A CBA method reagent for detecting GFAP antibody by fusing expression of GFAP alpha and GFAP epsilon and a detection method

Abstract

The application discloses a CBA method reagent for detecting GFAP antibody by fusing expression of GFAP alpha and GFAP epsilon and a detection method, and belongs to the technical field of neuroimmunological diagnosis. The reagent comprises a host cell which is recombined to express a fusion protein. The fusion protein comprises the following functional domains in sequence: a secretion signal peptide, a GFAP alpha partial sequence, a GFAP epsilon partial sequence, a transmembrane sequence and a fluorescent label. The core of the application is that the fusion cell is constructed by using the partial sequences of GFAP alpha and GFAP epsilon, and the co-expression is realized in the host cell by using the co-transfection technology. The sensitivity and the specificity of the detection of the CFAP antibody in the CBA method (cellular immunofluorescence method) can be significantly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a CBA method reagent for detecting GFAP antibody by fusing expression of GFAP alpha and GFAP epsilon and a detection method, in particular to a method for detecting GFAP antibody by fusing expression of GFAP alpha and GFAP epsilon and a detection method, and belongs to the technical field of neuroimmunological diagnosis. BACKGROUND

[0002] Glial fibrillary acidic protein (GFAP) is a monomer Ⅲ type intermediate filament protein with a molecular weight of about 50 kDa, which is highly specifically expressed in astrocytes and is a classic skeletal marker of this type of cells. It forms a dynamic connection between the cell nucleus and the cell membrane, not only participates in the reorganization and remodeling of the cytoskeleton, but also mediates cell adhesion, maintains central nervous system myelin formation and neuronal structure stability, and as a key branch point of multiple signal transduction pathways, regulates the response of cells to changes in the external microenvironment.

[0003] Under normal physiological conditions, GFAP maintains the steady state morphology and function of astrocytes, and provides metabolic, ionic and synaptic environment support for neurons; when the CNS is subjected to mechanical damage, infectious or non-infectious inflammation and other pathological stimuli, astrocytes rapidly enter reactive proliferation (reactive astrogliosis), and the expression level of GFAP is significantly up-regulated, and therefore it is recognized as a core biological marker for evaluating the degree of nerve tissue damage and inflammatory activity. In addition, the abnormal expression profile of GFAP is closely related to a variety of nervous system diseases (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, brain tumors and hereditary leukodystrophy, etc.), and becomes an important molecular target for disease mechanism research and clinical diagnosis and monitoring.

[0004] GFAP alpha is the most common and most in-depth researched subtype in the GFAP family, and is the main intermediate filament protein in mature astrocytes of the central nervous system. This subtype plays a core role in maintaining the structural integrity of astrocytes, regulating cell polarity, morphological plasticity and migration after injury. Abnormal expression or mutation of GFAP alpha is closely related to the occurrence and progression of astrocytoma, Alexander disease (AxD) and various neuroinflammatory diseases.

[0005] Glial fibrillary acidic protein epsilon (GFAP epsilon) is a unique isoform of GFAP gene generated by alternative splicing, which is characterized by an extension of the C-terminal domain: an additional functional fragment of 42 amino acid residues (basic region rich in arginine / lysine) is inserted downstream of the standard GFAP alpha sequence. This region has significant protein-protein interaction potential and may be involved in regulating the cross-linking of GFAP networks with other intracellular / extracellular molecules, thereby affecting the functional remodeling of astrocytes in specific pathological situations.

[0006] Autoimmune GFAP astrocytopathy was first systematically defined in 2016 as a CNS autoimmune disease mediated by GFAP autoantibodies (mainly IgG type). IgG autoantibodies against GFAP alpha can be detected in both cerebrospinal fluid (CSF) and serum of patients, and the positive predictive value of CSF is significantly higher than that of serum. Currently, tissue-based assay (TBA) and cell-based assay (CBA) are recommended by international guidelines as complementary strategies for GFAP antibody detection. It is worth noting that GFAP astrocytopathy can be combined with tumors, suggesting the existence of potential paraneoplastic immune mechanisms; however, there is still a lack of clear evidence of a causal relationship between viral infection and the disease. GFAP antibodies do not directly induce tissue damage, but rather serve as biomarkers of the immune inflammatory process, and their detection value lies in suggesting potential immunopathological processes.

[0007] Because the GFAP gene can produce at least eight isoforms through alternative splicing, the technical complexity and cost of CBA are high if all isoform antibodies are detected simultaneously. The Mayo Clinic study of 102 patients for GFAP-IgG showed that all patients (100%) showed specific reaction with GFAPa isoform (2017, Glial fibrillary acidic protein immunoglobulin G as biomarker of autoimmune astrocytopathy: Analysis of 102 patients). A series of studies of 22 patients in a tertiary referral center in Europe also confirmed that 100% of patients' serum bound to GFAPa, and 63.6% of patients recognized both GFAPa and GFAPd, while the incidence of GFAPd alone was 0 (2017, Clinical and immunological characteristics of the spectrum of GFAP autoimmunity: a case series of 22 patients). Therefore, the CBA method for detecting GFAPa antibody can be preferred in clinical routine. However, a study in China including 19 cerebrospinal fluid samples found that 14 cases were only GFAPa-IgG positive, and 5 cases were only GFAPe-IgG positive, suggesting that GFAPe isoform antibody may have unique clinical relevance in the Chinese population, which is worthy of further sample verification (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 human serum cerebrospinal fluid GFAP antibody. The method constructs HEK-293T cell models overexpressing GFAPα and GFAPε as target cells for detection, and at the same time constructs a patient characteristic information database (including basic information and clinical symptoms, etc.), and uses a random forest machine learning algorithm to establish an artificial intelligence prediction model. The model can predict the probability of GFAP antibody positive according to the clinical characteristics of the patient, thereby realizing the clinical auxiliary diagnosis process from sample collection, positive preliminary screening, cell model diagnosis, to final data analysis and model prediction. However, in the cell model of this method, GFAPα and GFAPε are expressed in the form of full-length in the cells, which is easy to form filamentous protein aggregates in the cytoplasm. When detecting serum samples with low dilution multiple, other non-GFAP antibodies in the sample may be non-specifically bound to the aggregated proteins, resulting in false positive results. Such cases usually need to be verified by tissue immunofluorescence for the second time, which not only increases the detection steps, time and cost, but also may introduce the risk of misjudgment. Therefore, its application in routine clinical detection may be limited. SUMMARY

[0009] The present application aims to provide a GFAPα and GFAPε fusion expression CBA method reagent for detecting GFAP antibody and a detection method. The core is to construct a fusion cell using part of the sequence of GFAPα and GFAPε, and to realize co-expression in host cells through co-transfection technology, which can significantly improve the sensitivity and specificity of detecting CFAP antibody in CBA method (cellular immunofluorescence method).

[0010] The present application is realized by the following technical scheme: a GFAPα and GFAPε fusion expression CBA method reagent for detecting GFAP antibody, the reagent comprising a host cell recombined to express a fusion protein, the fusion protein comprising the following functional domains in sequence: a secretion signal peptide, a GFAPα partial sequence with an amino acid sequence as shown in SEQ ID NO: 1, a GFAPε partial sequence with an 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 as shown in SEQ ID NO: 3.

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

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

[0014] The fusion protein further comprises a first connecting peptide connecting the GFAP alpha partial sequence and the GFAP epsilon partial sequence, and the amino acid sequence is shown as SEQ ID NO: 6.

[0015] The fusion protein further comprises a second connecting peptide connecting the GFAP epsilon partial sequence and the transmembrane sequence, and the amino acid sequence is shown as SEQ ID NO: 7.

[0016] The fusion protein further comprises a third connecting peptide connecting the transmembrane sequence and the fluorescent label, and the amino acid sequence is shown as SEQ ID NO: 8.

[0017] The recombinant construction is to construct the gene encoding the fusion protein into a lentivirus expression vector, and then transfect into CHO cells.

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

[0019] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0020] (1) The present application constructs a structure-optimized fusion protein by intercepting specific partial sequences of GFAP alpha and GFAP epsilon, connecting the GFAP alpha partial sequence with the help of the secretion signal peptide to guide expression, and combining the connecting peptide to fuse and express it with the transmembrane sequence and the fluorescent label, which can enhance the localization and epitope exposure of the antigen on the cell surface, and lay a foundation for high-specificity detection of GFAP antibody.

[0021] (2) The present application uses secretion signal peptide, GFAP alpha partial sequence, GFAP epsilon partial sequence, transmembrane sequence and fluorescent label to construct a fusion protein, combines lentivirus vector transfection with CHO cells to form a stable expression cell line, successfully prepares a high-sensitivity GFAP antibody detection product, and further establishes a supporting cell immunofluorescence detection method (CBA), which shows excellent sensitivity and specificity when applied to human serum or cerebrospinal fluid sample detection, and provides reliable technical support for clinical precise diagnosis of autoimmune GFAP astrocyte disease. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure is a structural schematic diagram of the lentivirus vector of the present application.

[0023] Figure 2 The figure is an immunofluorescence image of a positive sample in Example 4 of the present application.

[0024] Figure 3 The figure is an immunofluorescence image of a negative sample in Example 4 of the present application. DETAILED DESCRIPTION

[0025] The invention purposes, technical solutions and beneficial effects will be further explained in detail below.

[0026] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed, and all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the invention belongs, unless otherwise specified.

[0027] The core of the present application is to provide a high-sensitivity and high-specificity GFAP antibody detection reagent and method, by intercepting the specific partial sequence of GFAPa and GFAPe, and guiding the expression by means of secretory signal peptide, combining with the connecting peptide to orderly fuse it with the transmembrane sequence and fluorescent label, a structure-optimized fusion protein is constructed, the coding gene of the fusion protein is cloned into the lentivirus vector, and after transfecting CHO cells, a stable expression cell line is obtained, and then a high-performance detection product (including detection reagent or kit) can be prepared, and a matching CBA detection method is established based on the detection product, when applied to autoimmune GFAP antibody detection, it can show excellent sensitivity and specificity.

[0028] Specifically, for the fusion protein of the present application, it sequentially includes the following functional domains: secretory signal peptide, GFAPa partial sequence, GFAPe partial sequence, transmembrane sequence and fluorescent label, wherein,

[0029] The GFAPa partial sequence is from the 43-432 amino acid sequence of transcription isomer 1, see SEQ ID NO: 1, and the amino acid sequence is:

[0030] PPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.

[0031] The GFAPe partial sequence is from amino acids 375-431 of transcript isoform 2, see SEQ ID NO: 2, which has the amino acid sequence:

[0032] NRITIPVQTFSNLQIRGGKSTKDGEN HKVTRYLKSLTIRVIPIQAHQIVNGTPPARG.

[0033] The secretory signal peptide has the amino acid sequence: MKWVTFISLLFLFSSAYS, see SEQ ID NO: 3.

[0034] The transmembrane sequence is the 183-206 amino acid sequence of the CD8a hinge, i.e., the hinge region of CD8a, see SEQ ID NO: 4, which has the amino acid sequence: IYIWAPLAGTCGVLLLSLVITLYC.

[0035] The fluorescent tag is mCherry, see SEQ ID NO: 5, which has the amino acid sequence:

[0036] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0037] 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:

[0038] See SEQ ID NO: 6, the amino acid sequence of the first linker peptide is: SGGGGSGGGGSGGGGS.

[0039] See SEQ ID NO: 7, the amino acid sequence of the second linker is: GSTGGGGSGGGGGSGGGGSGAASR.

[0040] See SEQ ID NO: 8, the amino acid sequence of the third linker is: ASGGGGSGGGGSSG.

[0041] 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.

[0042] Example 1: Construction of a lentiviral expression vector for fusion protein

[0043] This embodiment relates to the process of constructing a lentiviral vector for the fusion protein GFAPα-GFAPε-CD8a hinge-mCherry.

[0044] The structure of the fusion protein comprises, in order, a secretion signal peptide (SEQ ID NO: 3), a GFAPa partial sequence (SEQ ID NO: 1), a first connecting peptide (SEQ ID NO: 6), a GFAPe partial sequence (SEQ ID NO: 2), a second connecting peptide (SEQ ID NO: 7), a transmembrane sequence (SEQ ID NO: 4), a third connecting peptide (SEQ ID NO: 8), and a fluorescent tag (SEQ ID NO: 5). Each functional domain is connected in series by the connecting peptide to form a complete fusion protein coding sequence. The coding sequence is cloned into a lentivirus expression vector to successfully construct a recombinant plasmid. The structural diagram of the constructed lentivirus vector is shown in Figure 1 .

[0045] Example 2: Preparation of a GFAP antibody detection product

[0046] This example relates to the preparation process of an autoimmune GFAP antibody detection product.

[0047] The lentivirus vector prepared in Example 1 is transfected into CHO cells, and the transfected cells are then screened by limiting dilution to obtain monoclonal cells expressing the GFAPa-GFAPe-CD8a hinge-mCherry fusion protein. Then, the culture dish covered with the transfected GFAPa-GFAPe-CD8a hinge-mCherry cells is digested with trypsin-EDTA solution to form a single cell suspension. The cell density is calculated using a cell counting plate, and the cells are diluted with complete culture medium to 4 / 100 uL, 2 / 100 uL, and 1 / 100 uL using the limiting dilution method. Then, 100 uL / well is inoculated into a 96-well plate, and the wells with single cell groups are screened. The wells with strong red fluorescence are selected for freezing, primary antibody immunofluorescence detection. The cells that meet the requirements can be used to prepare detection reagents for subsequent immunofluorescence experiments.

[0048] Cell sheet inoculation: the decompartmentalized CHO-GFAPa-GFAPe-CD8a hinge-mCherry monoclonal cells and control empty cells are mixed uniformly at a ratio of 1:2, and inoculated into a 96-well plate or cell climbing sheet at a total density of 1x2x10 5 / mL. The cells are cultured in F12K+10%FBS+NEAA, and the culture is stopped when the confluence is 90-100% after about 48 hours of growth.

[0049] Example 3: Cell immunofluorescence detection method of GFAP antibody

[0050] This example relates to the specific process of detecting GFAP antibody by cell immunofluorescence method, which includes the following operations:

[0051] Prepare blood or cerebrospinal fluid samples, and the sample diluent contains the following components:

[0052] Phosphate buffer (pH 7.0-7.4), BSA (2%);

[0053] Dilute the sample with the sample diluent for the first time. The dilution ratio of serum is 1:10, and the cerebrospinal fluid is used as the original solution (without dilution).

[0054] Sample incubation: incubate at 37°C for 1 hour, and wash with PBS for 3 times;

[0055] Dilute the secondary antibody with the sample diluent: use goat anti-human IgG (H+L) with a dilution ratio of 1:500, incubate at room temperature for 45 minutes, and wash with PBS for 3 times;

[0056] Interpretation: interpret the sample under the microscope.

[0057] Example 4:

[0058] According to the methods of Example 1 and Example 2, the constructed CHO-GFAPα-GFAPε-CD8a hinge-mCherry fusion expression detects GFAP antibody, and the screened stable cells and wild type CHO cells are mixed and plated in a 96-well cell culture plate. After 48 hours of culture, they are fixed by polyethylene glycol.

[0059] Comparative Example 1:

[0060] This comparative example uses the full sequence of GFAPα (1-432aa) (see NM_002055.5) to express the detection of GFAP antibody.

[0061] By constructing a eukaryotic transient expression vector containing the above sequence, CHO cells are transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, they are fixed by polyethylene glycol and permeabilized by Triton X-100.

[0062] Comparative Example 2:

[0063] This comparative example uses the full sequence of GFAPε (1-431aa) (see NM_001131019.3) to express the detection of NMDAR antibody.

[0064] By constructing a eukaryotic transient expression vector containing the above sequence, CHO cells are transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, they are fixed by polyethylene glycol and permeabilized by Triton X-100.

[0065] Comparative Example 3:

[0066] This example is the 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.

[0067] The eukaryotic transient expression vector containing the above sequence was constructed, and CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, fixation was performed with polyethylene glycol, and permeabilization was performed with Triton X-100.

[0068] Example 4:

[0069] This example is the 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.

[0070] The eukaryotic transient expression vector containing the above sequence was constructed, and CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, fixation was performed with polyethylene glycol.

[0071] Example 5:

[0072] This example is the process of fusion expression of secretory signal peptide + GFAPα full sequence (1-432aa) + first linker peptide + GFAPε partial sequence (375-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0073] The eukaryotic transient expression vector containing the above sequence was constructed, and CHO cells were transiently transfected with Lipofectamine™ 3000 transfection reagent. After 48 hours of culture, fixation was performed with polyethylene glycol.

[0074] Example 6:

[0075] This example is the process of fusion expression of secretory signal peptide + GFAPα partial sequence (43-432aa) + first linker peptide + GFAPε full sequence (1-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry to detect GFAP antibody.

[0076] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, fixed by polyethylene glycol.

[0077] Comparative Example 7:

[0078] This comparative example is the process of fusion expression of GFAP antibody by using secretory signal peptide + GFAP alpha partial sequence (43-432aa) + first linker peptide + GFAP epsilon partial sequence (225-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry.

[0079] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, fixed by polyethylene glycol.

[0080] Comparative Example 8:

[0081] This comparative example is the process of fusion expression of GFAP antibody by using secretory signal peptide + GFAP alpha partial sequence (43-432aa) + first linker peptide + GFAP epsilon partial sequence (225-431aa) + second linker peptide + CD8a hinge (183-206aa) + third linker peptide + mCherry.

[0082] The eukaryotic transient expression vector containing the above sequence was constructed, CHO and 293 cells were transiently transfected with Lipofectamine™ 3000 transfection reagent, and after 48 hours of culture, fixed by polyethylene glycol.

[0083] The fixed cells prepared in the above Example 4, Comparative Example 1 to Comparative Example 8 were added with 96 positive samples clinically diagnosed as GFAP antibody disease, respectively, for immunofluorescence detection.

[0084] The dilution ratio of serum was 1:10, 1:32, 1:100; the cerebrospinal fluid was used as a stock solution without dilution, incubated (incubated at 37 degrees Celsius for 1 hour), washed the wells with PBS for 3 times, added secondary antibody (sheep anti-human IgG, with a dilution ratio of 1:500) and incubated (at room temperature for 45 minutes), washed with PBS for 3 times, then added a small amount of PBS to soak the cells, and judged under a microscope.

[0085] The immunofluorescence detection results are shown in Figure 2 and Table 1 below: Comparison of GFAP antibody disease positive samples.

[0086] Figure 2For the immunofluorescence detection results of the positive sample in Example 4, Figure A is the red light fluorescence of the GFAPα and GFAPε fusion protein itself, B is the green fluorescence produced by the secondary antibody after the positive sample and the secondary antibody are incubated, C is the superimposed image of A and B, showing that the red and green fluorescence coincides in situ, indicating that the sample detection result is positive.

[0087] Table 1: Comparison of detection rates of anti-GFAP antibody positive samples

[0088]

[0089] The fixed cells prepared in Example 4, Comparative Example 1 to Comparative Example 8 above were added to 78 healthy human serum samples and 142 other patient serum samples and 64 cerebrospinal fluid samples that were not GFAP, respectively, and immunofluorescence detection was performed (the immunofluorescence detection method is the same as above).

[0090] The serum dilution ratio was 1:10, 1:32, 1:100; the cerebrospinal fluid was used as a stock solution without dilution, incubated (incubated at 37°C for 1 hour), washed the wells with PBS 3 times, added secondary antibody (goat anti-human IgG, with a dilution ratio of 1:500) and incubated (at room temperature for 45 minutes), after washing with PBS 3 times, a small amount of PBS was added to soak the cells, and microscopic interpretation was performed.

[0091] The immunofluorescence detection results are shown in Figure 3 and Table 2 below: Comparison of healthy people and non-GFAP other patient negative samples.

[0092] Figure 3 For the immunofluorescence detection results of the negative sample in Example 4, Figure A is the red light fluorescence of the GFAPα and GFAPε fusion protein itself, B is the negative sample without obvious fluorescence signal, C is the superimposed image of A and B, only showing red fluorescence, indicating that the sample detection result is negative.

[0093] Table 2: Comparison of healthy people and non-GFAP other patient negative samples

[0094]

[0095] In summary, the method of the present application can be used for detection of GFAP autoimmune antibodies by fusion expression of specific partial sequences of GFAPa and GFAPe, which has specificity and sensitivity, and its detection effect is better than that of Comparative Examples 1 to 8. In Comparative Examples 1 to 8, Comparative Examples 1 and 2 are detection methods of separately expressing full-length GFAP (GFAPa or GFAPe transcript), Comparative Examples 3 and 4 are methods of fusion expression of full-length GFAPa and GFAPe, and Comparative Examples 5 to 8 are fusion expression of partial or full-length sequences of GFAPa and GFAPe, but the fusion sequences are not the same as those of Example 4, which belong to the fusion sequences of the lengthening or shortening of the partial sequences of GFAPa and GFAPe in Example 4.

[0096] In summary, the present application constructs a recombinant protein for detecting autoimmune GFAP antibodies and a matching cell immunofluorescence detection system by fusion expression of specific sequences of GFAPa (43-432 aa) and GFAPe (375-431 aa). The experimental results show that the detection system shows excellent detection sensitivity in analyzing 96 GFAP antibody disease positive samples, whether in cerebrospinal fluid or in different dilutions of serum; no false positive results appear in 284 healthy people and patients with non-GFAP related diseases, showing good specificity.

[0097] Compared with other construction strategies listed in Comparative Examples 1 to 8, the fusion expression of partial sequences of GFAPa and GFAPE adopted by the present application achieves higher positive detection rate under multiple dilution conditions, and does not introduce non-specific reaction. Specifically, although Comparative Examples 1 to 8 use full-length GFAPa or GFAPe sequences for separate or co-expression, their detection sensitivity is lower than that of the present application in different sample types; although Comparative Examples 5 to 8 use partial sequences for fusion expression, due to the difference in selected sequence segments, their detection performance is also significantly weaker than that of the system constructed in Example 4 of the present application.

[0098] Therefore, the CBA method reagent and detection method for GFAP antibody detection provided by the present application are superior to the existing conventional construction system in terms of sensitivity and specificity, and are suitable for high-confidence screening and detection of GFAP autoimmune antibodies in clinical samples.

[0099] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made according to the technical essence of the present application to the above embodiment falls within the protection scope of the present application.

Claims

1. A CBA method reagent for detecting a GFAP antibody using a GFAPα and GFAPε fusion expression, characterized by: The reagent comprises a host cell recombined and constructed to express a fusion protein, which consists of, in sequence, a secretion signal peptide, a GFAP alpha partial sequence, a first linker peptide, a GFAP epsilon partial sequence, a second linker peptide, a transmembrane sequence, a third linker peptide, and a fluorescent tag, ​ The amino acid sequence of the secretion signal peptide is shown as SEQ ID NO:

3. The amino acid sequence of the GFAP alpha partial sequence is shown as SEQ ID NO:

1. The amino acid sequence of the first linker peptide is shown as SEQ ID NO:

6. The amino acid sequence of the GFAP epsilon partial sequence is shown as SEQ ID NO:

2. The amino acid sequence of the second linker peptide is shown as SEQ ID NO:

7. The transmembrane sequence is a CD8a hinge, and the amino acid sequence is shown as SEQ ID NO:

4. The amino acid sequence of the third linker peptide is shown as SEQ ID NO:

8. The fluorescent tag is mCherry, and the amino acid sequence is shown as SEQ ID NO:

5.

2. The CBA method reagent according to claim 1, characterized in that: The recombination and construction is to construct a gene encoding the fusion protein into a lentiviral expression vector, and then transfect into CHO cells.

3. A CBA assay for detecting GFAP antibodies for non-diagnostic or non-therapeutic purposes, characterized in that: The reagent of any one of claims 1 or 2 is used to detect the GFAP antibody in the sample to be tested by CBA method.

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