A cell fixing solution for a myasthenia gravis antibody spectrum (IgG) detection kit (cellular immunofluorescence method), and products and applications thereof

Abstract

The application provides a cell fixing solution for a myasthenia gravis antibody spectrum (IgG) detection kit (cellular immunofluorescence method) and a product and application thereof, and relates to the technical field of in-vitro detection. The cell fixing solution provided by the application is composed of polyoxymethylene, formaldehyde, acetic acid, raffinose and a buffer solution, can effectively fix the cell morphology, maximally retains the natural spatial conformation and antigen epitope integrity of myasthenia gravis related antigens AChR, Titin, RyR1, MuSK and LRP4, reduces the problems of antigen denaturation, epitope shielding and protein loss in the fixing process, can efficiently and accurately realize the synchronous joint detection of various specific antibodies, and the myasthenia gravis antibody spectrum (IgG) detection kit (cellular immunofluorescence method) prepared by the application has stable detection results, and has important clinical application value and industrial popularization prospect.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, and relates to cell fixation methods, specifically to a cell fixation solution for a myasthenia gravis antibody spectrum (IgG) detection kit (cell immunofluorescence assay), its products, and applications. Background Technology

[0002] In the field of in vitro detection, cell slides are an indispensable experimental technology carrier. The core of their function is to uniformly seed and adhere cultured cells onto the surface of a specially designed glass or plastic sheet (i.e., a cell slide), allowing the cells to maintain their natural growth morphology and physiological state, facilitating subsequent cellular observation and analysis. Cell slides effectively eliminate interference from the complex in vivo environment, enabling researchers to conveniently and precisely intervene in cell experiments. They are widely used in various experimental scenarios such as immunofluorescence (IF), in situ hybridization (FISH), apoptosis detection (TUNEL), and immunohistochemistry. They are key tools for revealing cell morphology, motility, intercellular interactions, and the expression, distribution, and function of biomolecules, providing an important experimental foundation for disease mechanism research, drug screening, and clinical pathological diagnosis. Commonly used cell slides include ordinary coverslips and specialized slides treated with TC (tissue culture). Specialized slides, through surface modification to enhance cell adhesion, are suitable for more precise cellular research.

[0003] The preparation of cell slides involves several key steps, including slide pretreatment, cell seeding, culture, fixation, permeabilization, staining, and mounting. Cell fixation is the core step in protecting cell morphology and internal structure, ensuring the reliability of subsequent detections. Its effectiveness directly determines the detection quality and the accuracy of experimental data. The core purpose of cell fixation is to "freeze" the physiological state of cells through physical or chemical methods, preventing the degradation of intracellular biomolecules and the deformation of cell morphology, while preserving the activity and localization of target substances such as antigens and nucleic acids, providing a stable sample basis for subsequent staining, hybridization, and other detection steps.

[0004] Currently, the cell fixation methods commonly used in cell slide preparation are mainly divided into two categories: cross-linking agent fixation and organic solvent fixation. The mechanisms of action, applicable scenarios, and effects of different fixation methods vary significantly, and their impact on the detection results of cell slides also differs. Among them, cross-linking agent fixation commonly uses paraformaldehyde (PFA) and 4% neutral formaldehyde (10% neutral formalin) as reagents. Its mechanism of action is to cross-link intracellular protein molecules to form a stable antigen network, which can better preserve cell morphology and antigen positions. It is suitable for most experimental scenarios such as immunoelectron microscopy, immunohistochemistry, and immunofluorescence, and its damage to fluorescent proteins is relatively mild. However, it has problems such as poor penetration, potential masking of some cell epitopes, and increased cell autofluorescence, which in turn affect the subsequent antibody binding efficiency and the clarity of the fluorescence detection signal. Organic solvent fixation methods commonly use cold acetone, 75%-95% ethanol, and methanol. Their mechanism of action is to precipitate and fix intracellular proteins onto the cell structure through dehydration, while simultaneously achieving cell membrane permeability. Among these methods, methanol fixation is more sensitive to phosphorylated epitopes and is suitable for the detection of phosphorylated proteins. However, these methods can destroy some surface antigens and fluorescent proteins (such as GFP), alter cell scattering characteristics, increase detection background, and require strict temperature control during operation, which can easily lead to cell morphology deformation or membrane protein loss. Therefore, they are not suitable for experimental scenarios that only detect cell surface markers.

[0005] Chinese patent CN107299111A discloses the preparation of a cell slide assembly for a CBA kit for detecting MOG-IgG, wherein the cell fixative used in preparing the cell slide is 4% w / v paraformaldehyde; Chinese patent CN102043047A discloses a rapid and economical immunofluorescence method based on cell slides, wherein the cell fixative used in preparing the cell slide is 3% v / v formaldehyde solution. The core problem with existing cell fixation methods lies in poor cell fixation, leading to poor detection results from cell slides and failing to meet the precision requirements of advanced experiments and clinical diagnosis. Therefore, optimizing the cell fixation method in the cell slide preparation process to solve the problems of poor fixation, low detection accuracy, and low repeatability in existing technologies has become an urgent technical problem to be solved. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a cell fixation solution for a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay), as well as the product and its application.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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, and 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 positive 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 the accuracy and excellent specificity of the CBA (cell immunofluorescence assay).

[0015] 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 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.

[0016] 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.

[0017] 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).

[0018] 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.

[0019] The technical solution of this invention is as follows: On one hand, the present invention provides a cell fixation solution for a myasthenia gravis antibody spectrum detection kit, wherein the cell fixation solution is composed of paraformaldehyde, formaldehyde, acetic acid, raffinose and buffer solution.

[0020] Specifically, the cell fixative contains 2.5%-3.5% w / v paraformaldehyde. More specifically, the cell fixative contains 2.5%-2.6%, 2.6%-2.7%, 2.7%-2.8%, 2.8%-2.9%, 2.9%-3.0%, 3.0%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, or 3.4%-3.5% w / v paraformaldehyde.

[0021] Preferably, the cell fixative contains 3.0%-3.1%, 3.1%-3.2%, 3.2%-3.3%, 3.3%-3.4%, or 3.4%-3.5% w / v paraformaldehyde.

[0022] More preferably, the cell fixative contains 3.0% w / v paraformaldehyde.

[0023] Specifically, the cell fixative contains 0.4%-0.8% v / v formaldehyde.

[0024] More specifically, the cell fixative contains 0.4%-0.5%, 0.5%-0.6%, 0.6%-0.7%, or 0.7%-0.8% v / v formaldehyde.

[0025] Preferably, the cell fixative contains 0.4%-0.5% or 0.5%-0.6% v / v formaldehyde.

[0026] More preferably, the cell fixative contains 0.6% v / v formaldehyde.

[0027] Specifically, the cell fixative contains 0.3%-0.5% w / v raffinose.

[0028] More specifically, the cell fixative contains 0.3%-0.4% or 0.4%-0.5% w / v raffinose.

[0029] Preferably, the cell fixative contains 0.3%-0.4% w / v raffinose.

[0030] More preferably, the cell fixative contains 0.4% w / v raffinose.

[0031] Specifically, the cell fixative contains 0.10%-0.16% v / v acetic acid.

[0032] More specifically, the cell fixative contains 0.10%-0.11%, 0.11%-0.12%, 0.12%-0.13%, 0.13%-0.14%, 0.14%-0.15%, or 0.15%-0.16% v / v acetic acid.

[0033] Preferably, the cell fixative contains 0.13%-0.14%, 0.14%-0.15%, or 0.15%-0.16% v / v acetic acid.

[0034] More preferably, the cell fixative contains 0.13% v / v acetic acid.

[0035] Specifically, the buffer solution includes any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer.

[0036] In some specific embodiments of the present invention, the buffer solution is a 1× phosphate buffer with a pH of 7.2-7.4.

[0037] In some specific embodiments of the present invention, the cell fixative contains 3% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.4% w / v raffinose, 0.13% v / v acetic acid and the remainder PBS buffer.

[0038] In some specific embodiments of the present invention, the cell fixative contains 2.5% w / v paraformaldehyde, 0.8% v / v formaldehyde, 0.5% w / v raffinose, 0.10% v / v acetic acid and the remainder PBS buffer.

[0039] In some specific embodiments of the present invention, the cell fixative contains 3.5% w / v paraformaldehyde, 0.4% v / v formaldehyde, 0.3% w / v raffinose, 0.16% v / v acetic acid and the remainder PBS buffer.

[0040] In another aspect, the present invention provides a method for preparing the cell fixative as described in any of the above claims. The preparation method includes: taking an appropriate amount of buffer solution, preheating it, adding paraformaldehyde, raffinose, formaldehyde aqueous solution, and acetic acid, stirring evenly, adding buffer solution to make up to the target volume, mixing evenly, filtering, and obtaining the cell fixative.

[0041] In another aspect, the present invention provides the application of the cell fixative described in any of the above claims, the application including: (1) application in cell fixation; (2) application in the preparation of cell slides.

[0042] Specifically, the cell smears are used to prepare a myasthenia gravis antibody profile detection kit.

[0043] Preferably, the myasthenia gravis antibody includes any one or more of the following: AChR antibody, Titin antibody, RyR1 antibody, Musk antibody, and LRP4 antibody.

[0044] Preferably, the myasthenia gravis antibody spectrum detection kit contains a biochip.

[0045] More preferably, the biochip includes a chip body; the biochip body is provided with a reaction groove, a sample inlet, a sample suction port and a labeling area; the reaction groove contains cell smears expressing different myasthenia gravis-related antigen proteins.

[0046] More preferably, the myasthenia gravis-related antigens include any one or more of the following: AChR antigen, Titin antigen, RyR1 antigen, Musk antigen, and LRP4 antigen.

[0047] In another aspect, the present invention provides a method for preparing cell slides, the method comprising using the cell fixative described in any of the preceding claims.

[0048] Specifically, the method includes the following steps: S1. Inoculate cells into a culture plate containing cell slides, transfect with the target plasmid, and incubate. S2. After cell incubation, discard the culture medium, rinse, add cell fixation solution, and fix. S3. After fixation, discard the fixative, rinse, seal, rinse again, blot dry, and dry to obtain cell slides.

[0049] Preferably, the cells described in step S1 comprise mammalian cells that overexpress myasthenia gravis-associated antigens.

[0050] More preferably, the mammalian cells include any one or more of HEK293 cells, CHO cells, Vero cells, HeLa cells, COS-7 cells, and BHK-21 cells.

[0051] In certain specific embodiments of the present invention, HEK293 cells are selected as mammalian cells and overexpress any one or more of the following myasthenia gravis-related antigens labeled with mCherry: acetylcholine receptor (AChR), titin, ryneline receptor 1 (RyR1), muscle-specific receptor tyrosine kinase (MuSK), and low-density lipoprotein receptor-associated protein 4 (LRP4). mCherry is used as a positive reporter tag for transfection to visually determine cell transfection efficiency.

[0052] Preferably, the fixation in step S3 includes fixing at room temperature for 20-40 minutes.

[0053] The beneficial effects of this invention are as follows: The cell fixative disclosed in this invention for a myasthenia gravis antibody profile (IgG) detection kit (cell immunofluorescence assay) utilizes a synergistic cross-linking system formed by combining paraformaldehyde and formaldehyde, and is scientifically formulated with specific amounts of raffinose and acetic acid. Compared to single-component fixatives or improperly formulated fixatives in the prior art, this invention offers the following significant advantages: (1) Excellent antigen retention effect: While effectively fixing cell morphology and preventing cell shrinkage and shedding, it can retain the natural spatial conformation and epitope integrity of myasthenia gravis-related antigens such as AChR, Titin, RyR1, Musk, and LRP4 to the greatest extent, effectively reducing antigen denaturation, epitope masking and protein loss during fixation, and significantly improving the binding efficiency of antigens with specific IgG antibodies in patient serum, providing core guarantee for the accuracy of subsequent immunofluorescence detection.

[0054] (2) Simple operation and good repeatability: The cell fixation solution of the present invention has mild fixation conditions, without the need for strict low temperature control or complicated operation procedures. Fixation can be completed at room temperature (20-25℃) and the fixation time is only 20-40 minutes. It is convenient to operate and has a short time consumption, which can effectively reduce the difficulty of experimental operation, improve the repeatability of the detection process, and meet the needs of large-scale clinical sample detection and industrial production.

[0055] (3) Strong adaptability and excellent detection performance: This cell fixative can be widely used in the preparation of mammalian cell slides overexpressing myasthenia gravis-related antigens such as HEK293 and CHO. It is also compatible with the fixation process of biochip antigen slides in myasthenia gravis antibody profile detection kits, enabling simultaneous detection of AChR, Titin, RyR1, Musk, and LRP4-related antibodies. Experimental verification shows that cell slides prepared using the fixative of this invention have a positive cell rate of over 78%, and the fluorescence density value is significantly higher than that of existing fixatives, effectively solving the technical problems of poor fixation effect and insufficient detection accuracy in existing technologies.

[0056] (4) High industrialization and clinical application value: The cell fixation solution of this invention has a simple formula, easy-to-obtain raw materials, and simple preparation process, and can be produced on a large scale; the cell smears and matching kits prepared by it have efficient detection process and accurate results, which can meet the clinical needs of myasthenia gravis classification diagnosis, differential diagnosis and disease assessment, while reducing detection costs, and have broad industrial promotion prospects and important clinical application value. Attached Figure Description

[0057] Figure 1 The results are from the assay of the antigenicity retention capacity of the target antigen.

[0058] Figure 2This is a schematic diagram showing the presence of anti-Titin antibodies in the sample.

[0059] Figure 3 This is a schematic diagram showing that the sample does not contain anti-Titin antibodies. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Example 1: A cell fixative A cell fixative comprises paraformaldehyde, formaldehyde, raffinose, acetic acid, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.4% w / v raffinose, 0.13% v / v acetic acid, and the balance being PBS buffer.

[0070] The cell fixative is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde, raffinose, formaldehyde aqueous solution, and acetic acid, stir well, then add PBS buffer to bring the volume to the target level and mix thoroughly. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixative.

[0071] Example 2 A cell fixative A cell fixative consists of paraformaldehyde, formaldehyde, raffinose, acetic acid, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 2.5% w / v paraformaldehyde, 0.8% v / v formaldehyde aqueous solution, 0.5% w / v raffinose, 0.10% v / v acetic acid, and the balance being PBS buffer. The preparation method of the cell fixative is as described in Example 1.

[0072] Example 3 A cell fixative A cell fixative consists of paraformaldehyde, formaldehyde, raffinose, acetic acid, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3.5% w / v paraformaldehyde, 0.4% v / v formaldehyde aqueous solution, 0.3% w / v raffinose, 0.16% v / v acetic acid, and the balance being PBS buffer. The preparation method of the cell fixative is as described in Example 1.

[0073] Comparative Example 1: A cell fixative A cell fixative comprises paraformaldehyde, formaldehyde, acetic acid, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3.4% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.13% v / v acetic acid, and the balance PBS buffer.

[0074] The cell fixative is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde, formaldehyde aqueous solution, and acetic acid, stir until homogeneous, then add PBS buffer to bring the volume to the target level and mix well. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixative.

[0075] Comparative Example 2: A cell fixative A cell fixative consists of paraformaldehyde, formaldehyde, raffinose, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3% w / v paraformaldehyde, 0.73% w / v formaldehyde, 0.4% w / v raffinose, and the balance PBS buffer.

[0076] The cell fixative is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde, raffinose, and formaldehyde aqueous solution, stir well, then add PBS buffer to bring the volume to the target level and mix thoroughly. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixative.

[0077] Comparative Example 3: A cell fixative A cell fixative consists of paraformaldehyde, formaldehyde, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3.4% w / v paraformaldehyde, 0.73% v / v formaldehyde, and the balance PBS buffer.

[0078] The cell fixation solution is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde and formaldehyde aqueous solution, stir until homogeneous, then add PBS buffer to bring the volume to the target level and mix well. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixation solution.

[0079] Comparative Example 4: A cell fixative A cell fixative comprises paraformaldehyde, formaldehyde, trehalose, acetic acid, and 1×PBS buffer at pH 7.2-7.4. The cell fixative contains 3% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.4% w / v trehalose, 0.13% v / v acetic acid, and the balance being PBS buffer.

[0080] The cell fixative is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde, trehalose, formaldehyde aqueous solution, and acetic acid, stir until homogeneous, then add PBS buffer to bring the volume to the target level and mix well. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixative.

[0081] Comparative Example 5: A cell fixative A cell fixative is a 1×PBS buffer containing 4% w / v paraformaldehyde at pH 7.2-7.4.

[0082] The cell fixative is prepared as follows: Place an appropriate amount of PBS buffer in a clean container, preheat to 50-60°C, add paraformaldehyde, stir until homogeneous, then add PBS buffer to bring the volume to the target level and mix well. Filter the solution through a 0.22 μm sterile membrane to obtain the cell fixative.

[0083] Experimental Example 1: Cell fixative used in the preparation of cell slides 1. Cell slide fixation After completing the incubation according to the method described in Basic Experiment Example 2, discard the culture medium from the mCherry-Titin cell smears, rinse the cell smears three times with sterile PBS buffer, 5 min each time; add 1 mL of cell fixation solution to each well (add cell fixation solutions from Examples 1-3 or Comparative Examples 1-5 respectively), and fix at room temperature for 30 min.

[0084] 2. Post-processing of cell slides Discard the fixative and rinse the slides three times with sterile PBS buffer for 5 min each time; 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 forceps, place them on sterile filter paper, blot off the moisture at the edges, and dry them to obtain the cell slides for Titin antibody detection in Examples 1-3 or Comparative Examples 1-5, respectively.

[0085] 3. Verification of the ability to retain the antigenicity of the target antigen (1) Preparation The cell slides prepared above for Titin 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.

[0086] (2) Sample incubation Add 200 μL of specific positive samples against Titin 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 antibodies in the samples fully bind to the antigen on the antigen slide.

[0087] (3) Cleaning Remove the cell slide and gently rinse it three times with sterile PBS buffer, 5 minutes each time.

[0088] (4) Secondary antibody incubation Add 200 μL of fluorescently labeled secondary antibody to the cleaned cell slide, ensuring the secondary antibody evenly covers the cell slide surface. Incubate 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.

[0089] (5) Secondary cleaning After the secondary antibody incubation is complete, remove the fluorescently labeled secondary antibody and gently rinse the slide three times with sterile PBS buffer, 5 minutes each time.

[0090] (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-Titin 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-Titin antibody.

[0091] (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.

[0092] 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.

[0093] The measurement results are shown in Table 1 and Figure 1 As shown: Table 1

[0094] The cell fixation solutions of Examples 1-3 of this invention can gently fix cells, maintaining the overlap of green and red fluorescence at over 78%, and achieving an average optical density of 0.820-0.851 for green fluorescence (high binding strength). The positive cell rate is significantly higher than that of the other pairs, maximizing the preservation of the natural conformation and epitope integrity of the Titin target antigen. Its antigenicity retention ability is significantly better than that of the other pairs, meeting the accuracy requirements for anti-Titin antibody detection in myasthenia gravis and providing a reliable guarantee for the efficient detection of subsequent myasthenia gravis antibody profiles.

[0095] Experiment Example 2: Assembly and Use of a Biochip for Detecting Myasthenia Gravis Antibody Profile (IgG) 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 cell fixation solution (the cell fixation solution from Example 1) to each well, and fix at room temperature for 30 min.

[0096] Discard the fixative and rinse the cell slides three times with sterile PBS buffer for 5 min each time. 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 fixed cell slides.

[0097] 2. 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 slides. Fixed cell slides (mCherry-AChR, mCherry-Titin, mCherry-RyR1, mCherry-MuSK, or mCherry-LRP4) and blank control antigen slides are arranged sequentially from left to right within the reaction chamber, enabling parallel detection of multiple antibodies.

[0098] 3. 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 packed into aluminum foil bags and sealed. They are stored in a dark and moisture-proof environment for easy storage, transportation and clinical use.

[0099] 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).

[0100] 4. Instructions for use of biochips It is recommended to conduct the experiment at room temperature to ensure the accuracy of the results.

[0101] (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.

[0102] (2) Sample incubation: Add 70 μL of the sample to be tested to each reaction zone and incubate at room temperature for 1 hour.

[0103] (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.

[0104] (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).

[0105] (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.

[0106] (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.

[0107] (7) Interpretation criteria Taking Titin 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 fluorescence signal overlaps with the location emitting the red fluorescence signal, and the characteristics are basically the same, then it can be determined that the sample contains anti-Titin 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-Titin antibody. Figure 2 The green and red fluorescent signals overlap in position and have essentially the same characteristics, indicating that the sample contains anti-Titin 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-Titin antibodies.

[0108] Similarly, in addition to Titin, which is transfected with fluorescent proteins, the reagent also includes one or more of AChR, RyR1, Musk, and LRP4, which are transfected with fluorescent proteins, allowing for the simultaneous detection and interpretation of multiple antibodies in the sample. If any one or more of Titin, AChR, RyR1, Musk, and LRP4 are found to be positive, the sample is diagnosed as a myasthenia gravis antibody spectrum disease sample.

[0109] 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 fixative for a myasthenia gravis antibody profile detection kit, characterized in that, The cell fixative is composed of paraformaldehyde, formaldehyde, acetic acid, raffinose, and buffer solution; the cell fixative contains 2.5%-3.5% w / v paraformaldehyde, 0.4%-0.8% v / v formaldehyde, 0.3%-0.5% w / v raffinose, and 0.10%-0.16% v / v acetic acid.

2. The cell fixative according to claim 1, characterized in that, The cell fixative contains 3.0%-3.5% w / v paraformaldehyde, 0.4%-0.6% v / v formaldehyde, 0.3%-0.4% w / v raffinose and 0.13%-0.16% v / v acetic acid.

3. The cell fixative according to claim 1, characterized in that, The buffer solution includes any one or more of phosphate buffer, Tris-HCl buffer, and HEPES buffer.

4. The method for preparing the cell fixative according to any one of claims 1-3, characterized in that, The preparation method includes: taking an appropriate amount of buffer solution, preheating it, adding paraformaldehyde, raffinose, formaldehyde aqueous solution, and acetic acid, stirring evenly, adding buffer solution to make up to the target volume, mixing well, filtering, and obtaining cell fixative.

5. The application of the cell fixative according to any one of claims 1-3, characterized in that, The applications include: (1) application in cell fixation; and (2) application in the preparation of cell slides.

6. The application according to claim 5, characterized in that, The cell smears described herein are used to prepare an antibody profile detection kit for myasthenia gravis.

7. The application according to claim 6, characterized in that, The myasthenia gravis antibodies include any one or more of the following: AChR antibody, Titin antibody, RyR1 antibody, Musk antibody, and LRP4 antibody.

8. The application according to claim 6, 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.

9. The application according to claim 8, characterized in that, The myasthenia gravis-related antigens include any one or more of the following: AChR antigen, Titin antigen, RyR1 antigen, MuSK antigen, and LRP4 antigen.

10. A method for preparing cell-spreading slides, characterized in that, The method includes using the cell fixative according to any one of claims 1-3.

11. The method according to claim 10, characterized in that, The method includes the following steps: S1. Inoculate cells into a culture plate containing cell slides, transfect with the target plasmid, and incubate. S2. After cell incubation, discard the culture medium, rinse, add cell fixation solution, and fix. S3. After fixation, discard the fixative, rinse, seal, rinse again, blot dry, and dry to obtain cell slides.

12. The method according to claim 11, characterized in that, The cells described in step S1 include mammalian cells that overexpress myasthenia gravis-associated antigens.

13. The method according to claim 12, characterized in that, The mammalian cells mentioned include any one or more of the following: HEK293 cells, CHO cells, Vero cells, HeLa cells, COS-7 cells, and BHK-21 cells.

14. The method according to claim 11, characterized in that, The fixation mentioned in step S2 includes fixing at room temperature for 20-40 minutes.

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