An antibody against glial fibrillary acid protein and its preparation method and application

Abstract

The present invention discloses an antibody against glial fibrillary acid protein, and a preparation method and application thereof. The antibody of the present invention has a high recognition ability for glial fibrillary acid protein, and can detect the level of glial fibrillary acid protein in serum, so that it can be used for scientific research or clinical immunohistochemical detection of glial fibrillary acid protein expression, and further can be used for early diagnosis and prognosis of patients with traumatic craniocerebral injury, acute cerebral stroke and ischemic cerebral stroke.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and immunology, and in particular to an antibody targeting glial fibrillary acid protein and a preparation method and application thereof. Background Art

[0002] Glial fibrillary acid protein (GFAP) is a component of the intermediate filament protein superfamily type III. It is expressed in mature glial cells and is highly sensitive and specific to glial cell damage. The molecular weight of this protein is about 50 kDa. It not only regulates cell metabolism, forms and maintains the blood-brain barrier, produces and releases neurotrophic factors, but also plays an important role in maintaining the morphology and function of astrocytes. GFAP participates in cytoskeleton reorganization, adhesion and stabilization of neuronal structure, maintains intracranial myelin formation and serves as a cell signal transduction pathway. It is composed of four a-helical segments (1A, 1B, 2A and 2B) to form a rod-shaped dimer, flanked by non-helical N-terminal head and C-terminal tail domains.

[0003] At present, there is still a need to develop specific antibodies against GFAP, so as to achieve early diagnosis and prognosis of patients with traumatic brain injury, acute stroke and ischemic stroke based on GFAP immunoassay.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention provides an antibody against glial fibrillary acid protein and a preparation method and application thereof. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can target glial fibrillary acid protein, and the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III):

[0008] (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8;

[0009] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0010] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0011] In certain embodiments, according to the antibody or antigen-binding fragment thereof of the present invention, the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment comprises a Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc fragment.

[0012] The second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof described in the present invention.

[0013] The third aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0014] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule or vector molecule of the present invention.

[0015] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which is prepared by artificial synthesis or genetic engineering.

[0016] A sixth aspect of the present invention provides a product for detecting glial fibrillary acid protein, which comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0017] In certain embodiments, the detection product according to the present invention comprises a kit, a test paper or a protein chip.

[0018] The seventh aspect of the present invention provides use of the antibody or antigen-binding fragment thereof described in the present invention in the preparation of a glial fibrillary acid protein detection kit.

[0019] The present invention develops an antibody that can target glial fibrillary acidic protein, which can specifically bind to glial fibrillary acidic protein, thereby achieving early diagnosis and prognosis of patients with traumatic brain injury, acute cerebral stroke and ischemic cerebral stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the recombinant expression vector used in the preparation scheme of the GFAP antibody.

[0021] Figure 2 GFAP antigen purification and renaturation are shown.

[0022] Figure 3 Antibody activity verification results are shown.

[0023] Figure 4 The results of the enzyme-linked immunosorbent assay for serum samples from healthy and clinical populations are shown. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] Antibodies or antigen-binding fragments thereof

[0028] In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which can bind to glial fibrillary acid protein with high affinity and targeting. Without being bound by any theory, the antibody or antigen-binding fragment thereof of the present invention comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or light chain CDR1-3 shown in SEQ ID NO.4-6.

[0029] In a specific embodiment, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0030] (I) the heavy chain variable region sequence shown in SEQ ID NO.7 and / or the light chain variable region sequence shown in SEQ ID NO.8;

[0031] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0032] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0034] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region interspersed with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0035] As used herein, the terms "homology" and "sequence identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required for achieving optimal alignment in the full-length sequence being compared.

[0036] Herein, the antibody sequences obtained by modification also belong to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to a change in an amino acid sequence that results in an increase of one or more amino acids compared to a naturally occurring molecule. It should be noted that in the modified antibodies provided by the present invention, the modification preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0037] In the present invention, the antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a mouse antibody.

[0038] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoantibody" or Ab, refers to an immunoglobulin obtained from a pure cell line, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies against different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture and are not mixed with other immunoglobulins. This characteristic is in contrast to polyclonal antibody preparations, which generally include antibodies against different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0039] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy chain or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light chain and the heavy chain are all from the variable regions of antibodies of one animal species (such as mice, rats, etc.), while the constant portion is homologous to the antibody sequence from another species (such as humans). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, while the constant regions combined therewith are from humans. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith. At the same time, since the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response during injection is lower than that of using antibodies with non-human constant regions.

[0040] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.

[0041] Herein, the term "murine antibody" generally refers to an antibody whose variable region framework and CDR region are derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, it is also derived from mouse germline immunoglobulin sequences. In the present invention, the murine antibody may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo.

[0042] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab, Fab' or F(ab')2.

[0043] In the present invention, the terms "directed against", "binding", "immunobinding", "specific binding" and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific for the immunoglobulin. The strength or affinity of the immunological binding interaction can be expressed in terms of a dissociation constant (Kd), with a smaller Kd representing a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be described in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the art and described herein.

[0044] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or nucleic acid that has been extracted from its natural environment. An antibody or fragment thereof, nucleic acid that has been "isolated" thus includes an antibody or fragment thereof, nucleic acid purified by standard purification methods. The term also includes antibodies or fragments thereof, nucleic acids prepared by recombinant expression in a host cell, and chemically synthesized antibodies or fragments thereof, nucleic acids.

[0045] Nucleic acid molecules

[0046] In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0047] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, and also including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0048] Once the coding sequence of the antibody of the present invention is isolated, the antibody can be obtained in large quantities by recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then separate it from the propagated host cells by conventional methods.

[0049] Carrier molecules

[0050] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0051] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0052] Host cells

[0053] In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0054] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. A host cell includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0055] Preparation method

[0056] In one aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0057] In certain embodiments, the antibodies of the present invention are obtained by artificial synthesis. Methods for artificially synthesizing antibodies are known in the art, for example, the antibodies of the present invention or antigen-binding fragments thereof are obtained by direct amino acid synthesis.

[0058] In certain embodiments, the antibodies of the present invention are obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems. In a preferred embodiment, the antibodies of the present invention can be prepared by the following steps:

[0059] (1) constructing a recombinant vector expressing the antibody heavy chain of the amino acid sequence shown in SEQ ID NO.7 and / or a recombinant vector expressing the antibody light chain of the amino acid sequence shown in SEQ ID NO.8;

[0060] (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression;

[0061] (3) Collect and purify antibodies.

[0062] In another preferred embodiment, the antibody of the present invention is prepared by immunization with a recombinant antigen, wherein the recombinant antigen has the amino acid sequence shown in SEQ ID No. 9. In a specific embodiment, the method for preparing the antibody of the present invention comprises:

[0063] (1') constructing a recombinant vector expressing the antigen sequence shown in SEQ ID No.9, and obtaining a recombinant antigen after expression;

[0064] (2') inoculating the antigen into mice to prepare monoclonal antibodies;

[0065] (3') Using the antigen to screen positive hybridoma cells.

[0066] In the step (1') of the present invention, the full-length GFAP gene is inserted into vectors such as pET28a, pET32a, pET22b, pET30a, etc., preferably pET32a. The nucleotide sequence of the constructed GFAP prokaryotic expression vector is shown in SEQ ID NO.10.

[0067] In the step (1') of the present invention, the host bacteria used to express the recombinant antigen is Rosetta (DE3) or BL21 (DE3). Preferably, a surfactant such as TritonX-100, Tween-20, Sarkosyl, NP40, etc. is used to denature the GFAP antigen in the form of inclusion bodies, preferably Sarkosyl is used as a denaturant, and the concentration of the denaturant is not particularly limited. Preferably, the optimal concentration of the denaturant is between 0.5% and 1.5%.

[0068] In the step (1') of the present invention, the denatured recombinant GFAP protein refolding solution contains 10% glycerol, Tris, NaCl, Sarkosyl, and EDTA.

[0069] In the step (2') of the present invention, the amount of antigen inoculated into the experimental mice is 200 μg, and the immune adjuvant used for the antigen is Freund's complete adjuvant.

[0070] In the steps (1') and (3') of the present invention, the protein sequence of the full-length antigen used is shown in SEQ ID NO.9.

[0071] Testing products

[0072] The present invention further provides a detection product, which is used to detect (or quantify) glial fibrillary acidic protein, and comprises the antibody or antigen-binding fragment thereof described in the present invention, and instructions on how to implement the detection method of the present invention on glial fibrillary acidic protein.

[0073] In a preferred embodiment, the detection product includes a kit, a test paper or a protein chip.

[0074] As used herein, the term "kit" refers to a combination of reagents and other materials. The kit is expected to include reagents such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or antigen binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials, for example, the kit may also include instructions for using the reagents. The kit may be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for performing detection instructions. The kit may be prepared by a variety of methods known in the art.

[0075] In certain embodiments, the kit may further include at least one of a cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. The substrate solution may use a known substrate, and examples thereof include, but are not limited to, a chromogenic substrate, a fluorescent substrate, a luminescent substrate, and the like. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, a BSA solution, a trehalose solution, an animal serum, and the like.

[0076] use

[0077] The present invention further provides the use of a detection reagent in the preparation of a glial fibrillary acid protein detection kit, wherein the detection kit is used for the prediction, diagnosis and / or prognosis of GFAP-related diseases, and "GFAP-related diseases" refer to abnormal expression of GFAP in a subject, for example, a significantly upregulated expression level compared to the normal expression of GFAP in a healthy population. GFAP-related diseases include, but are not limited to, traumatic craniocerebral injury, acute cerebral stroke and ischemic cerebral stroke.

[0078] Example 1

[0079] This example demonstrates the synthesis of a monoclonal antibody.

[0080] 1. Immunogen Preparation

[0081] Since GFAP belongs to the intermediate filament protein superfamily, proteins will stick to each other to form flocculent insoluble substances, so it is difficult to prepare soluble GFAP antigen. In this embodiment, the preparation scheme of GFAP antibody uses prokaryotic recombinant GFAP antigen by adding TrxA tag on PET32a carrier ( Figure 1 ) to enhance the protein structure stability, solubility and immunogenicity, solve the problem of aggregation and degradation caused by the nature of the antigen itself, and further improve the success rate of antibody preparation.

[0082] The correctly sequenced pET32a-GFAP-Rosetta (DE3) strain was inoculated in 10 ml LB+Amp medium at 1:100. After overnight culture, it was inoculated in 1L LB+Amp medium at 1:100. When OD600≈0.6, 0.5 mM IPTG was added. After overnight culture at 220rpm and 20℃, the bacteria were collected and the fermentation product was added to 250 ml 50mM Tris-HCl (pH8.0) + 500 mM NaCl to be completely resuspended. After being precooled on ice for 10 min, ultrasonic lysis was performed at 300 W, 1 s, 1 s interval, and a total ultrasonic time of 30 min. It was identified that GFAP was expressed as inclusion bodies in prokaryotic expression.

[0083] The inclusion body precipitate after bacterial cell disruption was resuspended with 25 mM Tris-HCl (pH8.0), 125 mM NaCl, 0.5% Tween-20, 1 mM EDTA, and 1 M UREA, vertically mixed for 30 min, centrifuged at 12000 rpm for 10 min, discarded the supernatant, and repeated washing twice. The precipitate was washed with 25 mM Tris-HCl (pH8.0) and 125 mM NaCl, and after sufficient resuspending, centrifuged at 12000 rpm for 10 min to fully remove the residual UREA.

[0084] After the inclusion bodies were washed, they were dissolved in 25 mM Tris-HCl (pH8.0), 125 mM NaCl, and 1.5% SLS dissolution buffer. After complete dissolution, the undissolved impurities were removed by centrifugation at 12,000 rpm for 20 min. The remaining supernatant was dialyzed into 25 mM Tris-HCl (pH8.0), 125 mM NaCl, 0.1% Tween-20, and 50% glycerol. The purity of the purified recombinant GFAP protein was about 90% ( Figure 2 ).

[0085] 2. Mouse Immunization

[0086] After obtaining the immune antigen, purebred BALB / C mice were used for immunization. The immunization method can be selected from three methods: chemical adjuvant, water adjuvant or electroporation to introduce expression plasmid. In this embodiment, Freund's adjuvant was used for conventional immunization.

[0087] For the first immunization, 100 μg of GFAP antigen was added to Freund's complete adjuvant and emulsified by syringe push method, then injected subcutaneously at multiple points on the back.

[0088] ↓2 weeks later

[0089] The second immunization was 50 μg antigen, which was fully emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points on the back;

[0090] ↓2 weeks later

[0091] The third immunization scheme is the same as the second immunization, and blood is collected 5-7 days later to measure the titer by ELISA;

[0092] ↓2 weeks later

[0093] The mice with the highest titer were selected for booster immunization, and 100 μg / mouse GFAP antigen was diluted with PBS and injected intraperitoneally;

[0094] ↓3 days later

[0095] The spleen of mice was removed for cell fusion.

[0096] Table 1 Results of the third immune titer determination

[0097]

[0098] The experimental results showed that the titers of 1#, 3#, 5#, and 6# were 200,000, and the titers of 2# and 4# were 100,000. 1# mice were selected for booster immunization and fusion.

[0099] 3. Cell Fusion

[0100] Generally, there are two alternatives for cell fusion: chemical method (PEG fusion) and electric fusion. This embodiment adopts electric fusion.

[0101] First, blood from the eyeball was collected as a positive control for screening. Then, the epidermis and peritoneum were peeled off under a sterile environment, and the mouse spleen was picked out. After washing with DMEM culture medium, it was placed in a cell sieve for grinding. The spleen cells were collected and washed three times in a centrifuge tube.

[0102] After the mouse myeloma cells SP / 20 and spleen cells were washed, they were counted and washed once with a mixture of SP / 20 and spleen cells at a ratio of 1:2, and then washed three times with preheated electrofusion buffer. After diluting the cells with electrofusion buffer at a concentration of 2,000,000, they were prepared for electrofusion.

[0103] Add the cell mixture to the cleaned electrode cup, run the electrofusion program after the resistance test is normal, wait for 3 minutes after the program is completed, and then add it to the preheated HAT culture medium. After the cells recover for 1 hour, plate them into a 96-well plate containing feeder layer cells, 100 μL per well.

[0104] 4. Cloning and screening of hybridoma cells

[0105] GFAP was coated with an antigen concentration of 1 μg / ml, and after blocking, the cells were placed in a 4°C refrigerator for later use.

[0106] After the fusion cells have grown for one week, 100 μL of the cell supernatant is added to the coated GFAP antigen plate for ELISA screening, and the reserved serum is used as a positive control. The positive wells with an OD value of more than 2.0 are selected for subcloning, and the subcloning is carried out by limiting dilution.

[0107] Repeat the above process until only one cell line remains in the 96-well plate, then expand the culture to a 10 cm plate, freeze and prepare ascites samples. The collected ascites is purified and tested using a ProA column.

[0108] 5. Antibody Activity Verification

[0109] (1) Coating: Dilute GFAP antigen to 1 μg / ml in PBS, 100 μl / well, and incubate at 37°C for 2 h.

[0110] (2) Blocking: Use CEA blocking solution for blocking, 200 μl per well, incubate at 37°C for 2 h.

[0111] (3) Primary antibody: GFAP monoclonal antibody was serially diluted from 10 μg / ml using PBS, 100 μl / well, incubated at 37°C for 1 h, and then washed.

[0112] (4) Secondary antibody: HRP-labeled goat anti-mouse was diluted with CCP enzyme diluent at a ratio of 1:1000, 100 μl / well, and incubated at 37°C for 30 min.

[0113] (5) Color development: Mix solution A and solution B in a 1:1 ratio, 100 μl / well, and incubate at room temperature for 5 min.

[0114] (6) Stopping solution: 50 μl / well.

[0115] (7) Reading: Read the value using an ELISA reader.

[0116] Table 2 Antibody activity verification

[0117]

[0118] The results are shown in Table 2 and Figure 3 As shown, antibody #9 has the highest activity, indicating that both have the strongest ability to bind to GFAP antigen.

[0119] The antibody sequence sequencing results of antibody #9 showed that it had heavy chain CDR1-3 as shown in SEQ ID NO.1-3 and light chain CDR1-3 as shown in SEQ ID NO.4-6. The amino acid sequence of the heavy chain variable region of the antibody was shown in SEQ ID NO.7, and the light chain variable region sequence was shown in SEQ ID NO.8.

[0120] Example 2

[0121] This example shows the comparative detection of serum samples of healthy people and people with traumatic brain injury, acute stroke and ischemic stroke by using the anti-GFAP antibody screened in Example 1 using an enzyme-linked immunosorbent assay.

[0122] The prepared GFAP antibody was diluted to 2 μg / ml with coating buffer, 100 μl was added to each well, placed at 4℃ overnight, 200 μl of blocking solution was added and placed at 37℃ for 2 h, GFAP antigen was diluted to 1 ng / ml, three-fold gradient dilution, 7 gradients were diluted, placed at 37℃ for 1 h, the plate was washed, 100 μl of GFAP-HRP secondary antibody with a dilution ratio of 1000 times was added to each well, the plate was washed, the color development solution was added for 5 min, the stop solution was added, and the ELISA plate was used to read and the standard curve was established. The GFAP content in the serum of healthy controls (HC) and people with clinical backgrounds (patients) was detected, and the number of samples was 30 people each.

[0123] Test results such as Figure 4 As shown, the results indicate that the antibody GFAP protein of the present invention has a high recognition ability and can detect the GFAP level in serum, so it can be used for scientific research or clinical immunohistochemistry to detect GFAP expression, and further can be used for early diagnosis and prognosis of patients with traumatic brain injury, acute cerebral stroke and ischemic cerebral stroke.

[0124] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that It can target glial fibrillary acid protein, and the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 shown in SEQ ID NO.1-3 and light chain CDR1-3 shown in SEQ ID NO.4-6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence shown in SEQ ID NO.7 and a light chain variable region sequence shown in SEQ ID NO.

8.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody includes a monoclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody, and the antigen-binding fragment includes Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc.

4. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier molecule, characterized in that It comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that It comprises the nucleic acid molecule according to claim 4 or the vector molecule according to claim 5.

7. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: Prepared by artificial synthesis or genetic engineering.

8. A product for detecting glial fibrillary acidic protein, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the detection product is a kit, a test paper or a protein chip.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a glial fibrillary acid protein detection kit.

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