CN106978400A - Anti- PD L1 protein monoclonal antibodies and application thereof - Google Patents
Anti- PD L1 protein monoclonal antibodies and application thereof Download PDFInfo
- Publication number
- CN106978400A CN106978400A CN201611134214.3A CN201611134214A CN106978400A CN 106978400 A CN106978400 A CN 106978400A CN 201611134214 A CN201611134214 A CN 201611134214A CN 106978400 A CN106978400 A CN 106978400A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibodies
- protein
- hybridoma
- cgmcc
- protein monoclonal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to biological technical field, anti-PD L1 protein monoclonal antibodies hybridoma and its anti-PD L1 monoclonal antibodies of generation and application are disclosed.Anti- PD L1 protein monoclonal antibody hybridomas deposit number of the present invention is CGMCC NO：12287.The hybridoma that the present invention is provided can stably excreting produce anti-PD L1 protein monoclonal antibodies, and combined with PD L1 protein-specifics, and with nearly 10000 kinds other albumen no cross reactions on protein chip, specificity, accuracy and the reliability of PD L1 protein immunizations detection are significantly improved, the detection of PD L1 in various tumor tissues and its microenvironment is widely used in.
Description
Technical field
The present invention relates to biological technical field, and in particular to anti-PD-L1 protein monoclonal antibodies hybridoma and its production
Raw anti-PD-L1 monoclonal antibodies and application.
Background technology
The ligand 1 of programmed cell death factors 1 (processed death-1 ligand 1, PD-L1) is also known as B7-
H1, CD274, are an inhibition costimulatory moleculeses by CD274 gene codes, are that Dong is equal to 1999 from human gene bank
Find and confirm in placenta cdna library when middle search B7.1 and B7.2 immune globulin variable regions and constant region homolgous molecule
's.PD-L1 belongs to I type transmembrane glycoproteins, there is 290 amino acid, including extracellular region, hydrophobic transmembrane and afterbody cytoplasmic domain.PD-L1
Then wide expression, in lymphocyte, such as activates T and B cell, macrophage, BMDC, thymic epithelial cell is thin albumen
The position such as born of the same parents and heart and placenta, while also high expression in the immunocyte in kinds of tumor cells and tumor microenvironment,
Such as lung cancer, breast cancer, the cancer of the esophagus, lymthoma.
PD-L1 plays more crucial role in immunological regulation.The PD-L1 of tumor cells expression is with expression in activation T
Negativity costimulatory molecules PD-1 on cell is combined, and can suppress T cell propagation, and promote the apoptosis of activating T cell.In recent years,
Substantial amounts of research shows that PD-L1 can be examined as a kind of targeted therapy of important molecular marker in various tumours and prognosis
Played an important role in disconnected.
At present clinically generally with immunohistochemistry (Immunohistochemistry, IHC) experiment detection tumor group
The expression situation of PD-L1 in inner tumour cell is knitted, the core of its experimental method is specific binding PD-L1 monoclonal antibody,
The quality of its performance directly decides the sensitivity and specificity entirely detected.Therefore, a kind of binding specificity is developed higher
The monoclonal antibody for PD-L1 albumen have great importance.
The content of the invention
In view of this, it is an object of the invention to provide anti-PD-L1 protein monoclonal antibodies hybridoma and its generation
Anti- PD-L1 monoclonal antibodies and application, improve the binding specificity of monoclonal antibody and PD-L1 albumen that the hybridoma is produced
And be applied in the preparation of Related product.
To achieve the above object, the present invention provides following technical scheme：
Anti- PD-L1 protein monoclonal antibodies hybridoma, deposit number is CGMCC NO：12287.
Anti- PD-L1 protein monoclonal antibodies hybridoma of the present invention is prepared by following methods：
(1) structure of recombinant expression carrier：According to PD-L1 ORF nucleotide sequences, (PD-L1 ORF nucleotide sequences are such as
Shown in SEQ ID NO.1,870bp；PD-L1 amino acid sequences are as shown in SEQ ID NO.2) design primer enter performing PCR amplification, base
Because both sides introduce restriction endonuclease sites SgfI and MluI respectively, expression vector pCMV6-Entry (article No.s are inserted
PS100001, Origene company), build PD-L1 recombinant expression plasmids RC213071；
(2) expression and purification of PD-L1 recombinant proteins：PD-L1 recombinant expression plasmids are transfected into HEK293T, cracking centrifugation
Supernatant is taken, the purifying of DDK affinity columns obtains the PD-L1 recombinant proteins of purifying；
(3) screening and preparation of monoclonal antibody：BALB/c mouse is immunized using the PD-L1 recombinant proteins of above-mentioned purifying,
Mouse spleen cells are taken to be merged with SP2/0 cells, limiting dilution assay obtains monoclonal, ELISA method screening positive hybridoma
Cell, acquisition can secrete the hybridoma cell strain of anti-PD-L1 specific antibodies；Antibody is prepared by serum free medium, passed through
Affinity column purifying obtains PD-L1 monoclonal antibodies.By immunohistochemical experiment verify the monoclonal antibody sensitivity and
Specificity.
After prepared by the above method, the hybridoma for being capable of the anti-PD-L1 monoclonal antibodies of stably excreting is filtered out, is ordered
Entitled UMAB228, hypotype is accredited as IgG1, and is preserved in Chinese microorganism strain preservation conservator on April 27th, 2016
Meeting common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, is protected
It is CGMCC NO to hide numbering：12287.
Meanwhile, the present invention also provides a kind of anti-PD-L1 protein monoclonal antibodies, is CGMCC NO by deposit number：12287
Hybridoma secretion produce.The art conventional method can be used by preparing anti-PD-L1 protein monoclonal antibodies, such as be passed through
Serum free medium prepares antibody, and PD-L1 monoclonal antibodies are obtained by affinity column purifying.
Deposit number of the present invention is CGMCC NO：12287 hybridoma chromosome stabilityX, it secretes what is produced
Anti- PD-L1 protein monoclonal antibodies are IgG1 types, and potency is higher.The SABC testing result of the monoclonal antibody shows,
It can be in specific recognition cancerous lung tissue, breast cancer tissue, human esophageal carcinoma, lymphoma tissue, normal tonsil
PD-L1 albumen.
Meanwhile, the specific test for detecting the monoclonal antibody using OriGene high-density proteins chip shows, of the present invention
Monoclonal antibody and nearly 10000 kinds other albumen no cross reactions.
Therefore, it is CGMCC NO present invention also offers deposit number：12287 hybridoma is preparing anti-PD-
Application and secreted anti-PD-L1 protein monoclonal antibodies in L1 protein monoclonal antibodies are preparing detection PD-L1 albumen
Immune detection product in application.
Preferably, the immune detection product is kit, test paper or chip.
The present invention also provides a kind of kit of SABC detection, including deposit number is CGMCC NO：12287 it is miscellaneous
The anti-PD-L1 protein monoclonal antibodies for handing over oncocyte secretion to produce.The immunity detection reagent can detect tumor tissues and its
PD-L1 expression situation in microenvironment.
In addition, it is CGMCC NO that the present invention, which also provides deposit number,：The anti-PD- that 12287 hybridoma secretion is produced
Application of the L1 protein monoclonal antibodies in the kit for preparing marked tumor tissue and its microenvironment medium size lymphocyte.
Preferably, the tumour includes lung cancer, lymthoma, the cancer of the esophagus, liver cancer and breast cancer.
The hybridoma that the present invention is provided can stably excreting produce anti-PD-L1 protein monoclonal antibodies, and with PD-L1 eggs
White specific binding, and with nearly 10000 kinds other albumen no cross reactions on protein chip, significantly improve PD-L1 albumen and exempt from
Specificity, accuracy and the reliability of epidemic disease detection, are widely used in the detection of PD-L1 in various tumor tissues and its microenvironment.
Biological deposits information explanation
UMAB228, Classification And Nomenclature is the strain of Programmed death 1 (PD-L1) monoclonal antibody hybridoma cell, in 2016
On April 27, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, deposit number is CGMCC NO：12287.
Brief description of the drawings
Fig. 1 show cloning site figure in embodiment 1；
Fig. 2 show PD-L1 albumen Western blot testing result figures in embodiment 2, and PD-L1 is detected with anti-DDK
Expression of the albumen in HEK293T cells, wherein swimming lane L are that the HEK293T cell pyrolysis liquids of transfection empty carrier are the inspection of antigen
Survey the testing result of result, swimming lane R for the HEK293T cell pyrolysis liquid antigens of transfection pCMV6-PD-L1 plasmids；
Fig. 3 show PD-L1 protein SDS-PAGEs result figure in embodiment 2；
(primary antibody is that UMAB228 secretes the PD-L1 produced to the cancerous lung tissue SABC testing result figure of embodiment illustrated in fig. 44
Monoclonal antibody, 1:50)；
(primary antibody is that UMAB228 secretes the PD- produced to the lymphoma tissue SABC testing result figure of embodiment illustrated in fig. 54
L1 monoclonal antibodies, 1:50)；
(primary antibody is that UMAB228 secretes the PD- produced to the human esophageal carcinoma SABC testing result figure of embodiment illustrated in fig. 64
L1 monoclonal antibodies, 1:50)；
(primary antibody is that UMAB228 secretes the PD- produced to the breast cancer tissue's SABC testing result figure of embodiment illustrated in fig. 74
L1 monoclonal antibodies, 1:50)；
(primary antibody is that UMAB228 secretes the PD- produced to the tonsil SABC testing result figure of embodiment illustrated in fig. 84
L1 monoclonal antibodies, 1:50)；
(primary antibody is that UMAB228 secretes the PD-L1 produced to embodiment illustrated in fig. 9 5origene protein chip qualification results figure
Monoclonal antibody, 1:100；Secondary antibody is the donkey anti-mouse IgG that Alexa 647- are marked, 1:500).
Embodiment：
The invention discloses anti-PD-L1 protein monoclonal antibodies hybridoma and its anti-PD-L1 monoclonals of generation are anti-
Body and application, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular
It is that all similar replacements and change are apparent to those skilled in the art, they are considered as being included in this
Invention.The product of the present invention and application are described by preferred embodiment, and related personnel can substantially not depart from this
Method described herein and application are modified in the content of the invention, spirit and scope or suitably change are with combining, come realize with
Using the technology of the present invention.
The anti-PD-L1 protein monoclonal antibodies hybridoma and its anti-PD-L1 of generation provided below with regard to the present invention is mono-
Clonal antibody and application are described further.
Embodiment 1：The structure of PD-L1 recombinant expression plasmids
According to PD-L1 cDNA sequence synthetic gene, design two primers and introduce restriction enzyme site SgfI and MluI respectively,
Expression vector pCMV6-Entry is cloned into, the eukaryotic expression recombinant plasmid of PD-L1 full-length proteins is set up.Cloning site is designed such as
Shown in Fig. 1.
Embodiment 2：The expression and purification of PD-L1 recombinant proteins
1st, HEK293T cells are transfected：HEK293T cells are with 1:3, which reach continuation in culture dish, cultivates；Take 7.5mLDMEM (nothings
Serum and antibiotic) into 50mL pipes, add 300 μ LPEI MegaTran 1.0 and mix；Add 75 μ g PD-L1 recombinant plasmids
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 respectively2Trained in incubator
Support.After transfection 24 hours, 25 μ L2M sodium butyrates are added per ware cell to final concentration 5mM.
2nd, cell lysis：After transfection 48 hours, cell cracking is carried out.Culture medium is sucked, 1mLPBS is added and is rinsed, inhaled
Remove PBS.1mL lysis buffers are added, preceding addition protease inhibitors PI and PMSF is used.It is placed in ice chest and is shaken on shaking table
Swing, collect and lysate is obtained in all culture dishes, supernatant is collected in 4 degree of centrifugations.
3rd, DDK affinity columns are purified：With 0.45 μM, the lysate supernatant after 33mm pvdf membrane filter filter centrifugations is simultaneously
15mL pipes are transferred to, the Beads 1mL that mix is added, is put into after sealing in 360 degree of vortex mixers, in 4 DEG C of combinations 2 hours；Take out
15mL is managed, and lysate is poured into BIO-RAD chromatographic columns, and is caught and penetrated liquid, drop to the greatest extent after penetrate liquid sampling WB detections, see Fig. 2；
Post material is rinsed 1-2 times with lysis buffer, Beads is rinsed 3 times with TBST again after drop is most, 0.1M Glycine are used after dripping to the greatest extent
PH3.5 is eluted, for the first time 200 μ L, and drop is not collected to the greatest extent, second and third each 500 μ L, and 250 μ L of third time are collected to a 1.5mL
In Tube, and it is rapidly added NaH2PO4(pH=11.0) pH7.0 or so is neutralized to, often pipe adds glycerine to final concentration of 10%,
Tween-80 to final concentration of 0.1%.PD-L1 albumen after purification is identified with SDS-PAGE, sees Fig. 3.
From Fig. 2 results, tag antibody anti-DDK can detect transfection pCMV6-PD-L1 recombinant plasmid
PD-L1 recombinant proteins (R) in HEK293T cell pyrolysis liquids, because PD-L1 is I type transmembrane glycoproteins, therefore its egg size is 45
Multi-ribbon between~55kDa, is consistent with expection, shows that PD-L1 recombinant proteins are correctly expressed.
From Fig. 3 results, purified by DDK affinity columns, Glycine elutions can obtain the PD-L1 weights of high-purity
Histone.
Embodiment 3：The preparation screening of anti-PD-L1 monoclonal antibody hybridoma cells and its monoclonal antibody
The total length PD-L1 recombinant proteins (hereinafter referred to as PD-L1 antigens) of the purifying produced according to standard method with restructuring
For B6/C57 mouse (Beijing Vital River Experimental Animals Technology Co., Ltd.) to be immunized.Specific method is as follows：
1st, animal immune：Purified PD-L1 antigens are emulsified with complete Freund's adjuvant, using subcutaneous or intraperitoneal injection side
6-8 week old BALB/c mouses are immunized in method, and for 50 μ g/ only, interval carries out being immunized for second immunizing dose after two weeks, with not exclusively not
Family name's adjuvant emulsion, immunizing dose is 50 μ g/.It is immune to take tail blood to determine serum titer with ELISA method gradient dilution afterwards twice；Root
Determine whether booster immunization according to result, choose antibody titer highest mouse and carry out cell fusion.
2nd, cell fusion：Myeloma cell uses the sp2/0 that BALB/c originates, and exponential phase is in during fusion；Take
Immune mouse spleen, is made lymphocyte single cell suspension；Mouse spleen lymphocyte is with myeloma cell with 1:5-1:10 mixing,
37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, incomplete culture medium and remaining terminate liquid is added, centrifugation, which is abandoned, adds HAT after supernatant
Culture medium, which suspends, to be mixed, and MC constant volumes are dispensed into 3.5cm culture dishes, are put in wet box to 50mL, are placed in 37 DEG C, 5%CO2It is permanent
Cultivated in warm incubator.
3rd, screen and clone：Fusion selects cell clone in 7-10 days, is carried out using the PD-L1 recombinant proteins of purifying
ELISA is tested.Mark cell line number.Positive hole cell is carried out to determine ELISA within 5-6 days after limiting dilution, each limiting dilution
Value, the higher monoclonal hole of picking OD280 positive values carries out limiting dilution, until it is sun that ELISA, which determines the complete hardened fruit of 96 orifice plates,
Property.The high monoclonal singling of picking positive value.Its correspondence fusion plate cell line is UMAB228.
4th, the preparation and purification of cell conditioned medium monoclonal antibody：The DMEM culture mediums containing 15% serum are used to train cell line UMAB228
Support and cultivated in 10cm culture dishes, spread cultivation to about 4 × 107When individual, 800rpm centrifugation 5min abandon supernatant and cell are transferred into 2L
In rolling bottle, serum free medium is added, it is about 3 × 10 to make cell density5Individual/ml.Continue after cultivating 1~2 week, work as cell death
(now cell density is about 1-2 × 10 when rate reaches 60%-70%6Individual/ml), collect cell suspension 6000rpm at a high speed from
Heart 20min, takes supernatant, and affinity chromatography carries out supernatant purifying, and corresponding post material, cell line UMAB228 are selected according to antibody subtype
The monoclonal antibody hypotype of generation is IgG1, is purified using protein G.Monoclonal antibody concentration mensuration after purification, packing (100uL/
Pipe, concentration is 1mg/ml), be stored in 4-8 DEG C.
Embodiment 4：The monoclonal antibody that UMAB228 secretions are produced detects for the SABC of primary antibody
(1) experimental method：
1st, take lung cancer, lymthoma, the cancer of the esophagus, breast cancer and tonsil block to carry out FFPE respectively, use
SAKURA histotomes are cut into slices, and tissue thickness is 4 μm.
2nd, dewaxing and aquation：Analyze pure 3 × 10min of dimethylbenzene, absolute ethyl alcohol 3 × 10min, 95% ethanol 5min, 85%
Ethanol 5min, 75% ethanol 5min, deionized water immersion 3min × 3 time
3rd, antigen retrieval buffers (1mM EDTA, pH8.5 10mM Tris-HCL buffer solutions) pressure cooker hot high pressure is added to repair
2.5min, when high pressure pot temperature is down to about 90 DEG C, opens pressure cooker, takes out sample, then naturally cool to room temperature.Deionization
Water soaks 3min × 3 time.
4th, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is soaked
5min × 3 time.
5th, plus confining liquid (Normal Goat Serum of PBS+5% skimmed milk powers+5%), 37 DEG C of incubation 60min.
6th, confining liquid is removed, is not rinsed, PD-L1 monoclonal antibodies (UMAB228 secretions are produced), thinner ratio is added：1:50, use envelope
Liquid is closed to be diluted.It is placed in wet box, 37 DEG C of incubation 60min.PBST (0.1%Tween-20) is washed 2 times, every time washing
5min.PBST (0.02%Tween-20) is washed 1 time, and 5min is washed every time.
7th, reagent PV6000 (Catlog No.PV-6000) in polymer HRP staining kits is added dropwise, 37 DEG C are incubated 15 points
Clock.Washed 3 times using PBS, each 5min.
8th, developed the color using DAB solution (Zhong Shan Golden Bridge ZLI-9019), develop the color 3~10min.Distill water washing.
9th, haematoxylin redyeing nucleus 2min, distilled water rinsing, the differentiation of 1% hydrochloric acid.Distilled water is rinsed 3 times, is stored at room temperature
1min。
10th, it is dehydrated and transparent：75% ethanol 5min, 100% ethanol 5min x 3 times, 85% ethanol 5min, 95% ethanol
5min, 100% 3 × 5min of ethanol；3 × 5min of dimethylbenzene, neutral gum mounting.
11st, microscopy, is shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8.
(2) experimental result：
From Fig. 4 results, PD-L1 albumen is in specific cell film expression, such as figure arrow in the tumour cell of cancerous lung tissue
Tumour cell shown in head.
From Fig. 5 results, PD-L1 albumen is in specific cell film expression on the lymphocyte in lymphoma tissue,
Lymphoma cell as indicated in the figures by an arrow.
From Fig. 6 results, PD-L1 albumen is in specific cell film expression in the tumour cell of human esophageal carcinoma, is such as schemed
Tumour cell shown in arrow.
From Fig. 7 results, PD-L1 albumen is in specific cell film expression in the tumour cell of breast cancer tissue, is such as schemed
Tumour cell shown in arrow.
From Fig. 8 results, PD-L1 albumen is in specific cell film table on the lymphocyte in normal tonsil
Reach, as indicated in the figures by an arrow lymphocyte.
As a result show that the monoclonal antibody that UMAB228 of the present invention is produced being capable of specific recognition lung cancer, lymphoma tissue, food
PD-L1 albumen in pipe cancer, breast cancer and tonsil.
Embodiment 5：The specific proteins chip detection for the monoclonal antibody that UMAB228 secretions are produced
Lysate is overexpressed comprising 10000 HEK293T cell proteins on OriGene high-density protein chips, per hatching egg
White lysate has the repetition of two copies on chip.Protein lysate is by trace on nitrocellulose filter.Each clock egg
The positioning of white lysate can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, each
There are some references on sub- matrix, by referring to can quantify the content of albumen on each chip point, monitor each immune response number
According to repeatability, and positioning positive signal direction.It is using OriGene albumen (OriGene Cat PA100001) below
The method that chip carries out the Identification of Monoclonal Antibodies experiment that UMAB228 secretions are produced：
1st, a protein chip is placed in 50mL centrifuge tubes, infiltrates chip using 40mL deionized waters, be placed on shaking table,
Mixed at room temperature 30 minutes.Deionized water is discarded, chip is balanced using 10mLPBST.Room temperature treatment 10 minutes.
2nd, add 40mL5% skim milks (being diluted with PBST) into 50mL centrifuge tubes to be placed on shaking table, room temperature envelope
Close 30 minutes.
3rd, primary antibody UMAB228 is diluted using confining liquid (5% skim milk), thinner ratio is classified as 1：100.
4th, clean sealed membrane is pasted on experimental bench, 250-300 μ L primary antibodies is added dropwise on sealed membrane.
5th, protein chip is extracted out from confining liquid, by the one of protein chip NC films down, contacted from one side of chip
Antibody, is slowly slided, by surface tension of liquid, and antibody will slowly infiltrate chip NC films, until whole NC films infiltration is in primary antibody
In solution.Whole operation process avoids producing bubble.Chip is moved on under 4 DEG C of environment, stood, primary antibody is incubated overnight.In chip
Upper capping culture dish lid, sticks a hygenic towelette thereon, causes antibody to evaporate to prevent from being incubated for a long time.
6th, chip was moved in 50mL centrifuge tubes in second day, chip is rinsed twice using PBST, remove unnecessary antibody.Make
Chip is washed with 40mL PBST (0.1%Tween-20), is placed on shaking table and is well mixed, wash three times, 5min is washed every time.
7th, the anti-mouse IgG of donkey of secondary antibody Alexa 647- marks is diluted using confining liquid (5% skim milk), dilution ratio is
1：500.
8th, according to above-mentioned steps 4, step 5 carries out secondary antibody and is incubated operation.Incubation at room temperature 1 hour.Aluminium foil is used above chip
Paper is covered, to prevent signal bleaching.
9th, according to above-mentioned steps 6, chip is washed using PBST.
10th, using deionized water rinsing chip, to remove remnants salinity and denaturant.
11st, drying at room temperature chip, it is ensured that chip is completely dried.
12nd, fluorescence signal is read using chip scanner.
13rd, chip direction and the site of positive signal are determined according to BSA-Cy3 and BSA-Cy5.
14th, correspondence protein lysate ID is found out according to positive signal site, according to lysate database information, found pair
Answer protein name, NCBI typings number (accession number), protein I D, the information such as albumen size.As a result Fig. 8 is seen.
Due to not containing PD-L1 albumen on the protein chip, therefore UMAB288 antibody should not have any on the chip
Binding signal.Fig. 8 results show that all albumen on protein chip secrete the monoclonal antibody produced without knot with UMAB228
Signal is closed, shows monoclonal antibody of the present invention and other nearly 10000 kinds of albumen no cross reactions.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>Anti- IDO1 protein monoclonal antibodies and application thereof
<210> 1
<211>1212
<212> DNA
<213>Artificial sequence
<400> 1
ORIGIN
1 ATGGCACACG CTATGGAAAA CTCCTGGACA ATCAGTAAAG AGTACCATAT TGATGAAGAA
61 GTGGGCTTTG CTCTGCCAAA TCCACAGGAA AATCTACCTG ATTTTTATAA TGACTGGATG
121 TTCATTGCTA AACATCTGCC TGATCTCATA GAGTCTGGCC AGCTTCGAGA AAGAGTTGAG
181 AAGTTAAACA TGCTCAGCAT TGATCATCTC ACAGACCACA AGTCACAGCG CCTTGCACGT
241 CTAGTTCTGG GATGCATCAC CATGGCATAT GTGTGGGGCA AAGGTCATGG AGATGTCCGT
301 AAGGTCTTGC CAAGAAATAT TGCTGTTCCT TACTGCCAAC TCTCCAAGAA ACTGGAACTG
361 CCTCCTATTT TGGTTTATGC AGACTGTGTC TTGGCAAACT GGAAGAAAAA GGATCCTAAT
421 AAGCCCCTGA CTTATGAGAA CATGGACGTT TTGTTCTCAT TTCGTGATGG AGACTGCAGT
481 AAAGGATTCT TCCTGGTCTC TCTATTGGTG GAAATAGCAG CTGCTTCTGC AATCAAAGTA
541 ATTCCTACTG TATTCAAGGC AATGCAAATG CAAGAACGGG ACACTTTGCT AAAGGCGCTG
601 TTGGAAATAG CTTCTTGCTT GGAGAAAGCC CTTCAAGTGT TTCACCAAAT CCACGATCAT
661 GTGAACCCAA AAGCATTTTT CAGTGTTCTT CGCATATATT TGTCTGGCTG GAAAGGCAAC
721 CCCCAGCTAT CAGACGGTCT GGTGTATGAA GGGTTCTGGG AAGACCCAAA GGAGTTTGCA
781 GGGGGCAGTG CAGGCCAAAG CAGCGTCTTT CAGTGCTTTG ACGTCCTGCT GGGCATCCAG
841 CAGACTGCTG GTGGAGGACA TGCTGCTCAG TTCCTCCAGG ACATGAGAAG ATATATGCCA
901 CCAGCTCACA GGAACTTCCT GTGCTCATTA GAGTCAAATC CCTCAGTCCG TGAGTTTGTC
961 CTTTCAAAAG GTGATGCTGG CCTGCGGGAA GCTTATGACG CCTGTGTGAA AGCTCTGGTC
1021 TCCCTGAGGA GCTACCATCT GCAAATCGTG ACTAAGTACA TCCTGATTCC TGCAAGCCAG
1081 CAGCCAAAGG AGAATAAGAC CTCTGAAGAC CCTTCAAAAC TGGAAGCCAA AGGAACTGGA
1141 GGCACTGATT TAATGAATTT CCTGAAGACT GTAAGAAGTA CAACTGAGAA ATCCCTTTTG
1201 AAGGAAGGTT AA
//
<210> 2
<211>403
<212> PRT
<213>Artificial sequence
<400> 2
ORIGIN
1 MAHAMENSWT ISKEYHIDEE VGFALPNPQE NLPDFYNDWM FIAKHLPDLI ESGQLRERVE
61 KLNMLSIDHL TDHKSQRLAR LVLGCITMAY VWGKGHGDVR KVLPRNIAVP YCQLSKKLEL
121 PPILVYADCV LANWKKKDPN KPLTYENMDV LFSFRDGDCS KGFFLVSLLV EIAAASAIKV
181 IPTVFKAMQM QERDTLLKAL LEIASCLEKA LQVFHQIHDH VNPKAFFSVL RIYLSGWKGN
241 PQLSDGLVYE GFWEDPKEFA GGSAGQSSVF QCFDVLLGIQ QTAGGGHAAQ FLQDMRRYMP
301 PAHRNFLCSL ESNPSVREFV LSKGDAGLRE AYDACVKALV SLRSYHLQIV TKYILIPASQ
361 QPKENKTSED PSKLEAKGTG GTDLMNFLKT VRSTTEKSLL KEG
//
Claims (8)
1. anti-PD-L1 protein monoclonal antibodies hybridoma, it is characterised in that deposit number is CGMCC NO：12287.
2. deposit number is CGMCC NO：12287 hybridoma answering in anti-PD-L1 protein monoclonal antibodies are prepared
With.
3. anti-PD-L1 protein monoclonal antibodies, it is characterised in that by deposit number be CGMCC NO：12287 hybridoma
Secretion is produced.
4. deposit number is CGMCC NO：The anti-PD-L1 protein monoclonal antibodies that 12287 hybridoma secretion is produced are in system
Application in the immune detection product of standby detection PD-L1 albumen.
5. apply according to claim 4, it is characterised in that the immune detection product is kit, test paper or chip.
6. a kind of kit of SABC detection, it is characterised in that including deposit number be CGMCC NO：12287 hybridization
The anti-PD-L1 protein monoclonal antibodies that oncocyte secretion is produced.
7. deposit number is CGMCC NO：The anti-PD-L1 protein monoclonal antibodies that 12287 hybridoma secretion is produced are in system
Application in the kit of standby marked tumor tissue and its microenvironment medium size lymphocyte.
8. apply according to claim 7, it is characterised in that the tumour includes knot lymthoma, lung cancer, liver cancer, the cancer of the esophagus
And breast cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611134214.3A CN106978400A (en) | 2016-12-13 | 2016-12-13 | Anti- PD L1 protein monoclonal antibodies and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611134214.3A CN106978400A (en) | 2016-12-13 | 2016-12-13 | Anti- PD L1 protein monoclonal antibodies and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106978400A true CN106978400A (en) | 2017-07-25 |
Family
ID=59340438
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611134214.3A Pending CN106978400A (en) | 2016-12-13 | 2016-12-13 | Anti- PD L1 protein monoclonal antibodies and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106978400A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975554A (en) * | 2019-03-04 | 2019-07-05 | 宁波美晶医疗技术有限公司 | The detection method and its special agent of cell PD-L1 protein expression in a kind of body fluid sample |
| US10669339B2 (en) | 2015-03-13 | 2020-06-02 | Cytomx Therapeutics, Inc. | Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof |
| CN113045662A (en) * | 2021-05-31 | 2021-06-29 | 西宝生物科技（上海）股份有限公司 | Nano antibody for specifically recognizing PD-L1 and application thereof |
| US11168144B2 (en) | 2017-06-01 | 2021-11-09 | Cytomx Therapeutics, Inc. | Activatable anti-PDL1 antibodies, and methods of use thereof |
| CN114195897A (en) * | 2021-12-07 | 2022-03-18 | 广州兆瑞医学生物科技有限公司 | PD-L1 monoclonal antibody, heavy chain and light chain variable regions, monoclonal cell strain, application and kit |
| CN115947848A (en) * | 2022-10-18 | 2023-04-11 | 济南诚艾准凌生物科技有限公司 | Anti-cancer composition prepared from DC cells and monoclonal antibody |
| CN116535511A (en) * | 2023-06-30 | 2023-08-04 | 苏州驾玉生物医药有限公司 | Immunohistochemical antibody for detecting PD-L1 and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104640A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human PD-L1 monoclonal antibody and application thereof |
| EP1899379A2 (en) * | 2005-06-20 | 2008-03-19 | Medarex, Inc. | Cd19 antibodies and their uses |
| CN105461808A (en) * | 2015-12-24 | 2016-04-06 | 长春金赛药业有限责任公司 | Monoclonal antibody and application thereof |
| CN105968200A (en) * | 2016-05-20 | 2016-09-28 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-l1 humanized monoclonal antibody and application thereof |
| CN106632676A (en) * | 2017-01-20 | 2017-05-10 | 新乡学院 | Mouse anti-swine PD-L1 (Programmed Death 1 Ligand 1) monoclonal antibody and application thereof |
-
2016
- 2016-12-13 CN CN201611134214.3A patent/CN106978400A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1899379A2 (en) * | 2005-06-20 | 2008-03-19 | Medarex, Inc. | Cd19 antibodies and their uses |
| CN101104640A (en) * | 2006-07-10 | 2008-01-16 | 苏州大学 | Preparation for anti human PD-L1 monoclonal antibody and application thereof |
| CN105461808A (en) * | 2015-12-24 | 2016-04-06 | 长春金赛药业有限责任公司 | Monoclonal antibody and application thereof |
| CN105968200A (en) * | 2016-05-20 | 2016-09-28 | 瑞阳(苏州)生物科技有限公司 | Anti-human pd-l1 humanized monoclonal antibody and application thereof |
| CN106632676A (en) * | 2017-01-20 | 2017-05-10 | 新乡学院 | Mouse anti-swine PD-L1 (Programmed Death 1 Ligand 1) monoclonal antibody and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| YUICHIRO OHIGASHI等: "Clinical Significance of Programmed death-1 ligand-1 and Programmed death-1 ligand-2 Expression in Human Esophageal Cancer", 《CLIN CANCER RES》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10669339B2 (en) | 2015-03-13 | 2020-06-02 | Cytomx Therapeutics, Inc. | Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof |
| US11174316B2 (en) | 2015-03-13 | 2021-11-16 | Cytomx Therapeutics, Inc. | Anti-PDL1 antibodies, activatable anti-PDL1 antibodies, and methods of use thereof |
| US11168144B2 (en) | 2017-06-01 | 2021-11-09 | Cytomx Therapeutics, Inc. | Activatable anti-PDL1 antibodies, and methods of use thereof |
| CN109975554A (en) * | 2019-03-04 | 2019-07-05 | 宁波美晶医疗技术有限公司 | The detection method and its special agent of cell PD-L1 protein expression in a kind of body fluid sample |
| CN113045662A (en) * | 2021-05-31 | 2021-06-29 | 西宝生物科技（上海）股份有限公司 | Nano antibody for specifically recognizing PD-L1 and application thereof |
| CN113045662B (en) * | 2021-05-31 | 2021-08-13 | 西宝生物科技（上海）股份有限公司 | Nano antibody for specifically recognizing PD-L1 and application thereof |
| CN114195897A (en) * | 2021-12-07 | 2022-03-18 | 广州兆瑞医学生物科技有限公司 | PD-L1 monoclonal antibody, heavy chain and light chain variable regions, monoclonal cell strain, application and kit |
| CN114195897B (en) * | 2021-12-07 | 2023-10-27 | 广州兆瑞医学生物科技有限公司 | PD-L1 monoclonal antibody, heavy chain, light chain variable region, monoclonal cell strain, application and kit |
| CN115947848A (en) * | 2022-10-18 | 2023-04-11 | 济南诚艾准凌生物科技有限公司 | Anti-cancer composition prepared from DC cells and monoclonal antibody |
| CN115947848B (en) * | 2022-10-18 | 2023-11-10 | 深圳市启至健康管理有限公司 | Anti-cancer composition prepared from DC cells and monoclonal antibodies |
| CN116535511A (en) * | 2023-06-30 | 2023-08-04 | 苏州驾玉生物医药有限公司 | Immunohistochemical antibody for detecting PD-L1 and application thereof |
| CN116535511B (en) * | 2023-06-30 | 2023-09-12 | 苏州驾玉生物医药有限公司 | Immunohistochemical antibody for detecting PD-L1 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106978400A (en) | Anti- PD L1 protein monoclonal antibodies and application thereof | |
| CN108467432A (en) | The monoclonal antibody and its cell strain, preparation method and application of anti-E-cadherin albumen | |
| CN107188962A (en) | Anti- CK7 protein monoclonal antibodies and application thereof | |
| CN105238762A (en) | Anti-PD-1 protein monoclonal antibody hybridomas cell, anti- PD-1 monoclonal antibody generated by same and application | |
| CN111454360A (en) | anti-CD 61 protein monoclonal antibody, cell line, preparation method and application thereof | |
| CN105131114B (en) | The anti-p16 monoclonal antibodies and application of anti-p16 protein monoclonal antibodies hybridoma and its generation | |
| CN106188292B (en) | Anti- CD4 protein monoclonal antibody and application thereof | |
| CN102827281B (en) | Monoclonal antibody against CD2 protein and application thereof | |
| CN107188965A (en) | Anti- DOG1 protein monoclonal antibodies and application thereof | |
| CN108997501A (en) | Anti- VWF protein monoclonal antibody and application thereof | |
| CN106749668A (en) | Anti- IDO1 protein monoclonal antibodies and application thereof | |
| CN106957823A (en) | Anti- PD L2 protein monoclonal antibodies and application thereof | |
| CN102924594A (en) | Anti-tumor-protein-P63 monoclonal antibody and purpose thereof | |
| CN106978401A (en) | Anti- ROS1 protein monoclonal antibodies and application thereof | |
| CN109096400A (en) | Anti- PDPN protein monoclonal antibody and application thereof | |
| CN107446046A (en) | Anti- CD20 protein monoclonal antibodies and application thereof | |
| CN106977605A (en) | Anti- CD19 protein monoclonal antibodies and application thereof | |
| CN107266572A (en) | Anti- PD L1 protein monoclonal antibodies and application thereof | |
| CN108998423A (en) | Anti- vimentin (Vimentin) monoclonal antibody and application thereof | |
| CN113845592B (en) | anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application | |
| CN106854245A (en) | Protein monoclonal antibody of AntiCD3 McAb 0 and application thereof | |
| CN107236040A (en) | Anti- MSH6 protein monoclonal antibodies and application thereof | |
| CN105274060B (en) | The anti-GATA3 monoclonal antibodies and application of anti-GATA3 protein monoclonal antibodies hybridoma and its generation | |
| CN107058238A (en) | Anti- CD10 protein monoclonal antibodies and application thereof | |
| CN107043419A (en) | Anti- BTLA protein monoclonal antibodies and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170725 |