An anti-human gfap monoclonal antibody and products and uses based thereon

Abstract

The application discloses an anti-human GFAP monoclonal antibody and products and applications based thereon, and relates to a monoclonal cell strain capable of continuously producing the anti-human GFAP monoclonal antibody, which is obtained by immunizing mice with a full-length human GFAP protein expressed by E. coli as an antigen, and then performing fusion and several times of cloning, and the monoclonal antibody is secreted by the cell strain. The antibody comprises a light chain and a heavy chain, the light chain belongs to kappa, and the heavy chain belongs to IgG2b; wherein: the amino acid sequences of three complementarity determining regions of the light chain variable region are respectively shown as SEQ ID No. 1, SEQ ID No. 18 and SEQ ID No. 2; and the amino acid sequences of three complementarity determining regions of the heavy chain variable region are respectively shown as SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11. The monoclonal antibody can specifically recognize a target antigen human GFAP protein, has high affinity and sensitivity, and can be used for detection of human GFAP and development of an in vitro diagnostic kit.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody technology, specifically relating to a monoclonal antibody against glial fibrillary acidic protein (GFAP), products based thereon, and their applications. Background Technology

[0002] GFAP is an important intermediate filament protein in cells, participating in the formation of the cytoskeleton of glial cells and closely related to astrocyte regeneration and synaptic reconstruction. At the same time, GFAP plays an important role in central nervous system diseases such as neurodegenerative diseases, traumatic brain injury, and stroke.

[0003] Alzheimer's disease is a slowly progressing but irreversible neurological disorder, and it is also the most common neurodegenerative disease. Because glial cell damage is inevitable during the progression of Alzheimer's disease, and GFAP was initially considered a specific marker of astrocytes, when these cells are damaged, GFAP leaks out in large quantities from the ruptured cells into the cerebrospinal fluid and crosses the blood-brain barrier into the peripheral circulation system.

[0004] Traumatic brain injury (TBI) is the most dangerous type of brain injury, often leading to very severe and persistent damage, and is the most common cause of death after accidents such as car crashes. Current evidence suggests that changes in peripheral blood GFAP levels can more sensitively (compared to CT scans) identify the presence and extent of brain injury, and that dynamic monitoring of GFAP levels in the blood can predict disease progression and patient prognosis.

[0005] Stroke is classified into hemorrhagic stroke and ischemic stroke. The causes and initial treatment methods differ, making rapid identification of the type after stroke symptoms appear crucial. Studies show that GFAP release occurs earlier in hemorrhagic strokes. In hemorrhagic strokes, blood GFAP levels begin to rise significantly 3-4 hours after symptom onset, while in ischemic strokes, the increase is more delayed, generally occurring 24-48 hours after onset.

[0006] Although anti-GFAP antibodies have been reported, reagents / kits for detecting GFAP using these antibodies suffer from insufficient sensitivity or low specificity. Therefore, highly sensitive and specific methods for detecting GFAP levels are urgently needed. Highly specific and high-affinity anti-GFAP antibodies and antibody pairs are key to developing GFAP immunoassay reagents and kits. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the present invention aims to provide an anti-human GFAP monoclonal antibody, products based thereon, and applications, so as to solve the problem that the reagents / kits for detecting GFAP in the prior art have insufficient sensitivity or low specificity.

[0008] To achieve the above objectives, the anti-human GFAP monoclonal antibody (named 2E9-10B8) provided by the present invention comprises a light chain and a heavy chain, wherein the light chain belongs to kappa and the heavy chain belongs to IgG2b; the three complementarity-determining regions LCDR1, LCDR2 and LCDR3 of the variable region of the light chain of the monoclonal antibody 2E9-10B8 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 18 and SEQ ID No. 2, respectively; and the three complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the variable region of the heavy chain have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

[0009] Furthermore, the framework regions LFR1, LFR2, LFR3, and LFR4 of the light chain variable region of the monoclonal antibody 2E9-10B8 have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively, and the framework regions HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively.

[0010] Furthermore, the light chain variable region of the monoclonal antibody 2E9-10B8 has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 7 or the nucleotide sequence shown in SEQ ID No. 8, preferably 95% sequence identity; the heavy chain variable region has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 16 or the nucleotide sequence shown in SEQ ID No. 17, preferably 95% sequence identity.

[0011] This invention provides a product for detecting GFAP levels, comprising:

[0012] a) Nucleic acid molecule: The nucleic acid molecule encodes monoclonal antibody 2E9-10B8 or a functional fragment thereof;

[0013] b) Recombinant expression vector: the recombinant expression vector comprises the nucleic acid molecule described in a);

[0014] c) Host cell: The host cell contains the recombinant expression vector described in b).

[0015] Furthermore, the recombinant expression vector has a signal peptide operatively linked to the antibody and contains a transcriptional regulatory element; the host cell is selected from mammalian cells, such as 293T cells, CHO cells, or Expi293F cells. TM cell.

[0016] Furthermore, the product also includes reagents for performing antigen-antibody reactions or reagents for detecting reactions.

[0017] Furthermore, reagents used to perform antigen-antibody reactions include buffers, salts, diluents, etc.

[0018] Furthermore, the product includes detection reagents, kits, or test strips, wherein the kits include: colloidal gold immunoassay kits, chemiluminescence immunoassay kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits, fluorescence immunoassay kits, and microfluidic chips.

[0019] The method for preparing the monoclonal antibody 2E9-10B8 of the present invention is as follows: culturing the above-mentioned host cells, and isolating and purifying the monoclonal antibody from the host cells and / or the culture medium in which the host cells are grown.

[0020] This invention also provides the application of the above-mentioned product for detecting GFAP levels in the preparation of products for diagnosing GFAP-related diseases, characterized in that the product includes diagnostic reagents, kits, or diagnostic test strips. The diseases related to GFAP protein are any one of Alzheimer's disease, traumatic brain injury, and stroke.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention uses recombinant human full-length GFAP protein expressed in *E. coli* as the antigen to immunize mice. Through cell fusion and several cloning processes, a monoclonal cell line that continuously secretes anti-human GFAP monoclonal antibodies was obtained. The anti-human GFAP monoclonal antibody 2E9-10B8 was obtained through the secretion of this cell line. The anti-human GFAP monoclonal antibody of this invention has high affinity and sensitivity, and can specifically recognize the target antigen GFAP. It can be used for the detection of human GFAP and the development of in vitro diagnostic reagents and kits. Attached Figure Description

[0023] Figure 1 These are the results of tail blood titer assays in GFAP-immunized mice. The treatment mouse was a GFAP-immunized mouse, and the control mouse was a normal mouse.

[0024] Figure 2 These are the electrophoresis results of monoclonal antibody 2E9-10B8; M is the marker; 1-4 and 5-8 are the ascites fluid and purification results of two mice, respectively, where 1 and 5 are ascites fluid; 2 and 6 are flow-through; 3 and 7 are after purification; 4 and 8 are after purification concentration.

[0025] Figure 3 This is the result of the identification of the monoclonal antibody 2E9-10B8 subtype.

[0026] Figure 4 This is the result of ELISA verification of the specificity of monoclonal antibody 2E9-10B8.

[0027] Figure 5 The results are from immunofluorescence detection of monoclonal antibody 2E9-10B8.

[0028] Figure 6 This is an ELISA graph showing the binding activity of monoclonal antibody 2E9-10B8. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0030] Example 1

[0031] 1. Preparation of GFAP

[0032] The human GFAP gene sequence was synthesized into the PCMV vector, and then the target gene was cloned into the PET-24a vector. The plasmid was extracted, identified by double enzyme digestion, and sequenced for alignment. The constructed prokaryotic expression plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium, incubated at 37°C until the OD value reached 0.5–1, and then IPTG was added and incubated overnight at 28°C to induce expression. The bacterial cells were collected, sonicated, centrifuged, and the supernatant was collected. The protein purified by Ni column affinity chromatography was dialyzed and concentrated to obtain a high concentration of GFAP.

[0033] 2. Obtaining hybridoma cells

[0034] Step 1: Immunize mice with GFAP

[0035] Three female Balb / c mice aged 5–8 weeks were used. After one week of acclimatization, 50 μg of GFAP was dissolved in 500 μL of PBS and mixed with Freund's complete adjuvant at a 1:1 volume ratio. The mixture was then emulsified to a water-in-oil state and injected subcutaneously at four points to complete the first immunization. Three weeks later, 50 μg of GFAP was dissolved in 500 μL of PBS and mixed with Freund's incomplete adjuvant at a 1:1 volume ratio. The mixture was then emulsified to a water-in-oil state and injected subcutaneously at four points to complete the second immunization. Three weeks after that, 50 μg of GFAP was dissolved in 500 μL of PBS and injected intraperitoneally to complete the third immunization.

[0036] Step 2: ELISA detection of tail tip blood titer in immunized mice

[0037] Three weeks after the third immunization, tail blood was collected from mice to collect serum, and its titer was detected by ELISA. The specific steps were as follows: GFAP was diluted to 1 ng / μL and added to the ELISA plate, 100 μL / well, and incubated overnight at 4°C; washed once with TBST for 2 min each time, and patted dry; 200 μL of 3% BSA-TBST was added to the well and blocked at 37°C for 1 h; washed twice with TBST for 2 min each time, and patted dry; 100 μL of serially diluted mouse serum was added to the well and incubated at 37°C for 1 h; washed three times with TBST for 2 min each time, and patted dry; 100 μL of goat anti-mouse IgG-HRP was added to the well and incubated at 37°C for 40 min; washed five times with TBST for 2 min each time, and patted dry; 100 μL of chromogenic solution was added to the well and incubated at room temperature for 10 min, then H2SO4 was added to stop the incubation. The absorbance was measured on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. Figure 1 The antibody titer test results of the immunized mice used in subsequent experiments are presented. The results show that, compared to unimmunized normal mice, the antibody titer of the immunized mice was higher than 1:128,000. This result indicates that the immunized mice developed an immune response to the immunogen GFAP and can be used for subsequent experiments.

[0038] Step 3: Cell Fusion

[0039] SP2 / 0 myeloma cells were acclimated to 20% fetal bovine serum for one week. One immunized mouse was given a shock immunization three days prior to fusion, with 50 μg GFAP mixed with 500 μL PBS and injected intraperitoneally. On the day of fusion, the mouse was sacrificed, and the spleen was aseptically harvested, ground, and washed twice with serum-free medium. Spleen cells were mixed with SP2 / 0 cells at a ratio of 10:1, centrifuged at 1200 rpm for 6 min, the supernatant was discarded, and the bottom of the centrifuge tube was gently tapped to loosen the cell pellet. Within 30 seconds, 1 mL of 45% PEG solution preheated to 37°C was slowly added. After standing at room temperature for 90 seconds, incomplete medium preheated to 37°C was slowly added, mixed, and centrifuged at 800 rpm for 6 min. The supernatant was discarded, and 40 mL of complete medium preheated to 37°C was added, mixed, and then transferred to a 96-well cell culture plate inoculated with peritoneal macrophages. The plate was then incubated at 37°C in a 5% CO2 incubator. HAT and HT were added every other day from day 2 to day 7 after fusion to screen fused cells.

[0040] Step 4: ELISA detection of positive hybridoma cells and cloning of hybridoma cells

[0041] On the 7th day after fusion, the growth of hybridoma cells was observed. When the cells in the well exceeded 1 / 3 of the bottom of the well, the secretion of hybridoma antibodies was detected. The detection steps were as follows: GFAP was diluted and added to the ELISA plate at 100 μL / well, and incubated overnight at 4°C; TBST was used to wash once, 2 min each time, and the plate was patted dry; 3% BSA-TBST was added at 200 μL / well, and the plate was blocked at 37°C for 1 h; TBST was used to wash twice, 2 min each time, and the plate was patted dry; hybridoma cell supernatant was added at 100 μL / well, and the plate was incubated at 37°C for 1 h; TBST was used to wash three times, 2 min each time, and the plate was patted dry; goat anti-mouse IgG-HRP was added at 100 μL / well, and the plate was incubated at 37°C for 40 min; TBST was used to wash five times, 2 min each time, and the plate was patted dry; chromogenic solution was added at 100 μL / well, and the plate was incubated at room temperature for 10 min, then H2SO4 was added to stop the incubation. The absorbance was measured on a microplate reader at a wavelength of 450 nm, and the reference wavelength was 630 nm. Clones with high OD values ​​were selected for subcloning, and ELISA screening was performed again after 7–10 days. After three cloning processes, one positive clone was finally selected and named hybridoma cell 10B8.

[0042] 3. Preparation of monoclonal antibody 2E9-10B8 from ascites fluid

[0043] 500 μL of ascites adjuvant was injected into the peritoneal cavity of 10-week-old Balb / c mice. Hybridoma cells were cultured in 10B8 culture to the logarithmic growth phase, and the cell density was adjusted to 2 × 10⁸. 6 The ascites fluid was collected 7 days after injection of the ascites adjuvant, followed by intraperitoneal injection of 500 μL, and then collected 7 days later.

[0044] 4. Purification of monoclonal antibody 2E9-10B8

[0045] The collected ascites fluid was diluted with binding / wash buffer (pH 7.0), centrifuged, and the supernatant was added. Protein A was added, mixed, and incubated overnight at 4°C on a shaker. The chromatography column was equilibrated twice with binding / wash buffer. The overnight binding buffer was added to the column, and after the protein A beads settled to the bottom, the column was washed twice with binding / wash buffer. The antibody bound to protein A was then eluted with elution buffer (pH 3.0), and 1 / 10 volume of neutralizing buffer (pH 8.5) was immediately added to the eluent. The mixture was mixed, and the resulting elution and neutralization buffer was concentrated using an ultrafiltration tube. The concentrated elution and neutralization buffer was then gradually replaced with PBS (pH 7.2) to obtain monoclonal antibody 2E9-10B8. After determining the concentration using the BCA method, the antibody was simultaneously subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining with the original ascites fluid. The results are as follows: Figure 2 As shown, the heavy and light chain bands of the antibody can be clearly seen, and the amount of impurities is significantly reduced after purification, indicating a significant improvement in antibody purity.

[0046] The monoclonal antibody 2E9-10B8 obtained above was identified and its performance was tested. The specific experiments are as follows:

[0047] 1. Identification of monoclonal antibody 2E9-10B8 subtype

[0048] Add 100 ng GFAP / well to the ELISA plate and coat overnight at 4°C; wash once with TBST for 2 min each time, then blot dry; add 200 μL 3% BSA-TBST / well and block at 37°C for 1 h; wash twice with TBST for 2 min each time, then blot dry; add 100 μL hybridoma cell supernatant / well and incubate at 37°C for 1 h; wash three times with TBST for 2 min each time, then blot dry; add 100 μL HRP-labeled rabbit anti-mouse secondary antibody (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, kappa, lambda) / well and incubate at 37°C for 40 min; wash five times with TBST for 2 min each time, then blot dry; add 100 μL chromogenic solution / well and incubate at room temperature for 10 min, then stop with H2SO4. Measure the absorbance on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. Experimental results are as follows Figure 3 As shown, the OD values ​​of 2E9-10B8 IgG2b and kappa types are significantly higher than those of other subtypes and the negative control. Therefore, the heavy chain of the monoclonal antibody 2E9-10B8 is of the IgG2b type, and the light chain is of the kappa type.

[0049] 2. ELISA verification of the specificity of monoclonal antibody 2E9-10B8

[0050] The purified GFAP from Example 1 and the laboratory-available NFL and BSA were diluted to 1 μg / mL with coating buffer and added to ELISA plates, along with the diluent, at a concentration of 100 μL / well. The plates were incubated overnight at 4°C. The plates were washed once with TBST for 2 min each time and patted dry. 3% GFAP was then added to the ELISA plates. BSA-TBST, 200 μL / well, blocked at 37℃ for 2 h; TBST washed twice, 2 min each time, then patted dry; 100 μL / well of hybridoma cell supernatant added, incubated at 37℃ for 1 h; TBST washed three times, 2 min each time, then patted dry; goat anti-mouse IgG-HRP added, 100 μL / well, incubated at 37℃ for 40 min; TBST washed five times, 2 min each time, then patted dry; chromogenic solution added, 100 μL / well, incubated at RT for 10 min, then H2SO4 added to stop the incubation. Absorbance was measured on a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. Results are shown in Table 1 and [Table data missing]. Figure 4 .

[0051] Table 1. ELISA validation of the specificity of monoclonal antibody 2E9-10B8

[0052]

[0053]

[0054] Table 1 and Figure 4 The experimental results show that the monoclonal antibody 2E9-10B8 can specifically recognize the target antigen GFAP.

[0055] 3. Application of monoclonal antibody 2E9-10B8 in immunofluorescence detection

[0056] U251 and U87 cells were seeded onto slides, fixed with paraformaldehyde, washed three times with TBST, and then incubated at room temperature for 1 h with 5% BSA-TBST (containing 0.1% Triton X-100). After washing once with TBST, the slides were incubated at room temperature for 2 h with diluted monoclonal antibody 2E9-10B8. After washing three times with TBST, the slides were incubated at room temperature for 1 h with Alexa Fluor 549-labeled goat anti-mouse IgG antibody. After washing with TBST, the slides were mounted with mounting medium containing DAPI. The staining results were observed under a fluorescence microscope. Figure 5 The experimental results showed that after incubation with antibodies, significant red coloration could be observed under a microscope in both cell types. Figure 5 GFAP) and DAPI-stained blue cell nuclei ( Figure 5DAPI (in Chinese), and the two can overlap well ( Figure 5 The results show that the monoclonal antibody 2E9-10B8 can specifically stain for GFAP (DAPI / GFAP), and can be used to detect GFAP in immunofluorescence staining samples.

[0057] 4. ELISA detection of monoclonal antibody 2E9-10B8 binding activity

[0058] GFAP was coated onto ELISA plates at 0.1 μg / well and incubated overnight at 4°C. The plates were washed once with TBST for 2 min each time and then patted dry. 200 μL of 3% BSA-TBST was added to each well, and the plates were blocked at 37°C for 2 h. The plates were then washed twice with TBST for 2 min each time and then patted dry. Serially diluted monoclonal antibody 2E9-10B8 was added and incubated at 37°C for 1 h. The plates were washed three times with TBST for 2 min each time and then patted dry. 100 μL of goat anti-mouse IgG-HRP was added to each well, and the plates were incubated at 37°C for 40 min. The plates were washed five times with TBST for 2 min each time and then patted dry. 100 μL of chromogenic reagent was added to each well, and the plates were incubated at RT for 10 min. H₂SO₄ was added to stop the incubation. The absorbance was measured using a microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm. Figure 6 The experimental results showed that the monoclonal antibody 2E9-10B8 could bind well to GFAP protein in a concentration-dependent manner, and EC50 was significantly reduced. 50 It is 0.0546 μg / mL.

[0059] 5. Sequencing of monoclonal antibody 2E9-10B8

[0060] Total RNA was extracted from hybridoma cells and reverse transcribed to obtain cDNA. The variable regions of the antibody heavy and light chains were amplified using the 5' RACE method. The amplified fragments were subcloned into the pEASY-Blunt vector, and the plasmid was extracted and sequenced to obtain the sequenced light and heavy chain sequences of the monoclonal antibody 2E9-10B8. The complementarity-determining regions of the antibody amino acid sequence were labeled using the Kabat method.

[0061] The amino acid sequence of the light chain variable region of monoclonal antibody 2E9-10B8 is shown in SEQ ID No. 7, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ ID No. 1, SEQ ID No. 18, and SEQ ID No. 2, respectively, and the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively. The gene sequence is shown in SEQ ID No. 8.

[0062] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2E9-10B8 is shown in SEQ ID No. 16, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11, respectively; and the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 are shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively. The gene sequence is shown in SEQ ID No. 17.

[0063] SEQ ID No. 1: KSVSTSGYSY

[0064] SEQ ID No. 2: QHILCLTR

[0065] SEQ ID No.3: DIVLTQSPASLAVSLGQRATISYRAS

[0066] SEQ ID No.4:MHWNQQKPGQPPRLLIY

[0067] SEQ ID No.5: NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC

[0068] SEQ ID No. 6: SEGGPSWKN

[0069] SEQ ID No.7: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQ QKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIL CLTRSEGGPSWKN

[0070] SEQ ID No.8:gacattgtgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcat acagggccagcaaaagtgtcagtacatctggctatagttatatgcactggaaccaacagaaaccaggacagccacccagactcctcatctatcttgtatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattctctgccttacacgttcggaggggggaccaagctggaaaaac

[0071] SEQ ID No.9:GFSLTSYD

[0072] SEQ ID No.10:IWTGGGT

[0073] SEQ ID No.11:VRRFTAMDY

[0074] SEQ ID No.12:QVQLKESGPGLVAPSQSLSITCTVS

[0075] SEQ ID No.13:ISWIRQPPGKGLEWLGV

[0076] SEQ ID No.14:NYNSAFMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYC

[0077] SEQ ID No.15:WGQGTSVTVSS

[0078] SEQ ID No.16:QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYDISWIRQPPG KGLEWLGVIWTGGGTNYNSAFMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYC VRRFTAMDYWGQGTSVTVSS

[0079] SEQ ID No.17: caggtgcaactgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattac ctgcactgtctctgggttctcattaaccagctatgatataagctggattcgccagccaccaggaaagggtctggagtggcttggagtaatatggactggtggaggcacaaattataattcagctttcatgtccagactgag catcagcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatattactgtgtaagacggtttactgctatggactactggggtcaaggaacctcagtcaccgtctcctca

[0080] SEQ ID No. 18: LVS

[0081] In summary, the monoclonal antibody 2E9-10B8 provided in this invention has good binding activity with GFAP and high specificity, and can be used for the detection of GFAP.

Claims

1. A monoclonal antibody against human GFAP, characterized in that, It includes light chains and heavy chains; the light chains belong to kappa, and the heavy chains belong to IgG2b; among which: The amino acid sequences of the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID No. 1, SEQ ID No. 18, and SEQ ID No. 2, respectively. The amino acid sequences of the three complementarity-determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11, respectively.

2. The anti-human GFAP monoclonal antibody according to claim 1, characterized in that, The framework regions LFR1, LFR2, LFR3, and LFR4 of the light chain variable region of the anti-human GFAP monoclonal antibody have at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively. The heavy chain variable region of the anti-human GFAP monoclonal antibody, specifically the framework regions HFR1, HFR2, HFR3, and HFR4, has at least 90% sequence identity with the amino acid sequences shown in SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively.

3. The anti-human GFAP monoclonal antibody according to claim 2, characterized in that, The light chain variable region of the anti-human GFAP monoclonal antibody has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No. 7; the heavy chain variable region of the anti-human GFAP monoclonal antibody has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.

16.

4. The anti-human GFAP monoclonal antibody according to claim 3, characterized in that, The light chain variable region of the anti-human GFAP monoclonal antibody has 95% sequence identity with the amino acid sequence shown in SEQ ID No. 7; the heavy chain variable region of the anti-human GFAP monoclonal antibody has 95% sequence identity with the amino acid sequence shown in SEQ ID No.

16.

5. A product for detecting GFAP levels, characterized in that, include: a) Nucleic acid molecule: the nucleic acid molecule encodes the monoclonal antibody according to any one of claims 1 to 4; b) Recombinant expression vector: the recombinant expression vector comprises the nucleic acid molecule described in a); c) Host cell: The host cell contains the recombinant expression vector described in b).

6. The product for detecting GFAP levels according to claim 5, characterized in that, The recombinant expression vector has a signal peptide linked to an antibody and contains a transcriptional regulatory element; the host cell is any one of 293T cells, CHO cells, or Expi293F cells.

7. The product for detecting GFAP levels according to claim 5, characterized in that, The product also includes reagents for performing antigen-antibody reactions or for detecting reactions; the reagents for performing antigen-antibody reactions include buffers, salts, and diluents.

8. The product for detecting GFAP levels according to claim 5, characterized in that, The product includes detection reagents, kits, or test strips, wherein the kit is any one of colloidal gold immunoassay kits, chemiluminescence immunoassay kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits, fluorescence immunoassay kits, and microfluidic chips.

9. The use of the product for detecting GFAP levels as described in claim 5 in the preparation of products for diagnosing GFAP-related diseases, characterized in that, The products include diagnostic reagents, kits, or test strips; the diseases associated with GFAP are any one of Alzheimer's disease, traumatic brain injury, or stroke.

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