CN117820471A

CN117820471A - GFAP specific antibody and application thereof in GFAP detection kit

Info

Publication number: CN117820471A
Application number: CN202410247761.0A
Authority: CN
Inventors: 曹林; 冯速; 颜丹; 赵驰浩
Original assignee: Hunan Novozan Medical Technology Co ltd; Vazyme Medical Co ltd
Current assignee: Hunan Novozan Medical Technology Co ltd; Vazyme Medical Co ltd
Priority date: 2024-03-05
Filing date: 2024-03-05
Publication date: 2024-04-05
Anticipated expiration: 2044-03-05
Also published as: CN117820471B

Abstract

The application discloses a GFAP specific antibody and application thereof in a GFAP detection kit, and belongs to the technical field of biological medicines. The present application provides an antibody or antigen-binding fragment thereof that specifically binds to GFAP, and also provides polynucleotides encoding the antibodies or antigen-binding fragments, vectors comprising the polynucleotides, host cells comprising the vectors, methods of making the antibodies or antigen-binding fragments, and kits for detecting GFAP proteins. The detection kit provided by the application can be used for auxiliary diagnosis of diseases related to GFAP.

Description

GFAP specific antibody and application thereof in GFAP detection kit

Technical Field

The application belongs to the technical field of biological medicines, and relates to an antibody targeting GFAP, a preparation method and a kit for detecting GFAP.

Background

GFAP is rich in glutamate and aspartate, has little distribution in chondrocytes, fibroblasts, myoepithelial cells, lymphocytes, hepatic stellate cells, but is mainly present in AST of the central nervous system, being the main framework protein in AST. GFAP is specifically expressed in AST and is a marker protein thereof, and is considered as a specific marker for AST maturation activation. It forms a link between the nucleus and the cell membrane, and is involved in cytoskeletal reorganization, cell adhesion, maintenance of structures of myelination and neurons in the brain, and also involved in cell movement migration and cell proliferation as a cell signal involved in transduction pathways, etc. Has the functions of maintaining stable AST morphology, participating in blood brain barrier and myelination, regulating synaptic function and the like.

Several studies have shown that abnormal levels of GFAP expression are associated with various genetic and psychiatric disorders, levels of GFAP are associated with stroke, and gliosis is accompanied by increased GFAP expression. GFAP levels can reflect the size of cerebral infarction lesions, with higher GFAP levels in plasma leading to greater infarct lesions and positive correlation of both. Furthermore, GFAP levels are also related to the extent of nerve function damage, the higher the GFAP level in plasma, the more severe the nerve function damage.

The most commonly used detection methods for GFAP in clinic are enzyme-linked immunosorbent assay (ELISA) and Chemiluminescence (CLIA), electrochemiluminescence and liquid chromatography. The performance of the GFAP specific antibodies used in the detection method directly determines the sensitivity and specificity of the detection.

Therefore, it is found that a monoclonal antibody against GFAP protein having high binding specificity is of great importance for detecting the expression level of GFAP.

Disclosure of Invention

The application provides a monoclonal antibody for recognizing glial fibrillary acidic protein and application thereof in a chemiluminescent immunoassay kit, and provides a tool for auxiliary diagnosis of diseases related to the glial fibrillary acidic protein (namely GFAP).

In one aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds GFAP.

In some embodiments, the antibody or antigen binding fragment comprises at least one, two, three, four, five, or six CDRs selected from the group consisting of: (a) comprises a sequence identical to SEQ ID NO:1, CDR-H1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (b) comprises a sequence identical to SEQ ID NO:2, CDR-H2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (c) comprises a sequence identical to SEQ ID NO:3, CDR-H3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (d) comprises a sequence identical to SEQ ID NO:4, CDR-L1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (e) Comprising CDR-L2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of RAS; and (f) comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CDR-L3.

In some embodiments, the antibody or antigen-binding fragment comprises at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) comprises a sequence identical to SEQ ID NO:1, CDR-H1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (b) comprises a sequence identical to SEQ ID NO:2, CDR-H2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; and (c) comprises a sequence identical to SEQ ID NO:3, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CDR-H3. In some embodiments, the antibody or antigen binding fragment comprises (a) a polypeptide comprising SEQ ID NO:1, CDR-H1 of the amino acid sequence of seq id no; (b) a polypeptide comprising SEQ ID NO:2, CDR-H2 of the amino acid sequence of seq id no; and (c) a polypeptide comprising SEQ ID NO:3, and CDR-H3 of the amino acid sequence of seq id no.

In some embodiments, the antibody or antigen binding fragment comprises at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) comprises a sequence identical to SEQ ID NO:4, CDR-L1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (b) Comprising CDR-L2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of RAS; and (c) comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CDR-L3. In some embodiments, the antibody or antigen binding fragment comprises (a) a polypeptide comprising a sequence identical to SEQ ID NO:4, CDR-L1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (b) Comprising CDR-L2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of RAS; and (c) comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CDR-L3.

In some embodiments, the antibody or antigen-binding fragment comprises (a) a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (i) comprises a sequence identical to SEQ ID NO:1, CDR-H1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (ii) comprises a sequence identical to SEQ ID NO:2, CDR-H2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (iii) comprises a sequence identical to SEQ ID NO:3, CDR-H3 having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and (b) a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (i) comprises a sequence identical to SEQ ID NO:4, CDR-L1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of seq id no; (ii) Comprising CDR-L2 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of RAS; and (iii) comprises a sequence identical to SEQ ID NO:5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CDR-L3.

In some embodiments, the antibody or antigen binding fragment comprises (a) a polypeptide comprising SEQ ID NO:1, CDR-H1 of the amino acid sequence of seq id no; (b) a polypeptide comprising SEQ ID NO:2, CDR-H2 of the amino acid sequence of seq id no; (c) a polypeptide comprising SEQ ID NO:3, CDR-H3 of the amino acid sequence of seq id no; (d) a polypeptide comprising SEQ ID NO:4, CDR-L1 of the amino acid sequence of seq id no; (e) CDR-L2 comprising the amino acid sequence of RAS; and (f) comprises a sequence selected from the group consisting of SEQ ID NOs: 5, and CDR-L3 of the amino acid sequence of seq id no.

In some embodiments, the antibody or antigen binding fragment comprises a sequence that hybridizes to SEQ ID NO:6, a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-GFAP antibodies comprising the sequences retain the ability to bind GFAP. In one embodiment, the VH comprises one, two or three CDRs selected from the group consisting of: (a) a polypeptide comprising SEQ ID NO:1, CDR-H1 of the amino acid sequence of seq id no; (b) a polypeptide comprising SEQ ID NO:2, CDR-H2 of the amino acid sequence of seq id no; and (c) a polypeptide comprising SEQ ID NO:3, and CDR-H3 of the amino acid sequence of seq id no.

In some embodiments, the antibody or antigen binding fragment comprises a sequence that hybridizes to SEQ ID NO:7, a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, VL sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but anti-GFAP antibodies comprising the sequences retain the ability to bind GFAP. In some embodiments, the VL comprises one, two, or three CDRs selected from the group consisting of: (a) a polypeptide comprising SEQ ID NO:4, CDR-L1 of the amino acid sequence of seq id no; (b) CDR-L2 comprising the amino acid sequence of RAS; and (c) a polypeptide comprising SEQ ID NO:5, and CDR-L3 of the amino acid sequence of seq id no.

In some embodiments, the antibody or antigen-binding fragment comprises a VH in any of the embodiments provided above, and a VL in any of the embodiments provided above. In some embodiments, the antibody comprises the sequences as set forth in SEQ ID NO:6 and SEQ ID NO:7, including post-translational modifications of those sequences.

In one aspect, the present application provides a polynucleotide encoding an antibody or antigen-binding fragment of the present application.

In one aspect, the present application provides a vector comprising a polynucleotide of the present application.

In some embodiments, an isolated nucleic acid encoding an anti-GFAP antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of an antibody and/or amino acid sequences comprising the VH of an antibody (e.g., the light chain and/or heavy chain of an antibody). In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided.

In one aspect, the present application provides a host cell comprising a polynucleotide or vector of the present application.

In some embodiments, the host cell comprises: (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In some embodiments, the host cell is a eukaryotic cell and the host cell is a 293 cell. In some embodiments, a method of making an anti-GFAP antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

In one aspect, the present application provides a kit for detecting GFAP.

In some embodiments, the kit comprises an antibody or antigen binding fragment of the present application. In some embodiments, the kit detects GFAP in a non-diagnostic purpose of immunodetection. In some embodiments, the kit is a chemiluminescent method, an electrochemiluminescent method, an ELISA. In some embodiments, the kit is a chemiluminescent immunoassay kit of the double antibody sandwich principle comprising: magnetic beads coated with a primary antibody targeting GFAP; a secondary antibody labeled with a chemiluminescent agent that targets GFAP. In some embodiments, the chemiluminescent agent is selected from at least one of acridinium ester, alkaline phosphatase (ALP), horseradish peroxidase (HRP).

In some embodiments, one antibody of the pair of first and second antibodies that targets GFAP is selected from the antibodies or antigen binding fragments as described above. In some embodiments, the heavy chain CDRH1-CDRH3 amino acid sequence of the other antibody in the pair of antibodies comprising the first antibody and the second antibody that targets GFAP is as set forth in SEQ ID NO:18-SEQ ID NO:20, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:21, the amino acid sequence of the light chain CDRL2 is DAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO: shown at 22. In some embodiments, the other antibody of the pair of first and second antibodies that targets GFAP comprises an amino acid sequence as set forth in SEQ ID NO:16 and a heavy chain variable region VH as set forth in SEQ ID NO:17, and a light chain variable region VL.

In some embodiments, the second antibody is an antibody or antigen-binding fragment described herein, and the heavy chain CDRH1-CDRH3 amino acid sequence of the first antibody is as set forth in SEQ ID NO:18-SEQ ID NO:20, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:21, the amino acid sequence of the light chain CDRL2 is DAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO: shown at 22. In some embodiments, the second antibody is an antibody or antigen-binding fragment described herein, and the first antibody comprises an amino acid sequence as set forth in SEQ ID NO:16 and a heavy chain variable region VH as set forth in SEQ ID NO:17, and a light chain variable region VL.

In one aspect, the application provides the use of an antibody or antigen binding fragment as described above in the preparation of a reagent or kit for detecting GFAP. In one aspect, the application provides the use of an antibody or antigen binding fragment as described above in the preparation of a kit for aiding in the diagnosis of a GFAP related disease.

In one aspect, the present application provides a method for preparing an antibody or antigen binding fragment as described above, specifically prepared using a single B cell antibody cloning technique, comprising the steps of:

(1) Immunizing rabbits with GFAP as immunogen, collecting spleen after the immunization is finished, and separating spleen cells;

(2) Screening specific B lymphocytes, extracting total RNA of the B lymphocytes, synthesizing cDNA (complementary deoxyribonucleic acid), and obtaining antibody variable region and constant region sequences by PCR (polymerase chain reaction);

(3) Inserting the amplified antibody genes into a recombinant expression vector, and transfecting 293 cells;

(4) Collecting and purifying culture supernatant to obtain monoclonal antibody.

Further, the amino acid sequence of the GFAP in the step 1) is shown as SEQ ID NO: shown at 8.

Further, the primer sequence used in the PCR in the step 2) is shown in SEQ ID NO: 9-11.

The heavy chain coding region gene of the antibody is amplified, and the forward primer sequence is as follows:

caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc(SEQ ID NO：9)。

the heavy chain coding region gene of the antibody is amplified, and the reverse primer sequence is as follows:

tagtggatccgagctcggtacctcatttacccggagagcg(SEQ ID NO：10)。

the antibody light chain coding region gene is amplified, and the forward primer sequence is as follows:

caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc(SEQ ID NO：9)。

the antibody light chain coding region gene is amplified, and the reverse primer sequence is as follows:

tagtggatccgagctcggtacctcaacagtcacccctattg(SEQ ID NO：11)。

further, the purification of step 4) is specifically: the supernatant was purified by a protease A affinity column.

In one aspect, the present application provides a paired screening method for antibodies or antigen-binding fragments that specifically bind GFAP, specifically using a fixed heavy chain variable region as set forth in SEQ ID NO:16 and the light chain variable region is set forth in SEQ ID NO:17, and screening antibodies or antigen binding fragments by sandwich ELISA assay pairing.

Advantageous effects of the present application

The application provides a novel GFAP-targeted recombinant antibody and a kit containing the recombinant antibody, and the recombinant antibody has high affinity, high sensitivity and obviously shortened production period, and is more suitable for being used as a core raw material in the field of in-vitro diagnostic reagents.

Drawings

FIG. 1 is a graph showing the results of antigen-specific single B cell flow sorting during the preparation of the anti-GFAP monoclonal antibodies of the present application.

FIG. 2 is a graph showing the correlation between the detection result and clinical value of the anti-GFAP monoclonal antibody used for chemiluminescence detection of a quantitative gradient clinical sample.

Detailed Description

The following detailed description of the present disclosure is provided in connection with examples, but the implementation of the present disclosure is not limited thereto, and it is apparent that the examples described below are only some examples of the present disclosure, and that other similar examples are within the scope of protection of the present disclosure without inventive faculty for those skilled in the art.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents, patent applications, and publications cited throughout the disclosure are incorporated herein by reference in their entirety. If there are multiple definitions for terms herein, those in this section control.

The technical solutions provided by the present disclosure are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present disclosure.

Example 1: preparation of anti-glial fibrillary acidic protein (Glial fibrillary acidic protein, GFAP) monoclonal antibody

1. Antigen preparation

GFAP, mammalian cell 293F cell expression system was selected in this example, with His tag at C-terminal. The immunogen and the sorting source are the same and are GFAP-His recombinant proteins.

GFAP, sequence:

MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRVDFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVMGGGGSHHHHHH（SEQ ID NO：8）。

to obtain a GFAP rabbit monoclonal antibody, a New Zealand white rabbit was immunized with 500ug of each of the GFAP-His protein developed independently as an immunogen. For the first immunization, the immunogen is mixed with equivalent Freund's adjuvant to prepare an emulsifier, the emulsifier is prepared by taking 250ug of the immunogen and equivalent Freund's adjuvant at intervals of 3 weeks, and the emulsifier is prepared by subcutaneous multipoint injection and boosting twice. PBMC samples are collected after three immunizations, GFAP-His protein is used as a sorting original package plate, serum titer is measured by ELISA method, and the rabbit with high serum titer is taken, and after 250ug immunogen is subjected to subcutaneous multipoint injection for enhancing immunization once, the spleen of the rabbit is taken.

2. Single B cell suspension preparation

The separation and preparation of rabbit spleen sample lymphocytes is to prepare single cell suspension by physically grinding rabbit spleen and filtering through a porous filter screen. Single cell suspensions were used for cell staining and flow sorting work.

3. Antigen-specific single B cell sorting

Antigen specific single B cell sorting is based on specific recognition of lymphocyte B cell surface characteristic markers by using a flow cell sorting technology, and the acquisition of specific single B cells in lymphocyte suspension is completed.

In this example, the raw coupled FITC dye was selected.

Anti-rabbit IgG secondary antibodies are self-grinding type antibodies. And coupling PE dye.

At the time of cell labeling, DAPI dye was added to differentiate Dead/living cells (Dead/Live cells).

Cell labeling protocol, lymphocyte B cell sorting protocol: read/Live-/IgG+/anti+.

Cell labeling operation: the rabbit lymphocyte suspension is centrifuged for 5min at 300g, 5mL buffer solution is added, the mixture is stirred upside down, and centrifuged for 5min at 300 g. The supernatant was discarded and repeated 1 time, and 30uL of the cell suspension was taken for cell counting, and 40uL of the cell suspension was taken for blank control and single-stained tubes to be labeled. The remaining cell fluid was used as a sample tube, centrifuged at 300g for 5min, and resuspended in a small amount of PBS. The blank tube was left untreated. Single-stained tubes, supplemented with PBS to 100uL, and 2uL PE, 2uL FITC, 2uL DAPI dye were added, respectively. Sample tubes according to PE dye 1.5uL/10 ⁶ Cells, FITC dye 2ug/10 ⁶ The cells were counted, the corresponding antibodies were added in the dark, and left at 4℃for 30min. After the antibody incubation, 2mL buffer solution was added, mixed gently, centrifuged at 300g for 5min, and washed repeatedly 3 times. 1mL buffer solution was resuspended, and after cell filtration, the cells were waited for sorting on the machine.

After fluorescence compensation adjustment, based on the living cell population, PE and FITC double-positive signal cell populations are defined (figure 1), antigen-specific B cells are sorted into 96-well plates, only 1 cell is in each well, the sorted well plates need to be stored at low temperature immediately, and a dry ice box is arranged in the experiment of the embodiment, and the wells can be placed for a short time. The wells contained cell lysates and the sorted 96-well PCR plates were directly subjected to single cell antibody gene amplification.

4. Preparation of rabbit single B cell cDNA

Single B cell cDNA library was prepared based on SMART 5' RACE technology using reagents of Nanjinozan Biotechnology Co., ltd (VAZYME) N711 kit, commercially available. The amplification systems involved in the experiments in the examples are referred to the N711 kit instructions.

Single B cell RNA reverse transcription: after the sorting of the 96-well plates is completed, the plates are thawed and placed in a PCR instrument to run a program, and the plates are placed on ice for 2min after the program is finished.

Single B cell cDNA single-stranded synthesis: after the completion of the reverse transcription reaction procedure, a single-stranded synthesis system may be added. After the system is added, the pore plate is mixed evenly, the mixture is placed in a PCR instrument to run a program, and after the program is finished, the pore plate sample is placed on ice for 2min.

Single B cell DNA double strand synthesis: after the synthesis reaction of the cDNA single-stranded product is completed, a double-stranded synthesis system may be added. After the system is added, the pore plate is mixed evenly, centrifuged and then placed in a PCR instrument to run a program, and after the program is finished, the pore plate sample is placed on ice for standing.

5. Rabbit single B cell PCR technology amplified antibody coding gene

A single B cell cDNA library can be used for preparing the natural paired heavy chain and light chain coding genes of antibody.

The reagents used for the amplification of the coding gene are all kits for the Nanjinozan biotechnology Co., ltd (VAZYME) P515# and are available in the market. The amplification system involved in the experiments in the examples can be referred to the P515 kit instructions.

The upstream primer contains homologous arms overlapping with the 3' -end of the CMV gene sequence of the promoter, so that the antibody coding gene can be directly used for constructing a linear expression frame after being called.

The downstream primer for the heavy chain coding gene of the antibody is positioned at the position of the constant region and comprises a homologous arm overlapped with the BGH-polyA gene sequence.

The downstream primer of the light chain coding gene is positioned at the position of the constant region and contains a homologous arm complementary with the BGH-polyA gene sequence, so that the antibody coding gene can be directly used for constructing a linear expression frame after being called.

caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc（SEQ ID NO：9）。

tagtggatccgagctcggtacctcatttacccggagagcg（SEQ ID NO：10）。

caagctggctagcgtttaaacttgccaccagtcgtatgaagctaagagatc（SEQ ID NO：9）。

tagtggatccgagctcggtacctcaacagtcacccctattg（SEQ ID NO：11）。

modulation of antibody light chain and heavy chain coding genes: adding a PCR amplification system according to the specification, mixing the pore plates lightly, placing the mixture into a PCR instrument to run a program, and standing the pore plate sample on ice after the program is finished.

In the embodiment, the amplification product pairing positive rate of the antibody light chain and heavy chain coding genes in the same 96-well plate is more than 80%, and the detection strips are clear by agarose gel electrophoresis, so that the single B cell flow sorting and coding gene amplification experiments are effective. Amplification products, used for construction of recombinant expression plasmids.

6. Construction and expression of recombinant expression plasmids for heavy and light chains of antibody

Recombinant expression vector pcDNA3.1 (Invitrogen) was selected and purchased at ThermoFisher SCIENTIFIC. Prior to recombinant construction, hindIII restriction enzyme single enzyme was selected to linearize the expression vector and restriction enzymes were purchased on a New England Biolabs functional network.

The vector and the coding gene are recombined efficiently, a seamless cloning kit is selected, and a C115# kit is purchased in a VAZYME functional network.

Construction of recombinant expression plasmids: the amplified products of the heavy chain and the light chain of the antibody are cyclized and connected with pcDNA3.1 linearization vector by using a seamless cloning technology, then Escherichia coli DH5 alpha competent cells are transformed, LB fixed culture medium plates are coated, and the plates are inverted and cultured at 37 ℃ overnight.

Recombinant positive clone selection: the heavy chain and the light chain of the primary screening antibody are respectively picked into 8 single colonies, and the colony positive rate is determined after PCR bacterial detection.

Bacterial detection PCR of the recombinant plasmid, and the upstream primer sequence of the recombinant plasmid is as follows: caagctggctagcgtttaaactt (SEQ ID NO: 12).

The primer sequence of the antibody heavy chain bacterial PCR downstream is as follows: ctcatttacccggagagcg (SEQ ID NO: 13).

The sequence of the primer downstream of the PCR for the light chain bacterial detection of the antibody is as follows: acctcaacagtcacccctattg (SEQ ID NO: 14).

Recombinant positive clones were sent: and 5 bacterial PCR positive clones are selected from the heavy chain and the light chain of the antibody and sent to Shanghai biological company for sequencing.

Analysis of rabbit antibody gene sequences: and (3) finishing gene determination of the antibody sequence V region by using an IMGT database, finishing analysis of antibody sequences by using antibody heavy chain and light chain CDR1/CDR2/CDR3 regions and the like, and deriving and determining correct sequence numbers of bacterial detection PCR positive clones.

Small expression of recombinant expression plasmids: cloning the correct sequence, and extracting the light chain and heavy chain plasmids of the antibody from the bacterial liquid in a small amount. The plasmids were co-transfected into HEK293 mammalian cells after mixing. Cell supernatants were collected by centrifugation 10 days after cell transfection. The supernatant was subjected to antigen-specific evaluation, and cell supernatant purification was arranged after waiting for ELISA primary screening results.

In this example, 100 plasmids were transfected per round, i.e., 100 monoclonal antibodies were obtained per round of transfection. A 5-cycle rotation experiment was performed in total.

7. Recombinant expression supernatant antigen specificity evaluation

The antigen coated plate is prepared, the screening sources are GFAP-His protein and GST-His tag protein, which are recombinant expression of 293F cells, and the C terminal carries His tag.

Screening protocol for monoclonal antibodies to GFAP protein: antibodies that react with the GFAP-His protein and do not react with the GST-His protein, i.e., initially designated as GFAP-specific monoclonal antibodies.

Screening of the original GST-His with the sequence of

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKGGGGSHHHHHH（SEQ ID NO：15）。

The cell supernatant was detected by indirect ELISA, and the goat Anti-Rabbit polyclonal antibody (HRP) detection was performed using self-produced Rabbit secondary antibody-Rabbit IgG mAb, and the reactive OD >1, indicating that the recombinant plasmid was normally expressed on 293 cells.

Indirect ELISA was used to detect cell supernatants, and reactivity was evaluated on each of the 2 antigen-coated plates to obtain primary screening results for well plate supernatants (Table 1 shows exemplary detection data for 50 cell supernatants).

Table 1: antigen-antibody affinity data sheet for partial antibodies

Indirect ELISA detection results: the GFAP-His antibody is subjected to primary screening, cotransfection and recombination to obtain 500 monoclonal antibodies, the antibodies react with GFAP-His protein and do not react with GST-His protein, and 100 monoclonal antibodies are primarily screened.

The cell supernatant determined by preliminary screening is purified by protein A to obtain a small amount of monoclonal antibody, and the average amount of each strain is 1-3mg.

8. ELISA paired screening of recombinant antibodies

The paired antibodies of GFAP proteins are screened, and the development of GFAP high-affinity specific antibodies is finished early, so that the embodiment selects and fixes the Rabbit anti-GFAP mAb, the sandwich ELISA experiment paired detection is carried out on the preliminarily screened 100 monoclonal antibodies, and the better monoclonal antibody which can be paired with the Rabbit anti-GFAP mAb is screened.

The sequence of the variable region of the heavy chain of the Rabbit anti-GFAP mAb is as follows:

QSLEESGGRLVTPGGSLTLTCTVSGIDLTTYTMTWVRQAPGKGLEWIGIISGSGTTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARDLYGSSDIYNIWGPGTLVTVST（SEQ ID NO：16）；

（CDRH1：GIDLTTYT（SEQ ID NO：18）；CDRH2：ISGSGTT（SEQ ID NO：19）；CDRH3：ARDLYGSSDIYNI（SEQ ID NO：20））

the light chain variable region sequences are:

DIVMTQTPASVSAAVGGTVTINCQASESIRNYLSWYQQKPGQPPKLLIFDASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQTYYGVSGFGNAFGGGTEVVVK（SEQ ID NO：17）；

（CDRL1：ESIRNY（SEQ ID NO：21）；CDRL2：DAS；CDRL3：QTYYGVSGFGNA（SEQ ID NO：22））

the ELISA platform synchronous detection data of the clinical positive sample show that the ELISA pairing experiment can select GFAP-His as a detection source.

The 100 monoclonal antibodies screened initially are subjected to detection of GFAP-His by a sandwich ELISA experiment, the first 10 strains with high and low paired detection signal values are finally screened out, and the clinical sample detection condition of the antibodies is verified by a chemiluminescent platform (Table 2 shows the detection results of 20 paired antibodies in an illustrative manner).

Table 2: detection data (part) of GFAP-His paired antibody

9. Recombinant antibody chemiluminescent platform screening

And (3) detecting the reactivity and the specificity of the antibody screened in the step (8) on a chemiluminescent platform, wherein the specific operation is as follows:

and (3) coating magnetic beads: the Rabbit anti-GFAP mAb is fixed as a coating antibody (the sequence information of the variable region of the antibody is shown in the step 8), 2mg of magnetic beads are taken, an activated buffer is washed for 2 times, a certain amount of EDC is added for shaking and mixing, and the supernatant is removed by magnetic attraction. Adding 1000uL of coupling buffer into the precipitate, adding 40ug of antibody, shaking and mixing for 2h, adding 100uL of blocking solution, shaking and mixing and blocking for 3h. Finally, 1000uL TBST is added to clean the magnetic beads, and 1000uL preservation solution is added.

Acridinium ester labeling: taking the antibody screened in the step 8 as a labeled antibody, and taking 100- (100/C) _Ab ) uL coupling Buffer to 0.5 mL brown EP tube, add (100/C _Ab ) The uL Ab was placed in a 0.5 mL brown EP tube to give a final concentration of 1mg/mL antibody label, 5mM acridine ester was added to the 0.5 mL brown EP tube, the mixture was homogenized by vortex mixer, and then reacted with a vertical kneader at room temperature (20-25 ℃) for 2 hours, followed by purification to remove the free acridine ester.

The screening antigen is selected from GFAP-His protein and GST-His protein, the specific sequence information of the screening antigen is displayed before, and the sample diluent is detected as a control.

The preparation of the coated antibody, the labeled antibody and the screening antigen is respectively completed according to the method, and each screening antigen is detected according to a set program by using a full-automatic chemiluminescence apparatus. The detection result is reacted with GFAP-His protein, and the antibody which does not react with GST-His protein is the preferable antibody.

10 monoclonal antibodies screened by sandwich ELISA experiments are detected by a chemiluminescent platform, and 1 GFAP specific antibody is preferably selected. Table 3 shows the detection results of 4 pairs of paired antibodies by way of example.

Table 3: GFAP specific antibody chemiluminescence platform pairing detection data (part)

The screened specific antibody was designated as Anti-GFAP-rImab-1, and the heavy chain variable region sequence was:

QSLEESGGRLVTPGTPLTLTCTSSGFSLSSYYIQWVRQAPGKGLEWIGIIYASGSTYYASWAKGRFTISKTSATVDLKITSPTQEDTATYFCARNDYDSNLIMSIWGPGTLVTVSS（SEQ ID NO：6）；

the light chain variable region sequences are:

AYDMTQTPASMEVAVGGTVTINCQASQNIGSSLAWYQQKPGQRPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISGLQCDDAATYYCQQHFSIGAVQNVFGGGTEVVVR（SEQ ID NO：7）。

table 4: amino acid sequence of anti-GFAP antibody

Example 2: application of anti-Glial Fibrillary Acidic Protein (GFAP) monoclonal antibody in chemiluminescence detection of clinical samples

The optimized GFAP specific antibody Anti-GFAP-rRNA-1 is applied to a chemiluminescence detection experiment, matched with an antibody capable of being matched with the antibody, a constant value gradient clinical sample is detected, and the correlation between a detection result and a clinical constant value is compared.

Gradient clinical samples GFAP concentrations in samples were quantified using simoa single-molecule immunization, specific concentrations are shown in table 5:

table 5: clinical sample GFAP concentration meter

The concentration of GFAP in samples P1 to P9 and sample dilutions was measured using a full-automatic chemiluminescence apparatus (step same as in example 1 (9)) using the antibody pair consisting of the preferred GFAP-specific antibody Anti-GFAP-rRNA-1 and the antibody Rabbit Anti-GFAP mAb paired with the above, and the results are shown in Table 6:

table 6: chemiluminescent detection result table for clinical samples

And drawing a scatter diagram of the GFAP concentration of each sample by taking the GFAP concentration detected by a simoa single-molecule immunoassay as a horizontal axis and the GFAP concentration detected by a chemiluminescence method as a vertical axis, and calculating a correlation coefficient of the GFAP concentration value between the two detection methods.

As shown in fig. 2, the correlation coefficient r=0.9954, R of the two detection methods ² When applied to chemiluminescent detection experiments, the detection result has strong correlation with simoa single molecule immunization, namely, a preferred GFAP specific antibody Anti-GFAP-rRNA-1 is matched with an antibody capable of being paired with the antibody, and the preferred antibody can be used for preparing a downstream kit.

While embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the application, the scope of which is defined by the claims and their equivalents.

Claims

1. An antibody or antigen binding fragment thereof specifically binding to GFAP, having the amino acid sequences of heavy chain CDRH1-CDRH3 as set forth in SEQ ID NO:1-SEQ ID NO:3, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:4, wherein the amino acid sequence of the light chain CDRL2 is RAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO: shown at 5.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising: as set forth in SEQ ID NO:6 and a heavy chain variable region VH as set forth in SEQ ID NO:7, and a light chain variable region VL.

3. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-2.

4. A kit for detecting GFAP, comprising a magnetic bead coated with a first antibody that targets GFAP and a second antibody that targets GFAP labeled with a chemiluminescent agent, wherein one of the antibodies in the pair of first and second antibodies that targets GFAP is the antibody or antigen binding fragment thereof of any one of claims 1-2.

5. The kit of claim 4, wherein the heavy chain CDRH1-CDRH3 amino acid sequences of the other antibody in the pair of antibodies comprising the first antibody and the second antibody are set forth in SEQ ID NOs: 18-SEQ ID NO:20, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:21, the amino acid sequence of the light chain CDRL2 is DAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO: shown at 22.

6. The kit of claim 5, the other antibody of the pair of first and second antibodies comprising: as set forth in SEQ ID NO:16 and a heavy chain variable region VH as set forth in SEQ ID NO:17, and a light chain variable region VL.

7. The kit of claim 4, wherein the second antibody is the antibody or antigen-binding fragment thereof of any one of claims 1-2, and the heavy chain CDRH1-CDRH3 amino acid sequences of the first antibody are set forth in SEQ ID NOs: 18-SEQ ID NO:20, the amino acid sequence of the light chain CDRL1 is shown in SEQ ID NO:21, the amino acid sequence of the light chain CDRL2 is DAS, and the amino acid sequence of the light chain CDRL3 is shown in SEQ ID NO: shown at 22.

8. The kit of claim 4, wherein the second antibody is the antibody or antigen-binding fragment thereof of any one of claims 1-2, and the first antibody comprises the amino acid sequence set forth in SEQ ID NO:16 and a heavy chain variable region VH as set forth in SEQ ID NO:17, and a light chain variable region VL.

9. The kit according to claim 4, wherein the chemiluminescent agent is at least one selected from the group consisting of acridinium esters, alkaline phosphatase, and horseradish peroxidase.

10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-2 in the preparation of a kit for aiding in the diagnosis of a GFAP-related disease.