A cell climbing sheet processing reagent for a lupus nephritis / paranephritis antibody spectrum detection kit (cellular immunofluorescence method), a method thereof and application thereof

Abstract

The application provides a cell climbing sheet processing reagent for a Ranvier node / paranode antibody spectrum detection kit (cellular immunofluorescence method), a method and application thereof, and relates to the technical field of in-vitro detection. The cell climbing sheet processing reagent comprises a cell fixing solution with a specific composition and an antigen repair composition after cell fixing, wherein the cell fixing solution is composed of paraformaldehyde, formaldehyde, acetic acid, raffinose and a buffer solution, and the antigen repair composition after cell fixing is composed of ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin 300 and a buffer solution. Through the synergistic effect of the two, the natural conformation of the target antigen is maximally retained, and non-specific interference is reduced, so that precise and efficient detection of the Ranvier node / paranode antibody spectrum NF155, NF186, CNTN1, CASPR1 and MAG can be realized, the clinical diagnosis demand is met, and reliable technical support is provided for precise typing of autoimmune Ranvier node disease.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, specifically relating to a cell smear processing reagent, method, and application for a Langefier nodal / paranodal antibody profile detection kit (cell immunofluorescence assay). Background Technology

[0002] Autoimmune Nodo-paranodopathies (AN) are a type of autoimmune peripheral neuropathy whose core pathogenic mechanism involves autoantibodies targeting key structural proteins in the nodes of Ranvier and paranodelar regions of peripheral nerves. They are an important subtype of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and can also be seen in acute peripheral neuropathy spectrum diseases such as Guillain-Barré syndrome (GBS). Nodes of Ranvier are tiny functional regions with interrupted myelin sheaths in myelinated nerve fibers. They consist of a complex of nodes, paranodes, and perinodes, forming a precise molecular complex that forms the structural basis for the skip-transmission of nerve impulses. When autoantibodies attack proteins in this region, they disrupt the stability of myelin-axon connections, dissociate ion channel clusters, and cause nerve conduction block. Clinically, this manifests primarily as progressive motor and sensory disturbances, ataxia, tremor, and autonomic dysfunction. In severe cases, respiratory muscles may also be affected. Furthermore, patients with different antibody subtypes show significant differences in their response to immunotherapy (such as intravenous immunoglobulin IVIG, glucocorticoids, and rituximab). Accurate subtyping and diagnosis are crucial for individualized clinical treatment.

[0003] Currently, clinical detection methods for AN are centered on serum / cerebrospinal fluid antibody detection, supplemented by neurophysiological, cerebrospinal fluid biochemistry and imaging examinations. Existing mainstream detection technologies have obvious limitations: (1) Western blotting (WB): Based on the qualitative / semi-quantitative detection of antigen-antibody specific binding, it can screen multiple target antigens at the same time. However, the antigen is a denatured protein and cannot simulate the natural conformation, which easily leads to false positives / false negatives. Moreover, it cannot achieve antigen localization, and the detection sensitivity and specificity are insufficient, making it difficult to distinguish low-affinity non-pathogenic antibodies. (2) Enzyme-linked immunosorbent assay (ELISA): It is simple to operate and can be quantified, but it mostly uses recombinant single antigen coating, which cannot restore the natural spatial structure of the RANVIE junction protein complex. It has a high false negative rate for conformation epitope-dependent antibodies (such as NF155 and CNTN1 antibodies), and the single antigen detection mode cannot meet the needs of multi-antibody joint detection. (3) Indirect Immunofluorescence (IIF): Using tissue sections (such as rat sciatic nerve and cerebellum sections) as antigen matrix, it can detect natural conformational antibodies with high specificity. However, the antigen components of tissue sections are complex, which can easily lead to non-specific fluorescence and cannot accurately distinguish different target antigens, making it difficult to achieve simultaneous multi-antibody typing detection. (4) Cell-based Assay (CBA): By expressing a single target antigen on the cell surface through transfection technology, it can detect conformational epitope antibodies. It is one of the "gold standard" methods for AN antibody detection. However, existing technologies are mostly single-antigen single-well detection, which is cumbersome, has low throughput, and high cost, and cannot meet the high-efficiency needs of clinical multi-antibody joint detection.

[0004] Cell slides are monolayer slides formed by seeding adherent cells onto glass slides treated with poly-L-lysine and gelatin, and culturing them until the cells adhere and fuse. They are commonly used carriers in cell biology and immunology assays. Their core advantages are: precise expression of single or multiple target antigens on the cell surface through gene transfection technology, maximally restoring the native spatial conformation and post-translational modifications of the antigens, and preserving the integrity of conformational epitopes; clear cell morphology and a clean background, facilitating observation of specific immunofluorescence signals under a fluorescence microscope; and the ability to customize antigen combinations according to needs, providing a technical basis for multi-antibody assays.

[0005] The existing Langfi nodal / paranodal antibody detection technology and cell smear application have the following core defects, which cannot meet the clinical needs for accurate and efficient detection: (1) Single detection mode, unable to detect multiple antibodies: The mainstream detection methods (WB, ELISA, single antigen CBA) are all single antigen detection modes. When clinical patients need to screen 10 targets such as NF155, NF186, CNTN1, etc. at the same time, the operation is cumbersome, time-consuming, requires a large amount of samples, and has high detection costs, making it difficult to achieve rapid subtyping diagnosis. (2) Insufficient natural conformation reduction, limiting detection accuracy: Traditional WB and ELISA use denatured or single recombinant antigens, which cannot reduce the natural spatial structure of the Langfi nodal protein complex, resulting in a high rate of missed detection for conformation epitope-dependent antibodies such as NF155 and CNTN1; Although tissue section IIF retains the natural conformation, the antigen components are complex and non-specific interference is strong, making it impossible to accurately distinguish multiple targets. (3) Cell crawling technology does not achieve multi-antigen co-expression and joint detection: The existing cell crawling-CBA can only express a single target antigen and cannot simultaneously express multiple targets of Ranvier's knot / paranodes on the same cell crawling sheet, resulting in low detection throughput and efficiency. (4) Complex operation process and poor clinical applicability: Single antigen detection requires multiple transfections and multiple incubations, which requires high technical skills from operators and has a long detection cycle, making it difficult to meet the needs of large-scale clinical sample screening and rapid diagnosis.

[0006] In summary, cell-smear-CBA technology, with its advantages of natural conformation antigen expression, high specificity, and visual detection, has become the preferred technology for antibody detection in autoimmune diseases. However, its current application in the field of AN is still limited to single antigen detection, failing to achieve simultaneous detection of multiple antibodies at or near the nodal junction of Ranvier's node. Therefore, existing technologies lack a simple, accurate, and simultaneous detection method for multiple antibodies at or near the nodal junction of Ranvier's node, which cannot meet the clinical needs of precise AN subtyping and personalized treatment. There is an urgent need to develop a multi-antigen co-expression detection technology based on cell-smear to achieve simultaneous, efficient, and accurate detection of multiple target antibodies. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a cell smear processing reagent, method, and application for the Langfi's nodal / paranodal antibody profiling detection kit (cell immunofluorescence assay).

[0008] the term: The term "Node of Ranvier / Paranodal Antibody Profile" as used in this invention refers to a group of autoantibodies targeting key structural proteins of the nodes of Ranvier and their paranodal regions in myelinated nerve fibers. This antibody profile includes autoantibodies against fasciculation protein 155 (NF155), fasciculation protein 186 (NF186), contactin 1 (CNTN1), contactin-associated protein 1 (CASPR1), and myelin-associated glycoprotein (MAG).

[0009] The term "NF155" used in this invention refers to Neurofascin 155, a cell adhesion molecule mainly expressed in the paranodal region of myelinated nerve fibers, mediating septum-like junction formation, and belonging to the immunoglobulin superfamily. NF155 plays a crucial role in maintaining the structure of the paranodal region and the segmental distribution of sodium-potassium channels, and its autoantibodies are one of the core detection targets in Ranvier's nodal / paranodal antibody profile.

[0010] The term "NF186" used in this invention refers to Neurofascin 186, a cell adhesion molecule mainly expressed at the node of Ranvier in myelinated nerve fibers, involved in sodium channel clustering and anchoring, and also a member of the immunoglobulin superfamily. NF186 interacts with neuronal sodium channels, which is essential for the stability of the node structure and the hopping conduction of action potentials. Its autoantibodies are another core detection target in the node / paranodecal antibody profile.

[0011] The term "CNTN1" used in this invention refers to Contactin-1, a glycosylphosphatidylinositol-anchored cell adhesion molecule that is enriched and expressed in the paranodelar region of myelinated nerve fibers. It forms a complex with CASPR1 and NF155, jointly maintaining the integrity of septal junctions and axon-glial cell signal transduction. CNTN1 autoantibody detection is of great significance for the diagnosis of paranodelar diseases.

[0012] The term "CASPR1" used in this invention refers to Contactin-associated protein 1, a transmembrane scaffold protein that nonvalently binds to CNTN1 in the paranodelar region and stabilizes the structure of the septum-like junction complex by interacting with cytoskeletal proteins. CASPR1 autoantibodies are one of the key detection targets in Langefier's nodal / paranodelar antibody profile.

[0013] The term "MAG" used in this invention refers to myelin-associated glycoprotein, a glycoprotein expressed on the innermost layer of the myelin sheath and the surface of the axonal membrane, which participates in mediating the myelin-axon signaling pathway and maintaining myelin stability. Anti-MAG antibodies are closely related to peripheral neuropathy such as distal acquired demyelinating symmetrical neuropathy (DADS) and are an important supplementary detection target for Ranvier's node / paranodecal antibody profile.

[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-NF155 plasmid, mCherry-NF186 plasmid, mCherry-CNTN1 plasmid, mCherry-CASPR1 plasmid, and mCherry-MAG plasmid of this invention, mCherry is independently expressed along with the target antigens NF155, NF186, CNTN1, CASPR1, and MAG. 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 and CF488A-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. The above design is key to ensuring accurate interpretation and excellent specificity of the CBA method (cell immunofluorescence assay).

[0015] The mCherry-NF155, mCherry-NF186, mCherry-CNTN1, mCherry-CASPR1, or mCherry-MAG plasmids used in this invention are not particularly limited in origin. 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 generate 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 Ranvier's knot / paranodeficiency-associated 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 antibodies used in the embodiments of this invention were purchased from ThermoFisher Scientific (catalog numbers A11013, A21215) and Biotium (catalog number BNC881345); 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 inoculating 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 blocking. It can be used for immunofluorescence detection of Langefier knot / para-knot antibody profiles.

[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 in the Langfi knot / next-knot antibody spectrum. It has the characteristics of low sample volume, simple operation, and high detection efficiency.

[0019] The technical solution of this invention is as follows: On the one hand, the present invention provides a cell smear treatment reagent, which is composed of a cell fixation solution and a cell fixation antigen retrieval composition; The cell fixative is composed of paraformaldehyde, formaldehyde, acetic acid, raffinose, and buffer solution. The cell fixation antigen retrieval composition consists of ammonium sulfate, hydroxylamine hydrochloride, Brij 78, TCEP, ProClin300, 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] Preferably, the method for preparing the cell fixative includes: taking an appropriate amount of buffer solution, preheating it, adding paraformaldehyde, raffinose, formaldehyde, and acetic acid, stirring evenly, adding buffer solution to make up to the target volume, mixing well, filtering, and obtaining the cell fixative.

[0038] Specifically, the cell fixation antigen retrieval composition contains 70-130 mM ammonium sulfate.

[0039] 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.

[0040] 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.

[0041] More preferably, the cell fixation antigen retrieval composition contains 100 mM ammonium sulfate.

[0042] Specifically, the cell fixation antigen repair composition contains 50-100 mM hydroxylamine hydrochloride.

[0043] 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.

[0044] 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.

[0045] More preferably, the cell fixation antigen repair composition contains 75 mM hydroxylamine hydrochloride.

[0046] Specifically, the cell fixation antigen retrieval composition contains 0.25%-0.55% v / v Brij 78.

[0047] 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.

[0048] 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.

[0049] More preferably, the cell fixation antigen retrieval composition contains 0.40% v / v Brij 78.

[0050] Specifically, the cell fixation antigen retrieval composition contains 0.65-1.25 mM TCEP.

[0051] 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.

[0052] 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.

[0053] More preferably, the cell fixation antigen retrieval composition contains 1 mM TCEP.

[0054] Specifically, the cell fixation antigen retrieval composition contains 0.02%-0.05% v / v ProClin 300.

[0055] 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.

[0056] Preferably, the cell fixation antigen retrieval composition contains 0.04%-0.05% v / v ProClin 300.

[0057] More preferably, the cell fixation antigen retrieval composition contains 0.04% v / v ProClin 300.

[0058] Specifically, the buffer solution includes, but is not limited to, one or more of the following: phosphate buffer, Tris-HCl buffer, and HEPES buffer.

[0059] In some specific embodiments of the present invention, the buffer solution is a 1× phosphate buffer with a pH of 7.2-7.4.

[0060] 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.

[0061] Preferably, the Brij 78 is melted by heating in a water bath at 50℃-60℃ and then slowly dripped into the system.

[0062] Preferably, the pH adjustment is to adjust the pH to 7.0-7.5.

[0063] In another aspect, the present invention provides a method for processing cell smears, wherein the method includes using the cell smear processing reagent described in any of the preceding claims.

[0064] Specifically, the cell smear processing 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, add the cell fixation antigen retrieval composition, and incubate. S4. After incubation, discard the antigen retrieval composition after cell fixation, rinse, block, rinse again, blot dry, and dry to obtain the treated cell slide.

[0065] Preferably, the target plasmid in step S1 includes a plasmid that overexpresses Ranvier's knot / paranodecal antibody-associated antigen.

[0066] More preferably, the Langefier nodal / paranodal antibody-related antigen includes any one or more of NF155, NF186, CNTN1, CASPR1, and MAG.

[0067] Preferably, the target plasmid also expresses a fluorescent protein, which serves as a positive transfection reporter tag for visually determining transfection efficiency.

[0068] Preferably, the cells mentioned in step S1 include any one or more of HEK293 cells, CHO cells, Vero cells, HeLa cells, COS-7 cells, and BHK-21 cells.

[0069] Preferably, the fixing conditions described in step S2 include: fixing at room temperature for 20-40 minutes.

[0070] Preferably, the incubation in step S3 includes: incubation at room temperature for 20-40 minutes.

[0071] In another aspect, the present invention provides the application of the cell smear treatment reagent described in any of the above claims in the preparation of the Ranvier knot / para-knot antibody profile detection kit.

[0072] In another aspect, the present invention provides a Langfi's nodal / paranodal antibody profile detection kit, wherein the Langfi's nodal / paranodal antibody profile detection kit contains the cell smear treatment reagent described in any one of the above claims.

[0073] Specifically, the Langfi's nodal / paranodal antibody profiling detection kit also includes any one or more of the following: a biochip, a fluorescently labeled secondary antibody, a working solution, and a mounting medium.

[0074] 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; and a cell slide containing target cells is fixed in the reaction groove.

[0075] The technical effects of this invention are as follows: This invention innovatively constructs a synergistic treatment system consisting of a cell fixation solution in a specific ratio and an antigen repair composition after cell fixation. The two are not simply additive components, but rather produce a synergistic effect of 1+1>2. Compared with the comparison of a single fixation solution, a key component replacement, or a missing repair system, it can significantly improve the positive cell rate of target antigen and the average fluorescence density value, and greatly optimize antigen retention capacity and antibody binding stability. With the matching limited processing parameters, the operation is simple, the conditions are mild, and it has strong versatility.

[0076] This invention establishes a standardized cell smear processing method that can stably prepare cell smears overexpressing multiple target antigens at or near the nodes of Langefeller. Combined with mCherry fluorescent reporter tags, it can intuitively determine cell transfection efficiency and positive expression areas, providing an accurate interpretation benchmark for antibody detection by cell immunofluorescence and enabling simultaneous detection of five core antibodies: NF155, NF186, CNTN1, CASPR1, and MAG.

[0077] This invention can be integrated into a cell smear processing reagent, a reagent kit, and an integrated biochip. The chip integrates functional structures such as a reaction chamber, a sample inlet, and a labeling area, realizing the integration of sample addition, antigen-antibody reaction, and parallel detection of multiple targets. In clinical applications, the detection sensitivity reaches 100.00%, specificity 95.00%, and overall accuracy 96.88%, with extremely low non-specific interference. It can accurately distinguish between patients with autoimmune Ranvier's disease and healthy individuals, providing reliable technical support for precise disease subtyping and individualized clinical immunotherapy, and is suitable for the needs of large-scale clinical sample screening and rapid diagnosis. Attached Figure Description

[0078] Figure 1 To investigate the effect of different treatment methods on the percentage of NF155 antibody-positive cells.

[0079] Figure 2 The cell smear processing system and method of this invention are used for the detection of Langefier knot / para-knot antibody profiles.

[0080] Figure 3 This is a schematic diagram of the sample containing anti-NF155 antibody. The positions of the green fluorescent signal and the red fluorescent signal overlap and their characteristics are basically the same.

[0081] Figure 4 This is a schematic diagram showing that the sample does not contain anti-NF155 antibody. The location of the red fluorescent signal is where there is no fluorescent signal in the green fluorescent signal area. Detailed Implementation

[0082] 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.

[0083] 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.

[0084] Basic Example 1: Preparation of cell crawling slides containing different antigens 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 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.

[0085] (2) Immerse the cleaned glass slide 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 slide and place it in a clean bench to cool to room temperature to obtain sterile glass slide.

[0086] (3) Perform the mounting operation in the clean bench: Place the sterile glass slides stably at 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.

[0087] (4) Add 100 μg / mL polylysine solution to each culture well where a glass slide is placed, ensuring that the solution completely covers the surface of the glass slide. Soak for 2 hours to allow polylysine to be evenly adsorbed on the surface of the glass slide. After soaking, use a sterile pipette to remove excess polylysine solution from the well. Rinse twice with sterile purified water. Place the culture plate in a clean bench, open the culture plate cover, and allow it to air dry naturally until the surface of the glass slide is completely dry. This is the prepared cell spreader, which can be used directly for cell inoculation experiments or sealed and stored at 4°C for later use.

[0088] 2. 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.

[0089] Add 2 mL of single-cell suspension of adjusted concentration to each well of a 6-well culture plate containing pretreated cell slides. Gently shake the culture plate to distribute the cells evenly on the surface of the slides. Place the plate in a 37°C, 5% CO2 incubator and incubate for 24 h for subsequent transfection.

[0090] 3. Recombinant plasmid transfection and incubation Using mCherry-NF155, mCherry-NF186, mCherry-CNTN1, mCherry-CASPR1, and mCherry-MAG plasmids as target plasmids, 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 then added dropwise to 2 mL of the prepared solution (1 × 10⁻⁶). 5 The cells were mixed gently by shaking in a single-cell suspension (number of cells / mL) and then transfected in a 37°C, 5% CO2 incubator for 48 hours to obtain mCherry-NF155 cell slides, mCherry-NF186 cell slides, mCherry-CNTN1 cell slides, mCherry-CASPR1 cell slides, or mCherry-MAG cell slides for subsequent experiments.

[0091] Example 1: A cell smear processing system and method 1. Preparation of cell fixative Cell fixation solution: containing 3% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.4% w / v raffinose, and 0.13% v / v acetic acid, in 1×PBS buffer at pH 7.2-7.4.

[0092] The preparation method is as follows: Place an appropriate amount of 1×PBS buffer in a clean container, preheat to 50-60℃, add paraformaldehyde, raffinose, formaldehyde, and acetic acid, stir evenly, add PBS buffer to make up to the target volume, mix well, and filter sterilize using a 0.22μm sterile filter membrane. The filtrate is the cell fixation solution.

[0093] 2. Preparation of antigen retrieval composition after cell fixation The cell fixation antigen retrieval composition contains 100 mM ammonium sulfate, 75 mM hydroxylamine hydrochloride, 0.4% v / v Brij78, 1 mM TCEP, and 0.04% v / v ProClin 300, in 1×PBS buffer (pH 7.2-7.4). Preparation method: Using 1×PBS buffer as the solvent, add ammonium sulfate and hydroxylamine hydrochloride sequentially, stirring until completely dissolved; melt Brij78 in a 50°C water bath, then slowly add it dropwise to the system while stirring thoroughly; adjust the pH to 7.4; subsequently add TCEP and ProClin 300, mix well, and filter through a 0.22 μm microporous membrane for sterilization. The filtrate is the cell fixation antigen retrieval composition.

[0094] 3. Cell slide processing methods (1) After completing the incubation according to the method described in Basic Experiment Example 1, discard the culture medium from the mCherry-NF155 cell smears, rinse the cell smears three times with sterile PBS buffer for 5 min each time; add 1 mL of cell fixative to each well, fix at room temperature for 30 min, discard the fixative, and rinse the smears three times with sterile PBS buffer for 5 min each time.

[0095] (2) Take 1 mL of the antigen retrieval composition after cell fixation and incubate at room temperature for 30 min to perform antigen retrieval. After retrieval, discard the antigen retrieval composition after cell fixation and wash the cell slides three times with sterile PBS buffer for 5 min each time, gently shaking to ensure that the residual retrieval solution is completely removed.

[0096] (3) 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. Use sterile tweezers to remove the coverslips, place them on sterile filter paper, blot dry the edges, and dry to obtain the treated mCherry-NF155 cell slides.

[0097] Example 2: A cell smear processing system and method 1. Preparation of cell fixative Cell fixation solution: containing 2.5% w / v paraformaldehyde, 0.8% v / v formaldehyde, 0.5% w / v raffinose, and 0.10% v / v acetic acid, in 1×PBS buffer at pH 7.2-7.4. Preparation method is as described in Example 1.

[0098] 2. Preparation of antigen retrieval composition after cell fixation Antigen retrieval composition after cell fixation: Contains 130 mM ammonium sulfate, 50 mM hydroxylamine hydrochloride, 0.55% v / v Brij78, 0.65 mM TCEP, and 0.02% v / v ProClin 300, in 1×PBS buffer at pH 7.2-7.4. Preparation method is as described in Example 1.

[0099] 3. Cell slide processing methods Refer to Example 1.

[0100] Example 3: A cell smear processing system and method 1. Preparation of cell fixative Cell fixation solution: containing 3.5% w / v paraformaldehyde, 0.4% v / v formaldehyde, 0.3% w / v raffinose, and 0.16% v / v acetic acid, in 1×PBS buffer at pH 7.2-7.4. Preparation method is as described in Example 1.

[0101] 2. Preparation of antigen retrieval composition after cell fixation Antigen retrieval composition after cell fixation: Contains 70 mM ammonium sulfate, 100 mM hydroxylamine hydrochloride, 0.25% v / v Brij78, 1.25 mM TCEP, and 0.05% v / v ProClin 300, in 1×PBS buffer at pH 7.2-7.4. Preparation method is as described in Example 1.

[0102] 3. Cell slide processing methods Refer to Example 1.

[0103] Comparative Example 1: A cell smear processing system and method The only difference between Comparative Example 1 and Example 1 is: "1. Preparation of cell fixative".

[0104] 1. Preparation of cell fixative Cell fixation solution: containing 3% w / v paraformaldehyde, 0.6% v / v formaldehyde, 0.4% w / v trehalose, and 0.13% v / v acetic acid, in 1×PBS buffer at pH 7.2-7.4.

[0105] The preparation method is as follows: Take an appropriate amount of PBS buffer and place it in a clean container. Preheat it to 50-60℃, add paraformaldehyde, trehalose, formaldehyde and acetic acid, stir evenly, add PBS buffer to make up to the target volume, mix well, and filter it with a 0.22μm sterile filter membrane for sterilization. The filtrate is the cell fixation solution.

[0106] Comparative Example 2: A cell smear processing system and method The only difference between Comparative Example 2 and Example 1 is: "1. Preparation of cell fixative".

[0107] 1. Preparation of cell fixative Cell fixation solution: containing 4% w / v paraformaldehyde, in 1×PBS buffer at pH 7.2-7.4.

[0108] The preparation method is as follows: Place an appropriate amount of 1×PBS buffer in a clean container, preheat to 50-60℃, add paraformaldehyde, stir well, then add PBS buffer to bring the volume to the target volume and mix well. Use a 0.22μm sterile filter membrane for filtration sterilization.

[0109] Comparative Example 3: A cell smear processing system and method The only difference between Comparative Example 3 and Example 1 is: "2. Preparation of antigen repair composition after cell fixation".

[0110] 2. Preparation of antigen retrieval composition after cell fixation Antigen retrieval composition after cell fixation: Contains 175 mM ammonium sulfate, 0.4% v / v Brij 78, 1 mM TCEP, and 0.04% v / v ProClin 300, in 1×PBS buffer (pH 7.2-7.4). Preparation method: Using 1×PBS buffer as the solvent, add ammonium sulfate sequentially and stir until completely dissolved; melt Brij 78 in a 50°C water bath, then slowly add it dropwise to the system while stirring thoroughly; adjust the pH of the system to 7.4; subsequently add TCEP and ProClin 300, mix well, and filter through a 0.22 μm microporous membrane for sterilization. The filtrate is the antigen retrieval composition after cell fixation.

[0111] Comparative Example 4: A cell smear processing system and method The only difference between Comparative Example 4 and Example 1 is: "2. Preparation of antigen repair composition after cell fixation".

[0112] 2. Preparation of antigen retrieval composition after cell fixation Antigen retrieval composition after cell fixation: Contains 176 mM ammonium sulfate, 0.4% v / v Brij 78, and 0.04% v / v ProClin 300, in 1×PBS buffer (pH 7.2-7.4). Preparation method: Ammonium sulfate is added to 1×PBS buffer and stirred until completely dissolved; Brij 78 is melted by heating in a 50°C water bath and then slowly added dropwise to the system while stirring thoroughly; the pH of the system is adjusted to 7.4; then ProClin 300 is added, mixed well, and filtered through a 0.22 μm microporous membrane for sterilization to obtain the antigen retrieval composition after cell fixation.

[0113] Comparative Example 5: A cell smear processing system and method 1. Preparation of cell fixative Cell fixation solution: containing 4% w / v paraformaldehyde, in 1×PBS buffer at pH 7.2-7.4.

[0114] The preparation method is as follows: Place an appropriate amount of 1×PBS buffer in a clean container, preheat to 50-60℃, add paraformaldehyde, stir well, then add PBS buffer to bring the volume to the target volume and mix well. Use a 0.22μm sterile filter membrane for filtration sterilization.

[0115] 2. Preparation of antigen retrieval composition after cell fixation Antigen retrieval composition after cell fixation: Contains 176 mM ammonium sulfate, 0.4% v / v Brij 78, and 0.04% v / v ProClin 300, in 1×PBS buffer (pH 7.2-7.4). Preparation method: Ammonium sulfate is added to 1×PBS buffer and stirred until completely dissolved; Brij 78 is melted by heating in a 50°C water bath and then slowly added dropwise to the system while stirring thoroughly; the pH of the system is adjusted to 7.4; then ProClin 300 is added, mixed well, and filtered through a 0.22 μm microporous membrane for sterilization to obtain the antigen retrieval composition after cell fixation.

[0116] 3. Cell slide processing methods Refer to Example 1.

[0117] Experiment Example 1: Effect of different treatment methods on the percentage of NF155 antibody-positive cells 1. Preparation The treated mCherry-NF155 cell slides prepared in Examples 1-3 and 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.

[0118] 2. Sample incubation Add 200 μL of a specific positive sample against the NF155 antigen to each cell slide, and set up a negative sample (PBS buffer) as a control. Incubate at room temperature for 1 hour to ensure that the specific antibodies in the samples fully bind to the antigen on the antigen slide.

[0119] 3. Cleaning Remove the cell slide and gently rinse it three times with sterile PBS buffer, 5 minutes each time.

[0120] 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.

[0121] 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.

[0122] 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-NF155 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-NF155 antibody.

[0123] 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.

[0124] 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.

[0125] The measurement results are shown in Table 1 and Figure 1 As shown: Table 1

[0126] As can be clearly seen from the experimental data in Table 1, Examples 1-3, which use the cell fixative with the cell fixation antigen repair composition of the present invention in combination, show significant advantages in both positive cell rate and average optical density of green fluorescence (IOD / Area) compared to replacing fixative components alone, simplifying fixative formulation, deleting / reducing key components of antigen repair composition, and discarding the ratios of each pair of optimized dual-system formulations.

[0127] Comparative Example 5 lacked both the cell fixation medium and the antigen retrieval composition formulated specifically for this invention, resulting in the worst positive cell rate of only 74.18%. Compared to Comparative Example 5, Comparative Example 4, which only replaced the cell fixation medium with the specific formulation of this invention while still using the defective antigen retrieval system, only saw a 9.17% increase in the positive cell rate; Comparative Example 2, which only replaced the cell fixation composition with the specific formulation of this invention while still using a standard single fixation medium, saw a 12.35% increase in the positive cell rate.

[0128] In Example 1 of this invention, a cell fixation solution with a specific composition and an antigen retrieval composition after cell fixation are used together to form a complete treatment system. The positive cell rate is as high as 98.30%, which is a significant increase of 24.12% compared with Comparative Example 5. This increase is much greater than the sum of the increases from using the fixation solution alone and using the antigen retrieval composition alone, with an additional gain of 7.07%. This fully demonstrates that the two are not simply superimposed, but produce a significant synergistic effect, achieving an unexpected technical effect of 1+1>2. Neither can be dispensed with; only by using them together can the natural conformation of the target antigen be preserved to the maximum extent and the positive detection rate be improved.

[0129] Experimental Example 2: The cell smear processing system and method of the present invention were used for the detection of Langfi's knot / para-knot antibody profiles. 1. mCherry-NF155 cell slides, mCherry-NF186 cell slides, mCherry-CNTN1 cell slides, mCherry-CASPR1 cell slides, or mCherry-MAG cell slides were prepared using the cell slide treatment system and method described in Example 1. The resulting cell slides included treated mCherry-NF155 cell slides, treated mCherry-NF186 cell slides, treated mCherry-CNTN1 cell slides, treated mCherry-CASPR1 cell slides, and treated mCherry-MAG cell slides.

[0130] 2. Preparation: Cut the processed cell slides into 2.5×2.5mm pieces, attach them to glass slides with UV adhesive, and cure. Place them in a humidified chamber and soak them in sterile PBS buffer for 10 minutes. After soaking, discard the sterile PBS buffer.

[0131] 3. Sample incubation: Add 200 μL of specific positive samples against NF155 antigen, NF186 antigen, CNTN1 antigen, CASPR1 antigen or MAG antigen respectively to the cell slides. At the same time, set up a negative sample (PBS buffer) as a control. Incubate at room temperature for 1 hour to ensure that the specific antibodies in the samples fully bind to the antigens on the antigen slides.

[0132] 4. Washing: Remove the cell slide and gently rinse it three times with sterile PBS buffer for 5 minutes each time.

[0133] 5. 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 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.

[0134] 6. Fluorescence detection and result interpretation (1) Add 200 μL of mounting medium, place the cell smear under a fluorescence inverted microscope, and observe the emitted fluorescence signals at wavelengths of 480-500 nm and 540-565 nm respectively. 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 where the green fluorescent signal is emitted overlaps with the location where the red fluorescent signal is emitted, and the characteristics are basically the same, then it can be determined that the sample contains the corresponding antibody; (3) If a green fluorescent signal cannot be emitted; or the position where the green fluorescent signal is emitted does not overlap with the position where the red fluorescent signal is emitted; or the position where the green fluorescent signal is emitted overlaps with the position where the red fluorescent signal is emitted, but the two have large differences in characteristics; then it is determined that the sample does not contain the corresponding antibody.

[0135] The measurement results are shown in Table 2 and Figure 2 As shown: Table 2

[0136] The results showed that the cell smears prepared using the cell smear processing system and method of Example 1 of this invention for the five target antigens (NF155, NF186, CNTN1, CASPR1, MAG) all exhibited excellent detection performance when used for the detection of corresponding specific positive samples. They can effectively achieve accurate detection of the five core antibodies in the Langfi nodal / paranodal antibody spectrum and meet the clinical needs for multi-antibody combined detection.

[0137] Experiment Example 3: Assembly and Use of a Biochip for Langefier Nodal / Parallel Antibody Spectroscopy Detection 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 side. The front side 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 side has a reaction chamber for fixing the cell antigen plates. Biochip 1 is formed by sequentially arranging the treated mCherry-NF186 cell crawlers, treated mCherry-CASPR1 cell crawlers, and blank control antigen plates from left to right within the reaction chamber; biochip 2 is formed by sequentially arranging the treated mCherry-NF155 cell crawlers, treated mCherry-CNTN1 cell crawlers, and blank control antigen plates from left to right within the reaction chamber; and biochip 3 is formed by sequentially arranging the treated mCherry-MAG cell crawlers and blank control antigen plates from left to right within the reaction chamber, enabling parallel detection of multiple antibodies.

[0138] 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.

[0139] This chip integrates sample mixing, multi-channel reaction, QR code traceability, and sealed packaging, enabling simultaneous detection of five antibodies related to Langefier's nodal / paranodal antibody profile.

[0140] 3. Instructions for use of biochips It is recommended to conduct the experiment at room temperature to ensure the accuracy of the results.

[0141] (1) Warming and washing: Take out 3 biochips and place them in a humidified chamber (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 and discard the working solution.

[0142] (2) Sample incubation: Add 70 μL of the sample to be tested to each reaction zone and incubate at room temperature for 1 hour.

[0143] (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.

[0144] (4) Secondary antibody incubation: Add 70 μL of diluted fluorescently labeled goat anti-human IgG to the reaction area of ​​biochip 1, add 70 μL of diluted fluorescently labeled mouse anti-human IgG4 to the reaction area of ​​biochip 2, and add 70 μL of diluted fluorescently labeled goat anti-human IgM to the reaction area of ​​biochip 3. Incubate at room temperature in the dark for 30 minutes. (From this step onwards, keep out of the light throughout the entire process).

[0145] (5) Cleaning: Remove the fluorescent secondary antibody from the 3 biochips, 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.

[0146] (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.

[0147] (7) Interpretation criteria Taking NF155 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, it can be determined that the sample contains anti-NF155 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 be free of anti-NF155 antibody. Figure 3 The green fluorescence signal and the red fluorescence signal overlap in position and have basically the same characteristics, indicating that the sample contains anti-NF155 antibody; Figure 4 The absence of a red fluorescent signal at the location of the green fluorescent signal indicates that the sample does not contain anti-NF155 antibody.

[0148] Similarly, in addition to NF1551-transfected cells labeled with fluorescent proteins, the reagent also includes one or more of NF186, CNTN, CASPR1, and MAG-transfected cells labeled with fluorescent proteins, allowing for the simultaneous detection and interpretation of multiple antibodies in the sample. If any one or more of NF155, NF186, CNTN1, CASPR1, and MAG are found to be positive, the sample is diagnosed as a Langerhans node / paranodal disease sample.

[0149] 4. Biochips are used for clinical sample testing. Peripheral blood serum samples were collected from 12 patients clinically diagnosed with Ranvier's node / perinodecal disease and 20 healthy volunteers. After collection, samples were frozen at -20℃, thawed at 4℃ before use, and centrifuged (3000 rpm, 10 min) to remove precipitate. The supernatant was then used. The samples were analyzed according to the biochip instructions. The results are shown in Table 3. Table 3

[0150] When the cell smear processing method of Example 1 of this invention is used for clinical sample testing, the positive detection rate of 100.00% for samples from 12 patients with Ranvier's node / paranodal disease is excellent, effectively avoiding missed detections; the specificity of 95.00% for samples from 20 healthy volunteers is only one case of false positive, with extremely low non-specific interference; the overall detection accuracy is 96.88%, and the detection results are stable and reliable, accurately distinguishing between patients with Ranvier's node / paranodal disease and healthy individuals, fully meeting the needs of accurate detection of Ranvier's node / paranodal antibody profiles in clinical practice, and providing reliable technical support for the clinical diagnosis of the disease.

[0151] 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 smear treatment reagent, characterized in that, The cell smear treatment reagent consists of a cell fixation solution and a cell fixation antigen retrieval composition. The cell fixative consists of 2.5%-3.5% w / v paraformaldehyde, 0.4%-0.8% v / v formaldehyde, 0.10%-0.16% v / v acetic acid, 0.3%-0.5% w / v raffinose, and buffer solution. The cell fixation antigen retrieval composition consists of 70-130mM ammonium sulfate, 50-100mM hydroxylamine hydrochloride, 0.25%-0.55% v / v Brij 78, 0.65-1.25mM TCEP, 0.02%-0.05% v / v ProClin 300, and buffer solution.

2. The cell smear treatment reagent 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; 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. A method for processing cell smears, wherein the method comprises using the cell smear processing reagent according to any one of claims 1-2.

4. The cell smear processing method according to claim 3, characterized in that, The cell smear processing 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, add the cell fixation antigen retrieval composition, and incubate. S4. After incubation, discard the antigen retrieval composition after cell fixation, rinse, block, rinse again, blot dry, and dry to obtain the treated cell slide.

5. The cell smear processing method according to claim 4, characterized in that, The target plasmid mentioned in step S1 includes a plasmid that overexpresses Langefier knot / para-knot antibody-associated antigen.

6. The cell smear processing method according to claim 5, characterized in that, The aforementioned Langefier nodal / paranodal antibody-related antigens include any one or more of NF155, NF186, CNTN1, CASPR1, and MAG.

7. The cell smear processing method according to claim 5, characterized in that, The target plasmid also expresses a fluorescent protein, which serves as a positive transfection reporter tag for visually determining transfection efficiency.

8. The cell smear processing method according to claim 4, characterized in that, The cells mentioned in step S1 include any one or more of HEK293 cells, CHO cells, Vero cells, HeLa cells, COS-7 cells, and BHK-21 cells.

9. The cell smear processing method according to claim 4, characterized in that, The fixing conditions mentioned in step S2 include: fixing at room temperature for 20-40 minutes; the incubation mentioned in step S3 includes: incubation at room temperature for 20-40 minutes.

10. The use of the cell smear processing reagent according to any one of claims 1-2 in the preparation of the Ranvier's knot / para-knot antibody profiling kit.

11. A Langefier nodal / paranodal antibody profiling detection kit, characterized in that, The Langfi's nodal / paranodal antibody profiling kit contains the cell smear treatment reagent as described in any one of claims 1-2.

12. The Langfi's nodal / paranodal antibody profiling detection kit according to claim 11, characterized in that, The Langfi's nodal / paranodal antibody profiling kit also includes any one or more of the following: a biochip, a fluorescently labeled secondary antibody, a working solution, and a mounting medium.

13. The Langfi's nodal / paranodal antibody profiling detection kit according to claim 12, characterized in that, 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 a cell slide containing target cells.

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