Use of Anti-gelsolin3 antibody related to demyelinating guillain-barrÉ syndrome

Abstract

Disclosed in the present invention is the use of an anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome. The use mainly comprises the following steps: collecting the serum from a GBS patient and a control individual, performing co-immunoprecipitation with a sciatic nerve protein to obtain an immune complex, separating and identifying the immune complex by using a mass spectrometer to obtain a candidate protein sequence, inserting a candidate protein coding gene into a vector, transfecting cells followed by immunofluorescence verification to determine the localization and expression of the candidate protein, constructing a candidate protein expression plasmid, expressing and purifying a recombinant protein in Escherichia coli, performing co-immunoprecipitation verification on the purified recombinant protein by using the serum from the GBS patient to determine the presence of an anti-Gelsolin3 IgG antibody, further verifying the expression of the anti-Gelsolin3 IgG antibody in the serum from the GBS patient by using a Western blot method, and verifying the binding of the serum from an anti-Gelsolin3 IgG antibody-positive patient to the sciatic nerve by using a single-fiber immunofluorescence method.

Description

Application of anti-Gelsolin3 antibodies in demyelinating Guillain-Barré syndrome Technical Field

[0001] This invention relates to the field of biological antibody technology, specifically to the application of anti-Gelsolin3 antibodies associated with demyelinating Guillain-Barré syndrome. Background Technology

[0002] Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy. Its common clinical manifestation is limb weakness with or without sensory disturbances. In severe cases, it can lead to respiratory muscle weakness and endanger life. The clinical diagnosis of this disease mainly relies on typical clinical manifestations, cerebrospinal fluid analysis, and electrophysiological examinations. However, some patients do not show obvious early symptoms, which leads to delayed diagnosis and affects clinical recovery. The existing clinical treatments for this disease include gamma globulin and plasma exchange. Clinical data show that only 65% ​​of patients respond to the above treatments, about 20% of patients still have varying degrees of disability after aggressive immunotherapy, and about 5% of patients die from serious complications such as lung infections. Finding early biomarkers to assist in the diagnosis of GBS is of great significance.

[0003] Based on their different pathological characteristics, GBS can be divided into demyelinating and axonal types. Current research at home and abroad has fully demonstrated that the basic mechanism of GBS pathogenesis is that autoimmune antibodies attack the myelin sheath or axon of peripheral nerves, thereby inducing complement deposition and the formation of membrane attack complexes. Antiganglioside antibodies are the most common pathogenic antibodies in GBS. Since the 1990s, a series of antiganglioside antibodies, such as antiGM1, antiGQ1b, antiGD1a, and antiGalNAc-GD1a, have been found to be closely related to the pathogenesis of axonal GBS. Among them, antiGM1, antiGD1a, and antiGalNAc-GD1a antibodies are associated with the pathogenesis of axonal GBS, while antiGQ1b antibody is associated with the pathogenesis of Miller-Fischer syndrome (a variant of GBS). The pathogenicity of these antibodies has been confirmed in relevant animal models. However, the pathogenic antibodies for demyelinating GBS are still unclear.

[0004] The search for new biomarkers and pathogenic antibodies in GBS, especially pathogenic antibodies against demyelinating GBS, has been a hot topic in research both domestically and internationally, and is also an urgent problem to be solved in the clinical diagnosis and treatment of GBS. In the past 20 years, Professor Jerome Devaux of France has detected antibodies such as anti-NF155, gliomedin, and NF186 in GBS. However, these antibodies are not specific and are also present in patients with other immune peripheral neuropathy, such as chronic inflammatory demyelinating polyneuropathy. Currently, commonly used antibody detection methods include single-fiber immunofluorescence, cell immunofluorescence, immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an application for anti-Gelsolin3 antibodies related to demyelinating Guillain-Barré syndrome. Based on a GBS patient sample library, the invention focuses on the exploration of novel biomarkers and pathogenic antibodies. When screening for pathogenic antibodies using single-fiber immunofluorescence, significant antibodies binding to the paraganglionic region of the sciatic nerve in mouse serum were found. The fluorescence distribution morphology of these antibodies was significantly different from previously discovered antibodies such as anti-NF155, gliomedin, NF186, CNTN1, and Caspr1. Using immunoprecipitation combined with mass spectrometry, 41 differentially binding antibodies to sciatic nerve proteins were identified in this patient, control patients, and normal controls. Further cellular immunoassay confirmed that the significantly binding antibody was anti-Gelsolin3. Gelsolin3 antibody was detected in the serum of GBS patients using immunoprecipitation and Western blot screening. Local injection of serum from patients carrying this antibody into the sciatic nerve of mice revealed significant immunofluorescence binding in the paranodal loop region. This antibody was not detected in normal human serum, patients with neuromyelitis optica, or controls of chronic inflammatory demyelinating polyneuropathy. Since immunofluorescence localization confirmed that Gelsolin3 is mainly located in the paranodal loop region of the myelin sheath, and the electrophysiological manifestations of patients carrying this antibody are primarily demyelinating, this confirms that anti-Gelsolin3 antibody is a pathogenic antibody for demyelinating GBS, and detection of this antibody in GBS patients can be performed.

[0006] The technical solution provided by this invention is the application of anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome. The experimental reagents include: 4% paraformaldehyde, trypsin, acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol, iodoacetamide, ultrapure water, anti-fluorescence attenuation agent for mounting, polylysine solution, gamma globulin, cholera toxin B, TBS, Tween 20, goat serum, anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, MH1031 or anti-human IgG4 antibody, ECL luminescent solution, BCA protein quantification kit, normal human serum, GBS patient and control serum, 1M Tris-HCl buffer, RIPA lysis buffer and 0.1M glycine solution.

[0007] Furthermore, the application of the anti-Gelsolin3 antibody associated with demyelinating Guillain-Barré syndrome mainly includes the following steps: collecting serum from GBS patients and control individuals, performing immunoprecipitation with sciatic nerve protein to obtain immune complexes, separating and identifying the immune complexes using mass spectrometry to obtain candidate protein sequences, inserting the candidate protein encoding gene into a vector, performing immunofluorescence verification after cell transfection to determine the location and expression of the candidate protein, constructing a candidate protein expression plasmid, expressing and purifying the recombinant protein in E. coli, performing immunoprecipitation verification on the purified recombinant protein using GBS patient serum to determine the presence of anti-Gelsolin3 IgG antibody, further verifying the expression of anti-Gelsolin3 IgG antibody in GBS patient serum using Western blot, and verifying the binding of anti-Gelsolin3 IgG antibody-positive patient serum to the sciatic nerve using single-fiber immunofluorescence.

[0008] Preferably, the specific steps for obtaining the immune complex are as follows:

[0009] S1. After anesthetizing mice, the sciatic nerve was harvested and RIPA lysis buffer (containing PMSF + cocktail protease inhibitor + phosphatase inhibitor) was added. The mixture was ground at low temperature, sonicated, and continued to lyse on ice for 20 min. The mixture was then centrifuged at 14000g for 30 min. The supernatant was transferred to a new 1.5 ml centrifuge tube and protein quantification was performed using the BCA method.

[0010] S2. Add an appropriate amount of Protein A / G Beads to a 1.5ml centrifuge tube, centrifuge at 1000rpm for 1min, discard the supernatant, and wash twice with PBS.

[0011] S3. Add 10-20 μL of serum (normal human serum, GBS patient serum, or other control serum) to Protein A / G Beads, incubate on a shaker at 4°C for 2 hours, and wash 3 times with PBS.

[0012] S4. Add sciatic nerve protein to the above mixture, incubate overnight on a shaker at 4°C, and wash 3 times with PBS;

[0013] S5. Add 2X SDS loading buffer to the above reactants, centrifuge at 1000 rpm for 1 min, take the supernatant and load it for SDS-PAGE, and stain with Coomassie Brilliant Blue.

[0014] S6. Gel Cutting and Enzymatic Hydrolysis: Cut the gel strip into small pieces. Decolorize the gel pieces using 50% acetonitrile containing 50mM ammonium bicarbonate. Dehydrate the gel pieces by incubating with 100% acetonitrile for 5 minutes. Then remove the liquid phase from the system and add a 10mM dithiothreitol solution. Incubate at 37°C for 60 minutes. Dehydrate again by incubating with 100% acetonitrile. After removing the liquid phase, add 55mM iodoacetamide and incubate at room temperature. After incubating with light for 45 minutes, the sample was washed with ammonium bicarbonate to a final concentration of 50 mM, then incubated again with 100% acetonitrile for dehydration. Finally, the gel was resuspended in 50 mM ammonium bicarbonate containing 10 ng / μl trypsin and incubated on ice for 1 hour. After removing excess solution from the sample, the gel was enzymatically digested overnight at 37°C. The peptides obtained from the digestion were extracted sequentially with 50% acetonitrile / 5% formic acid and 100% acetonitrile to extract the gel. The peptide solution was then freeze-dried for later use.

[0015] Furthermore, the specific steps for obtaining the candidate protein sequence are as follows:

[0016] K1 and peptide fragments were dissolved in mobile phase A of liquid chromatography and then separated using an EASY-nLC1000 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were as follows: 0-16 min, 9%–25% B phase; 16-22 min, 25%–40% B phase; 22-26 min, 40%–80% B phase; 26-30 min, 80% B phase. The flow rate was maintained at 450 rpm.

[0017] K2 peptides were separated by an ultra-high performance liquid chromatography (UHPLC) system and then injected into an NSI ion source for ionization before being analyzed by a Thermo Scientific™ Q Exactive mass spectrometer. The ion source voltage was set to 2.2 kV. The peptide precursor ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z with a scan resolution of 70,000; the secondary scan resolution was set to 17,500. The data acquisition mode used was a data-dependent scan (DDA) procedure, in which the top 20 peptide precursor ions with the highest signal intensity were selected after the primary scan and sequentially injected into the HCD collision cell for fragmentation at 28% of the fragmentation energy. The secondary mass spectrometry analysis was also performed sequentially. To improve the efficiency of the mass spectrometry, the automatic gain control (AGC) was set to 5e4, the signal value was set to 1e4 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 15.0 s to reduce the repeated scanning of precursor ions.

[0018] K3. Data Search: The secondary mass spectrometry data were searched using Proteome Discoverer 1.3 with the following parameters: the database was set to a combined search of Homo Sapiens (SwissProt, 20366 sequences) and Musculus (SwissProt, 17045 sequences); the restriction enzyme method was set to Trypsin / P; the missed cleavage site was set to 2; the mass error tolerance for primary precursor ions was set to 10 ppm; the mass error tolerance for secondary fragment ions was set to 0.02 Da; the fixed modification was set to cysteine ​​alkylation; the variable modification was set to methionine oxidation and protein N-terminus acetylation; the peptide ion score was required to be higher than 20; and the peptide confidence of the identification results was set to High.

[0019] Preferably, the specific steps of the immunofluorescence verification are as follows: Based on the protein localization, Gelsolin3 is selected as the candidate protein. The full-length cDNA encoding Gelsolin3 is inserted into a pcDNA3.1(+) vector containing an enhanced green fluorescent protein (EGFP) tag at the C-terminus. Human embryonic kidney 293 cells are seeded in 24-well plates with Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution and incubated for 24 hours. According to the manufacturer's instructions (Invitrogen), the cells are transfected with a plasmid expressing gelsolin3-EGFP using Lipofectamine 2000. 48 hours later, the transfected cells are fixed with 4% paraformaldehyde for 10 minutes and then incubated at room temperature with 0.5% Triton X-ray solution. After X-100 and 5% normal goat serum permeation / blocking for 20 minutes, and washing three times with PBS, the cells were incubated overnight at 4°C with diluted serum (1:100) and anti-coagulant antibody (1:250). The cells were then washed with PBS and incubated for 1 hour at room temperature with Alexa Fluor 647-conjugated goat anti-human IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG. After washing, the cells were mounted with DAPI-containing anti-fading mounting medium and images were taken using an Axio Imager Z2 microscope (Jena Zeiss, Germany).

[0020] Preferably, the specific steps for purifying the recombinant protein are as follows: First, a Gelsolin3 expression plasmid is constructed. Total RNA is extracted from the sciatic nerve of C56BL / 6J mice using TRIzol reagent (Invitrogen, CA, USA). Complementary DNA (cDNA) is synthesized using the PrimeScript™ II first-strand cDNA synthesis kit (Takara, Beijing, China). The full-length Gelsolin3 gene is amplified from the cDNA by PCR and then cloned into the pET-28a(+) vector (Novagen). This vector provides a 6×His tag for the recombinant protein at the C-terminus. The recombinant vector is then transformed into host *E. coli* BL21(DE3) for protein expression. The culture conditions are optimized, fermenting at 37°C until the OD600 reaches 0.8. Then, the cells are induced at 16°C with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 hours. The cells are harvested and suspended in a solution containing 20 mM Tris and 500 mM Tris at pH 7.5. The protein was lysed in NaCl buffer and sonicated at 200W for 30 minutes (1 second on, 2 seconds off). The lysate was centrifuged at 14000g for 20 minutes at 4°C. The supernatant was purified using a nickel chelation column (TransGen, Beijing). The protein was eluted with lysis buffer containing 500mM imidazole. The protein sample was extensively dialyzed at pH 7.5 with a buffer containing 20mM Tris, 100mM NaCl and 2mM EDTA. The protein was identified by SDS-PAGE and Western blot.

[0021] Further, the specific steps for the immunoprecipitation verification are as follows: Protein immunoprecipitation (IP) analysis is performed on purified gelsolin 3 (GSN3) protein using patient serum. The negative control group is normal serum without the target antibody, used to assess non-specific binding. First, the purified protein and serum are incubated at 4°C for 4 hours. Protein A / G magnetic beads (Smart-Lifesciences Biotechnology Co., Ltd., SA032005) are washed three times with PBS-T. Then, the antigen-antibody complex is added to the Protein A / G magnetic beads and incubated overnight at 4°C. After washing the magnetic beads five times with PBS-T, the immunoprecipitated protein is eluted with 2×SDS loading buffer, separated with 7.5% SDS-PAGE, transferred to a PVDF membrane, and then washed with 5% SDS-PAGE. The membrane was blocked with BSA for 2 hours, then incubated overnight at 4 degrees Celsius with primary antibody GSN (11644-2-AP, Proteintech, China). After washing, it was incubated at room temperature for 1 hour with enzyme-labeled goat anti-rabbit IgG (H+L) (AS014, Abclonal), and then developed with ECL luminescent solution (BKLS0100, Millipore).

[0022] Preferably, the specific steps for further verifying the expression of anti-Gelsolin3 IgG antibody in the serum of GBS patients using the Western blot method are as follows: 40 μg of purified GSN3 protein is taken, diluted with 5x loading buffer, denatured at 95°C for 5 minutes, loaded into a 7.5% polyacrylamide gel, and electrophoresed at a constant voltage of 80V. Then, the protein is transferred to a PVDF membrane at a constant current of 200mA for 100 minutes. The membrane is blocked with 5% skim milk powder in TBS-T for 2 hours. The patient serum is diluted with protein-free blocking buffer (1:50) and incubated overnight at 4°C. After washing, the membrane is coated with anti-human IgG antibody (… Anti-human IgG1 antibody (BA1070, Boster Biological, Wuhan, China), anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031 or anti-human IgG4 antibody (A-10654, Invitrogen, USA) were incubated at room temperature for 1 hour. Finally, color development was performed using ECL luminescent solution (Millipore, BKLS0100). As shown in the figure below, the expression of anti-GSN3 IgG antibody can be detected in GBS patients.

[0023] Further, the specific steps for verifying the binding of anti-Gelsolin3IgG antibody-positive patient serum to the sciatic nerve using the single-fiber immunofluorescence method are as follows: The sciatic nerve is dissected from adult C57BL / 6J mice and fixed with 4% paraformaldehyde for 40 minutes at room temperature. Then, the sciatic nerve fibers on a glass slide are combed with a fine needle. The slide is air-dried overnight at room temperature and kept at -20°C until use. For this experiment, the combed fibers are blocked for 2 hours at room temperature with PBS containing 10% normal goat serum and 0.3% Triton X-100. Then, the slide is incubated overnight at 4°C with diluted patient serum (1:200) and rabbit anti-Caspr1 (1:300) in PBS. The slide is washed and then incubated at room temperature with goat anti-human IgG conjugated to DyLight 650 (1:500, Abcam, USA) and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, USA). After incubating with Fisher for 2 hours, the slide was encapsulated with anti-fluorescence attenuation mounting medium (Solarbio) and images were taken using a laser confocal microscope. Significant fluorescence binding was observed in the paramyelin loop area of ​​the sciatic nerve fiber.

[0024] Preferably, the sciatic nerve protein is derived from mice;

[0025] Furthermore, the immunofluorescence verification was performed using human embryonic kidney 293 cells for transfection and verification;

[0026] Preferably, the Western blot method is validated using anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, and anti-human IgG4 antibody;

[0027] Preferably, the anti-Gelsolin3IgG antibody is specifically expressed in patients with Guillain-Barré syndrome (GBS).

[0028] The advantages of this invention compared to the prior art are:

[0029] A novel anti-Gelsolin3 IgG antibody was identified from a GBS patient using immunoprecipitation, mass spectrometry, cell transfection, and immunofluorescence. Then, using a dual-validation method of single-fiber immunofluorescence and Western blot, the anti-Gelsolin3 IgG antibody was detected in 17 out of 415 GBS patients, and the electrophysiological subtype of these antibody-positive patients was predominantly demyelinating. This antibody was not found in 415 normal controls or 113 patients with chronic inflammatory demyelinating polyneuropathy. Therefore, this invention discloses that the anti-Gelsolin3 IgG antibody is a newly discovered antibody expressed in demyelinating GBS patients and provides a detection method for this antibody. In particular, the single-fiber immunofluorescence method allows for the observation of significant fluorescent binding in the paramyelin loop region of the sciatic nerve fibers, providing direct evidence for the diagnosis of GBS. Attached Figure Description

[0030] Figure 1 is a schematic diagram of single-fiber immunofluorescence assay showing the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention.

[0031] Figure 2 is a schematic diagram of the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention, which is used to identify differential protein binding between GBS173, GBS225 (control) and normal control sera.

[0032] Figure 3 is a schematic diagram showing that 41 differentially expressed proteins were identified between the GBS173 and control serum groups in the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention.

[0033] Figure 4 is a schematic diagram of the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention, incubating HEK293 cells transfected with pcDNA3.1(+) vector containing full-length GSN3 cDNA and enhanced green fluorescent protein (EGFP) tag with GBS173 serum.

[0034] Figure 5. Schematic diagram of the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention, showing the incubation of frozen sections of the sciatic nerve of C57BL / 6 mice with GBS173 serum and commercial rabbit IgG anti-gelatin antibody.

[0035] Figure 6. Schematic diagram of the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome of the present invention in screening 415 GBS patients using single-fiber immunofluorescence.

[0036] Figure 7 shows the application of the anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome in this invention. For positive patients, we used Western blot to further verify the expression of the antibody and the IgG subtype.

[0037] Among them, Figure 1 shows a novel and typical binding of IgG in the para-circular area of ​​the sciatic nerve in the GBS173 patient;

[0038] Figure 2 shows total protein extracted from the sciatic nerve of C57BL / 6 mice, incubated with different sera, followed by 10% SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Gels located in the white box were collected for mass spectrometry detection of the target antigen.

[0039] Figure 3 shows Gelsolin 3 (GSN3) located in the paraganglionic region of the peripheral nerve Ranvier ganglion, which was further validated using a cell-based assay.

[0040] Figure 4 shows that immunostaining in GBS173 serum clearly binds to HEK293 cells expressing GSN3;

[0041] Figure 5 shows that immunostaining of GBS173 IgG (green) and commercial IgG anti-colloidin antibody (red) has consistent binding sites in the delusion region. Immunostaining of Caspr1 (purple) was used to locate IgG binding.

[0042] Figure 6 shows a patient who tested positive for antibodies.

[0043] Figure 7 shows that the patient was positive for total IgG and IgG1 subtype. Detailed Implementation

[0044] The substantive content of the present invention will be described in detail below with reference to the embodiments. Due to space limitations, the description of the experimental process cannot be very detailed. All parts not described in detail in the experiment are conventional operations well known to those skilled in the art.

[0045] Example: As shown in Figures 1-7, the experimental reagents in this example include: 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd., P1110), trypsin (Promega), acetonitrile (Fisher Chemical), formic acid (Fluka), ammonium bicarbonate (Sigma), dithiothreitol (Sigma), iodoacetamide (Sigma), ultrapure water, anti-fluorescence attenuation agent mounting (Abcam, USA, ab104139), polylysine solution (Sigma, USA, P8920); gamma globulin (IVIg, Hualan Biological Engineering Co., Ltd., China); cholera toxin B (Sigma, USA, C1655); TBS, Tween 20, goat serum, anti-human IgG antibody (BA1070, Boster Biological, Wuhan, China), and anti-human IgG1 antibody (A1). 0648, Invitrogen, USA), anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031 or anti-human IgG4 antibody (A-10654, Invitrogen, USA), ECL luminescent solution (Millipore, BKLS0100, USA), BCA protein quantification kit (Jiangsu Beyotime), normal human serum, GBS patient and control serum, 1M Tris-HCl buffer (pH=9.0): Beijing Solarbio Science & Technology Co., Ltd. (#T1160); RIPA lysis buffer: Beijing Solarbio Science & Technology Co., Ltd. (#R0020); 0.1M glycine solution (pH2.7): Zeye Biotechnology (#ZY61990G).

[0046] Experimental animals: 6-8 week old C57BL / 6 mice.

[0047] I. Immunoprecipitation of sciatic nerve protein in patient serum:

[0048] S1. After anesthetizing mice, the sciatic nerve was harvested and RIPA lysis buffer (containing PMSF + cocktail protease inhibitor + phosphatase inhibitor) was added. The mixture was ground at low temperature, sonicated, and continued to lyse on ice for 20 min. The mixture was then centrifuged at 14000g for 30 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube for protein quantification using the BCA method.

[0049] S2. Add an appropriate amount of Protein A / GBeads to a 1.5ml centrifuge tube, centrifuge at 1000rpm for 1min, discard the supernatant, and wash twice with PBS.

[0050] S3. Add 10-20 μL of serum (normal human serum, GBS patient serum, or other control serum) to Protein A / GBeads, incubate on a shaker at 4°C for 2 hours, and wash 3 times with PBS.

[0051] S4. Add sciatic nerve protein to the above mixture, incubate overnight on a shaker at 4°C, and wash 3 times with PBS;

[0052] S5. Add 2XSDS loading buffer to the above reactants, centrifuge at 1000 rpm for 1 min, take the supernatant and load it for SDS-PAGE, and stain with Coomassie Brilliant Blue.

[0053] S6. Gel Cutting and Enzymatic Hydrolysis: Cut the gel strip into small pieces. Decolorize the gel pieces using 50% acetonitrile containing 50mM ammonium bicarbonate. Dehydrate the gel pieces by incubating with 100% acetonitrile for 5 minutes. Then remove the liquid phase from the system and add a 10mM dithiothreitol solution. Incubate at 37°C for 60 minutes. Dehydrate again by incubating with 100% acetonitrile. After removing the liquid phase, add 55mM iodoacetamide and incubate at room temperature. Incubate with light for 45 minutes; then wash with ammonium bicarbonate to a final concentration of 50 mM, and incubate again with 100% acetonitrile for dehydration. Finally, resuspend the gel block with 50 mM ammonium bicarbonate containing 10 ng / μl trypsin, incubate on ice for 1 hour, remove excess solution from the sample, and enzymatically digest the gel block overnight at 37°C. Extract the peptide fragments from the gel block sequentially with 50% acetonitrile / 5% formic acid and 100% acetonitrile. Freeze-dry the peptide solution for later use.

[0054] II. Mass Spectrometry Identification:

[0055] The analysis was performed using a Thermo Scientific TMO Exactive mass spectrometer, and the specific procedure is as follows:

[0056] K1 and peptide fragments were dissolved in mobile phase A of liquid chromatography and then separated using an EASY-nLC1000 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings were as follows: 0-16 min, 9%–25% phase B; 16-22 min, 25%–40% phase B; 22-26 min, 40%–80% phase B; 26-30 min, 80% phase B. The flow rate was maintained at 450 rpm.

[0057] K2 peptides were separated by an ultra-high performance liquid chromatography (UHPLC) system and then injected into an NSI ion source for ionization before being analyzed by a ThermoScientificTMQExactive mass spectrometer. The ion source voltage was set to 2.2 kV. The peptide precursor ions and their secondary fragments were detected and analyzed using high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z with a scan resolution of 70,000; the secondary scan resolution was set to 17,500. The data acquisition mode used was a data-dependent scan (DDA) procedure, in which the top 20 peptide precursor ions with the highest signal intensity were selected after the primary scan and sequentially injected into the HCD collision cell for fragmentation at 28% of the fragmentation energy. The secondary mass spectrometry analysis was performed sequentially. To improve the efficiency of the mass spectrometry, the automatic gain control (AGC) was set to 5e4, the signal value was set to 1e4 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 15.0 s to reduce the repeated scanning of precursor ions.

[0058] K3. Data Search: Secondary mass spectrometry data were searched using ProteomeDiscoverer 1.3 with the following parameters: Database: HomoSapiens (SwissProt, 20366 sequences) and Musculus (SwissProt, 17045 sequences) combined for search; Enzyme digestion method: Trypsin / P; Missed cleavage site: 2; Primary precursor ion mass error tolerance: 10 ppm; Secondary fragment ion mass error tolerance: 0.02 Da; Fixed modification: Cysteine ​​alkylation; Variable modification: Methionine oxidation and N-terminal acetylation; Peptide ion score requirement: higher than 20; Peptide confidence: High.

[0059] III. Verification of candidate proteins using cell immunofluorescence;

[0060] Based on protein localization, Gelsolin3 was selected as a candidate protein. The full-length cDNA encoding Gelsolin3 was inserted into a pcDNA3.1(+) vector containing an enhanced green fluorescent protein (EGFP) tag at the C-terminus. Human embryonic kidney 293 cells were seeded in 24-well plates with Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution and incubated for 24 hours. Following the manufacturer's instructions (Invitrogen), the cells were transfected with the gelsolin3-EGFP plasmid using Lipofectamine 2000. After 48 hours of cell staining, the transfected cells were fixed with 4% paraformaldehyde for 10 minutes and then permeated / blocked with 0.5% Triton X-100 and 5% normal goat serum for 20 minutes at room temperature. After washing three times with PBS, the cells were incubated overnight at 4°C with diluted serum (1:100) and anti-coagulant antibody (1:250). The cells were then washed with PBS and incubated for 1 hour at room temperature with AlexaFluor 647-conjugated goat anti-human IgG and AlexaFluor 568-conjugated goat anti-rabbit IgG. After washing, the cells were mounted with DAPI-containing anti-fading mounting medium and images were taken using an AxioImager Z2 microscope (Jena Zeiss, Germany).

[0061] IV. Expression and purification of recombinant Gelsolin3:

[0062] First, a Gelsolin3 expression plasmid was constructed. Total RNA was extracted from the sciatic nerve of C56BL / 6J mice using TRIzol reagent (Invitrogen, CA, USA). Complementary DNA (cDNA) was synthesized using the PrimeScript™ II first-strand cDNA synthesis kit (Takara, Beijing, China). The full-length Gelsolin3 gene was amplified from the cDNA by PCR and then cloned into the pET-28a(+) vector (Novagen). This vector provided a 6×His tag for the recombinant protein at the C-terminus. The recombinant vector was then transformed into host *E. coli* BL21(DE3) for protein expression. Culture conditions were optimized, and fermentation was carried out at 37°C until OD600 reached a certain value. Cells were induced at 16°C with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h. Cells were harvested and resuspended in a buffer containing 20 mM Tris and 500 mM NaCl at pH 7.5. Lysis was performed by sonication at 200 W for 30 min (1 second on, 2 seconds off). The lysate was centrifuged at 14000 g for 20 min at 4°C. The supernatant was purified using a nickel chelation column (TransGen, Beijing). Proteins were eluted with a lysis buffer containing 500 mM imidazole. Protein samples were extensively dialyzed at pH 7.5 with a buffer containing 20 mM Tris, 100 mM NaCl, and 2 mM EDTA. Proteins were identified by SDS-PAGE and Western blot.

[0063] V. Immunoprecipitation to verify the expression of anti-Gelsolin 3 IgG antibodies in the serum of GBS patients:

[0064] Protein immunoprecipitation (IP) analysis was performed on purified gelsolin 3 (GSN3) protein using patient serum. The negative control group consisted of normal serum without the target antibody, used to assess nonspecific binding. First, the purified protein and serum were incubated at 4°C for 4 hours. Protein A / G magnetic beads (Smart-LifesciencesBiotechnology Co., Ltd., SA032005) were washed three times with PBS-T. Then, the antigen-antibody complex was added to the Protein A / G magnetic beads and incubated at 4°C. After incubation overnight, the magnetic beads were washed 5 times with PBS-T, and the immunoprecipitated proteins were eluted with 2×SDS loading buffer. The mixture was then separated with 7.5% SDS-PAGE, transferred to a PVDF membrane, blocked with 5% BSA for 2 hours, and then incubated overnight at 4°C with primary antibody GSN (11644-2-AP, Proteintech, China). After washing, the mixture was incubated at room temperature for 1 hour with enzyme-labeled goat anti-rabbit IgG (H+L) (AS014, Abclonal), and then developed with ECL luminescent solution (BKLS0100, Millipore).

[0065] VI. Western blot verification of anti-Gelsolin 3 IgG antibody expression in the serum of GBS patients:

[0066] Take 40 μg of purified GSN3 protein, dilute with 5x loading buffer, denature at 95℃ for 5 min, load onto a 7.5% polyacrylamide gel and perform electrophoresis at a constant voltage of 80V. Then transfer the protein to a PVDF membrane at a constant current of 200mA for 100 min. Block the membrane with 5% skim milk powder in TBS-T for 2 hours. Incubate overnight at 4℃ with patient serum diluted 1:50 with protein-free blocking buffer. After washing, the membrane is coated with anti-human IgG antibody (BA1070, Boster, Wuhan, China) and anti-human IgG1 antibody (A10648). The antibody was incubated at room temperature for 1 hour with either anti-human IgG2 antibody (MH1722, Invitrogen, USA), anti-human IgG3 antibody (MA5-42730, Invitrogen, USA), MH1031, or anti-human IgG4 antibody (A-10654, Invitrogen, USA). Finally, the antibody was developed using ECL luminescent solution (Millipore, BKLS0100), as shown in the figure below. The expression of anti-GSN3 IgG antibody can be detected in GBS patients.

[0067] VII. Single-fiber immunofluorescence assay to verify the binding of serum from patients with positive anti-Gelsolin 3 IgG antibodies to the sciatic nerve:

[0068] The sciatic nerve was dissected from adult C57BL / 6J mice and fixed with 4% paraformaldehyde for 40 minutes at room temperature. The sciatic nerve fibers on a slide were then combed with a fine needle. The slide was air-dried overnight at room temperature and kept at -20°C until use. For this experiment, the combed fibers were blocked for 2 hours at room temperature with PBS containing 10% normal goat serum and 0.3% Triton X-100. The slides were then incubated overnight at 4°C with diluted patient serum (1:200) and rabbit anti-Caspr1 (1:300) in PBS. The slides were washed and incubated for 2 hours at room temperature with goat anti-human IgG conjugated to DyLight 650 (1:500, Abcam, USA) and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000, Thermo Fisher, USA). The slides were then mounted with anti-fluorescence attenuation mounting medium (Solarbio) and images were taken using a laser confocal microscope. Significant fluorescent binding was observed in the paramyeloid ring region of the sciatic nerve fibers.

[0069] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. In conclusion, if those skilled in the art are inspired by this description and design similar embodiments without departing from the spirit of the present invention, such embodiments should fall within the protection scope of the present invention.

Claims

1. Use of an anti-Gelsolin 3 antibody associated with a demyelinating Guillain-Barre syndrome, characterized in that, The main steps include: collecting serum from GBS patients and control individuals, performing immunoprecipitation with sciatic nerve protein to obtain immune complexes, separating and identifying the immune complexes using mass spectrometry to obtain candidate protein sequences, inserting the candidate protein encoding gene into a vector, performing immunofluorescence verification after cell transfection to determine the location and expression of the candidate protein, constructing a candidate protein expression plasmid, expressing and purifying the recombinant protein in E. coli, performing immunoprecipitation verification on the purified recombinant protein using GBS patient serum to determine the presence of anti-Gelsolin3 IgG antibody, further verifying the expression of anti-Gelsolin3 IgG antibody in GBS patient serum using Western blot, and verifying the binding of anti-Gelsolin3 IgG antibody-positive patient serum to the sciatic nerve using single-fiber immunofluorescence.

2. The use of an anti-Gelsolin 3 antibody associated with a demyelinating form of Guillain-Barre syndrome according to claim 1, characterized in that, The sciatic nerve protein was derived from mice.

3. The use of an anti-Gelsolin 3 antibody associated with demyelinating Guillain-Barre syndrome according to claim 1, characterized in that, The mass spectrometer in question is a Thermo Scientific TMO Exactive mass spectrometer.

4. The use of an anti-Gelsolin 3 antibody associated with a demyelinating form of Guillain-Barre syndrome according to claim 1, characterized in that, The immunofluorescence verification was performed using human embryonic kidney 293 cells for transfection and verification.

5. The use of an anti-Gelsolin 3 antibody associated with a demyelinating form of Guillain-Barre syndrome according to claim 1, characterized in that, The Western blot method was validated using anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, and anti-human IgG4 antibody.

6. The use of an anti-Gelsolin 3 antibody associated with a demyelinating form of Guillain-Barre syndrome according to claim 1, characterized in that, The anti-Gelsolin3 IgG antibody is specifically expressed in patients with Guillain-Barré syndrome (GBS).

7. The use of a demyelinating Guillain-Barre syndrome-associated anti-Gelsolin 3 antibody according to claim 1, characterized in that, The single-fiber immunofluorescence method is used for image acquisition and verification using a laser confocal microscope.

8. An experimental reagent, characterized in that, The aforementioned experimental reagent is used for the application of anti-Gelsolin3 antibodies related to demyelinating Guillain-Barré syndrome. The experimental reagent comprises 4% paraformaldehyde, trypsin, acetonitrile, formic acid, ammonium bicarbonate, dithiothreitol, iodoacetamide, ultrapure water, anti-fluorescence attenuation agent for mounting, polylysine solution, gamma globulin, cholera toxin B, TBS, Tween 20, goat serum, anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, MH1031 or anti-human IgG4 antibody, ECL luminescent solution, BCA protein quantification kit, normal human serum, GBS patient and control serum, 1M Tris-HCl buffer, RIPA lysis buffer, and 0.1M glycine solution.

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