CN116655789A - Anti-human GFAP monoclonal antibody, product based on same and application - Google Patents
Anti-human GFAP monoclonal antibody, product based on same and application Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
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Abstract
The invention discloses an anti-human GFAP monoclonal antibody, a product based on the same and application thereof, wherein the antibody is used for immunizing a mouse by taking a human GFAP full-length protein expressed by escherichia coli recombination as an antigen, and then the monoclonal cell strain capable of continuously generating the anti-human GFAP monoclonal antibody is obtained through fusion and cloning for a plurality of times, and the anti-human GFAP monoclonal antibody is obtained through secretion of the cell strain. The antibody comprises a light chain and a heavy chain, wherein the light chain belongs to kappa, and the heavy chain belongs to IgG2b; wherein: the amino acid sequences of the 3 complementarity determining regions of the light chain variable region are shown as SEQ ID No.1, SEQ ID No.18 and SEQ ID No.2 respectively; the amino acid sequences of the 3 complementarity determining regions of the heavy chain variable region are shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, respectively. The monoclonal antibody can specifically recognize target antigen human GFAP protein, has higher affinity and sensitivity, and can be used for detecting human GFAP and developing in-vitro diagnostic kits.
Description
Technical Field
The invention belongs to the technical field of monoclonal antibodies, and particularly relates to an anti-Glial Fiber Acid Protein (GFAP) monoclonal antibody, a product based on the same and application of the monoclonal antibody.
Background
GFAP is an important intermediate silk protein of cells, and is involved in the cytoskeleton constituting these glial cells, and has a close relationship with regeneration of astrocytes and reconstruction of synapses. Meanwhile, GFAP plays an important role in central nervous system diseases such as neurodegenerative diseases, traumatic brain injury, cerebral apoplexy and the like.
Alzheimer's disease is a slowly developing but irreversible neurological disorder, and is also the most common neurodegenerative disorder. Since Alzheimer's disease inevitably causes damage to glial cells during the course of the disease, and GFAP is considered to be a specific marker for astrocytes at the beginning of the discovery, when these cells are damaged, GFAP is shed from the ruptured cells into the cerebrospinal fluid in large amounts and penetrates the blood brain barrier into the peripheral circulatory system.
Traumatic craniocerebral injury is the most dangerous brain injury, often causes very serious sustained injury, and is the most common cause of death after accidents such as car accidents and the like. Existing evidence suggests that changes in GFAP content in peripheral blood can more sensitively (than CT) confirm whether brain damage has occurred, and the extent of brain damage, and that patient disease progression and patient prognosis can be predicted by dynamically detecting changes in GFAP content in blood.
Cerebral apoplexy is divided into hemorrhagic apoplexy and ischemic apoplexy, and the causes of the cerebral apoplexy and the ischemic apoplexy are different, and the early-stage treatment methods are also different, so that the rapid judgment of the type after the occurrence of cerebral apoplexy symptoms is very important. Studies have shown that GFAP release occurs earlier after hemorrhagic stroke. The former shows a marked increase in GFAP content in blood from 3 to 4 hours after symptoms, while the latter shows a more delayed change, typically from 24 to 48 hours after onset.
Although anti-GFAP antibodies have been reported at present, reagents/kits based on the anti-GFAP antibodies have the problem of insufficient sensitivity or low specificity. Therefore, a highly sensitive and highly specific method for detecting GFAP levels needs to be developed. The anti-GFAP antibody with high specificity and high affinity is key to developing GFAP immune detection reagents and kits.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide an anti-human GFAP monoclonal antibody, a product based on the anti-human GFAP monoclonal antibody and application thereof, so as to solve the defect that the reagent/kit for detecting GFAP in the prior art has insufficient sensitivity or low specificity.
In order to achieve the above object, the present invention provides an anti-human GFAP monoclonal antibody (designated as 2E9-10B 8) comprising a light chain and a heavy chain, wherein the light chain is kappa and the heavy chain is IgG2B; the 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 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.1, SEQ ID No.18 and SEQ ID No.2, respectively, and the 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region 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.
Further, the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region of said 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.
Further, the monoclonal antibody 2E9-10B8 light chain variable region has at least 90% sequence identity, preferably 95% sequence identity, with the amino acid sequence shown in SEQ ID No.7 or the nucleotide sequence shown in SEQ ID No. 8; the heavy chain variable region has at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence shown in SEQ ID No.16 or to the nucleotide sequence shown in SEQ ID No. 17.
The present invention provides a product for detecting GFAP levels comprising:
a) Nucleic acid molecules: the nucleic acid molecule encodes monoclonal antibody 2E9-10B8 or a functional fragment based thereon;
b) Recombinant expression vector: the recombinant expression vector comprises a) the nucleic acid molecule;
c) Host cell: the host cell comprises the recombinant expression vector of b).
Further, the recombinant expression vector has a signal peptide operably linked to an antibody and comprises a transcriptional regulatory element; the host cell is selected from mammalian cells such as 293T cells, CHO cells or Expi293F TM And (3) cells.
Further, the product may also include reagents for performing antigen-antibody reactions or reagents for detecting reactions.
Still further, reagents for performing antigen-antibody reactions include buffers, salts, diluents, and the like.
Further, the product comprises a detection reagent, a kit or a detection test paper, the kit comprising: colloidal gold immunoassay kit, chemiluminescent detection kit, radioimmunoassay kit, enzyme-linked immunoassay kit, fluorescent immunoassay kit and microfluidic chip.
The preparation method of the monoclonal antibody 2E9-10B8 comprises the following steps: culturing the above host cell, and isolating and purifying the monoclonal antibody from the host cell and/or a medium in which the host cell is grown.
The invention also provides application of the product for detecting the GFAP level in preparing a product for diagnosing diseases related to GFAP, which is characterized by comprising a diagnostic reagent, a kit or diagnostic test paper. The diseases related to the GFAP protein are any one of Alzheimer's disease, traumatic brain injury and cerebral apoplexy.
Compared with the prior art, the invention has the following beneficial effects:
the invention selects the human full-length GFAP protein expressed by escherichia coli recombination as an antigen to immunize a mouse, and obtains a monoclonal cell strain capable of continuously secreting the anti-human GFAP monoclonal antibody through cell fusion and several times of cloning, and the anti-human GFAP monoclonal antibody 2E9-10B8 is obtained through secretion of the cell strain. The anti-human GFAP monoclonal antibody has higher affinity and sensitivity, can specifically identify target antigen GFAP, and can be used for human GFAP detection and development of in-vitro diagnostic reagents and kits.
Drawings
FIG. 1 shows the results of the blood titer of the tail tip of a GFAP immunized mouse, and the treatment is a GFAP immunized mouse; control is a normal murine control.
FIG. 2 shows the results of the monoclonal antibody 2E9-10B8 electrophoresis; m is Marker; 1-4 and 5-8 are ascites and purification results of 2 mice respectively, wherein 1 and 5 are ascites; 2. 6 is a flow-through; 3. 7 is after purification; 4. 8 is concentrated after purification.
FIG. 3 shows the results of subtype 2E9-10B8 identification by monoclonal antibodies.
FIG. 4 is ELISA validation of monoclonal antibody 2E9-10B8 specificity results.
FIG. 5 shows the results of immunofluorescence detection of monoclonal antibodies 2E9-10B8.
FIG. 6 is a graph showing the binding activity of the monoclonal antibody 2E9-10B8 detected by ELISA.
Detailed Description
In order that the manner in which the invention may be better understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
1. Preparation of GFAP
And (3) synthesizing a human GFAP gene sequence onto a PCMV vector, and cloning a target gene into a PET-24a vector. Extracting plasmid, double enzyme cutting and identifying, and sequencing and comparing results. Transferring the constructed prokaryotic expression plasmid into competent cells of escherichia coli BL21 (DE 3), picking up monoclonal and inoculating the monoclonal into LB culture medium, shaking the bacteria at 37 ℃ until the OD value is 0.5-1, adding IPTG, and then carrying out overnight induction expression at 28 ℃. And (5) collecting thalli, and collecting a supernatant after ultrasonic crushing and centrifugation. And (3) dialyzing and concentrating the protein purified by Ni column affinity chromatography to obtain the high-concentration GFAP.
2. Acquisition of hybridoma cells
Step one, GFAP immunized mice
3 female Balb/c mice with the age of 5-8 weeks; after one week of adaptation feeding, 50 μg of GFAP is dissolved into 500 μl of PBS, mixed with Freund's complete adjuvant according to a volume ratio of 1:1, and injected subcutaneously at 4 points after being completely emulsified to a water-in-oil state to complete the first immunization; after three weeks, 50 mug of GFAP is dissolved into 500 mug LPBS, mixed with Freund's incomplete adjuvant according to the volume ratio of 1:1, and injected subcutaneously at 4 points after being completely emulsified to a water-in-oil state to complete the second immunization; after another three weeks, 50. Mu.g of GFAP was dissolved in 500. Mu.LPBS and the mixture was injected intraperitoneally to complete the third immunization.
Step two, ELISA detection of blood titer of the tip of the tail of the immune mouse
Three weeks after the third immunization, blood serum was collected from the rat tail and assayed for potency by ELISA. The method comprises the following specific steps: GFAP was diluted to 1 ng/. Mu.L, added to ELISA plate, 100. Mu.L/well, coated overnight at 4 ℃; washing with TBST for 1 time and 2 min/time, and drying; 3% BSA-TBST, 200. Mu.L/well, and blocking at 37℃for 1h; washing with TBST for 2 times and 2 min/time, and drying; adding mouse serum diluted in a gradient way, and incubating for 1h at 37 ℃ at 100 mu L/hole; washing with TBST for 3 times and 2 min/time, and drying; goat anti-mouse IgG-HRP was added, 100. Mu.L/well and incubated at 37℃for 40min; washing with TBST for 5 times and 2 min/time, and drying; adding color development liquid, 100 mu L/hole, incubating for 10min at room temperature, adding H 2 SO 4 The absorbance was measured on a microplate reader, wherein the measurement wavelength was 450nm and the reference wavelength was 630nm. Figure 1 gives the results of the immune mouse antibody titer test for the subsequent experiments, which shows that: compared with normal mice which are not immunized, the titer of the immunized mice is higher than 1:12.8 ten thousand. This result demonstrates that the immunized mice developed an immune response to the immunogen GFAP and can be used in subsequent experiments.
Step three, cell fusion
Myeloma cells SP2/0 were adapted to be fed with 20% fetal bovine serum for one week. 1 immunized mouse is taken, 50 mug GFAP is taken before fusion for 3 days, and is evenly mixed with 500 mug PBS and then injected into the abdominal cavity to finish impact immunization. On the day of fusion, mice were sacrificed aseptically to harvest the spleens, and after milling, washed 2 times with serum-free medium. Mixing spleen cells and SP2/0 cells at a number ratio of 10:1, centrifuging at 1200rpm for 6min, discarding supernatant, and flickingLoosening cell precipitate, slowly adding 1mL of 45% PEG solution preheated to 37deg.C for 30s, standing at room temperature for 90s, slowly adding incomplete culture medium preheated to 37deg.C, mixing, centrifuging at 800rpm for 6min, discarding supernatant, adding 40mL of complete culture medium preheated to 37deg.C, mixing, adding into 96-well cell culture plate inoculated with abdominal macrophages, and placing into 5% CO at 37deg.C 2 Culturing in an incubator. HAT and HT are supplemented every other day 2-7 days after fusion to screen fusion cells.
Step four, ELISA detection of positive hybridoma cells and cloning of hybridoma cells
On the 7 th day after fusion, the growth condition of the hybridoma cells is observed, and when the cells in the holes exceed 1/3 hole bottoms, the secretion condition of the hybridoma antibodies is detected. The detection steps are as follows: GFAP was diluted and then added to ELISA plates at 100. Mu.L/well, and coated overnight at 4 ℃; washing with TBST for 1 time and 2 min/time, and drying; 3% BSA-TBST, 200. Mu.L/well, and blocking at 37℃for 1h; washing with TBST for 2 times and 2 min/time, and drying; adding hybridoma cell supernatant, 100 mu L/well, and 37 ℃ for 1h; washing with TBST for 3 times and 2 min/time, and drying; goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP) was added at 100. Mu.L/well for 40min at 37 ℃; washing with TBST for 5 times and 2 min/time, and drying; adding color development liquid, 100 mu L/hole, incubating for 10min at room temperature, adding H 2 SO 4 The absorbance was measured on a microplate reader, wherein the measurement wavelength was 450nm and the reference wavelength was 630nm. Clones with high OD values were selected for subcloning and ELISA screening was performed again after 7-10 days. After 3 clones, 1 positive clone was finally selected and designated hybridoma cell 10B8.
3. Preparation of monoclonal antibody 2E9-10B8 from ascites
mu.L of ascites adjuvant was injected into the peritoneal cavity of 10 week old Balb/c mice. Culturing hybridoma cell 10B8 to logarithmic phase, and regulating cell density to 2×10 6 The ascites is collected 7 days after the ascites adjuvant is injected into the abdominal cavity by 500 mu L per mL.
4. Purification of monoclonal antibody 2E9-10B8
The collected ascites were diluted with binding/wash buffer (pH 7.0), centrifuged to obtain supernatant, protein A was added, and mixed well and then bound overnight at 4℃on a shaker. Balancing the chromatographic column with binding/wash buffer for 2 times, adding the overnight binding solution into the chromatographic column, washing the chromatographic column with binding/wash buffer for 2 times after protein A beads in the binding solution settle at the bottom of the chromatographic column, eluting the antibody bound on protein A with an Elutation buffer (pH 3.0), immediately adding 1/10 volume of neutralization buffer (pH 8.5) into the eluent, mixing, concentrating the obtained Elution neutralization solution with an ultrafiltration tube, and gradually replacing the Elution neutralization solution in the obtained concentrated solution with PBS (pH 7.2) to obtain monoclonal antibody 2E9-10B8. After the concentration is measured by BCA method, SDS-PAGE electrophoresis and Coomassie blue staining are carried out simultaneously with original ascites, the result is shown in figure 2, the heavy chain and light chain bands of the antibody can be clearly seen, the impurity protein is obviously reduced after purification, and the purity of the antibody is obviously improved.
The monoclonal antibodies 2E9-10B8 obtained above were respectively identified and tested for performance as follows:
1. identification of monoclonal antibody 2E9-10B8 subtype
100 ng/hole GFAP is added to the ELISA plate, and the ELISA plate is coated overnight at 4 ℃; washing with TBST for 1 time and 2 min/time, and drying; 3% BSA-TBST, 200. Mu.L/well, and blocking at 37℃for 1h; washing with TBST for 2 times and 2 min/time, and drying; adding 10B8 supernatant of the hybridoma cells, 100 mu L/hole, and incubating for 1h at 37 ℃; washing with TBST for 3 times and 2 min/time, and drying; HRP-labeled rabbit anti-mouse secondary antibodies (IgG 1, igG2a, igG2b, igG2c, igG3, igM, kappa, lambda), 100. Mu.L/well, incubated at 37℃for 40min; washing with TBST for 5 times and 2 min/time, and drying; adding color development liquid, 100 mu L/hole, incubating for 10min at room temperature, adding H 2 SO 4 The absorbance was measured on a microplate reader, wherein the measurement wavelength was 450nm and the reference wavelength was 630nm. As shown in FIG. 3, the OD values of the 2E9-10B8 IgG2b and kappa type were much higher than those of the other subtypes and negative control. Thus, the monoclonal antibody 2E9-10B8 heavy chain is of the IgG2B type and the light chain is of the kappa type.
2. ELISA for verifying specificity of monoclonal antibody 2E9-10B8
The purified GFAP expressed in example 1 and the laboratory existing NFL and BSA were diluted to 1. Mu.g/mL with coating buffer, respectively, and added to ELISA plates, and the dilutions were added to ELISA plates at the same time, 100. Mu.L/wellOvernight at 4 ℃; washing with TBST for 1 time and 2 min/time, and drying; 3% BSA-TBST, 200. Mu.L/well, and blocking at 37℃for 2h; washing with TBST for 2 times and 2 min/time, and drying; adding 10B8 supernatant of the hybridoma cells, 100 mu L/hole, and incubating for 1h at 37 ℃; washing with TBST for 3 times and 2 min/time, and drying; goat anti-mouse IgG-HRP was added, 100. Mu.L/well and incubated at 37℃for 40min; washing with TBST for 5 times and 2 min/time, and drying; adding color development liquid, 100 mu L/hole, incubating for 10min at RT, adding H 2 SO 4 The absorbance was measured on a microplate reader, wherein the measurement wavelength was 450nm and the reference wavelength was 630nm. The results are shown in Table 1 and FIG. 4.
TABLE 1ELISA verification of the specificity of monoclonal antibody 2E9-10B8
Table 1 and fig. 4 experimental results illustrate: monoclonal antibody 2E9-10B8 specifically recognizes the target antigen GFAP.
3. Application of monoclonal antibody 2E9-10B8 in immunofluorescence detection
Inoculating U251 and U87 cells to a climbing plate, fixing with paraformaldehyde, washing with TBST for 3 times, incubating with 5% BSA-TBST (containing 0.1% TritonX-100) at room temperature for 1h, washing with TBST for 1 time, and incubating diluted monoclonal antibody 2E9-10B8 for 2h at room temperature; after washing 3 times with TBST, the Alexa Fluor 549-labeled goat anti-mouse IgG antibody is incubated for 1h at room temperature; after TBST washing, DAPI-containing caplets were added. The staining results were observed under a fluorescence microscope. The experimental results in fig. 5 show that after 2 cells are incubated with the antibody, a significant red color (GFAP in fig. 5) and DAPI-stained blue nuclei (DAPI in fig. 5) can be observed under the microscope, and the two nuclei can overlap well (DAPI/GFAP in fig. 5), which indicates that monoclonal antibodies 2E9-10B8 can specifically stain GFAP and can be used for detection of GFAP in immunofluorescent stained samples.
4. ELISA detection of monoclonal antibody 2E9-10B8 binding Activity
GFAP coated ELISA plate, 0.1 μg/well, overnight at 4 ℃; washing with TBST for 1 time and 2 min/time, and drying; 3% BSA-TBST, 200. Mu.L/well, and blocking at 37℃for 2h; washing with TBST for 2 times and 2 min/time, and drying; adding the monoclonal antibody 2E9-10B8 after gradient dilution, and incubating for 1h at 37 ℃; washing with TBST for 3 times and 2 min/time, and drying; goat anti-mouse IgG-HRP was added, 100. Mu.L/well and incubated at 37℃for 40min; washing with TBST for 5 times and 2 min/time, and drying; adding color development liquid, 100 mu L/hole, incubating for 10min at RT, adding H 2 SO 4 The absorbance was measured on a microplate reader, wherein the measurement wavelength was 450nm and the reference wavelength was 630nm. The experimental results of fig. 6 show that: monoclonal antibody 2E9-10B8 can well bind GFAP protein and presents concentration dependence, EC 50 Is 0.0546 mug/mL.
5. Sequence determination of monoclonal antibody 2E9-10B8
Extracting total RNA of the hybridoma cells, and carrying out reverse transcription to obtain cDNA; amplifying antibody heavy chain and light chain variable region fragments by a 5' RACE method; subcloning the amplified fragment into pEASY-Blunt vector, extracting plasmid, sequencing to obtain the result of the sequenced monoclonal antibody 2E9-10B8 light chain and heavy chain sequence, and marking the complementarity determining region of the antibody amino acid sequence by using Kabat method.
The amino acid sequence of the light chain variable region of the monoclonal antibody 2E9-10B8 is shown as SEQ ID No.7, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID No.1, SEQ ID No.18 and SEQ ID No.2, respectively, and the amino acid sequences of framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively. The gene sequence is shown as SEQ ID No. 8.
The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E9-10B8 is shown as SEQ ID No.16, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, respectively; the amino acid sequences of the framework regions FR1, FR2, FR3 and FR4 are shown as SEQ ID No.12, SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, respectively. The gene sequence is shown as SEQ ID No. 17.
SEQ ID No.1：KSVSTSGYSY
SEQ ID No.2：QHILCLTR
SEQ ID No.3：DIVLTQSPASLAVSLGQRATISYRAS
SEQ ID No.4：MHWNQQKPGQPPRLLIY
SEQ ID No.5：NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC
SEQ ID No.6：SEGGPSWKN
SEQ ID No.7：DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQ QKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIL CLTRSEGGPSWKN
SEQ ID No.8：gacattgtgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcat acagggccagcaaaagtgtcagtacatctggctatagttatatgcactggaaccaacagaaaccaggacagccacccagactcctcatctatcttgtatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattctctgccttacacgttcggaggggggaccaagctggaaaaac
SEQ ID No.9：GFSLTSYD
SEQ ID No.10：IWTGGGT
SEQ ID No.11：VRRFTAMDY
SEQ ID No.12：QVQLKESGPGLVAPSQSLSITCTVS
SEQ ID No.13：ISWIRQPPGKGLEWLGV
SEQ ID No.14：NYNSAFMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYC
SEQ ID No.15：WGQGTSVTVSS
SEQ ID No.16：QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYDISWIRQPPG KGLEWLGVIWTGGGTNYNSAFMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYC VRRFTAMDYWGQGTSVTVSS
SEQ ID No.17：caggtgcaactgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattac ctgcactgtctctgggttctcattaaccagctatgatataagctggattcgccagccaccaggaaagggtctggagtggcttggagtaatatggactggtggaggcacaaattataattcagctttcatgtccagactgagcatcagcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatatattactgtgtaagacggtttactgctatggactactggggtcaaggaacctcagtcaccgtctcctca
SEQ ID No.18：LVS
In conclusion, the monoclonal antibody 2E9-10B8 provided by the invention has better binding activity with GFAP, has strong specificity, and can be used for detecting GFAP.
Claims (10)
1. An anti-human GFAP monoclonal antibody comprising a light chain and a heavy chain, the light chain being kappa and the heavy chain being IgG2b; wherein:
the 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region 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;
the 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region 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.
2. The anti-human GFAP monoclonal antibody of claim 1, wherein 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 to 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 framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region of the anti-human GFAP monoclonal antibody have at least 90% sequence identity to 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 of claim 2, wherein the light chain variable region of the anti-human GFAP monoclonal antibody has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID No.7 or the nucleotide sequence set forth in SEQ ID No. 8; 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 or the nucleotide sequence shown in SEQ ID No. 17.
4. The anti-human GFAP monoclonal antibody of claim 3, wherein the light chain variable region of the anti-human GFAP monoclonal antibody has 95% sequence identity to the amino acid sequence set forth in SEQ ID No.7 or the nucleotide sequence set forth in SEQ ID No. 8; 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 or the nucleotide sequence shown in SEQ ID No. 17.
5. A product for detecting GFAP levels comprising:
a) Nucleic acid molecules: the nucleic acid molecule encodes the monoclonal antibody or functional fragment thereof according to any one of claims 1-4;
b) Recombinant expression vector: the recombinant expression vector comprises a) the nucleic acid molecule;
c) Host cell: the host cell comprises the recombinant expression vector of b).
6. The product for detecting GFAP levels according to claim 5, wherein the recombinant expression vector has a signal peptide linked to an antibody and comprises a transcriptional regulatory element; the host cell is 293T cell, CHO cell, expi293F TM Any one of the cells.
7. The product for detecting GFAP levels according to claim 5, further comprising a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction; the reagent for carrying out antigen-antibody reaction comprises buffer, salt and diluent.
8. The product for detecting GFAP level according to claim 5, wherein the product comprises a detection reagent, a kit or a detection test paper, and the kit is any one of a colloidal gold immunoassay kit, a chemiluminescent assay kit, a radioimmunoassay kit, an enzyme linked immunoassay kit, a fluorescent immunoassay kit and a microfluidic chip.
9. Use of a product for detecting GFAP levels as claimed in claim 5 for the manufacture of a product for diagnosing a disease associated with GFAP, wherein the product comprises a diagnostic reagent, kit or diagnostic test strip.
10. The use according to claim 9, wherein the GFAP related disease is any one of alzheimer's disease, traumatic brain injury, cerebral stroke.
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