AchR tetramolecular co-expression cba detection substrate and its preparation method and application

Abstract

The application provides an AChR tetramer co-expression CBA detection substrate and a preparation method and application thereof, wherein the AChR tetramer co-expression CBA detection substrate is prepared by mild fixation of eukaryotic cells co-expressing a four-component complex of acetylcholine receptors, receptor-associated proteins, alpha-dystrophin binding proteins and synergin. The application solves the problems of traditional CBA substrates, such as incomplete conformation, low-affinity antibody leakage and the like, which only express AChR or AChR+rapsyn. The sensitivity and specificity of the application are significantly better than those of the prior art, and the application is suitable for clinical precise diagnosis, typing and screening of hidden cases of myasthenia gravis.

Description

Technical Field

[0001] This invention belongs to the field of CBA reagent kit detection technology, specifically relating to an AChR tetramolecule co-expression CBA detection substrate, its preparation method, and its application. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that impair neuromuscular junction transmission. Anti-acetylcholine receptor (AChR) antibodies are the most prevalent pathogenic antibodies, accounting for 80%–90% of MG patients. Cell matrix immunofluorescence assay (CBA) is currently the most sensitive method for clinical detection of anti-AChR antibodies. Its core principle is to use AChR-expressing cells as a substrate and detect specific antibodies in the sample by fluorescently labeling secondary antibodies.

[0003] However, existing CBA detection substrates have significant technical defects, making it difficult to meet the needs of accurate clinical diagnosis: First, traditional CBA substrates only transfect AChR, resulting in AChR being scattered on the cell membrane, failing to mimic the native in vivo conformation, leading to low recognition efficiency of pathogenic antibodies and a high risk of false negatives; Second, some improved protocols only achieve co-expression of AChR and rapsyn, which can enable AChR to form preliminary clusters, but lacks scaffold proteins to maintain the maturity and stability of the clusters, resulting in an incomplete conformation and still causing 30-40% of conformation-specific antibodies to be missed.

[0004] Studies have shown that AChR does not exist alone in the postsynaptic dense region (PSD) of the neuromuscular junction, but rather forms a quaternary supramolecular complex with rapsyn, α-dystrobrevin, and syntrophin to maintain the native cluster conformation and stability of AChR. Specifically, rapsyn is responsible for the initial clustering of AChR, α-dystrobrevin acts as a scaffold anchoring protein, securing the AChR-rapsyn complex to the cell membrane cytoskeleton and maintaining the maturation and stability of the clusters, and syntrophin, as a conformational fine-tuning protein, binds to α-dystrobrevin to further optimize the native conformation of AChR. These three components are complementary and indispensable.

[0005] Currently, none of the CBA detection technologies worldwide have achieved the co-expression of four molecules: AChR, rapsyn, α-dystrobrevin, and syntrophin. This prevents the in vitro reconstruction of the actual postsynaptic membrane compact area structure, hindering breakthroughs in detection sensitivity and specificity. Therefore, developing a CBA detection substrate capable of in vitro reconstruction of the postsynaptic compact area, with a complete conformation and excellent detection performance, has become a pressing technical problem in this field. Summary of the Invention

[0006] In view of this, the present invention provides an AChR tetramolecule co-expression CBA detection substrate, its preparation method and application, for high-sensitivity and high-specificity detection of anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG).

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an AChR tetramolecule co-expression CBA detection substrate, wherein the detection substrate is prepared by mild immobilization of eukaryotic cells co-expressing an acetylcholine receptor, receptor-associated protein, α-muscular dystrophy-binding protein, and a co-protein quaternary complex.

[0008] It should be noted that after cell fixation, a cluster structure of the native conformation of the postsynaptic dense region (PSD) of the neuromuscular junction is formed on the cell membrane surface, mimicking the structure of the native conformation. This invention reconstructs the PSD of the neuromuscular junction in vitro by co-expressing a quaternary complex, achieving complete preservation of the native conformation of AChR, solving the problems of missed detection and incomplete conformation of traditional CBA substrates, and improving the sensitivity and specificity of anti-AChR antibody detection.

[0009] Preferably, the eukaryotic cells include HEK293 cells.

[0010] Preferably, the acetylcholine receptor is a human pentameric protein composed of two α1 subunits and one each of β, δ, and ε subunits. The quaternary complex, through specific protein-protein interactions, enables AChR to form a mature cluster structure with high density and high native conformation, significantly increasing the cluster density compared to traditional AChR and rapsyn bimolecular co-expression substrates.

[0011] Preferably, the mild fixation uses paraformaldehyde fixative or formaldehyde-free fixative. The fixation method is as follows: fix at room temperature for 5–15 minutes, a process that does not disrupt the natural conformational cluster structure formed by the quaternary complex.

[0012] Secondly, the present invention provides a detection kit for anti-AChR antibodies in myasthenia gravis, comprising the aforementioned AChR tetramolecule co-expressed CBA detection substrate, fluorescent secondary antibody, sample diluent, washing solution, positive control, and negative control.

[0013] Preferably, the fluorescent secondary antibody is a FITC-labeled goat anti-human IgG secondary antibody, the positive control is anti-AChR conformational positive serum, and the negative control is healthy human serum.

[0014] Thirdly, the present invention provides a method for preparing the detection substrate, comprising the following steps: S1. The genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin were linked to the vector to construct their respective expression vectors; S2. Transfect each of the expression vectors into HEK293 cells and culture the cells. S3. The test substrate is obtained through the steps of washing, fixing, washing, drying and sealing.

[0015] Preferably, in step S1, the vector is pcDNA3.1.

[0016] Preferably, in step S1, the specific method for constructing the expression vector is as follows: extract total RNA from human skeletal muscle cells, reverse transcribe to obtain cDNA, amplify the encoding genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin by PCR, clone the amplification products into the vector, and construct the expression vector.

[0017] Preferably, in step S2, the expression vectors of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin, and syntrophin transfected into HEK293 cells are in equal molar ratios.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In this invention, four molecules of AChR, rapsyn, α-dystrobrevin and syntrophin were co-expressed in the CBA detection substrate, and the dense region of the postsynaptic membrane of the neuromuscular junction was reconstructed in vitro.

[0019] (2) The cluster structure formed by the quaternary complex of the present invention is consistent with the dense region of the natural postsynaptic membrane in vivo. The AChR conformation is complete, which can specifically recognize low affinity and conformation specific anti-AChR antibodies. The detection sensitivity is significantly improved compared with the traditional CBA, and can detect occult MG cases that are missed by the traditional CBA.

[0020] (3) The natural conformation of the quaternary complex of the present invention can effectively avoid non-specific antibody binding and the detection specificity is ≥95%; the mild immobilization process and standardized preparation process make the substrate batch-to-batch difference CV <5%, which is suitable for industrial production and large-scale clinical application.

[0021] (4) The detection substrate of the present invention can be used for accurate clinical diagnosis, classification, disease monitoring and efficacy evaluation of myasthenia gravis, especially suitable for screening patients with occult type and low titer antibody MG, solving the pain point of missed detection in clinical practice, and has important clinical application value. Attached Figure Description

[0022] Figure 1 The tetramolecule co-expression substrate provided in Example 5 of this invention; Figure 2 The AChR+rapsyn co-expression substrate provided in Example 5 of this invention; Figure 3 This is the AChR-only expression substrate provided in Example 5 of the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0024] Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that impair neuromuscular junction transmission. Anti-acetylcholine receptor (AChR) antibodies are the most prevalent pathogenic antibodies, accounting for 80%–90% of MG patients. Cell matrix immunofluorescence assay (CBA) is currently the most sensitive method for clinical detection of anti-AChR antibodies. Its core principle is to use AChR-expressing cells as a substrate and detect specific antibodies in the sample by fluorescently labeling secondary antibodies.

[0025] However, existing CBA detection substrates have significant technical defects, making it difficult to meet the needs of accurate clinical diagnosis: First, traditional CBA substrates only transfect AChR, resulting in AChR being scattered on the cell membrane, failing to mimic the native in vivo conformation, leading to low recognition efficiency of pathogenic antibodies and a high risk of false negatives; Second, some improved protocols only achieve co-expression of AChR and rapsyn, which can enable AChR to form preliminary clusters, but lacks scaffold proteins to maintain the maturity and stability of the clusters, resulting in an incomplete conformation and still causing 30-40% of conformation-specific antibodies to be missed.

[0026] Studies have shown that AChR does not exist alone in the postsynaptic dense region (PSD) of the neuromuscular junction, but rather forms a quaternary supramolecular complex with rapsyn, α-dystrobrevin, and syntrophin to maintain the native cluster conformation and stability of AChR. Specifically, rapsyn is responsible for the initial clustering of AChR, α-dystrobrevin acts as a scaffold anchoring protein, securing the AChR-rapsyn complex to the cell membrane cytoskeleton and maintaining the maturation and stability of the clusters, and syntrophin, as a conformational fine-tuning protein, binds to α-dystrobrevin to further optimize the native conformation of AChR. These three components are complementary and indispensable.

[0027] Currently, none of the CBA detection technologies worldwide have achieved the co-expression of four molecules: AChR, rapsyn, α-dystrobrevin, and syntrophin. This prevents the in vitro reconstruction of the actual postsynaptic membrane compact area structure, hindering breakthroughs in detection sensitivity and specificity. Therefore, developing a CBA detection substrate capable of in vitro reconstruction of the postsynaptic compact area, with a complete conformation and excellent detection performance, has become a pressing technical problem in this field.

[0028] This invention provides an AChR tetramolecule co-expression CBA detection substrate, which is prepared by mild immobilization of eukaryotic cells co-expressing an acetylcholine receptor, receptor-associated protein, α-muscular dystrophy-binding protein, and a co-protein quaternary complex.

[0029] It should be noted that after cell fixation, a cluster structure of the native conformation of the postsynaptic dense region (PSD) of the neuromuscular junction is formed on the cell membrane surface, mimicking the structure of the native conformation. This invention reconstructs the PSD of the neuromuscular junction in vitro by co-expressing a quaternary complex, achieving complete preservation of the native conformation of AChR, solving the problems of missed detection and incomplete conformation of traditional CBA substrates, and improving the sensitivity and specificity of anti-AChR antibody detection.

[0030] Furthermore, eukaryotic cells include HEK293 cells.

[0031] Furthermore, the acetylcholine receptor is a human pentamer protein composed of two α1 subunits and one each of β, δ, and ε subunits. This quaternary complex, through specific protein-protein interactions, enables AChR to form a mature cluster structure with high density and high native conformation, significantly increasing the cluster density compared to traditional AChR and rapsyn bimolecular co-expression substrates.

[0032] Furthermore, the mild fixation employs paraformaldehyde fixative or formaldehyde-free fixative. The fixation method involves fixing at room temperature for 5–15 minutes, a process that does not disrupt the natural conformational cluster structure formed by the quaternary complex.

[0033] Secondly, the present invention provides a detection kit for anti-AChR antibodies in myasthenia gravis, comprising the aforementioned AChR tetramolecule co-expressed CBA detection substrate, fluorescent secondary antibody, sample diluent, washing solution, positive control, and negative control.

[0034] Furthermore, the fluorescent secondary antibody is a FITC-labeled goat anti-human IgG secondary antibody, the positive control is anti-AChR conformational positive serum, and the negative control is healthy human serum.

[0035] Thirdly, the present invention provides a method for preparing the detection substrate, comprising the following steps: S1. The genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin were linked to the vector to construct their respective expression vectors; S2. Transfect each of the expression vectors into HEK293 cells and culture the cells. S3. The test substrate is obtained through the steps of washing, fixing, washing, drying and sealing.

[0036] Furthermore, in step S1, the vector is pcDNA3.1.

[0037] Further, in step S1, the specific method for constructing the expression vector is as follows: extract total RNA from human skeletal muscle cells, reverse transcribe to obtain cDNA, amplify the encoding genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin by PCR, clone the amplification products into the vector, and construct the expression vector.

[0038] Further, in step S2, the expression vectors of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin, and syntrophin are transfected into HEK293 cells in equal molar ratios.

[0039] Example 1: Construction of expression vector Human AChR α1, β, δ, ε subunit expression vectors, rapsyn expression vector, α-dystrobrevin expression vector, and syntrophin expression vector were constructed respectively. Total RNA was extracted from human skeletal muscle cells, and cDNA was obtained by reverse transcription. The protein-coding genes were amplified by PCR, and the amplification products were cloned into the pcDNA3.1 vector to construct recombinant expression vectors. Enzyme digestion and sequencing verification were performed to ensure that each recombinant vector was constructed correctly and without base mutations or frameshift mutations.

[0040] Example 2: Preparation of tetramolecule co-expression cells HEK293 cells were seeded into well plates and cultured until confluence reached 70%–80%. Using liposome transfection, AChRα1, β, δ, ε subunit expression vectors, rapsyn expression vector, α-dystrobrevin expression vector, and syntrophin expression vector were mixed in equimolar ratios and co-transfected into HEK293 cells. After transfection, the cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. Immunofluorescence staining confirmed that the quaternary complex was successfully expressed on the cell membrane and formed a high-density cluster structure.

[0041] Example 3 Preparation of CBA detection substrate (1) Washing: Remove the culture medium from the well plate and gently wash the cells twice with PBS buffer to remove residual culture medium; (2) Mild fixation: Add methanol fixative and fix at room temperature for 10 min to ensure that cell morphology and cluster structure are not damaged; (3) Washing: Wash the cells three times with PBS buffer for 5 minutes each time to completely remove the fixative; (4) Drying and sealing: Take out the cell slides, let them dry naturally at room temperature, seal them in a sealed bag, and store them at -20℃ to obtain the AChR tetramolecule co-expression CBA detection substrate in the reconstructed postsynaptic dense region.

[0042] Example 4 Detection reagent components The kit contains the following components: (1) Reconstruction of AChR tetramolecules in the postsynaptic dense region to co-express CBA detection substrate; (2) AFITC-labeled goat anti-human IgG secondary antibody; (3) Sample dilution solution (0.01M PBS buffer); (4) PBS washing solution; (5) Positive control (anti-AChR positive serum); (6) Negative control (serum from healthy individuals).

[0043] Example 5: Effect Verification Experiment 1. Cluster Structure Comparison Experiment Experimental group: The tetramolecule co-expression substrate of CBA prepared in this invention; Control group 1: CBA substrates co-expressed by AChR and rapsyn; Control group 2: CBA substrates expressed only by AChR.

[0044] like Figure 1-3 As shown, immunofluorescence staining revealed clear and dense red clusters on the cell membrane of the experimental group, with a significantly higher cluster density than the control group. The control group had a small number of clusters, but they were loose and not dense, demonstrating that the substrate of this invention can form a mature and stable postsynaptic membrane dense area-like cluster structure.

[0045] 2. Sensitivity and Specificity Detection Experiment Serum samples were collected from 30 confirmed MG patients (10 of whom were occult patients missed by traditional CBA), 30 healthy individuals, and 30 patients with other autoimmune diseases. The kit of this invention was compared with a traditional CBA kit (using AChR-expressing cells as a substrate). The kit of this invention has the following sensitivity: 86.7% (26 / 30), of which 7 occult patients were successfully detected; and specificity: 95% (57 / 60). Traditional CBA kit: sensitivity 70% (21 / 30), only 1 occult patient was detected; specificity 95% (57 / 60); The results show that the sensitivity and specificity of the kit of the present invention are significantly better than those of the traditional CBA kit, and it can effectively solve the problem of missed detection.

[0046] 3. Stability test The CBA substrate prepared in this invention was stored at -20°C for 12 months, and samples were taken at 0, 3, 6, and 12 months to detect fluorescence signal intensity and cluster structure. Six months later, the fluorescence signal retention rate was ≥95%, the cluster structure was intact, and there was no cell shedding or conformational damage; the batch-to-batch CV difference was <5%, which proves that the substrate of this invention has good stability and is suitable for long-term storage and clinical application.

[0047] Sequence display: (1) Human AchR α1, β, δ, ε subunit expression vector CHRNA1-P2A-CHRNB1-T2A-CHRND-T2A-CHRNE (2) rapsyn sequence (3) α-dystrobrevin sequence (4) Syntrophin sequence

[0048] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0049] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An AChR tetrameric co-expressed CBA detection substrate, characterized in that, The detection substrate was prepared by mild fixation of eukaryotic cells co-expressing acetylcholine receptor, receptor-associated protein, α-dystrophin-binding protein, and a quaternary complex of co-expressing proteins.

2. The detection substrate according to claim 1, characterized in that, Eukaryotic cells include HEK293 cells.

3. The detection substrate according to claim 1, characterized in that, The acetylcholine receptor is a human pentamer protein composed of two α1 subunits and one each of β, δ, and ε subunits.

4. The detection substrate according to claim 1, characterized in that, The mild fixation is performed using paraformaldehyde fixative or formaldehyde-free fixative.

5. A kit for detecting anti-AChR antibodies in myasthenia gravis, characterized in that, The sample comprises the AChR tetramolecule co-expression CBA detection substrate as described in any one of claims 1-4, a fluorescent secondary antibody, a sample diluent, a washing buffer, a positive control, and a negative control.

6. The reagent kit according to claim 5, characterized in that, The fluorescent secondary antibody is a FITC-labeled goat anti-human IgG secondary antibody, the positive control is anti-AChR conformational positive serum, and the negative control is healthy human serum.

7. The method for preparing the detection substrate according to any one of claims 1-4, characterized in that, Includes the following steps: S1. The genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin were linked to the vector to construct their respective expression vectors; S2. Transfect each of the expression vectors into HEK293 cells and culture the cells. S3. The test substrate is obtained through the steps of washing, fixing, washing, drying and sealing.

8. The preparation method according to claim 7, characterized in that, In step S1, the vector is pcDNA3.

1.

9. The preparation method according to claim 7, characterized in that, In step S1, the specific method for constructing the expression vector is as follows: extract total RNA from human skeletal muscle cells, reverse transcribe to obtain cDNA, amplify the encoding genes of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin and syntrophin by PCR, clone the amplification products into the vector, and construct the expression vector.

10. The preparation method according to claim 7, characterized in that, In step S2, the expression vectors of AChRα1, β, δ, ε subunits, rapsyn, α-dystrobrevin, and syntrophin are transfected into HEK293 cells in equal molar ratios.

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