CBA method reagent and detection method for detecting GFAP antibody through fusion expression of GFAP alpha and GFAP epsilon

Abstract

The invention discloses a CBA method reagent for detecting a GFAP antibody through fusion expression of GFAP alpha and GFAP epsilon and a detection method, and belongs to the technical field of neural immunodiagnosis. The fusion protein sequentially comprises the following functional domains: secretion signal peptide, a GFAP alpha partial sequence, a GFAP epsilon partial sequence, a transmembrane sequence and a fluorescent label. The core of the invention lies in that a fusion cell is constructed by using partial sequences of GFAP alpha and GFAP epsilon, and co-expression is realized in a host cell through a co-transfection technology, so that the sensitivity and specificity of detecting a CFAP antibody in a CBA method (cellular immunofluorescence method) can be remarkably improved.

Description

Technical Field

[0001] This invention relates to a CBA method reagent and detection method for detecting GFAP antibodies by fusion expression of GFAPα and GFAPε. Specifically, it relates to a method for detecting GFAP antibodies by fusion expression of partial sequences of GFAPα and GFAPε and then using cell immunofluorescence assay (CBA method) and the matching detection reagent, belonging to the field of neuroimmunodiagnostic technology. Background Technology

[0002] Glial fibrillary acidic protein (GFAP) is a monomeric type III intermediate filament protein with a molecular weight of approximately 50 kDa. It is highly specifically expressed almost exclusively in astrocytes and is a classic skeletal marker for this cell type. It forms a dynamic connection between the nucleus and the cell membrane, participating not only in cytoskeleton remodeling and remodeling but also mediating cell adhesion, maintaining myelin formation and neuronal structural stability in the central nervous system, and serving as a key fulcrum in multiple signal transduction pathways, regulating cellular responses to changes in the external microenvironment.

[0003] Under normal physiological conditions, GFAP maintains the homeostasis and function of astrocytes, providing metabolic, ionic, and synaptic support for neurons. When the central nervous system (CNS) suffers pathological stimuli such as mechanical injury, infectious or non-infectious inflammation, astrocytes rapidly enter reactive astrogliosis, and GFAP expression levels are significantly upregulated. Therefore, it is widely recognized as a core biological marker for assessing the degree of neural tissue damage and inflammatory activity. Furthermore, abnormal GFAP expression profiles are closely related to various neurological diseases (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, brain tumors, and hereditary leukodystrophy), making it an important molecular target for disease mechanism research and clinical monitoring.

[0004] GFAPα is the most common and best-studied subtype of the GFAP family, and it is a major intermediate filament protein in mature astrocytes of the central nervous system. This subtype plays a central role in maintaining the structural integrity of astrocytes, regulating cell polarity, morphological plasticity, and migration after injury. Aberrant expression or mutation of GFAPα is closely associated with the occurrence and progression of astrocytomas, Alexander's disease (AxD), and various neuroinflammatory diseases.

[0005] The glial fibrillary acidic protein epsilon (GFAPε) is a unique isoform of the GFAP gene produced through alternative splicing. Its most significant characteristic is the extension of its C-terminal domain: an additional 42 amino acid residues (a basic region rich in arginine / lysine) are inserted downstream of the standard GFAPα sequence. This region possesses significant protein-protein interaction potential and may participate in regulating the cross-linking of the GFAP network with other intracellular / extracellular molecules, thereby influencing the functional remodeling of astrocytes under specific pathological conditions.

[0006] Autoimmune glial fibrillary acidic protein astrocytopathy (GFAP astrocytopathy), first systematically defined in 2016, is a central nervous system autoimmune disease mediated by GFAP autoantibodies (primarily IgG). IgG autoantibodies against GFAPα can be detected in both cerebrospinal fluid (CSF) and serum of patients, with CSF showing a significantly higher positive predictive value than serum. Currently, tissue-based assays (TBA) and cell-based assays (CBA) are recommended by international guidelines as complementary strategies for GFAP antibody detection. Notably, GFAP astrocytopathy can coexist with tumors, suggesting a potential paratumor immune mechanism; however, a causal link between viral infection and the disease remains unclear. GFAP antibodies themselves do not directly induce tissue damage but exist as biomarkers of immune inflammatory processes; their detection value lies in indicating potential immunopathological processes.

[0007] Since the GFAP gene can generate at least eight isoforms through alternative splicing, the CBA method would be technically complex and costly to detect all isoform antibodies simultaneously. A Mayo Clinic study of 102 patients showed that all patients (100%) exhibited a specific reaction with the GFAPα isoform (2017, Glialfibrillary acidic protein immunoglobulin G as biomarker of autoimmune astrocytopathy: Analysis of 102 patients). A series of studies conducted at a European tertiary referral center on 22 patients also confirmed that 100% of patients' serum bound to GFAPα, 63.6% of patients simultaneously recognized both GFAPα and GFAPδ, and the incidence of recognizing GFAPδ alone was 0 (2017, Clinical and immunological characteristics of the spectrum of GFAP autoimmunity: a case series of 22 patients). Therefore, the CBA method is a preferred clinical approach for detecting GFAPα antibodies. However, a Chinese study that included 19 cerebrospinal fluid samples found that 14 cases were positive for GFAPα-IgG only, and 5 cases were positive for GFAPε-IgG only, suggesting that GFAPε isoform antibodies may have unique clinical relevance in the Chinese population and warrant further validation with a larger sample size (2018, Autoimmune glial fibrillary acidic protein astrocytopathy in Chinese patients: a retrospective study).

[0008] Chinese patent CN115993444A reports a two-color immunofluorescence detection method for GFAP antibodies in human serum and cerebrospinal fluid. This method uses a HEK-293T cell model overexpressing GFAPα and GFAPε as the target cells. It also constructs a patient characteristic information database (including basic information and clinical symptoms) and utilizes a random forest machine learning algorithm to establish an artificial intelligence prediction model. This model can predict the probability of a positive GFAP antibody test based on the patient's clinical characteristics, thus achieving a clinical auxiliary diagnostic process from sample collection, initial positive screening, cell model confirmation, to final data analysis and model prediction. However, in the cell model of this method, because GFAPα and GFAPε are expressed in their full-length form in cells, they easily form filamentous protein aggregates in the cytoplasm. When detecting serum samples at low dilutions, other non-GFAP antibodies in the sample may non-specifically bind to the aggregates, resulting in false positives. Such cases usually require secondary verification via tissue immunofluorescence, which not only increases the detection steps, time, and cost but also may introduce the risk of misjudgment. Therefore, its application in routine clinical testing may be limited. Summary of the Invention

[0009] This invention aims to provide a CBA method reagent and detection method for detecting GFAP antibodies by fusion expression of GFAPα and GFAPε. The core of this method is to construct fusion cells using partial sequences of GFAPα and GFAPε and achieve co-expression in host cells through co-transfection technology, which can significantly improve the sensitivity and specificity of detecting CFAP antibodies in the CBA method (cell immunofluorescence assay).

[0010] The present invention is achieved through the following technical solution: a CBA method reagent for detecting GFAP antibodies by fusion expression of GFAPα and GFAPε, wherein the reagent comprises a host cell recombinantly constructed to express the fusion protein, the fusion protein comprising the following functional domains in sequence: secretory signal peptide, a partial sequence of GFAPα amino acid sequence as shown in SEQ ID NO: 1, a partial sequence of GFAPε amino acid sequence as shown in SEQ ID NO: 2, a transmembrane sequence, and a fluorescent tag.

[0011] The amino acid sequence of the secretion signal peptide is shown in SEQ ID NO: 3.

[0012] The transmembrane sequence is CD8a hinge, and its amino acid sequence is shown in SEQ ID NO: 4.

[0013] The fluorescent tag is mCherry, and its amino acid sequence is shown in SEQ ID NO: 5.

[0014] The fusion protein also includes a first linker peptide that connects the GFAPα and GFAPε partial sequences, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0015] The fusion protein also includes a second linker peptide that connects the GFAPε partial sequence and the transmembrane sequence, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0016] The fusion protein also includes a third linker peptide that connects the transmembrane sequence and the fluorescent tag, the amino acid sequence of which is shown in SEQ ID NO: 8.

[0017] The recombinant construction involves constructing the gene encoding the fusion protein into a lentiviral expression vector and then transfecting it into CHO cells.

[0018] A CBA detection method for detecting GFAP antibodies for non-diagnostic purposes, using the above-mentioned reagents, detects GFAP antibodies in the sample to be tested by the CBA method.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention extracts specific partial sequences of GFAPα and GFAPε, and guides the expression of GFAPα partial sequence by linking it with secretory signal peptide. It then combines the linker peptide with transmembrane sequence and fluorescent tag to fuse and express the GFAPα partial sequence, thereby constructing a structurally optimized fusion protein. This fusion protein can enhance the localization and epitope exposure of antigen on the cell surface, laying the foundation for highly specific detection of GFAP antibodies.

[0020] (2) This invention utilizes secretory signal peptide, partial sequence of GFAPα, partial sequence of GFAPε, transmembrane sequence and fluorescent tag to construct a fusion protein, and combines it with lentiviral vector transfection of CHO cells to form a stable expression cell line, successfully preparing a highly sensitive GFAP antibody detection product. The supporting cell immunofluorescence detection method (CBA) further established exhibits excellent sensitivity and specificity when applied to the detection of human serum or cerebrospinal fluid samples, providing reliable technical support for the accurate clinical diagnosis of autoimmune GFAP astrocytosis. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the lentivirus vector of the present invention.

[0022] Figure 2 This is an immunofluorescence image of a positive sample in Example 4 of the present invention.

[0023] Figure 3 This is an immunofluorescence image of a negative sample in Example 4 of the present invention. Detailed Implementation

[0024] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] The core of this invention lies in providing a highly sensitive and specific GFAP antibody detection reagent and method. By extracting specific sequences of GFAPα and GFAPε and guiding their expression with a secretory signal peptide, and then combining them with a linker peptide to orderly fuse them with a transmembrane sequence and a fluorescent tag, a structurally optimized fusion protein is constructed. By cloning the encoding gene of this fusion protein into a lentiviral vector and transfecting it into CHO cells to obtain a stable expression cell line, a high-performance detection product (including detection reagents or kits) can be prepared. Based on this detection product, a matching CBA detection method can be established. When applied to the detection of autoimmune GFAP antibodies, it can exhibit excellent sensitivity and specificity.

[0027] Specifically, the fusion protein of the present invention comprises, in sequence, the following functional domains: a secretion signal peptide, a partial sequence of GFAPα, a partial sequence of GFAPε, a transmembrane sequence, and a fluorescent tag, wherein, The GFAPα partial sequence is derived from amino acid sequences 43-432 of transcriptional isoform 1, see SEQ ID NO: 1, whose amino acid sequence is as follows: PPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKE IRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.

[0028] The GFAPε partial sequence is derived from amino acid sequences 375-431 of transcriptional isoform 2, see SEQ ID NO: 2, whose amino acid sequence is as follows: NRITIPVQTFSNLQIRGGKSTKDGEN HKVTRYLKSLTIRVIPIQAHQIVNGTPPARG.

[0029] See SEQ ID NO: 3, the amino acid sequence of the secretion signal peptide is: MKWVTFISLLFLFSSAYS.

[0030] The transmembrane sequence is CD8a hinge, which is the amino acid sequence of the hinge region of CD8a from amino acid 183 to 206. See SEQ ID NO: 4, and its amino acid sequence is: IYIWAPLAGTCGVLLLSLVITLYC.

[0031] The fluorescent tag is mCherry, see SEQ ID NO: 5, its amino acid sequence is: MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0032] Preferably, the fusion protein of the present invention further includes a first linker peptide connecting the GFAPα partial sequence and the GFAPε partial sequence, a second linker peptide connecting the GFAPε partial sequence and the transmembrane sequence, and a third linker peptide connecting the transmembrane sequence and the fluorescent tag, specifically: See SEQ ID NO: 6, the amino acid sequence of the first linker peptide is: SGGGGSGGGGSGGGGS.

[0033] See SEQ ID NO: 7, the amino acid sequence of the second linker is: GSTGGGGSGGGGGSGGGGSGAASR.

[0034] See SEQ ID NO: 8, the amino acid sequence of the third linker is: ASGGGGSGGGGSSG.

[0035] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Example 1: Construction of a lentiviral expression vector for fusion protein This embodiment relates to the process of constructing a lentiviral vector for the fusion protein GFAPα-GFAPε-CD8a hinge-mCherry.

[0037] The fusion protein comprises, in sequence: a secretion signal peptide (SEQ ID NO: 3), a partial GFAPα sequence (SEQ ID NO: 1), a first linker peptide (SEQ ID NO: 6), a partial GFAPε sequence (SEQ ID NO: 2), a second linker peptide (SEQ ID NO: 7), a transmembrane sequence (SEQ ID NO: 4), a third linker peptide (SEQ ID NO: 8), and a fluorescent tag (SEQ ID NO: 5). Each functional domain is tandemly linked by the linker peptides to form a complete fusion protein coding sequence. This coding sequence was cloned into a lentiviral expression vector, successfully constructing a recombinant plasmid. A schematic diagram of the constructed lentiviral vector is shown below. Figure 1 .

[0038] Example 2: Preparation of GFAP antibody detection products This embodiment relates to the preparation process of an autoimmune GFAP antibody detection product.

[0039] The lentiviral vector prepared in Example 1 was transfected into CHO cells. The transfected cells were then screened using a limiting dilution method to obtain monoclonal cells expressing the GFAPα-GFAPε-CD8a hinge-mCherry fusion protein. Culture dishes confluent with these transfected cells were then digested into single-cell suspensions using trypsin-EDTA solution. Cell density was calculated using a cell counting chamber. The cells were then diluted with complete culture medium to 4 cells / 100 μL, 2 cells / 100 μL, and 1 cell / 100 μL using a limiting dilution method. Each well was then seeded at 100 μL / well into a 96-well plate. Wells with strong red fluorescence were selected for cryopreservation and primary antibody immunofluorescence detection. The qualified cells can be used to prepare detection reagents for subsequent immunofluorescence experiments.

[0040] Seeding of cell slices: Untreated CHO-GFAPα-GFAPε-CD8a hinge-mCherry monoclonal cells and control empty vector cells were mixed thoroughly at a monoclonal cell:control cell ratio of 1:2, and seeded at a total density of 1×2×10⁻⁶ cells / cells. 5Inoculate / mL into 96-well plates or cell slides, culture with F12K + 10% FBS + NEAA, and stop culture when the confluence reaches 90-100% after about 48 hours.

[0041] Example 3: Cellular Immunofluorescence Detection Method for GFAP Antibody This embodiment describes the specific process for detecting GFAP antibodies using cell immunofluorescence assay, including the following steps: Prepare blood or cerebrospinal fluid samples. The sample diluent should contain the following components: Phosphate buffer (pH 7.0-7.4), BSA (2%); The samples were initially diluted using sample diluent. The serum was diluted at a ratio of 1:10, while the cerebrospinal fluid was used as undiluted. Sample incubation: Incubate at 37 degrees Celsius for 1 hour, then wash 3 times with PBS; Dilute the secondary antibody with sample diluent: use goat anti-human IgG (H+L) at a dilution ratio of 1:500, incubate at room temperature for 45 minutes, and wash 3 times with PBS; Interpretation: Interpreting the positive or negative status of a sample under a microscope.

[0042] Example 4: Following the methods of Examples 1 and 2, the constructed CHO-GFAPα-GFAPε-CD8a hinge-mCherry was fused with an antibody for detecting GFAP. The stable cells selected by immobilization using the CBA method were mixed with wild-type CHO cells and seeded into a 96-well cell culture plate. After culturing for 48 hours, the cells were fixed with paraformaldehyde.

[0043] Comparative Example 1: This comparative example uses the full GFAPα sequence (1-432aa) (see NM_002055.5) to express the process for detecting GFAP antibodies.

[0044] By constructing a eukaryotic transient expression vector containing the above sequences, CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde and permeabilized with Triton X-100.

[0045] Comparative Example 2: This comparative example uses the full-length GFAPε sequence (1-431aa) (see NM_001131019.3) to express the process of detecting NMDAR antibodies.

[0046] By constructing a eukaryotic transient expression vector containing the above sequences, CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde and permeabilized with Triton X-100.

[0047] Comparative Example 3: This comparative example describes the process of constructing vectors using the full-length GFAPα sequence (1-432aa) and the full-length GFAPε sequence (1-431aa) respectively, and co-expressing them to detect GFAP antibodies.

[0048] By constructing a eukaryotic transient expression vector containing the above sequences, CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, the cells were fixed with paraformaldehyde and permeabilized with Triton X-100.

[0049] Comparative Example 4: This comparative example uses a process of fusion expression of secretory signal peptide + GFAPα full sequence (1-432aa) + first linker peptide + GFAPε full sequence (1-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0050] By constructing a eukaryotic transient expression vector containing the above sequences, CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent and cultured for 48 hours before being fixed with paraformaldehyde.

[0051] Comparative Example 5: This comparative example uses a process of fusion expression of secretory signal peptide + full GFAPα sequence (1-432aa) + first linker peptide + partial GFAPε sequence (375-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0052] By constructing a eukaryotic transient expression vector containing the above sequences, CHO was transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, it was fixed with paraformaldehyde.

[0053] Comparative Example 6: This comparative example uses a process of fusion expression of secretory signal peptide + partial GFAPα sequence (43-432aa) + first linker peptide + full GFAPε sequence (1-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0054] By constructing a eukaryotic transient expression vector containing the above sequences, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent and cultured for 48 hours before being fixed with paraformaldehyde.

[0055] Comparative Example 7: This comparative example involves the fusion expression of a secretory signal peptide, a partial GFAPα sequence (43-432aa), a first linker peptide, a partial GFAPε sequence (225-431aa), a second linker peptide, a CD8a hinge (183-206aa), a third linker peptide, and mCherry to detect GFAP antibodies.

[0056] By constructing a eukaryotic transient expression vector containing the above sequences, CHO was transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, it was fixed with paraformaldehyde.

[0057] Comparative Example 8: This comparative example uses a process of fusion expression of secretory signal peptide + GFAPα partial sequence (375-432aa) + first linker peptide + GFAPε partial sequence (375-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0058] By constructing a eukaryotic transient expression vector containing the above sequences, CHO was transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, it was fixed with paraformaldehyde.

[0059] The fixed cells prepared in Example 4 and Comparative Examples 1 to 8 were added to 96 positive samples clinically diagnosed with GFAP antibody disease and subjected to immunofluorescence detection.

[0060] Serum was diluted at 1:10, 1:32, and 1:100. Cerebrospinal fluid was used as stock solution without dilution and incubated (at 37°C for 1 hour). The cells were washed three times with PBS, and then incubated with secondary antibody (goat anti-human IgG diluted at 1:500) at room temperature for 45 minutes. After washing three times with PBS, a small amount of PBS was added to soak the cells, and the cells were then read under a microscope.

[0061] Immunofluorescence assay results are shown in Figure 2 Comparison with Table 1 below: GFAP antibody-positive samples.

[0062] Figure 2The image shows the immunofluorescence detection results of the positive sample in Example 4. In the figure, A is the red fluorescence of the GFAPα and GFAPε fusion protein itself, B is the green fluorescence produced by the secondary antibody after incubation of the positive sample and the secondary antibody, and C is the superimposed image of A and B, showing that the red and green fluorescence overlap in situ, indicating that the sample test result is positive.

[0063] Table 1: Comparison of detection rates of positive samples for anti-GFAP antibody disease

[0064] The fixed cells prepared in Example 4, Comparative Examples 1 to 8 were added to serum samples from 78 healthy individuals, serum samples from 142 other patients with non-GFAP, and cerebrospinal fluid samples from 64 individuals, and immunofluorescence detection was performed (the immunofluorescence detection method is the same as above).

[0065] Serum was diluted at 1:10, 1:32, and 1:100. Cerebrospinal fluid was used as stock solution without dilution and incubated (at 37°C for 1 hour). The cells were washed three times with PBS, and then incubated with secondary antibody (goat anti-human IgG diluted at 1:500) at room temperature for 45 minutes. After washing three times with PBS, a small amount of PBS was added to soak the cells, and the cells were then read under a microscope.

[0066] Immunofluorescence assay results are shown in Figure 3 And Table 2 below: Comparison of negative samples from healthy individuals and other patients without GFAP.

[0067] Figure 3 The image shows the immunofluorescence detection results of the negative sample in Example 4. In the figure, A is the red fluorescence of the GFAPα and GFAPε fusion protein itself, B is the negative sample with no obvious fluorescence signal, and C is the superimposed image of A and B, which only shows red fluorescence, indicating that the test result of the sample is negative.

[0068] Table 2: Comparison of negative samples from healthy individuals and other patients without GFAP

[0069] In summary, the method of the present invention, by employing the fusion expression of specific partial sequences of GFAPα and GFAPε, can be used for the detection of GFAP autoimmune antibodies, exhibiting specificity and sensitivity. Furthermore, its detection effect is superior to that of Comparative Examples 1 to 8. Among Comparative Examples 1 to 8, Comparative Examples 1 and 2 are detection methods that express the full-length GFAP (GFAPα or GFAPε transcripts) individually; Comparative Examples 3 and 4 are methods that fuse the full-length GFAPα and GFAPε; and Comparative Examples 5 to 8 are fusion expressions of partial or full-length sequences of GFAPα and GFAPε, but their fusion sequences are not the same as those in Example 4, belonging to the fusion sequences of GFAPα and GFAPε partially lengthened or shortened in Example 4. In summary, this invention constructs a recombinant protein and its accompanying immunofluorescence assay system for detecting autoimmune GFAP antibodies by fusing specific sequences of GFAPα (43-432aa) and GFAPε (375-431aa). Experimental results show that this assay system exhibits excellent detection sensitivity in both cerebrospinal fluid and serum at different dilutions in 96 GFAP antibody-positive samples; no false positives were observed in 284 negative samples from healthy individuals and patients without GFAP-related diseases, demonstrating good specificity.

[0070] Compared with other construction strategies listed in Comparative Examples 1 to 8, the GFAPa and GFAPE partial sequence fusion expression method adopted in this invention achieved a higher positive detection rate under multiple dilution conditions and did not introduce non-specific reactions. Specifically, although Comparative Examples 1 to 4 used full-length GFAPα or GFAPε sequences for single or co-expression, their detection sensitivity was lower than that of this invention in different sample types; although Comparative Examples 5 to 8 used partial sequences for fusion expression, their detection performance was also significantly weaker than the system constructed in Example 4 of this invention due to the different sequence segments selected.

[0071] Therefore, the CBA method reagent and detection method for GFAP antibody detection provided by this invention are superior to existing conventional construction systems in terms of sensitivity and specificity, and are suitable for high-reliability screening and detection of GFAP autoantibodies in clinical samples.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A CBA method reagent for detecting GFAP antibodies using GFAPα and GFAPε fusion expression, characterized in that: The reagent comprises a host cell recombinantly constructed to express a fusion protein, which sequentially includes the following functional domains: a secretory signal peptide, a partial GFAPα amino acid sequence as shown in SEQ ID NO: 1, a partial GFAPε amino acid sequence as shown in SEQ ID NO: 2, a transmembrane sequence, and a fluorescent tag.

2. The CBA method reagent according to claim 1, characterized in that: The amino acid sequence of the secretion signal peptide is shown in SEQ ID NO:

3.

3. The CBA method reagent according to claim 1, characterized in that: The transmembrane sequence is CD8a hinge, and its amino acid sequence is shown in SEQ ID NO:

4.

4. The CBA method reagent according to claim 1, characterized in that: The fluorescent tag is mCherry, and its amino acid sequence is shown in SEQ ID NO:

5.

5. The CBA method reagent according to claim 1, characterized in that: The fusion protein also includes a first linker peptide that connects the GFAPα and GFAPε partial sequences, the amino acid sequence of which is shown in SEQ ID NO:

6.

6. The CBA method reagent according to claim 1, characterized in that: The fusion protein also includes a second linker peptide that connects the GFAPε partial sequence and the transmembrane sequence, the amino acid sequence of which is shown in SEQ ID NO:

7.

7. The CBA method reagent according to claim 1, characterized in that: The fusion protein also includes a third linker peptide that connects the transmembrane sequence and the fluorescent tag, the amino acid sequence of which is shown in SEQ ID NO:

8.

8. The CBA method reagent according to claim 1, characterized in that: The recombinant construction involves constructing the gene encoding the fusion protein into a lentiviral expression vector and then transfecting it into CHO cells.

9. A CBA detection method for detecting GFAP antibodies for non-diagnostic purposes, characterized in that: Using the reagents described in any one of claims 1 to 8, the GFAP antibody in the sample to be tested was detected by the CBA method.

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