cell migration mechanism research. Specifically, they can be used for various experimental techniques such as immunohistochemistry, immunofluorescence, in situ hybridization, and frozen sectioning, providing an intuitive and reliable experimental basis for studying cell structure and function and revealing the mechanisms of disease development.

[0003] In the preparation of cell slides, cell fixation is an indispensable and crucial step. Its purpose is to rapidly terminate cellular physiological activities, maintain the integrity of cell morphology and structure, prevent cell autolysis and antigen degradation, and provide a stable sample basis for subsequent detection. Commonly used fixatives include formaldehyde, paraformaldehyde, and glutaraldehyde. Among these, 4% paraformaldehyde is the preferred fixative for most experiments due to its stable fixation effect and high cost-effectiveness. However, these aldehyde fixatives can form methylene bridges during fixation, causing cross-linking reactions of intracellular antigen molecules. This masks or blocks antigen epitopes, preventing them from effectively binding to specific antibodies, thus affecting the accuracy and sensitivity of subsequent detections and even leading to false negative results. Therefore, to improve the accuracy and specificity of antibody detection, antigen retrieval treatment is usually required after cell fixation. Its core function is to break the cross-linking state of antigen molecules through specific methods, expose the masked antigen epitopes, restore the immunogenicity of the antigen, and enable the antigen to specifically bind to the antibody, thereby ensuring the reliability of cell slide detection results. Therefore, the effectiveness of the antigen retrieval process directly determines the quality of subsequent cell slide detection and is one of the core critical steps affecting the accuracy and sensitivity of cell slide detection.

[0004] Chinese patent CN113740522B discloses an antigen retrieval solution and cell slides prepared using the same. The retrieval agent is a 1-1000 mM ammonium salt buffer solution, which is capable of reacting with aldehyde groups. The ammonium salt is (NH4)2SO4. Sodium dodecyl sulfate is added to the ammonium salt buffer solution, and the sodium dodecyl sulfate accounts for 0.025%-0.25% of the mass of the antigen retrieval solution. Chinese patent CN103409493B discloses a method for observing nerve cells, using a cell fixation retrieval agent, the component of which is 0.1% w / v trypsin antigen.

[0005] Currently, existing antigen retrieval methods for fixed cell slides are mainly divided into two categories: thermal retrieval and enzymatic retrieval. These two methods are widely used in immunohistochemical experiments using tissue as a carrier. However, in detection scenarios such as immunofluorescence using cells as a carrier, research and application still have many shortcomings, making it difficult to achieve ideal antigen retrieval effects, which in turn leads to poor detection results for cell slides. The specific problems are as follows: On the one hand, thermal retrieval usually uses citrate buffer (pH 6.0), EDTA buffer (pH 8.0-9.0), etc., as retrieval solutions, and retrieval is carried out by heating methods such as water bath, high pressure, and microwave. Although it can break antigen cross-linking and expose antigen epitopes to a certain extent, this method is very prone to cell slide detachment under high temperature conditions. Moreover, the operation process is cumbersome and time-consuming. The retrieval effect is affected by many factors such as heating temperature, heating time, and pH value of the retrieval solution, making it difficult to achieve a stable and controllable retrieval effect. Especially for special types of antigens such as nuclear antigens, the retrieval ability is limited, and problems such as false negatives or background stray light are prone to occur, affecting the accuracy of detection results.

[0006] On the other hand, enzymatic retrieval mainly uses enzymes such as trypsin, pepsin, and proteinase K to hydrolyze the cross-linked regions of antigen molecules. It is suitable for some antigens or fragile tissue samples that are easily destroyed by high temperatures. However, the retrieval effect of this method is easily affected by factors such as the type of enzyme, concentration, duration of action, and temperature. The retrieval conditions are difficult to control precisely, and it is easy to have insufficient enzymatic digestion leading to inadequate exposure of antigen epitopes, or excessive enzymatic digestion damaging cell morphology and antigen structure. This not only fails to guarantee the consistency of the retrieval effect, but may also further affect the accuracy of subsequent detection, leading to deviations in the test results.

[0007] In addition, existing technologies also use sodium dodecyl sulfate (SDS) for antigen retrieval in fixed cells, which exposes antigenic epitopes based on denaturation. However, this method has obvious drawbacks: SDS cannot effectively retrieve all types of antigenic epitopes, and if the SDS is not thoroughly washed away after retrieval, it will cause the antibodies incubated subsequently to denature, thereby interfering with the experimental results and reducing the accuracy of detection.

[0008] In summary, existing antigen retrieval methods used after cell fixation in cell slide preparation suffer from numerous drawbacks, including poor retrieval efficiency, insufficient stability, cumbersome operation, and potential cell damage or interference with detection. These issues lead to insufficient exposure of antigen epitopes and poor recovery of antigen immune activity, resulting in low sensitivity, poor accuracy, and poor reproducibility in subsequent cell slide detection. Consequently, these methods fail to meet the quality requirements of cell slide detection in basic research and clinical applications. Therefore, developing an antigen retrieval technology that can effectively address these problems is of great significance for improving the quality of cell slide preparation and detection results. Summary of the Invention

[0009] To address the aforementioned shortcomings, this invention provides a cell-fixed antigen retrieval composition for a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay), along with its product and applications. the term: The term "myasthenia gravis" used in this invention refers to an autoimmune disease mediated by autoantibodies, which is an acquired neuromuscular junction transmission disorder. The core of the lesion is located at the postsynaptic membrane connecting nerves and muscles. Due to damage to the relevant receptors in this area, nerve signals cannot be transmitted to the muscles normally, leading to a series of symptoms such as skeletal muscle weakness. It can affect multiple muscle groups throughout the body and can be life-threatening in severe cases. However, with proper treatment, most patients can maintain a high quality of life and have a normal lifespan.

[0010] The term "myasthenia gravis antibody profile" used in this invention refers to a group of characteristic autoantibodies closely related to the pathogenesis of myasthenia gravis, mainly including acetylcholine receptor (AChR) antibody, titin antibody, ryneline receptor 1 (RyR1) antibody, muscle-specific receptor tyrosine kinase (MuSK) antibody, low-density lipoprotein receptor-associated protein 4 (LRP4) antibody, etc. The combined detection of this antibody profile is the core laboratory basis for clinical myasthenia gravis subtyping diagnosis, differential diagnosis and disease assessment.

[0011] The term "AChR" used in this invention refers to the acetylcholine receptor, a protein complex located on the cell membrane that specifically binds to the neurotransmitter acetylcholine (ACh), mediating the transmission of nerve signals between synapses. It is a key molecule in the neuromuscular junction, the autonomic nervous system, and the central nervous system, and is mainly divided into muscarinic receptors (M receptors) and nicotinic receptors (N receptors). The nicotinic acetylcholine receptor (nAChR) on the skeletal muscle endplate membrane is closely related to myasthenia gravis. This protein is a key carrier of signal transmission at the neuromuscular junction, responsible for receiving acetylcholine released from nerve endings, triggering cell membrane depolarization, and subsequently triggering skeletal muscle contraction. In patients with myasthenia gravis, autoantibodies attack AChR, blocking the binding of acetylcholine to the receptor or accelerating receptor degradation, leading to impaired neuromuscular signal transmission, ultimately manifesting as typical symptoms such as skeletal muscle weakness and fatigue.

[0012] The term "Titin" used in this invention refers to titin, a giant structural protein mainly found in skeletal and cardiac muscle cells. It runs through the thick and thin filaments of the sarcomere, serving multiple functions including structural support, elastic buffering, and signal transduction. Its core role is to maintain the integrity and elasticity of the sarcomere, regulate the amplitude of muscle contraction and relaxation, and participate in muscle cell development, differentiation, and damage repair. In myasthenia gravis, titin is often recognized as an autoantigen by autoantibodies in the patient's body, triggering an immune response. This is especially true in patients with myasthenia gravis and muscular dystrophy, where the positive rate of anti-titin antibodies is high, further exacerbating muscle cell damage and worsening muscle weakness symptoms. Titin is also an important reference indicator for clinical diagnosis and disease assessment.

[0013] The term "RyR1" used in this invention refers to Ryanodine Receptor 1, a calcium channel protein located on the sarcoplasmic reticulum membrane of skeletal muscle. It is one of the largest known ion channel proteins, primarily responsible for regulating the release and reabsorption of calcium ions within the sarcoplasmic reticulum. When an excitatory signal is received at the neuromuscular junction, RyR1 is activated and opens, causing a large release of calcium ions from the sarcoplasmic reticulum into the cytoplasm, triggering myofibril sliding and achieving skeletal muscle contraction. After contraction, RyR1 closes, calcium ions are pumped back into the sarcoplasmic reticulum, and the muscle returns to a relaxed state. In patients with myasthenia gravis, autoantibodies may attack RyR1, leading to abnormal channel function and disordered calcium ion release, thereby affecting the coordination and strength of muscle contraction, exacerbating symptoms such as muscle weakness and limited mobility, especially associated with complications such as rhabdomyolysis in myasthenia gravis.

[0014] The term "MuSK" as used in this invention refers to muscle-specific receptor tyrosine kinase, a tyrosine kinase receptor mainly expressed at the neuromuscular junction of skeletal muscle. Primarily distributed on the endplate membrane of muscle fibers, it plays a crucial role in the development, maturation, and maintenance of the neuromuscular junction. Its core function is to mediate the adhesion between nerve endings and muscle fibers, promote the aggregation and stabilization of acetylcholine receptors on the endplate membrane, and ensure the normal transmission of neuromuscular signals. In some patients with myasthenia gravis (especially those with negative serum anti-acetylcholine receptor antibodies), autoantibodies specifically bind to MuSK, inhibiting its kinase activity. This leads to abnormal distribution and reduced number of acetylcholine receptors on the endplate membrane, damaging the structure and function of the neuromuscular junction and ultimately causing muscle weakness symptoms. MuSK is an important pathogenic target in this type of refractory myasthenia gravis.

[0015] The term "LRP4" used in this invention refers to Low-Density Lipoprotein Receptor-Related Protein 4 (LRP4), a membrane protein widely expressed in skeletal muscle, nerve tissue, and other sites. Belonging to the LRP4 superfamily, its core function is to participate in cell signaling, cell adhesion, and tissue development regulation. During neuromuscular junction formation, LRP4 can bind to Agrin protein released from nerve endings, activating downstream signaling pathways and recruiting MuSK and acetylcholine receptors, promoting the maturation and stability of the endplate membrane. In myasthenia gravis, LRP4 can act as an autoantigen recognized by autoantibodies in the patient's body. Once the antibody binds to LRP4, it blocks its interaction with Agrin and MuSK, disrupting the normal structure of the neuromuscular junction, leading to abnormal acetylcholine receptor aggregation, affecting neuromuscular signal transmission, and causing muscle weakness. It is also an important pathogenic protein in patients with serum anti-acetylcholine receptor antibody-negative myasthenia gravis.

[0016] The term "mCherry" used in this invention refers to a monomeric red fluorescent protein, which is also a widely used red fluorescent reporter tag in the fields of life sciences and medical diagnostics. In the mCherry-AChR plasmid, mCherry-Titin plasmid, mCherry-RyR1 plasmid, mCherry-MuSK plasmid, and mCherry-LRP4 plasmid of this invention, mCherry is independently expressed along with the target antigens AChR, Titin, RyR1, Musk, or LRP4. mCherry serves as a positive transfection reporter tag; the appearance of red fluorescence within cells indicates successful transfection and normal expression of the plasmid, providing a direct visual assessment of overall cell transfection efficiency. The green fluorescent signal (Alexa Fluor 488-labeled secondary antibody) corresponds to the specific binding site between the patient's antibody and the target antigen. The interpretation range is the transfected red fluorescent cell region; the presence of a specific green fluorescent signal within the red fluorescent cell region indicates a positive result, while the absence of a corresponding green fluorescent signal within the red fluorescent cell region indicates a negative result. This design is crucial for ensuring accurate and highly specific interpretation using the CBA method (cell immunofluorescence assay).

[0017] The mCherry-AChR plasmid, mCherry-Titin plasmid, mCherry-RyR1 plasmid, mCherry-MuSK plasmid, and mCherry-LRP4 plasmid used in this invention are not particularly limited in their source. They can be obtained through conventional commercial channels or constructed using conventional gene synthesis, molecular cloning, and other methods in the art. As is well known in the art, dual-expression recombinant plasmids that can simultaneously express fluorescent reporter proteins and target antigens are conventionally constructed vectors in this field. Even without detailed descriptions of their specific sources, those skilled in the art can clearly understand their intended use and implement them based on existing technology and the functions and uses described in this invention. Any recombinant expression vector that can stably express the corresponding target antigen in eukaryotic cells, normally produce mCherry red fluorescent signals to indicate the overall extent of transfected positive cells, and can be used for cell transfection, cell slide preparation, and specific recognition, binding, and immunofluorescence detection of myasthenia gravis-related antibodies, regardless of its specific construction method, backbone vector, or source of preparation, falls within the protection scope of this invention.

[0018] In this invention, the antibodies used for secondary antibody incubation are also purchased from commercially available sources. Different brands and catalog numbers can be selected according to the species of the primary antibody and the detection requirements. Examples of selections are as follows: if the primary antibody is a rabbit polyclonal antibody, goat anti-rabbit IgG secondary antibody can be used; if the primary antibody is a mouse monoclonal antibody, goat anti-mouse IgG secondary antibody can be used. The antibody used in this embodiment of the invention was purchased from ThermoFisher Scientific, catalog number A11013; the working solution used is phosphate buffer; and the main component of the mounting medium used is glycerol. Conventional working solutions or mounting medium types in the art can be used. The working solution or mounting medium can be routinely prepared or commercially purchased according to the type of secondary antibody and incubation conditions, and its specific components, concentrations, and sources are not limited.

[0019] The term "cell crawler" used in this invention refers to a product obtained by seeding and culturing host cells (such as HEK293 cells) transfected with the recombinant plasmid of this invention on a pretreated glass crawler as a solidification carrier, followed by transfection incubation, fixation and permeation, and blocking. It can be used for immunofluorescence detection in the myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence method).

[0020] The term "biochip" as used in this invention refers to a microchip device that integrates functions such as sample introduction, reaction, and detection. Its main body is equipped with a reaction tank, sample introduction port, sample aspiration port, and labeling area, which can realize the simultaneous detection of multiple targets of myasthenia gravis antibody spectrum. It has the characteristics of small sample volume, simple operation, and high detection efficiency.

[0021] The term "Brij 78" used in this invention refers to polyoxyethylene-20 cetyl ether, which is a nonionic surfactant with good solubilizing, wetting, emulsifying and cell membrane permeability effects. It can gently permeate cell structures, help relax the cross-linked and blocked antigen spatial conformation, promote the full exposure of antigen epitopes, and at the same time cause little damage to cell morphology and antigen activity, making it suitable for cell immunofluorescence detection systems.

[0022] The term "TCEP" used in this invention refers to tris(2-carboxyethyl)phosphine, which is a highly efficient and stable reducing agent that can gently reduce disulfide bonds within antigen protein molecules, reconstruct the native spatial conformation of the antigen, and further expose hidden antigenic epitopes. Compared with traditional reducing agents, it has stronger stability, fewer side effects, and does not affect the subsequent specific binding of antigen and antibody.

[0023] The term "ProClin 300" used in this invention refers to a broad-spectrum compound microbial preservative with antibacterial and bactericidal effects. It can effectively inhibit the growth of bacteria, molds and yeasts in the repair composition, maintain the stability of the reagent system for a long time, extend the shelf life of the reagent, and has no interference with cells, antigens and fluorescent immunoreaction.

[0024] In this invention, the term "SDS" refers to sodium dodecyl sulfate, an anionic denaturing surfactant. The term "DTT" refers to dithiothreitol, a protein disulfide bond reducing agent.

[0025] The technical solution of this invention is as follows: On one hand, the present invention provides a cell fixation antigen retrieval composition, which is composed of ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300 and buffer solution.

[0026] Specifically, the cell fixation antigen retrieval composition contains 70-130 mM ammonium sulfate.

[0027] More specifically, the cell-fixed antigen retrieval composition contains 70-71, 71-72, 72-73, 73-74, 74-75, 75-76, 76-77, 77-78, 78-79, 79-80, 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98, 98-99, 99-100, 100-101, 101-102, 102- 103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112, 112-113, 113-114, 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-121, 121-122, 122-123, 123-124, 124-125, 125-126, 126-127, 127-128, 128-129 or 129-130mM ammonium sulfate.

[0028] Preferably, the antigen retrieval composition after cell fixation contains 100-101, 101-102, 102-103, 103-104, 104-105, 105-106, 106-107, 107-108, 108-109, 109-110, 110-111, 111-112, 112-113, and 113-114. 114-115, 115-116, 116-117, 117-118, 118-119, 119-120, 120-121, 121-122, 122-123, 123-124, 124-125, 125-126, 126-127, 127-128, 128-129 or 129-130mM ammonium sulfate.

[0029] More preferably, the cell fixation antigen retrieval composition contains 100 mM ammonium sulfate.

[0030] Specifically, the cell fixation antigen retrieval composition contains 50-100 mM hydroxylamine hydrochloride.

[0031] More specifically, the cell-fixed antigen retrieval composition contains 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 71-72, 72-73, 73- 74, 74-75, 75-76, 76-77, 77-78, 78-79, 79-80, 80-81, 81-82, 82-83, 83-84, 84-85, 85-86, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 94-95, 95-96, 96-97, 97-98, 98-99 or 99-100mM hydroxylamine hydrochloride.

[0032] Preferably, the cell fixation antigen retrieval composition contains 50-51, 51-52, 52-53, 53-54, 54-55, 55-56, 56-57, 57-58, 58-59, 59-60, 60-61, 61-62, 62-63, 63-64, 64-65, 65-66, 66-67, 67-68, 68-69, 69-70, 70-71, 71-72, 72-73, 73-74 or 74-75 mM hydroxylamine hydrochloride.

[0033] More preferably, the cell fixation antigen repair composition contains 75 mM hydroxylamine hydrochloride.

[0034] Specifically, the cell fixation antigen retrieval composition contains 0.25%-0.55% v / v Brij 78.

[0035] More specifically, the cell-fixed antigen retrieval composition contains 0.25%-0.26%, 0.26%-0.27%, 0.27%-0.28%, 0.28%-0.29%, 0.29%-0.30%, 0.30%-0.31%, 0.31%-0.32%, 0.32%-0.33%, 0.33%-0.34%, 0.34%-0.35%, 0.35%-0.36%, 0.36%-0.37%, 0.37%-0.38%, 0.38%-0.39%, 0 0.39%-0.40%, 0.40%-0.41%, 0.41%-0.42%, 0.42%-0.43%, 0.43%-0.44%, 0.44%-0.45%, 0.45%-0.46%, 0.46%-0.47%, 0.47%-0.48%, 0.48%-0.49%, 0.49%-0.50%, 0.50%-0.51%, 0.51%-0.52%, 0.52%-0.53%, 0.53%-0.54% or 0.54%-0.55% v / v Brij 78.

[0036] Preferably, the cell-fixed antigen retrieval composition contains 0.40%-0.41%, 0.41%-0.42%, 0.42%-0.43%, 0.43%-0.44%, 0.44%-0.45%, 0.45%-0.46%, 0.46%-0.47%, 0.47%-0.48%, 0.48%-0.49%, 0.49%-0.50%, 0.50%-0.51%, 0.51%-0.52%, 0.52%-0.53%, 0.53%-0.54%, or 0.54%-0.55% v / v Brij 78.

[0037] More preferably, the cell fixation antigen retrieval composition contains 0.40% v / v Brij 78.

[0038] Specifically, the cell fixation antigen retrieval composition contains 0.65-1.25 mM TCEP.

[0039] More specifically, the antigen retrieval composition after cell fixation contains 0.65-0.66, 0.66-0.67, 0.67-0.68, 0.68-0.69, 0.69-0.70, 0.70-0.71, 0.71-0.72, 0.72-0.73, 0.73-0.74, 0.74-0.75, 0.75-0.76, 0.76-0.77, 0.77-0.78, and 0. 78-0.79, 0.79-0.80, 0.80-0.81, 0.81-0.82, 0.82-0.83, 0.83-0.84, 0.84-0.85, 0.85-0.86, 0.86-0.87, 0.87-0.88, 0.88-0.89, 0.89-0.90, 0.90-0.91, 0.91-0.92, 0.92-0.93, 0.93-0.9 4. 0.94-0.95, 0.95-0.96, 0.96-0.97, 0.97-0.98, 0.98-0.99, 0.99-1.00, 1.00-1.01, 1.01-1.02, 1.02-1.03, 1.03-1.04, 1.04-1.05, 1.05-1.06, 1.06-1.07, 1.07-1.08, 1.08-1.09, 1.09 -1.10, 1.10-1.11, 1.11-1.12, 1.12-1.13, 1.13-1.14, 1.14-1.15, 1.15-1.16, 1.16-1.17, 1. 17-1.18, 1.18-1.19, 1.19-1.20, 1.20-1.21, 1.21-1.22, 1.22-1.23, 1.23-1.24, 1.24-1.25mM TCEP.

[0040] Preferably, the antigen retrieval composition after cell fixation contains 1.00-1.01, 1.01-1.02, 1.02-1.03, 1.03-1.04, 1.04-1.05, 1.05-1.06, 1.06-1.07, 1.07-1.08, 1.08-1.09, 1.09-1.10, 1.10-1.11, and 1.11-1. 12. 1.12-1.13, 1.13-1.14, 1.14-1.15, 1.15-1.16, 1.16-1.17, 1.17-1.18, 1.18-1 .19, 1.19-1.20, 1.20-1.21, 1.21-1.22, 1.22-1.23, 1.23-1.24, 1.24-1.25mMTCEP.

[0041] More preferably, the cell fixation antigen retrieval composition contains 1 mM TCEP.

[0042] Specifically, the cell fixation antigen retrieval composition contains 0.02%-0.05% v / v ProClin 300.

[0043] More specifically, the cell fixation antigen retrieval composition contains 0.02%-0.03%, 0.03%-0.04%, or 0.04%-0.05% v / v ProClin 300.

[0044] Preferably, the cell fixation antigen retrieval composition contains 0.04%-0.05% v / v ProClin 300.

[0045] More preferably, the cell fixation antigen retrieval composition contains 0.04% v / v ProClin 300.

[0046] Specifically, the buffer solution includes, but is not limited to, one or more of the following: phosphate buffer, Tris-HCl buffer, and HEPES buffer.

[0047] In some specific embodiments of the present invention, the buffer solution is a 1× phosphate buffer with a pH of 7.2-7.4.

[0048] In another aspect, the present invention provides a method for preparing the antigen repair composition after cell fixation as described in any of the above claims.

[0049] Specifically, the preparation method includes: adding ammonium sulfate and hydroxylamine hydrochloride to a buffer solution and stirring until dissolved; adding Brij 78 and adjusting the pH; adding TCEP and ProClin 300; mixing and filtering to obtain the antigen retrieval composition after cell fixation.

[0050] Preferably, the Brij 78 is melted by heating in a water bath at 50℃-60℃ and then slowly dripped into the system.

[0051] Preferably, the pH adjustment is to adjust the pH to 7.0-7.5.

[0052] In another aspect, the present invention provides a cell fixation antigen repair product, wherein the cell fixation antigen repair product comprises the cell fixation antigen repair composition described in any one of the above claims.

[0053] Preferably, the cell fixation antigen retrieval product includes reagents and / or kits.

[0054] In another aspect, the present invention provides a method for antigen repair after cell fixation, the method comprising using the antigen repair composition or product for antigen repair after cell fixation as described in any of the preceding claims.

[0055] In another aspect, the present invention provides the application of the cell fixation antigen repair composition or cell fixation antigen repair product described in any one of the above claims, wherein the application includes any one or more of the following: (1) Application in cell fixation repair; (2) Application in the preparation of cell smears overexpressing antigens; (3) Application in the preparation of antibody detection products.

[0056] Preferably, the antigen is a myasthenia gravis-related antigen, which includes any one or more of the following: AChR antigen, Titin antigen, RyR1 antigen, Musk antigen, and LRP4 antigen.

[0057] Specifically, the antibody detection product is a myasthenia gravis antibody profile detection kit.

[0058] Preferably, the myasthenia gravis antibody spectrum detection kit contains a biochip, the biochip comprising a chip body; the biochip body is provided with a reaction groove, a sample inlet, a sample aspiration hole and a labeling area; the reaction groove contains immobilized cell smears expressing different myasthenia gravis-related antigen proteins.

[0059] The beneficial effects of this invention are as follows: The cell fixation antigen retrieval composition of the present invention comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and buffer solution in a synergistic manner. Each component plays a complementary role, which can effectively remove the cross-linking state of antigen molecules caused by aldehyde fixatives during cell fixation, fully expose the masked antigen epitopes, and efficiently protect the antigen's immune activity. At the same time, it avoids damage to cell morphology and does not interfere with the specific binding of subsequent antibodies to antigens. It fundamentally solves the core defects of existing antigen retrieval methods that easily damage cells and interfere with detection.

[0060] The antigen retrieval composition of this invention, obtained after cell fixation, is adaptable to the retrieval of various myasthenia gravis-related antigens. It can accurately achieve simultaneous detection of multiple specific antibodies, including AChR, Titin, RyR1, Musk, and LRP4. The myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay) prepared based on this composition provides stable detection results, is easy to operate, and requires no complex equipment. It effectively makes up for the shortcomings of existing detection technologies and provides reliable technical support for the accurate classification, differential diagnosis, and disease assessment of myasthenia gravis. It has important clinical application value, broad industrial promotion prospects, and significant practical value. Attached Figure Description

[0061] Figure 1The results are from the assay of the antigenicity retention capacity of the target antigen.

[0062] Figure 2 This is a schematic diagram showing the presence of anti-MuSK antibodies in the sample.

[0063] Figure 3 This is a schematic diagram showing that the sample does not contain anti-MuSK antibodies. Detailed Implementation

[0064] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0065] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0066] Basic Example 1: Pretreatment of Cell Slides 1. Take a square glass slide with a diameter of 24 mm as the slide substrate. Rinse it with purified water 3-5 times to initially remove surface impurities and dry it. Immerse the rinsed glass slide in concentrated sulfuric acid for 24 hours. After immersion, rinse it repeatedly with purified water 8-10 times.

[0067] 2. Immerse the cleaned glass slides in anhydrous ethanol for 6 hours. After immersion, rinse repeatedly with purified water 8-10 times and sterilize in a high-pressure steam sterilizer (121℃, 0.1MPa) for 20 minutes. After sterilization, remove the slides and cool them to room temperature in a clean bench to obtain sterile glass slides.

[0068] 3. Perform the slide placement operation in the clean bench: Place the sterile glass slides stably on the bottom of the wells of the sterile culture plate (6-well plate), ensuring that the glass slides are in close contact with the bottom of the wells, and place one glass slide in each culture well.

[0069] 4. Add 100 μg / mL poly-L-lysine solution to each well containing a glass slide, ensuring the solution completely covers the slide surface. Soak for 2 hours to allow the poly-L-lysine to be evenly adsorbed onto the slide surface. After soaking, use a sterile pipette to remove excess poly-L-lysine solution from the wells. Rinse twice with sterile purified water. Place the culture plate in a laminar flow hood, open the plate lid, and allow it to air dry naturally until the slide surface is completely dry. This is the prepared cell spreader, which can be used directly for cell seeding experiments or sealed and refrigerated at 4°C for later use.

[0070] Basic Example 2: Preparation of cell crawling slices containing different antigens 1. Host cell inoculation and culture HEK293 cells were cultured to the logarithmic growth phase. The old culture medium in the cell culture flask was discarded, and the cell surface was washed twice with PBS buffer. 0.25% trypsin digestion solution was added, and the cells were incubated at 37°C for 1 min. After observing the cells becoming rounded and detaching from the culture vessel under an inverted microscope, DMEM medium containing 10% FBS was added to terminate the digestion. The cells were gently pipetted to prepare a single-cell suspension, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 per mL.

[0071] Add 2 mL of single-cell suspension with adjusted concentration to each well of the 6-well culture plate containing the pretreated cell smear obtained in Basic Experiment Example 1. Gently shake the culture plate to distribute the cells evenly on the surface of the glass slide. Place the plate in a 37°C, 5% CO2 incubator and incubate statically for 24 h for subsequent transfection.

[0072] 2. Recombinant plasmid transfection and incubation Using mCherry-AChR, mCherry-Titin, mCherry-RyR1, mCherry-MuSK, or mCherry-LRP4 as the target plasmid, transfection was performed using PEI transfection reagent. Two EP tubes were prepared, each containing 125 μL of Opti-MEM medium. In one EP tube (tube ①), 3 μg of the target plasmid was added, and in the other EP tube (tube ②), 12 μL of PEI transfection reagent was added. The mixture was thoroughly mixed and allowed to stand for 10 min. The liquid from tube ① was then added to tube ②, thoroughly mixed, and allowed to stand for 5 min to obtain the transfection complex. The transfection complex was added dropwise to a 6-well plate containing cell spreaders, gently mixed, and incubated at 37°C in a 5% CO2 incubator for 48 hours to obtain mCherry-AChR cell spreaders, mCherry-Titin cell spreaders, mCherry-RyR1 cell spreaders, mCherry-MuSK cell spreaders, or mCherry-LRP4 cell spreaders, which were then used for subsequent experiments.

[0073] Example 1: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and PBS buffer. The final concentrations of each component in the antigen retrieval composition are: 100 mM ammonium sulfate, 75 mM hydroxylamine hydrochloride, 0.4% v / v Brij 78, 1 mM TCEP, and 0.04% v / v ProClin 300. The solvent is 1×PBS buffer at pH 7.2-7.4.

[0074] The preparation method of the antigen retrieval composition after cell fixation is as follows: using 1×PBS buffer as solvent, ammonium sulfate and hydroxylamine hydrochloride are added sequentially and stirred until completely dissolved; Brij 78 is heated and melted in a 50℃ water bath, then slowly added dropwise to the system and stirred thoroughly to disperse; the pH of the system is adjusted to 7.4; then TCEP and ProClin 300 are added, mixed well, and filtered through a 0.22μm microporous membrane for sterilization to obtain the antigen retrieval composition after cell fixation.

[0075] Example 2: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and PBS buffer. The final concentrations of each component in the antigen retrieval composition for cell fixation are: 130 mM ammonium sulfate, 50 mM hydroxylamine hydrochloride, 0.55% v / v Brij 78, 0.65 mM TCEP, and 0.02% v / v ProClin 300. The solvent is 1×PBS buffer with pH 7.2-7.4.

[0076] The preparation method of the antigen repair composition after cell fixation is as described in Example 1.

[0077] Example 3: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and PBS buffer. The final concentrations of each component in the antigen retrieval composition for cell fixation are: 70 mM ammonium sulfate, 100 mM hydroxylamine hydrochloride, 0.25% v / v Brij 78, 1.25 mM TCEP, and 0.05% v / v ProClin 300. The solvent is 1×PBS buffer with pH 7.2-7.4.

[0078] The preparation method of the antigen repair composition after cell fixation is as described in Example 1.

[0079] Comparative Example 1: An antigen retrieval composition after cell fixation A cell fixation antigen retrieval composition comprises hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and PBS buffer; the final concentrations of each component in the cell fixation antigen retrieval composition are: 175 mM hydroxylamine hydrochloride, 0.4% v / v Brij 78, 1 mM TCEP, and 0.04% v / v ProClin 300, and the solvent is 1×PBS buffer with pH 7.2-7.4.

[0080] The preparation method of the cell fixation antigen repair composition is as follows: 1×PBS buffer is used as the solvent, hydroxylamine hydrochloride is added and stirred until completely dissolved; Brij 78 is heated and melted in a 50℃ water bath, then slowly dripped into the system and stirred thoroughly to disperse; the pH of the system is adjusted to 7.4; then TCEP and ProClin 300 are added, mixed well, and filtered through a 0.22μm microporous membrane for sterilization to obtain the cell fixation antigen repair composition.

[0081] Comparative Example 2: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, Brij 78, TCEP, ProClin 300, and PBS buffer; the final concentrations of each component in the antigen retrieval composition are: 175 mM ammonium sulfate, 0.4% v / v Brij 78, 1 mM TCEP, and 0.04% v / v ProClin 300, and the solvent is 1×PBS buffer with pH 7.2-7.4.

[0082] The preparation method of the cell fixation antigen retrieval composition is as follows: ammonium sulfate is added to 1×PBS buffer as solvent and stirred until completely dissolved; Brij 78 is heated and melted in a 50℃ water bath, then slowly added dropwise to the system and stirred thoroughly to disperse; the pH of the system is adjusted to 7.4; then TCEP and ProClin 300 are added, mixed well, and filtered through a 0.22μm microporous membrane for sterilization to obtain the cell fixation antigen retrieval composition.

[0083] Comparative Example 3: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, ProClin 300, and PBS buffer; the final concentrations of each component in the antigen retrieval composition are: 100mM ammonium sulfate, 75mM hydroxylamine hydrochloride, 0.4% v / v Brij 78, and 0.04% v / v ProClin 300, and the solvent is 1×PBS buffer at pH 7.2-7.4.

[0084] The preparation method of the cell fixation antigen repair composition is as follows: using 1×PBS buffer as solvent, ammonium sulfate and hydroxylamine hydrochloride are added sequentially and stirred until completely dissolved; Brij 78 is heated and melted in a 50℃ water bath, then slowly added dropwise to the system and stirred thoroughly to disperse; the pH of the system is adjusted to 7.4; then ProClin 300 is added, mixed well, and filtered through a 0.22μm microporous membrane for sterilization to obtain the cell fixation antigen repair composition.

[0085] Comparative Example 4: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, Brij 78, ProClin 300, and PBS buffer; the final concentrations of each component in the antigen retrieval composition for cell fixation are: 176 mM ammonium sulfate, 0.4% v / v Brij 78, and 0.04% v / v ProClin 300, and the solvent is 1×PBS buffer with pH 7.2-7.4.

[0086] The preparation method of the cell fixation antigen retrieval composition is as follows: ammonium sulfate is added to 1×PBS buffer as solvent and stirred until completely dissolved; Brij 78 is heated and melted in a 50℃ water bath, then slowly added dropwise to the system and stirred thoroughly to disperse; the pH of the system is adjusted to 7.4; then ProClin 300 is added, mixed well, and filtered through a 0.22μm microporous membrane for sterilization to obtain the cell fixation antigen retrieval composition.

[0087] Comparative Example 5: An antigen retrieval composition after cell fixation An antigen retrieval composition for cell fixation comprises ammonium sulfate, hydroxylamine hydrochloride, SDS, DTT, ProClin 300, and PBS; the final concentrations of each component in the antigen retrieval composition for cell fixation are: 100mM ammonium sulfate, 75mM hydroxylamine hydrochloride, 0.4% w / v SDS, 1mM DTT, and 0.04% v / v ProClin 300, and the solvent is 1×PBS buffer at pH 7.2-7.4.

[0088] The preparation method of the antigen retrieval composition after cell fixation is as follows: using 1×PBS buffer as solvent, ammonium sulfate, hydroxylamine hydrochloride and SDS are added in sequence and stirred until completely dissolved; the pH of the system is adjusted to 7.4; then DTT and ProClin300 are added, mixed well and filtered through a 0.22μm microporous membrane for sterilization to obtain the antigen retrieval composition after cell fixation.

[0089] Experimental Example 1: Application of Antigen Retrieval Composition after Cell Fixation 1. Cell slide fixation After completing the incubation as described in Basic Experiment Example 2, discard the culture medium from the mCherry-MuSK cell slides, rinse the cell slides three times with sterile PBS buffer for 5 min each time, add 1 mL of 4% paraformaldehyde fixative to each well, fix at room temperature for 30 min, discard the fixative, and rinse the slides three times with sterile PBS buffer for 5 min each time.

[0090] 2. Antigen retrieval after cell fixation Take 1 mL of the cell fixation antigen retrieval composition prepared in Examples 1-3 or Comparative Examples 1-5 into each well, use PBS buffer as a blank control, and incubate at room temperature for 30 min to perform antigen retrieval. After retrieval, discard the cell fixation antigen retrieval composition, wash the cell slides three times with sterile PBS buffer for 5 min each time, and gently shake to ensure that residual retrieval solution is completely removed.

[0091] 3. Post-processing of cell slides Add 1 mL of 5% BSA blocking solution to each well and incubate at 37°C for 1 h. After blocking, rinse the slides three times with sterile PBS buffer for 5 min each time. Remove the coverslips with sterile tweezers, place them on sterile filter paper, blot off the moisture at the edges, and dry them to obtain the cell slides for Musk antibody detection in Examples 1-3 or Comparative Examples 1-5.

[0092] 4. Verification of the ability to retain the antigenicity of the target antigen (1) Preparation The cell slides prepared above for MuSK antibody detection in Examples 1-3 or Comparative Examples 1-5 were cut into 2.5×2.5mm pieces, glued onto glass slides with UV adhesive, and cured. They were then placed in a humidified chamber and soaked in sterile PBS buffer for 10 minutes. After soaking, the sterile PBS buffer was discarded.

[0093] (2) Sample incubation Add 200 μL of specific positive sample against MuSK antigen to each cell slide, and set up a negative sample (PBS buffer) as a control. Incubate at room temperature for 1 h to ensure that the specific antibody in the sample fully binds to the antigen on the antigen slide.

[0094] (3) Cleaning Remove the cell slide and gently rinse it three times with sterile PBS buffer, 5 minutes each time.

[0095] (4) Secondary antibody incubation Add 200 μL of fluorescently labeled secondary antibody working solution to the cleaned cell slide, ensuring the secondary antibody evenly covers the cell slide surface. Incubate the slide in a humidified chamber at room temperature in the dark for 30 minutes to allow the secondary antibody to specifically bind to the primary antibody bound to the target antigen. Note: This step must be performed in the dark from the beginning to avoid the fluorescent secondary antibody becoming ineffective due to light exposure.

[0096] (5) Secondary cleaning After the secondary antibody incubation is complete, remove the fluorescently labeled secondary antibody working solution and gently rinse the slide three times with sterile PBS buffer, 5 minutes each time.

[0097] (6) Fluorescence detection and result interpretation ① Add 200 μL of mounting medium, place the cell slide under a fluorescence inverted microscope, and observe the emitted fluorescence signals at wavelengths of 480-500 nm and 540-565 nm, respectively, to observe whether green and red fluorescence signals are emitted; compare the position and characteristics of the green fluorescence signal with the position of the red fluorescence signal to confirm whether they overlap. ② If the location emitting the green fluorescent signal overlaps with the location emitting the red fluorescent signal, and the characteristics are basically the same, then it can be determined that the sample contains anti-MuSK antibody; ③ If no green fluorescent signal is emitted; or the location of the green fluorescent signal does not overlap with the location of the red fluorescent signal; or the location of the green fluorescent signal overlaps with the location of the red fluorescent signal, but the two have significant differences in characteristics; then it is determined that the sample does not contain anti-MuSK antibody.

[0098] (7) Measurement results Each set of examples and comparative examples had 3 replicates, and 3 fields of view were selected for detection in each replicate. The positive cell rate of each group was calculated as follows: positive cell rate (%) = number of red-green overlapping cells / number of red fluorescent cells × 100%. A higher positive cell rate reflects that more target antigens are recognized and bound by the antibody, and the better the antigenicity is preserved.

[0099] Simultaneously, Image-Pro Plus software was used to quantitatively analyze the average optical density (IOD / Area) of green fluorescence in each group of positive fields of view. IOD (integrated optical density) refers to the total intensity of green fluorescence within the detection field, and Area is the area of ​​the detection field. The ratio of IOD / Area accurately reflects the intensity of green fluorescence per unit area. A higher average optical density value of green fluorescence indicates a greater number of primary and secondary antibodies bound to the target antigen, resulting in a stronger binding between the antibody and the antigen, thus indirectly evaluating the antigenicity retention capacity of the target antigen.

[0100] The measurement results are shown in Table 1 and Figure 1 As shown: Table 1

[0101] Experimental Example 2: Preparation of Antigen Repair Composition after Cell Fixation for Cell Crawling Slices Corresponding to Antigens in the Antibody Profile of Myasthenia Gravis 1. Fixation and post-processing of cell slides After completing the incubation as described in Basic Experiment Example 2, for mCherry-AChR cell slides, mCherry-Titin cell slides, mCherry-RyR1 cell slides, mCherry-MuSK cell slides, or mCherry-LRP4 cell slides, discard the culture medium, wash the cell slides three times with sterile PBS buffer, 5 min each time; add 1 mL of 4% paraformaldehyde fixative to each well, fix at room temperature for 30 min, discard the fixative, and wash the slides three times with sterile PBS buffer, 5 min each time.

[0102] 2. Antigen retrieval after cell fixation Add 1 mL of the antigen retrieval composition prepared in Example 1 to each well and incubate at room temperature for 30 min to perform antigen retrieval. After retrieval, discard the antigen retrieval composition and wash the cell slides three times with sterile PBS buffer for 5 min each time, gently shaking to ensure that any residual retrieval solution is completely removed.

[0103] 3. Post-processing of cell slides Add 1 mL of 5% BSA blocking solution to each well and incubate at 37°C for 1 h. After blocking, rinse the cell slides three times with sterile PBS buffer for 5 min each time. Remove the coverslips with sterile forceps, place them on sterile filter paper, blot off the moisture at the edges, and dry them to obtain the treated cell slides.

[0104] Experimental Example 3: Assembly and Use of a Biochip for Detecting Myasthenia Gravis Antibody Profile (IgG) 1. Composition of a biochip The biochip is a long, strip-shaped cell antigen plate with a clearly defined structural layout, divided into a front and a back panel. The front panel includes a sample inlet, aspiration port, a QR code identification label, and a mixing chamber, all integrated on the same plane for easy sample loading, aspiration, and traceability. The back panel features a reaction chamber for fixing the cell antigen plates. Processed cell slides (mCherry-AChR, mCherry-Titin, mCherry-RyR1, mCherry-MuSK, or mCherry-LRP4) and blank control antigen plates are arranged sequentially from left to right within the reaction chamber, enabling parallel detection of multiple antibodies.

[0105] 2. Cell Antigen Plate Assembly Process (1) Antigen patching: The antigen patches of each indicator cell and the blank control antigen patch are attached to the reaction tank in the set order from left to right; (2) Sealing and encapsulation: The reaction area is covered with a regular glass slide, and the reaction area with the antigen slide is sealed and encapsulated with UV adhesive. (3) Curing and fixing: Use a wafer baking machine to heat and cure the UV glue encapsulation area to firmly bond the glass slide to the chip body and ensure that the reaction area is sealed and leak-proof; (4) Identification and coding: A QR code and corresponding identification are printed on the label area using a marking machine to complete the chip identification marking; (5) Finished product packaging: The assembled and solidified cell antigen plates are sealed in aluminum foil bags, protected from light and moisture, for easy storage, transportation and clinical use.

[0106] This chip integrates sample mixing, multi-channel reaction, QR code traceability, and sealed packaging, enabling simultaneous detection of five antibodies related to the myasthenia gravis antibody spectrum (IgG).

[0107] 3. Instructions for use of biochips It is recommended to conduct the experiment at room temperature to ensure the accuracy of the results.

[0108] (1) Warming and washing: Take out the biochip and place it in a humidified box (self-provided) to equilibrate at room temperature for 10 minutes. Add 70 μL of working solution to each reaction zone and soak for 10 minutes, then aspirate the working solution.

[0109] (2) Sample incubation: Add 70 μL of the sample to be tested to each reaction zone and incubate at room temperature for 1 hour.

[0110] (3) Cleaning: Remove the sample to be tested, clean the biochip reaction area with working solution, add 70 μL of working solution to each reaction area, clean the reaction area quickly twice, add 70 μL of working solution for the third time, soak for 5 min and then remove the working solution.

[0111] (4) Secondary antibody incubation: Add 70 μL of diluted fluorescent secondary antibody (fluorescently labeled goat anti-human IgG) to each reaction zone and incubate at room temperature in the dark for 30 minutes. (From this step onwards, keep the entire process in the dark).

[0112] (5) Cleaning: Remove the fluorescent secondary antibody (fluorescently labeled goat anti-human IgG), clean the biochip reaction area with working solution, add 70 μL of working solution to each reaction area, clean the reaction area quickly twice, add 70 μL of working solution for the third time, soak for 5 min and then remove the working solution.

[0113] (6) Result interpretation: Add 70 μL of mounting medium to each reaction zone, observe the fluorescence signal under a 20x objective lens, and analyze the detection results.

[0114] (7) Interpretation criteria Take Musk as an example: 1) Use an inverted fluorescence microscope to observe the emitted fluorescence signals at wavelengths of 480-500nm and 540-565nm, respectively, and observe whether green and red fluorescence signals are emitted. 2) Compare the location and characteristics of the green fluorescent signal with the location of the red fluorescent signal to confirm whether they overlap; 3) If the location emitting the green fluorescent signal overlaps with the location emitting the red fluorescent signal, and the characteristics are basically the same, then it can be determined that the sample contains anti-MuSK antibody; 4) If no green fluorescent signal is emitted; or the location of the green fluorescent signal does not overlap with the location of the red fluorescent signal; or the location of the green fluorescent signal overlaps with the location of the red fluorescent signal, but the two have significant differences in characteristics; then the sample is determined to contain no anti-MuSK antibody.

[0115] Figure 2 The green and red fluorescent signals overlap in position and have basically the same characteristics, indicating that the sample contains anti-MuSK antibodies; Figure 3 The absence of a red fluorescent signal at the location of the green fluorescent signal indicates that the sample does not contain anti-MuSK antibodies.

[0116] Similarly, in addition to Musk transfecting cells with fluorescently labeled proteins, the reagent also includes one or more of Titin, AChR, RyR1K, and LRP4 transfecting cells with fluorescently labeled proteins, thus enabling the simultaneous detection and interpretation of multiple antibodies in the sample. If any one or more of Titin, AChR, RyR1K, Musk, and LRP4 are found to be positive, the sample is diagnosed as a myasthenia gravis antibody spectrum disease sample.

[0117] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A cell fixation antigen retrieval composition, characterized in that, The cell fixation antigen retrieval composition comprises ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300, and buffer solution; the cell fixation antigen retrieval composition contains 70-130 mM ammonium sulfate, 50-100 mM hydroxylamine hydrochloride, 0.25%-0.55% v / v Brij 78, 0.65-1.25 mM TCEP, and 0.02%-0.05% v / v ProClin 300.

2. The antigen retrieval composition after cell fixation according to claim 1, characterized in that, The cell fixation antigen retrieval composition contains 100-130 mM ammonium sulfate, 50-75 mM hydroxylamine hydrochloride, 0.40%-0.55% v / v Brij 78, 1.00-1.25 mM TCEP, and 0.04%-0.05% v / v ProClin 300.

3. The method for preparing the cell fixation antigen retrieval composition according to any one of claims 1-2, characterized in that, The preparation method includes: adding ammonium sulfate and hydroxylamine hydrochloride to a buffer solution and stirring until dissolved; adding Brij 78 and adjusting the pH; adding TCEP and ProClin 300; mixing well and filtering to obtain the antigen retrieval composition after cell fixation.

4. A cell fixation antigen retrieval product, characterized in that, The cell fixation antigen repair product comprises the cell fixation antigen repair composition according to any one of claims 1-2.

5. The cell fixation antigen repair product according to claim 4, characterized in that, The cell fixation antigen retrieval products include reagents and / or kits.

6. A method for antigen retrieval after cell fixation, characterized in that, The method includes using the cell fixation antigen repair composition according to any one of claims 1-2 or the cell fixation antigen repair product according to any one of claims 4-5.

7. The application of the cell fixation antigen repair composition according to any one of claims 1-2 or the cell fixation antigen repair product according to any one of claims 4-5, characterized in that, The applications include any one or more of the following: (1) Application in cell fixation repair; (2) Application in the preparation of cell smears overexpressing antigens; (3) Application in the preparation of antibody detection products.

8. The application according to claim 7, characterized in that, The antigen is a myasthenia gravis-related antigen, which includes any one or more of the following: AChR antigen, Titin antigen, RyR1 antigen, Musk antigen, and LRP4 antigen.

9. The application according to claim 7, characterized in that, The antibody detection product mentioned is a myasthenia gravis antibody profile detection kit.

10. The application according to claim 9, characterized in that, The aforementioned myasthenia gravis antibody spectrum detection kit contains a biochip, which includes a chip body; the biochip body is provided with a reaction groove, a sample inlet, a sample aspiration groove, and a labeling area; the reaction groove contains cell smears expressing different myasthenia gravis-related antigen proteins.

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