CN108997496A - Anti- GFAP protein monoclonal antibody and application thereof - Google Patents
Anti- GFAP protein monoclonal antibody and application thereof Download PDFInfo
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- CN108997496A CN108997496A CN201811015869.8A CN201811015869A CN108997496A CN 108997496 A CN108997496 A CN 108997496A CN 201811015869 A CN201811015869 A CN 201811015869A CN 108997496 A CN108997496 A CN 108997496A
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- gfap
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- 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
Abstract
The present invention relates to field of biotechnology, disclose a kind of hybridoma cell strain (deposit number is CGMCC No.15598), and the monoclonal antibody Umab129 that thus hybridoma cell strain generates.The invention further relates to monoclonal antibody Umab129 to prepare the application in the immune detection tool for detecting GFAP albumen, application of the immunohistochemical kit and monoclonal antibody Umab129 of the Umab129 containing monoclonal antibody in the kit that preparation is used for marked tumor.Monoclonal antibody of the present invention can in conjunction with GFAP protein-specific, and with other intracellular albumen no cross reactions, significantly improve specificity, the accuracy and reliability of the detection of GFAP protein immunization.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to can specific bond GFAP albumen monoclonal antibody Umab129,
Generate the cell strain of the monoclonal antibody and using the diagnostic method of the antibody and purposes.
Background technique
GFAP, that is, glial fibrillary acid protein is type III intermediate filament protein.GFAP and vimentin form star glue
Intermediate Filaments in cell plastid, and adjust its motility and shape.Specifically, vimentin fibril is in mesoderm growing early stage
Stage exists, and GFAP fibril is the feature of differentiation and mature cerebral astrocytic.
GFAP is commonly used for the marker of encephalic and intraspinal tumor from astrocyte, and GFAP median fiber exists in
In the Schwann cell that non-myelin is formed in peripheral nervous system.It is real that immunohistochemistry (IHC) pathology is clinically commonly used at present
The expression situation of albumen in detection tumour cell is tested, however the core of IHC experiment is the monoclonal antibody of binding proteins specific,
The superiority and inferiority of its performance directly decides the sensitivity and specificity entirely detected.Therefore, it is higher to develop a kind of binding specificity
The monoclonal antibody for GFAP albumen to IHC detection GFAP expression have great importance.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of monoclonals of the higher GFAP albumen of binding specificity to resist
Body, and its preparing the application in the immune detection tool for detecting GFAP albumen.
The present invention provides a kind of hybridoma cell strains, and it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers (referred to as CGMCC), the deposit date is on April 26th, 2018, deposit number was CGMCC No.15598.
The present invention also provides a kind of monoclonal antibody Umab129 for specifically binding GFAP albumen, by above-mentioned hybridoma
Cell strain generates.
Monoclonal antibody of the present invention the preparation method is as follows:
(1) building of recombinant expression carrier: according to GFAP ORF nucleotide sequence (GFAP ORF nucleotide sequence such as SEQ
Shown in ID NO.1,1299bp；GFAP amino acid sequence is as shown in SEQ ID NO.2)
Design primer PCR amplification GFAP ORF 1bp to 1299bp bit sequence, gene two sides introduce restricted respectively
Restriction enzyme site SgfI and MluI are inserted into expression vector pCMV6-Entry, construct GFAP amino acid sequence the 1st to the 432nd
Recombinant expression plasmid pCMV6-rGFAP；Upstream amplification primer sequence, SEQ ID NO.3:
CACGCGATCGCATGGAGAGGAGACGCATCACC downstream amplification primer sequence SEQ ID NO.4:
ACCGACGCGTCATCACATCCTTGTGCTCCTG
(2) expression and purification of GFAP recombinant protein: by GFAP recombinant expression plasmid convert HEK293T cell, crack from
The heart obtains supernatant, and DDK affinity chromatography column purification obtains the GFAP recombinant protein of purifying；
(3) screening and preparation of monoclonal antibody: Balb/c mouse is immunized using the GFAP recombinant protein of above-mentioned purifying, is taken
Mouse spleen cells are merged with SP2/0 cell, and limiting dilution assay obtains monoclonal, and it is thin that ELISA method screens positive hybridoma
Born of the same parents obtain the hybridoma cell strain that can secrete anti-GFAP specific antibody, are named as Umab129, subtype identification IgG1；Pass through
Free serum culture prepares antibody, obtains GFAP monoclonal antibody Umab129 by affinity chromatography column purification.Pass through respectively
Western Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.
The present invention also provides monoclonal antibody Umab129 in preparing the immune detection tool for detecting GFAP albumen
Application.
Specifically, the immune detection tool is kit, chip or test paper.
In the particular embodiment, the present invention provides a kind of immunologic combined detection reagent kit, including above-mentioned monoclonal are anti-
Body Umab129 can detect the expression situation of GFAP in histocyte.
The present invention also provides application of the said monoclonal antibody in the kit that preparation is used for marked tumor.Wherein institute
The tumour being proliferated and the expression of GFAP is closely related that tumour specifically refers to tumour cell is stated, including but not limited to astroglia is thin
Born of the same parents' tumor.
Compared with prior art, the present invention provides a kind of hybridoma cell strain (deposit number CGMCCNo.15598),
And the monoclonal antibody Umab129 that thus hybridoma cell strain generates.The present invention also provides monoclonal antibody Umab129 to exist
Prepare the application in the immune detection tool for detecting GFAP albumen, the immunohistochemistry reagent of the Umab129 containing monoclonal antibody
The application of box and monoclonal antibody Umab129 in the kit that preparation is used for marked tumor.Monoclonal of the present invention is anti-
Body can in conjunction with GFAP protein-specific, and with other intracellular albumen no cross reactions, significantly improve GFAP protein immunization
Specificity, the accuracy and reliability of detection, it is true to reflect GFAP protein expression level in tumour cell, it can be applied to star glue
The expression of GFAP in the tumor tissues such as cell plastid tumor.
Preservation information
The classification naming of hybridoma cell strain Umab129 for preservation are as follows: the hybridoma of GFAP mouse monoclonal antibody is thin
Born of the same parents' strain；
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Depositary institution's abbreviation: CGMCC；
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；
Preservation date: on April 26th, 2018；
Deposit number: CGMCC No.15598.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows 1 cloning site design drawing of embodiment, and wherein shading part is the area ORF；
Fig. 2 shows that embodiment 2 recombinates GFAP albumen Western blot testing result figure, with anti-DDK antibody test recombination
Expression of the GFAP albumen in HEK293T cell, wherein the HEK293T cell pyrolysis liquid that left side swimming lane is transfection empty carrier is anti-
The testing result of original, right lanes are that the HEK293T cell pyrolysis liquid of transfection pCMV6-rGFAP plasmid is the detection knot of antigen
Fruit；
Fig. 3 shows that embodiment 2 recombinates GFAP protein SDS-PAGE result figure, recombinates GFAP with anti-DDK affinity chromatography column purification
Albumen, albumen after purification pass through SDS-PAGE glue electricity arteries and veins, coomassie brilliant blue staining；
Fig. 4 shows that embodiment 3 identifies complete GFAP (Full length GFAP, GFAP- with monoclonal antibody Umab129
FL) the Western blot testing result figure of albumen.Swimming lane 1 is the cell pyrolysis liquid for transfecting pCMV6-Entry；Swimming lane 2 is to turn
Contaminate the cell pyrolysis liquid of pCMV6-GFAP-FL；
Fig. 5 shows embodiment 3 with endogenous GFAP in monoclonal antibody Umab129 identification SNB19 people's glioblastoma cells
The Western blot testing result figure of albumen.
Fig. 6 shows that embodiment 3 identifies endogenous GFAP egg in 10 kinds of different people's Tissue lysates with monoclonal antibody Umab129
White Western blot testing result figure.From left to right it is followed successively by 1: testis, 2: nethike embrane, 3: uterus, 4: mammary gland, 5: brain, 6:
Liver, 7: ovary, 8: colon, 9: spleen, 10: thyroid gland.
Fig. 7 shows embodiment 3 with endogenous GFAP albumen in monoclonal antibody Umab129 identification Mice brain tissues lysate
Western blot testing result figure.
Fig. 8 shows that 4 formalin of embodiment is fixed, (primary antibody is the adult brain tissue ImmunohistochemistryResults Results figure of paraffin embedding
GFAP monoclonal antibody Umab129)；
Fig. 9 show 4 formalin of embodiment fix, the Human embryo brain cortical tissue ImmunohistochemistryResults Results figure of paraffin embedding
(primary antibody is GFAP monoclonal antibody Umab129)；
Figure 10 show 4 formalin of embodiment fix, the Human embryo cerebellar tissue ImmunohistochemistryResults Results figure (primary antibody of paraffin embedding
For GFAP monoclonal antibody Umab129)；
Figure 11 shows that (green is primary antibody GFAP Dan Ke to 5 rat primary neuronal cell immunofluorescence dyeing result figure of embodiment
Grand antibody Umab129 dyeing, blue are that DAPI is dyed).
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The building of embodiment 1, GFAP recombinant expression plasmid
With the plasmid RC204548 (ORF1299bp containing GFAP) obtained from Biotechnology Co., Ltd of Aureal Dongyuan County of the U.S.
For template, designs two primers and introduce restriction enzyme site SgfI and MluI respectively, be cloned into expression vector pCMV6-Entry, build
Vertical GFAP recombinant expression plasmid.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, GFAP recombinant protein
1, transfect HEK293T cell: HEK293T cell is reached with 1:3 to be continued to cultivate in culture dish；Take 7.5mLDMEM (nothing
Serum and antibiotic) into 50mL pipe, 300 μ LPEI MegaTran1.0 are added and mix；75 μ g GFAP recombinant expression matter is added
Grain DNA is mixed into mixing liquid and is stood 30 minutes；Take 515 μ L into each culture dish in 37 DEG C of 5%CO respectively2In incubator
Culture.After transfection 24 hours, every ware cell adds 25 μ L2M sodium butyrates to final concentration 5mM.
2, lytic cell: after transfection 48 hours, cell cracking is carried out.Culture medium is sucked, 1mLPBS is added and is rinsed, inhales
Remove PBS.1mL lysis buffer is added, uses preceding addition protease inhibitors PI and PMSF.It is placed in ice chest and shakes on shaking table
It swings, collects the lysate in all culture dishes, supernatant is collected in 4 DEG C of centrifugations.Take a small amount of supernatant using the table of WB identification recombination GFAP
It reaches, result figure 2.
3, DDK affinity chromatography column purification: with 0.45 μM, the lysate supernatant after 33mm pvdf membrane filter filter centrifugation is simultaneously
It is transferred to 15mL pipe, the Beads 1mL mixed is added, is put into after sealing in 360 degree of vortex mixers, is combined 2 hours in 4 DEG C；It takes out
15mL pipe, lysate is poured into BIO-RAD chromatographic column, and catch and penetrate liquid, and liquid sampling WB detection is penetrated after drop is most；With cracking
Buffer rinses column material 1-2 times, is rinsed Beads 3 times with TBST after drop is most, is washed after dripping to the greatest extent with 0.1M Glycine pH3.5 again
De-, 200 μ L, drop are not collected to the greatest extent for the first time, second and third each 500 μ L, the 4th 250 μ L are collected to a 1.5mL centrifuge tube
In, and it is rapidly added NaH2PO4(pH=11.0) it is neutralized to pH7.0 or so, glycerol is added to final concentration of 10% in every pipe,
Tween-80 to final concentration of 0.1%.Recombination GFAP albumen after purification is identified with SDS-PAGE, sees Fig. 3.
By Fig. 2 result as it can be seen that in 52kD after WB detection in the HEK293T cell pyrolysis liquid of transfection pCMV6-rGFAP plasmid
There is apparent specific band at place, is consistent substantially with the theoretical molecular weight 50kD of polypeptide.Show that recombination GFAP albumen is special in cell
Expression.
Theory point by Fig. 3 result as it can be seen that the albumen of purifying has apparent specific band in PAGE glue 49kD, with polypeptide
Son amount 50kD is consistent substantially.Show to have obtained the preferable GFAP recombinant protein of purity.
The preparation and screening of embodiment 3, GFAP monoclonal antibody
According to the GFAP protein fragments of the standard method purifying of recombination generation, to Balb/c mouse, (it is real that tonneau China is tieed up in Beijing
Test zoo technical Co., Ltd) it is immunized.The specific method is as follows:
1, animal immune: purified GFAP antigen is emulsified with complete Freund's adjuvant, immune using subcutaneous injection method
6-8 week old Balb/c mouse, immunizing dose are 30 μ g/, and progress is immune for the second time after two weeks at interval, with incomplete Freund's adjuvant
Emulsification, immunizing dose are 30 μ g/.It is immune that tail blood is taken to measure serum titer with ELISA method gradient dilution afterwards twice；According to result
Determine whether booster immunization, chooses the highest mouse of antibody titer and carry out cell fusion.
2, cell fusion: myeloma cell uses the sp2/0 in the source Balb/c, and logarithmic growth phase is in when fusion；It takes
Immune mouse spleen, is made lymphocyte single cell suspension；Myeloma cell is mixed with mouse spleen lymphocyte with 1:5-1:10,
37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, incomplete culture medium terminate liquid and remaining terminate liquid is added, after supernatant is abandoned in centrifugation
HAT culture medium be added suspend and mix, MC constant volume to 50mL is dispensed into 3.5cm culture dish, is put in wet box, be placed in 37 DEG C,
5%CO2It is cultivated in constant incubator.
3, screen and clone: fusion selected cell clone in 7-10 days, carried out ELISA survey using GFAP purification of recombinant proteins
Examination.Mark cell strain number.Limiting dilution, 5-6 days measurement ELISA values after each limiting dilution, picking are carried out to positive hole cell
The OD280 positive is worth higher monoclonal hole and carries out limiting dilution, until ELISA measures 96 orifice plates, hardened fruit is the positive entirely.Picking
The positive is worth high monoclonal singling.It is Umab129 that it, which corresponds to fusion plate cell strain,.
4, the preparation and purification of free serum culture supernatant monoclonal antibody: monoclonal antibody hybridoma cell strain expands training in rolling bottle
It supports, when cell conditioned medium volume, which spreads cultivation, reaches 60%-70% to task amount and cell mortality, collects cell suspension 6000PRM
High speed centrifugation 20min, with the membrane filtration of 0.45um, affinity chromatography carries out supernatant purifying, is selected according to antibody subtype corresponding
Column material, the monoclonal antibody that cell strain Umab129 is generated are IgG1, are purified using protein G.Monoclonal antibody concentration after purification is surveyed
Fixed, WB is detected, dispenses, is frozen at -20 DEG C.Wherein WB testing result is shown in Fig. 4-7.
By Fig. 4 result as it can be seen that Umab129 can be good at identifying GFAP full-length proteins；By Fig. 5 result as it can be seen that Umab129
Identify the endogenous GFAP albumen in SNB19 people's glioblastoma cells；By Fig. 6-7 result as it can be seen that Umab129 specific recognition people
And the endogenous GFAP albumen in mouse tissue, molecular weight is consistent with expected molecular size range, shows monoclonal antibody Umab129 energy
Specifically Western blot detects complete GFAP albumen.
Embodiment 4, the immunohistochemistry that monoclonal antibody Umab129 is primary antibody detect
(1), experimental method:
1, the adult brain tissue for taking formalin fixed, Human embryo brain cortical tissue and Human embryo cerebellar tissue carry out stone
Wax embedding, is sliced using Finesse histotome, and tissue thickness is 6 μm.
2, dewaxing and aquation: analyzing pure 3 × 10min of dimethylbenzene, dehydrated alcohol 3 × 10min, 95% ethyl alcohol 5min, and 85%
Ethyl alcohol 5min, 75% ethyl alcohol 5min, deionized water impregnate 3min × 3 time.
3, antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker hot high pressure is added and repairs 3min, to height
When pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, takes out sample, then cooled to room temperature.Deionized water impregnates 3min
× 3 times.
4, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is impregnated
5min × 3 time.
5, confining liquid (+5% Normal Goat Serum of PBS+5% skimmed milk power) is added, 37 DEG C of incubation 60min.
6, confining liquid is removed, is not rinsed, is added GFAP monoclonal antibody (Umab129), thinner ratio: 1:100 is carried out using confining liquid
Dilution.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, washs 5min every time.PBST
(0.02%Tween-20) is washed 1 time, washs 5min every time.
7,1,37 DEG C of reagent of Polink- kit 2 (Catlog No.D37-15) incubation 10-20 minutes is added dropwise.Use PBS
Washing 3 times, each 5min.2,37 DEG C of reagent of Polink-2 kit (Catlog No.D37-15) incubation 10-20 minutes is added dropwise,
It is washed 3 times using PBS, each 5min.
8, it develops the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill water washing.
9, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water rinses 3 times, is stored at room temperature
1min。
10, it is dehydrated and transparent: 75% ethyl alcohol 5min, 100% ethyl alcohol 5min x 3 times, 85% ethyl alcohol 5min, 95% ethyl alcohol
5min, 100% 3 × 5min of ethyl alcohol；3 × 5min of dimethylbenzene, neutral gum mounting.
11, microscopy is shown in Fig. 8-10.
(2), experimental result:
It, can in Human embryo brain cortical tissue and Human embryo cerebellar tissue by Fig. 8-10 result as it can be seen that in adult brain tissue
See specific cytoplasmic dyeing.As a result consistent with GFAP positioning in the cell and tissue expression specificity, show that monoclonal is anti-
Body Umab129 can be used for the level of Immunohistochemical detection GFAP albumen.
Embodiment 5, the Immunofluorescence test that monoclonal antibody Umab129 is primary antibody
1), experimental method:
1, cell spreads version: taking the rat primary neuronal cell of in vitro culture to tile into 24 orifice plates, places incubator training
It supports 24 hours.
2, culture medium is removed, washed once with the PBS of preheating, cell monolayer culture is made.
3, fixed: 4% paraformaldehyde (PBS preparation) room temperature fixes 10 minutes.
4, fixer is removed, PBS is washed 3 times.
5, it penetrates: penetrating punching 5 minutes with 0.1%Triton-X-100 (PBS preparation) at room temperature, PBS is washed 3 times.
6, confining liquid (PBS+2%BSA) is added, 37 DEG C are incubated for 60 minutes.
7, GFAP monoclonal antibody (Umab129) is added, thinner ratio: 1:100 is diluted using confining liquid, and 37 DEG C are incubated for 60 points
Clock.PBS is washed 3 times.
8, Alexa is added- 488AffiniPure Goat Anti-Mouse IgG (H+L), thinner ratio: 1:
500, it is diluted using confining liquid, room temperature is protected from light incubation 60 minutes.PBS is washed 3 times.
9, DAPI (HH3342) redyes nucleus 3 minutes, microscopy.
As a result as shown in figure 11, the visible specific cytoplasmic dyeing in rat primary neuronal cell, shows monoclonal
Antibody Umab129 can be used for the level of Immunofluorescence test GFAP albumen.
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>anti-GFAP protein monoclonal antibody and application thereof
<210> 1
<211>1299
<212> DNA
<213>artificial sequence
<400> 1
1 ATGGAGAGGA GACGCATCAC CTCCGCTGCT CGCCGCTCCT ACGTCTCCTC AGGGGAGATG
61 ATGGTGGGGG GCCTGGCTCC TGGCCGCCGT CTGGGTCCTG GCACCCGCCT CTCCCTGGCT
121 CGAATGCCCC CTCCACTCCC GACCCGGGTG GATTTCTCCC TGGCTGGGGC ACTCAATGCT
181 GGCTTCAAGG AGACCCGGGC CAGTGAGCGG GCAGAGATGA TGGAGCTCAA TGACCGCTTT
241 GCCAGCTACA TCGAGAAGGT TCGCTTCCTG GAACAGCAAA ACAAGGCGCT GGCTGCTGAG
301 CTGAACCAGC TGCGGGCCAA GGAGCCCACC AAGCTGGCAG ACGTCTACCA GGCTGAGCTG
361 CGAGAGCTGC GGCTGCGGCT CGATCAACTC ACCGCCAACA GCGCCCGGCT GGAGGTTGAG
421 AGGGACAATC TGGCACAGGA CCTGGCCACT GTGAGGCAGA AGCTCCAGGA TGAAACCAAC
481 CTGAGGCTGG AAGCCGAGAA CAACCTGGCT GCCTATAGAC AGGAAGCAGA TGAAGCCACC
541 CTGGCCCGTC TGGATCTGGA GAGGAAGATT GAGTCGCTGG AGGAGGAGAT CCGGTTCTTG
601 AGGAAGATCC ACGAGGAGGA GGTTCGGGAA CTCCAGGAGC AGCTGGCCCG ACAGCAGGTC
661 CATGTGGAGC TTGACGTGGC CAAGCCAGAC CTCACCGCAG CCCTGAAAGA GATCCGCACG
721 CAGTATGAGG CAATGGCGTC CAGCAACATG CATGAAGCCG AAGAGTGGTA CCGCTCCAAG
781 TTTGCAGACC TGACAGACGC TGCTGCCCGC AACGCGGAGC TGCTCCGCCA GGCCAAGCAC
841 GAAGCCAACG ACTACCGGCG CCAGTTGCAG TCCTTGACCT GCGACCTGGA GTCTCTGCGC
901 GGCACGAACG AGTCCCTGGA GAGGCAGATG CGCGAGCAGG AGGAGCGGCA CGTGCGGGAG
961 GCGGCCAGTT ATCAGGAGGC GCTGGCGCGG CTGGAGGAAG AGGGGCAGAG CCTCAAGGAC
1021 GAGATGGCCC GCCACTTGCA GGAGTACCAG GACCTGCTCA ATGTCAAGCT GGCCCTGGAC
1081 ATCGAGATCG CCACCTACAG GAAGCTGCTA GAGGGCGAGG AGAACCGGAT CACCATTCCC
1141 GTGCAGACCT TCTCCAACCT GCAGATTCGA GAAACCAGCC TGGACACCAA GTCTGTGTCA
1201 GAAGGCCACC TCAAGAGGAA CATCGTGGTG AAGACCGTGG AGATGCGGGA TGGAGAGGTC
1261 ATTAAGGAGT CCAAGCAGGA GCACAAGGAT GTGATGTGA
//
<210> 2
<211>432
<212> PRT
<213>artificial sequence
<400> 2
ORIGIN
1 MERRRITSAA RRSYVSSGEM MVGGLAPGRR LGPGTRLSLA RMPPPLPTRV DFSLAGALNA
61 GFKETRASER AEMMELNDRF ASYIEKVRFL EQQNKALAAE LNQLRAKEPT KLADVYQAEL
121 RELRLRLDQL TANSARLEVE RDNLAQDLAT VRQKLQDETN LRLEAENNLA AYRQEADEAT
181 LARLDLERKI ESLEEEIRFL RKIHEEEVRE LQEQLARQQV HVELDVAKPD LTAALKEIRT
241 QYEAMASSNM HEAEEWYRSK FADLTDAAAR NAELLRQAKH EANDYRRQLQ SLTCDLESLR
301 GTNESLERQM REQEERHVRE AASYQEALAR LEEEGQSLKD EMARHLQEYQ DLLNVKLALD
361 IEIATYRKLL EGEENRITIP VQTFSNLQIR ETSLDTKSVS EGHLKRNIVV KTVEMRDGEV
421 IKESKQEHKD VM
//
Claims (7)
1. a kind of monoclonal antibody, which is characterized in that it specifically binds with glial fibrillary acid protein (GFAP)；The Dan Ke
Grand antibody is generated by hybridoma cell strain Umab129, and deposit number is CGMCC No.15598.
2. a kind of hybridoma cell strain, deposit number is CGMCC No.15598.
3. monoclonal antibody as described in claim 1 is in preparation for detecting the application in GFAP immune detection tool.
4. application according to claim 3, the immune detection tool is kit, chip or test paper.
5. a kind of immunologic combined detection reagent kit, including monoclonal antibody described in claim 1.
6. application of the monoclonal antibody as described in claim 1 in the kit that preparation is used for tagged tissue cell.
7. application according to claim 6, the histocyte is neuroastrocytoma, brain tissue etc..
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| CN201811015869.8A CN108997496A (en) | 2018-10-16 | 2018-10-16 | Anti- GFAP protein monoclonal antibody and application thereof |
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| CN108997496A true CN108997496A (en) | 2018-12-14 |
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ID=64590836
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633171A (en) * | 2018-12-29 | 2019-04-16 | 上海八通生物科技股份有限公司 | The quickly fluorescence immune chromatography test paper bar and its preparation and application of detection GFAP |
| CN110903389A (en) * | 2019-12-13 | 2020-03-24 | 福州迈新生物技术开发有限公司 | Monoclonal antibody and cell line for resisting GFAP protein, and preparation method and application thereof |
| CN112175079A (en) * | 2020-10-10 | 2021-01-05 | 武汉华美生物工程有限公司 | Preparation method and application of multifunctional anti-GFAP monoclonal antibody |
| CN114375902A (en) * | 2021-12-09 | 2022-04-22 | 广州医科大学附属第二医院 | Construction method of autoimmune GFAP astrocytosis rabbit animal model |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109633171A (en) * | 2018-12-29 | 2019-04-16 | 上海八通生物科技股份有限公司 | The quickly fluorescence immune chromatography test paper bar and its preparation and application of detection GFAP |
| CN110903389A (en) * | 2019-12-13 | 2020-03-24 | 福州迈新生物技术开发有限公司 | Monoclonal antibody and cell line for resisting GFAP protein, and preparation method and application thereof |
| CN112175079A (en) * | 2020-10-10 | 2021-01-05 | 武汉华美生物工程有限公司 | Preparation method and application of multifunctional anti-GFAP monoclonal antibody |
| CN114375902A (en) * | 2021-12-09 | 2022-04-22 | 广州医科大学附属第二医院 | Construction method of autoimmune GFAP astrocytosis rabbit animal model |
| CN114375902B (en) * | 2021-12-09 | 2023-08-18 | 广州医科大学附属第二医院 | Construction method of autoimmune GFAP astrocytopathy rabbit animal model |
| CN117820471A (en) * | 2024-03-05 | 2024-04-05 | 南京诺唯赞医疗科技有限公司 | GFAP specific antibody and application thereof in GFAP detection kit |
| CN117820471B (en) * | 2024-03-05 | 2024-05-10 | 南京诺唯赞医疗科技有限公司 | GFAP specific antibody and application thereof in GFAP detection kit |
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