CN110357964A - 1 protein monoclonal antibody of AntiCD3 McAb and application thereof - Google Patents

1 protein monoclonal antibody of AntiCD3 McAb and application thereof Download PDF

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CN110357964A
CN110357964A CN201910727944.1A CN201910727944A CN110357964A CN 110357964 A CN110357964 A CN 110357964A CN 201910727944 A CN201910727944 A CN 201910727944A CN 110357964 A CN110357964 A CN 110357964A
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monoclonal antibody
umab30
albumen
protein
cell
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扈晓敏
付伟
柳孟姣
任琪
王成林
何为无
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WUXI ORIGENE BIO-TECHNOLOGY CO LTD
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WUXI ORIGENE BIO-TECHNOLOGY CO LTD
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Abstract

The present invention relates to field of biotechnology, disclose a kind of hybridoma cell strain (deposit number is CGMCC No.18187), and the monoclonal antibody UMAB30 that thus hybridoma cell strain generates.The invention further relates to monoclonal antibody UMAB30 to prepare the application in the immune detection tool for detecting CD31 albumen, application of the immunohistochemical kit and monoclonal antibody UMAB30 of the UMAB30 containing monoclonal antibody in the kit that preparation is used for marked tumor.Monoclonal antibody of the present invention can be in conjunction with CD31 protein-specific, and with other intracellular albumen no cross reactions, specificity, the accuracy and reliability of the detection of CD31 protein immunization are significantly improved, can be used for immunohistochemical diagnosis and identifies good malignant angiogenic tumour.

Description

1 protein monoclonal antibody of AntiCD3 McAb and application thereof
Technical field
The present invention relates to field of biotechnology, and in particular to can specific bond CD31 albumen monoclonal antibody UMAB30, Generate the cell strain of the monoclonal antibody and using the diagnostic method of the antibody and purposes.
Background technique
Vascular cell adhesion molecule-1 (PECAM1) is also known as CD31, is the I type cross-film sugar of molecular weight about 130KD a kind of Albumen, contactin member.CD31 normal expression can be situated between in leucocyte, blood tubule and vascular endothelial cell Guided cell adherency, the function tool up-regulation effect to integrin, while in leucocyte across endothelial cell migration, adjusting blood platelet function Can, inhibit Apoptosis, mediate signal to go to etc. during play a significant role.Due to all blood in development and mature individual Endothelial cell all highly expresses CD31, and therefore, CD31 is often used as the marker of vascular endothelial cell.
CD31 plays an important role during kinds of tumors growth and metastases.In kinds of tumors tissue, such as kidney Cancer, liver cancer, lung cancer, cancer of pancreas and breast cancer, discovery CD31 are overexpressed.The unusual high levels of CD31 are considered pernicious with tumour Degree is directly proportional, and the degree of the expression response tumour growth of CD31 can be used for diagnosing and identifying good malignant angiogenic tumour.At present Clinically commonly use immunohistochemistry (IHC) Pathological experiment detection tumour cell in albumen expression situation, however IHC experiment Core is the monoclonal antibody of binding proteins specific, and the superiority and inferiority of performance directly decides the sensitivity that entirely detects and special Property.Therefore, a kind of higher monoclonal antibody for CD31 albumen of binding specificity is developed to IHC detection CD31 expression water It is flat to have great importance, and then can be used for immunohistochemical diagnosis and identify good malignant angiogenic tumour.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of monoclonals of the higher CD31 albumen of binding specificity to resist Body, and its preparing the application in the immune detection tool for detecting CD31 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 July 11st, 2019, deposit number was CGMCC No.18187.
It is thin by above-mentioned hybridoma the present invention also provides a kind of monoclonal antibody UMAB30 for specifically binding CD31 albumen Born of the same parents' strain generates.
Monoclonal antibody of the present invention the preparation method is as follows:
(1) building of recombinant expression carrier: according to CD31ORF nucleotide sequence (CD31ORF nucleotide sequence such as SEQ ID Shown in NO.1,2217bp；CD31 amino acid sequence is as shown in SEQ ID NO.2)
To 2217bp bit sequence, gene two sides are separated in restricted by design primer PCR amplification CD31ORF 1bp Enzyme cutting site SgfI and MluI is inserted into expression vector pCMV6-Entry, building CD31 amino acid sequence the 1st to the 738th Recombinant expression plasmid pCMV6-rCD31；Upstream amplification primer sequence, SEQ ID NO.3:CACGCGATCGCATGCAGCCGAGG TGGGCCCAA downstream amplification primer sequence SEQ ID NO.4:ACCGACGCGT CTAAGTTCCATCAAGGGAGCC
(2) expression and purification of CD31 recombinant protein: by CD31 recombinant expression plasmid convert HEK293T cell, crack from The heart obtains supernatant, and DDK affinity chromatography column purification obtains the CD31 recombinant protein of purifying；
(3) screening and preparation of monoclonal antibody: Balb/c mouse is immunized using the CD31 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 1 specific antibody of AntiCD3 McAb, are named as UMAB30, subtype identification IgG1；Pass through It collects ascites and prepares antibody, CD31 monoclonal antibody UMAB30 is obtained by affinity chromatography column purification.Pass through Western respectively Blot, immunohistochemical experiment verify the sensitivity and specificity of the monoclonal antibody.
Further the specificity of said monoclonal antibody is verified using OriGene high-density protein chip: Lysate, every kind of protein lysate are overexpressed comprising 17,000 HEK293T cell protein on OriGene high-density protein chip The repetition copied there are two having on chip.Protein lysate is by trace on nitrocellulose filter.Each protein lysate Positioning can be accurately positioned by Excel file.Albumen is divided into 40 sub- matrixes on protein chip, on each Asia matrix There are some references, by referring to can quantify the content of albumen on each chip point, monitor the repetition of immune response data every time Property, and the direction of positioning positive signal.
The monoclonal antibody UMAB30 is hybridized with said chip and determines positive signal site by the present invention, as the result is shown Monoclonal antibody UMAB30 of the present invention specifically binds CD31 albumen, and with other albumen no cross reactions.
The present invention also provides monoclonal antibody UMAB30 in preparing the immune detection tool for detecting CD31 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 UMAB30 can detect the expression situation of CD31 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 It states tumour and specifically refers to the proliferation of tumour cell and the tumour that transfer is closely related with the expression of CD31, including but not limited to kidney Cancer, liver cancer, lung cancer, cancer of pancreas and breast cancer.
Compared with prior art, the present invention provides a kind of hybridoma cell strain (deposit number CGMCC No.18187), and the thus monoclonal antibody UMAB30 that hybridoma cell strain generates.The present invention also provides monoclonal antibodies UMAB30 is preparing the application in the immune detection tool for detecting CD31 albumen, the immune group of the UMAB30 containing monoclonal antibody Change the application of kit and monoclonal antibody UMAB30 in the kit that preparation is used for marked tumor.List of the present invention Clonal antibody can in conjunction with CD31 protein-specific, and with other intracellular albumen no cross reactions, significantly improve CD31 egg Specificity, the accuracy and reliability of white immune detection, it is true to reflect CD31 protein expression level in tumour cell, it can be applied to The expression of CD31 in the associated tumor tissues such as kidney, liver cancer, lung cancer, cancer of pancreas and breast cancer.
Preservation information
The classification naming of hybridoma cell strain UMAB30 for preservation are as follows: the anti-human vascular cell adhesion molecule-1 of mouse (CD31) monoclonal hybridoma 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 July 11st, 2019；
Deposit number: CGMCC No.18187.
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 the design of 1 cloning site of embodiment as schemed, and wherein shading part is the area ORF；
Fig. 2 shows 2 recombinant C D31 albumen Western blot testing result figure of embodiment, with anti-DDK antibody test recombination Expression of the CD31 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-rCD31 plasmid is the detection knot of antigen Fruit；
Fig. 3 shows 2 recombinant C D31 protein SDS-PAGE result figure of embodiment, with anti-DDK affinity chromatography column purification recombinant C D31 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 CD31 (Full length CD31, CD31- with monoclonal antibody UMAB30 FL) the Western blot testing result figure of albumen.Wherein left side swimming lane is the cell pyrolysis liquid for transfecting pCMV6-Entry；It is right Breathing arm road is the cell pyrolysis liquid for transfecting pCMV6-CD31-FL；
Fig. 5 shows that embodiment 3 identifies endogenous CD31 egg in 10 kinds of different people's Tissue lysates with monoclonal antibody UMAB30 White Western blot testing result figure.From left to right it is followed successively by 1: liver, 2: ovary, 3: thyroid gland, 4: uterus, 5: lung, 6: Muscle, 7: the heart, 8: brain, 9: kidney, 10: colon.
Fig. 6 shows that 4 formalin of embodiment is fixed, (primary antibody is that CD31 is mono- to the human milk gland ImmunohistochemistryResults Results figure of paraffin embedding Clonal antibody UMAB30)；
Fig. 7 show 4 formalin of embodiment fix, human breast carcinoma ImmunohistochemistryResults Results figure (the primary antibody CD31 of paraffin embedding Monoclonal antibody UMABb30)；
Fig. 8 shows that 4 formalin of embodiment is fixed, (primary antibody is CD31 Dan Ke to people's kidney ImmunohistochemistryResults Results figure of paraffin embedding Grand antibody UMAB30)；
Fig. 9 shows that 4 formalin of embodiment is fixed, (primary antibody is that CD31 is mono- to people's kidney ImmunohistochemistryResults Results figure of paraffin embedding Clonal antibody UMAB30)；
Figure 10 shows that 4 formalin of embodiment is fixed, (primary antibody is that CD31 is mono- to the human lung cancer ImmunohistochemistryResults Results figure of paraffin embedding Clonal antibody UMAB30)；
Figure 11 shows that 4 formalin of embodiment is fixed, (primary antibody is that CD31 is mono- to the human liver cancer ImmunohistochemistryResults Results figure of paraffin embedding Clonal antibody UMAB30)；
Figure 12 shows that 4 formalin of embodiment is fixed, (primary antibody is that CD31 is mono- to the Human plactnta ImmunohistochemistryResults Results figure of paraffin embedding Clonal antibody UMAB30)；
Figure 13 shows that (primary antibody is CD31 monoclonal antibody UMAB30,1:100 to embodiment 5OriGene protein chip qualification result figure；Two Resist for DyLight649-conjugated AffiniPure Fragment Goat-anti-Mouse IgG, 1:400).
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, CD31 recombinant expression plasmid
It is with the plasmid RC208654 (containing CD31ORF2217bp) obtained from Biotechnology Co., Ltd, Aureal Dongyuan County, the U.S. Template, designs two primers and introduces restriction enzyme site SgfI and MluI respectively, and clone insertion expression vector pCMV6-Entry is built Vertical CD31 recombinant expression plasmid.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, CD31 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 CD31 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 recombinant C D31 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%.Recombinant C D31 albumen after purification is identified with SDS-PAGE, sees Fig. 3.
By Fig. 2 result as it can be seen that in 130kD after WB detection in the HEK293T cell pyrolysis liquid of transfection pCMV6-rCD31 plasmid There is apparent specific band at place, is consistent with the actual molecular weight of albumen.Show recombinant C D31 albumen specifically expressing in cell.
By Fig. 3 result as it can be seen that the albumen of purifying has apparent specific band in PAGE glue 130kD, the reality with albumen Molecular weight is consistent.Show to have obtained the preferable CD31 recombinant protein of purity.
The preparation and screening of embodiment 3, CD31 monoclonal antibody
According to the CD31 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 CD31 antigen is emulsified with complete Freund's adjuvant, immune using subcutaneous injection method 6-8 week old Balb/c mouse, immunizing dose are 60 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 CD31 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 O450 positive is worth higher monoclonal hole and carries out limiting dilution, until ELISA measures 96 orifice plates, hardened fruit is the positive entirely.Picking sun The property high monoclonal singling of value.It is UMAB30 that it, which corresponds to fusion plate cell strain,.
4, the preparation and purification of ascites monoclonal antibodies: the male Balb/c mouse peritoneal of 10-12 week old injects 0.5ml norphytane, Every mouse is injected intraperitoneally with 1mL syringe and wash the monoclonal cell suspension being resuspended through PBS after a week, cell dosage for 5 × 106/ only, every strain antibody makes a call to 2 mouse.Ascites, centrifuging and taking supernatant are collected after mouse ascites accumulation, affinity chromatography carries out abdomen Water purifying selects corresponding column material according to antibody subtype, and the monoclonal antibody that cell strain UMAB30 is generated is IgG1, using protein G into Row purifying.Monoclonal antibody concentration mensuration after purification, WB are detected, dispense, are frozen at -20 DEG C.Wherein WB testing result is shown in Fig. 4,5.
By Fig. 4 result as it can be seen that Umab30 can be good at identifying CD31 full-length proteins；By Fig. 5 result as it can be seen that UMAB30 knows Endogenous CD31 albumen in not a variety of people's tissues, molecular weight is consistent with expected molecular size range, shows monoclonal antibody UMAB30 Specifically complete CD31 albumen can be detected by Western blot.
Embodiment 4, the immunohistochemistry that monoclonal antibody UMAB30 is primary antibody detect
(1), experimental method:
1, the human milk gland for taking formalin fixed, breast cancer, kidney, kidney, lung cancer, liver cancer and placenta tissue carry out paraffin packet It buries, is sliced using Finesse histotome, 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 CD31 monoclonal antibody (UMAB30), 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. 6-12.
(2), experimental result:
By Fig. 6-12 result as it can be seen that in human breast carcinoma, kidney, lung cancer, liver cancer and the visible obvious specificity of placenta tissue Cell membrane dyeing.As a result consistent with CD31 positioning in the cell and tissue expression specificity, show monoclonal antibody UMAB30 It can be used for the level of Immunohistochemical detection CD31 albumen, CD31 dyeing proves there is endothelial cell tissue dyeing, can be used for Tumor Angiongesis is assessed, and then predicts tumour growth, deterioration, transfer and recurrence trend.By Fig. 6-8 result as it can be seen that in human milk Staining power in gland cancer, kidney is apparently higher than normal breast and nephridial tissue, and it is very high to show that monoclonal antibody UMAB30 has Sensibility can be used for immunohistochemical diagnosis and identify good malignant angiogenic tumour.
The specific detection of embodiment 5, monoclonal antibody UMAB30
Lysate, every hatching egg are overexpressed comprising 17,000 HEK293T cell protein on OriGene high-density protein chip White lysate have on chip there are two copy repetition.Protein lysate is by trace on nitrocellulose filter.Each hatching 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 immune response number every time According to repeatability, and positioning positive signal direction.The following are use OriGene albumen (OriGene Cat PA100001) The experimental method of chip progress UMAB30 Identification of the antibodies experiment:
1, a protein chip is placed in 50mL centrifuge tube, infiltrates chip using 40mL deionized water, is placed on shaking table, Mixed at room temperature 30 minutes.Deionized water is discarded, balances chip using 10mLPBST.Room temperature is handled 10 minutes.
2,40mL5% skim milk (being diluted with PBST) is added into 50mL centrifuge tube to be placed on shaking table, room temperature envelope It closes 30 minutes.
3, primary antibody UMAB30 is diluted using confining liquid (5% skim milk), thinner ratio is classified as 1:100.
4, clean sealed membrane is pasted on experimental bench, 250-300 μ L primary antibody is added dropwise on sealed membrane.
5, protein chip is extracted out from confining liquid, down by the one of protein chip NC film, from the contact on one side of chip Antibody slowly slides, and by surface tension of liquid, antibody will slowly infiltrate chip NC film, until whole NC film infiltration is in primary antibody In solution.Whole operation process avoids generating bubble.Chip is moved on under 4 DEG C of environment, is stood, primary antibody is incubated overnight.In chip Ware lid is cultivated in upper capping, sticks a hygenic towelette thereon, causes antibody to evaporate to prevent from being incubated for for a long time.
6, chip was moved in 50mL centrifuge tube in second day, twice using PBST rinsing chip, removes extra antibody.Make Chip is washed with 40mL PBST (0.1%Tween-20), is placed on shaking table and is uniformly mixed, washing three times, washs 5min every time.
7, secondary antibody DyLight649-conjugated AffiniPure is diluted using confining liquid (5% skim milk) Fragment Goat-anti-Mouse IgG, dilution ratio 1:400.
8, according to above-mentioned steps 4, step 5 carries out secondary antibody and is incubated for operation.Incubation at room temperature 1 hour.Aluminium foil is used above chip Paper covers, to prevent signal bleaching.
9, according to above-mentioned steps 6, chip is washed using PBST.
10, chip is rinsed using deionized water, to remove remaining salinity and denaturant.
11, drying at room temperature chip, it is ensured that chip is completely dried.
12, fluorescence signal is read using chip scanner.
13, the site of chip direction and positive signal is determined according to BSA-Cy3 and BSA-Cy5.
14, corresponding protein lysate ID is found out according to positive signal site, according to lysate database information, found pair Answer protein name, NCBI typing number (accession number), protein I D, the information such as albumen size.
As a result as shown in figure 13, monoclonal antibody UMAB30 specifically can only identify CD31 albumen on OriGene protein chip, and Other more than the 10000 kinds of unrelated proteins of nonrecognition show that monoclonal antibody UMAB30 has superelevation specificity.
Sequence table
<110>
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2217
<212> DNA
<213> Homo sapiens
<400> 1
atgcagccga ggtgggccca aggggccacg atgtggcttg gagtcctgct gacccttctg 60
ctctgttcaa gccttgaggg tcaagaaaac tctttcacaa tcaacagtgt tgacatgaag 120
agcctgccgg actggacggt gcaaaatggg aagaacctga ccctgcagtg cttcgcggat 180
gtcagcacca cctctcacgt caagcctcag caccagatgc tgttctataa ggatgacgtg 240
ctgttttaca acatctcctc catgaagagc acagagagtt attttattcc tgaagtccgg 300
atctatgact cagggacata taaatgtact gtgattgtga acaacaaaga gaaaaccact 360
gcagagtacc aggtgttggt ggaaggagtg cccagtccca gggtgacact ggacaagaaa 420
gaggccatcc aaggtgggat cgtgagggtc aactgttctg tcccagagga aaaggcccca 480
atacacttca caattgaaaa acttgaacta aatgaaaaaa tggtcaagct gaaaagagag 540
aagaattctc gagaccagaa ttttgtgata ctggaattcc ccgttgagga acaggaccgc 600
gttttatcct tccgatgtca agctaggatc atttctggga tccatatgca gacctcagaa 660
tctaccaaga gtgaactggt caccgtgacg gaatccttct ctacacccaa gttccacatc 720
agccccaccg gaatgatcat ggaaggagct cagctccaca ttaagtgcac cattcaagtg 780
actcacctgg cccaggagtt tccagaaatc ataattcaga aggacaaggc gattgtggcc 840
cacaacagac atggcaacaa ggctgtgtac tcagtcatgg ccatggtgga gcacagtggc 900
aactacacgt gcaaagtgga gtccagccgc atatccaagg tcagcagcat cgtggtcaac 960
ataacagaac tattttccaa gcccgaactg gaatcttcct tcacacatct ggaccaaggt 1020
gaaagactga acctgtcctg ctccatccca ggagcacctc cagccaactt caccatccag 1080
aaggaagata cgattgtgtc acagactcaa gatttcacca agatagcctc aaagtcggac 1140
agtgggacgt atatctgcac tgcaggtatt gacaaagtgg tcaagaaaag caacacagtc 1200
cagatagtcg tatgtgaaat gctctcccag cccaggattt cttatgatgc ccagtttgag 1260
gtcataaaag gacagaccat cgaagtccgt tgcgaatcga tcagtggaac tttgcctatt 1320
tcttaccaac ttttaaaaac aagtaaagtt ttggagaata gtaccaagaa ctcaaatgat 1380
cctgcggtat tcaaagacaa ccccactgaa gacgtcgaat accagtgtgt tgcagataat 1440
tgccattccc atgccaaaat gttaagtgag gttctgaggg tgaaggtgat agccccggtg 1500
gatgaggtcc agatttctat cctgtcaagt aaggtggtgg agtctggaga ggacattgtg 1560
ctgcaatgtg ctgtgaatga aggatctggt cccatcacct ataagtttta cagagaaaaa 1620
gagggcaaac ccttctatca aatgacctca aatgccaccc aggcattttg gaccaagcag 1680
aaggctagca aggaacagga gggagagtat tactgcacag ccttcaacag agccaaccac 1740
gcctccagtg tccccagaag caaaatactg acagtcagag tcattcttgc cccatggaag 1800
aaaggactta ttgcagtggt tatcatcgga gtgatcattg ctctcttgat cattgcggcc 1860
aaatgttatt ttctgaggaa agccaaggcc aagcagatgc cagtggaaat gtccaggcca 1920
gcagtaccac ttctgaactc caacaacgag aaaatgtcag atcccaatat ggaagctaac 1980
agtcattacg gtcacaatga cgatgtcaga aaccatgcaa tgaaaccaat aaatgataat 2040
aaagagcctc tgaactcaga cgtgcagtac acggaagttc aagtgtcctc agctgagtct 2100
cacaaagatc taggaaagaa ggacacagag acagtgtaca gtgaagtccg gaaagctgtc 2160
cctgatgccg tggaaagcag atactctaga acggaaggct cccttgatgg aacttag 2217

Claims (7)

1. a kind of monoclonal antibody, which is characterized in that it is in conjunction with CD31 protein-specific；The monoclonal antibody is by hybridoma Cell strain UMAB30 is generated, and deposit number is CGMCC No.18187.
2. a kind of hybridoma cell strain, deposit number is CGMCC No.18187.
3. monoclonal antibody as described in claim 1 is in preparation for detecting the application in CD31 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 the phases such as kidney, liver cancer, lung cancer, cancer of pancreas and breast cancer Close tumour.
CN201910727944.1A 2019-08-09 2019-08-09 1 protein monoclonal antibody of AntiCD3 McAb and application thereof Pending CN110357964A (en)

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CN116143921A (en) * 2022-11-16 2023-05-23 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at human CD31, and preparation method and application thereof
CN116813780A (en) * 2023-07-13 2023-09-29 武汉爱博泰克生物科技有限公司 Anti-human CD31 rabbit monoclonal antibody and application thereof

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WO2010000756A1 (en) * 2008-06-30 2010-01-07 Institut National De La Sante Et De La Recherche Medicale (Inserm) Detection of shed cd31, diagnosis of atherothrombosis and autoimmune disorders, and methods for analyzing signaling pathways
CN103275225A (en) * 2013-05-10 2013-09-04 洪建� Anti-CD31/CD146 double-specific monoclonal antibody with high affinity and application thereof
CN104371968A (en) * 2014-06-25 2015-02-25 北京农学院 Method for separating and purifying mouse lung microvascular endothelial cell
CN108588033A (en) * 2018-04-24 2018-09-28 富恩生物技术（成都）有限公司 Hybridoma cell strain, CD31 monoclonal antibodies, preparation method and application

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WO2010000756A1 (en) * 2008-06-30 2010-01-07 Institut National De La Sante Et De La Recherche Medicale (Inserm) Detection of shed cd31, diagnosis of atherothrombosis and autoimmune disorders, and methods for analyzing signaling pathways
CN103275225A (en) * 2013-05-10 2013-09-04 洪建� Anti-CD31/CD146 double-specific monoclonal antibody with high affinity and application thereof
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CN116143921B (en) * 2022-11-16 2023-08-29 武汉爱博泰克生物科技有限公司 Rabbit monoclonal antibody aiming at human CD31, and preparation method and application thereof
CN116813780A (en) * 2023-07-13 2023-09-29 武汉爱博泰克生物科技有限公司 Anti-human CD31 rabbit monoclonal antibody and application thereof
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