CN107058238A - Anti- CD10 protein monoclonal antibodies and application thereof - Google Patents
Anti- CD10 protein monoclonal antibodies and application thereof Download PDFInfo
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- CN107058238A CN107058238A CN201611224291.8A CN201611224291A CN107058238A CN 107058238 A CN107058238 A CN 107058238A CN 201611224291 A CN201611224291 A CN 201611224291A CN 107058238 A CN107058238 A CN 107058238A
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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
- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
Abstract
The present invention relates to biological technical field, anti-CD10 protein monoclonal antibodies hybridoma and its anti-CD10 monoclonal antibodies of generation and application are disclosed.Anti- CD10 protein monoclonal antibodies hybridoma deposit number of the present invention is CGMCC NO：13286.The hybridoma that the present invention is provided can stably excreting generation anti-CD10 protein monoclonal antibodies (clone number：UMAB235), and combined with CD10 protein-specifics, and with nearly 10000 kinds other albumen no cross reactions on protein chip, significantly improve CD10 protein immunizations detection specificity, accuracy and reliability, be widely used in the detection of CD10 in various tumor tissues and its microenvironment.
Description
Technical field
The present invention relates to biological technical field, and in particular to anti-CD10 protein monoclonal antibodies hybridoma and its generation
Anti- CD10 monoclonal antibodies and application.
Background technology
CDl0 molecules are found initially as common acute lymphocytic leukemia antigen (CALLA), by MME gene codes
A relative molecular mass containing 750aa single-stranded wears membrane glycoprotein for 90-110kDa II types.CDl0 is mainly expressed in marrow
Early stage B cell and fetal thymus a group cell on, mainly CD2-Cell.CDl0 can differentiate that the T of derived from bone marrow is thin
Born of the same parents' precursor, as cell enters the differentiation of T cell, CDl0 expression starts to reduce.In B cell, CDl0 expression is with thin
Born of the same parents obtain surface membrane immunoglobulin and start to reduce.CDl0 also expresses neutral grain in the B cell of centrum germinativum and ripe
On cell.In non-hematopoietic tissue, CDl0 molecules are also expressed on many histocytes, such as renal tubule and messangial cell, small
Enterocyte, marrow stromal cell, bile duct, musculoepithelia cell of mammary gland and salivary gland etc., synapses film in brain tissue, the fibre of culture
Dimension mother cell and some solid tumor cells fasten the expression for also having CDl0.
CDl0 molecules are widely distributed in various tissues, the function with neutral endopeptidase (NEP), and express not
Same tissue, its function is not quite similar.Be now widely used for the diagnosis of hematological system tumor and part entity tumour and
Index for diagnosis, is additionally operable to hematopoetic cell differentiation development research and the research of portion of tissue stem cell.
At present clinically generally with immunohistochemistry (Immunohistochemistry, IHC) experiment detection tumor group
The expression situation of CD10 in inner tumour cell is knitted, the core of its experimental method is specific binding CD10 monoclonal antibody, its
The quality of performance directly decides the sensitivity and specificity entirely detected.Therefore, a kind of binding specificity is developed higher
Monoclonal antibody for CD10 albumen has great importance.
The content of the invention
In view of this, it is an object of the invention to provide anti-CD10 protein monoclonal antibodies hybridoma and its generation
Anti- CD10 monoclonal antibodies and application, improve the monoclonal antibody of the hybridoma generation and the binding specificity of CD10 albumen and answer
In the preparation for using Related product.
To achieve the above object, the present invention provides following technical scheme：
Anti- CD10 protein monoclonal antibodies hybridoma, deposit number is CGMCC NO：13286.
Anti- CD10 protein monoclonal antibodies hybridoma of the present invention is prepared by following methods：
(1) structure of recombinant expression carrier：According to CD10 ORF nucleotide sequences (CD10 ORF nucleotide sequences such as SEQ
Shown in ID NO.1,2250bp；CD10 amino acid sequences are as shown in SEQ ID NO.2) design primer, with OriGene Products
CD10 total length plasmids RC224141 is template, enters the C-terminal 411-750aa fragments that performing PCR expands CD10, gene both sides are drawn respectively
Enter restriction endonuclease sites EcoRI and XhoI, insert expression vector pET-23a (+) (Novagen companies, article No. 69745-
3) CD10 recombinant expression plasmids pET-23a/rCD10, is built；
(2) expression and purification of CD10 recombinant proteins：CD10 recombinant expression plasmids are transformed into after E.Coli, amplification cultivation
Centrifuging and taking supernatant is cracked, the purifying of His affinity columns obtains the CD10 recombinant proteins of purifying；
(3) screening and preparation of monoclonal antibody：BALB/c mouse is immunized using the CD10 recombinant proteins of above-mentioned purifying, taken
Mouse spleen cells are merged with SP2/0 cells, and limiting dilution assay obtains monoclonal, and ELISA method screening positive hybridoma is thin
Born of the same parents, acquisition can secrete the hybridoma cell strain of anti-CD10 specific antibodies；Antibody is prepared by serum free medium, by affine
Column chromatography obtains CD10 monoclonal antibodies.The sensitivity of the monoclonal antibody is verified and special by immunohistochemical experiment
Property.
After prepared by the above method, the hybridoma for being capable of the anti-CD10 monoclonal antibodies of stably excreting is filtered out, is ordered
Entitled UMAB235, hypotype is accredited as IgG1, and is preserved in Chinese microorganism strain preservation conservator on November 22nd, 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：13286.
Meanwhile, the present invention also provides a kind of anti-CD10 protein monoclonal antibodies, is CGMCC NO by deposit number：13286
Hybridoma secretion produce.The art conventional method can be used by preparing anti-CD10 protein monoclonal antibodies, such as be passed through
Serum free medium prepares antibody, and CD10 monoclonal antibodies are obtained by affinity column purifying.
Deposit number of the present invention is CGMCC NO：13286 hybridoma chromosome stabilityX, it secretes what is produced
Anti- CD10 protein monoclonal antibodies are IgG1 types, and potency is higher.The protein blot (Western-Blot) of the monoclonal antibody
Testing result is shown in the CD10 albumen in K562, Ramos and Raji cells.
Simultaneously SABC testing result show, its can specific recognition renal clear cell carcinoma, breast cancer tissue,
CD10 albumen in lymphoma tissue, normal tonsillotome and nephridial tissue.
In addition, the specific test for detecting the monoclonal antibody using OriGene high-density proteins chip shows, it is 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：13286 hybridoma is preparing anti-CD10
Application and secreted anti-CD10 protein monoclonal antibodies in protein monoclonal antibody are preparing exempting from for detection CD10 albumen
Application in epidemic disease detection product.
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：13286 it is miscellaneous
The anti-CD10 protein monoclonal antibodies for handing over oncocyte secretion to produce.The immunity detection reagent can detect tumor tissues and its micro-
CD10 expression situation in environment.
In addition, it is CGMCC NO that the present invention, which also provides deposit number,：The anti-CD10 that 13286 hybridoma secretion is produced
Application of the protein monoclonal antibody in the kit for preparing marked tumor tissue and its microenvironment medium size lymphocyte.
Preferably, the tumour includes clear cell carcinoma of kidney, lymthoma and breast cancer.
The hybridoma that the present invention is provided can stably excreting produce anti-CD10 protein monoclonal antibodies, and with CD10 albumen
Specific binding, and with nearly 10000 kinds other albumen no cross reactions on protein chip, significantly improve CD10 protein immunizations inspection
Specificity, accuracy and the reliability of survey, are widely used in the detection of CD10 in various tumor tissues and its microenvironment.
Biological deposits information explanation
UMAB235, Classification And Nomenclature is the strain of CD10 monoclonal antibody hybridoma cells, on November 22nd, 2016 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of microbiology of the academy of sciences of state, deposit number is CGMCC NO：13286.
Brief description of the drawings
Fig. 1 show cloning site figure in embodiment 1；
Fig. 2 show CD10 protein SDS-PAGEs result figure in embodiment 2；
Fig. 3 show CD10 proteins marking Western-Blot result figures in embodiment 4, and (primary antibody is UMAB235 points
Secrete the CD10 monoclonal antibodies of generation, 1:500)；
(primary antibody is that UMAB235 secretes production to the renal clear cell carcinoma SABC testing result figure of embodiment illustrated in fig. 45
Raw CD10 monoclonal antibodies, 1:600)；
(primary antibody is that UMAB235 secretes what is produced to the lymphoma tissue SABC testing result figure of embodiment illustrated in fig. 55
CD10 monoclonal antibodies, 1:600)；
(primary antibody is that UMAB235 secretes what is produced to the breast cancer tissue's SABC testing result figure of embodiment illustrated in fig. 65
CD10 monoclonal antibodies, 1:600)；
(primary antibody is that UMAB235 secretes production to the normal tonsil SABC testing result figure of the people of embodiment illustrated in fig. 75
Raw CD10 monoclonal antibodies, 1:600)；
(primary antibody is that UMAB235 secretes what is produced to the people's normal kidney tissue SABC testing result figure of embodiment illustrated in fig. 85
CD10 monoclonal antibodies, 1:600)；
(primary antibody is that the CD10 that UMAB235 secretions are produced is mono- to embodiment illustrated in fig. 9 6origene protein chip qualification results figure
It is anti-, 1:100；Secondary antibody is the donkey anti-mouse IgG that Alexa 647- are marked, 1:500).
Embodiment：
The invention discloses anti-CD10 protein monoclonal antibodies hybridoma and its anti-CD10 monoclonal antibodies of generation
And application, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular,
All similar replacements and change are apparent to those skilled in the art, and they are considered as being included in this hair
It is bright.The product of the present invention and application are described by preferred embodiment, and related personnel can substantially not depart from this hair
Method described herein and application are modified in bright content, spirit and scope or suitably change is with combining, to realize and answer
Use the technology of the present invention.
The anti-CD10 protein monoclonal antibodies hybridoma and its anti-CD10 Dan Ke of generation that the present invention is provided below
Grand antibody and application are described further.
Embodiment 1：The structure of CD10 recombinant expression plasmids
According to CD10 full length cDNA sequence template RC224141 (Origene Products, article No. RC224141), for
Its C-terminal 411-750aa fragments design two primers and introduce restriction enzyme site EcoRI and XhoI respectively, are cloned into expression vector
PET-23a (+) (Novagen companies, article No. 69745-3), the restructuring for setting up CD10 C-terminal fragment 411-750aa albumen is former
Nuclear expression plasmid, the correctness of recombinant plasmid is determined through sequencing.Cloning site design is as shown in Figure 1.
Embodiment 2：The expression and purification of CD10 recombinant proteins
1st, Transformed E .Coli competence bacterium：A 1.5ml centrifuge tube is taken, 100 μ l competent cell suspensions is added, puts ice
On：2ul plasmid pET-23a/rCD10 are added, are softly mixed with pipettor.20min is stood on ice；Heat shock 90 in 42 DEG C of water-baths
Second, 3~5min on ice is then put rapidly；Add 1ml LB fluid nutrient mediums (being free of antibiotic), 37 DEG C of shaken cultivations after mixing
(180rpm) 1 hour；100ul bacterium solutions are taken, extremely on the LB solid mediums containing antibiotic Amp, coating is uniform；Treat that bacterium solution is trained
Support after base absorption, 37 DEG C are inverted culture 12~16 hours, colony growth is good and when not overlapping each other, takes out, then chooses list
Bacterium colony bacterial strain is chosen above-mentioned single bacterium colony bacterial strain in 5ml LB (Amp+) and turned after 5ml LB (Amp+) culture mediums 220rpm is cultivated 8 hours
Move to 1 liter of LB (Amp+) culture medium and expand culture, IPTG to final concentration of 0.25mM is added after it grows into OD280=0.6
Inducible protein is expressed, and continues 4 degree of 200rpm shaken cultivations after 12~14 hours, 4 degree of centrifugation bacterium solutions, collects bacterial sediment.
2nd, protein purification：The bacterial sediment that upper step is collected is with PBS gravity treatment and carries out ultrasonication (ultrasonic probe
For 6 Ф, power is 400W, whole working time 30min, and each circulating ultrasonic is spaced 5 seconds for 3 seconds), centrifugation (12000rpm,
5min), supernatant is collected, and (Novagen, Ni-NTA His-Bind is purified according to Novagen companies method for purifying proteins
Resin kits, article No.：70666-5), the recombinant protein rCD10/411-750aa for the band His labels that harvest is purified.
3rd, purifying protein is identified：The C-terminal 411-750 fragment albumen of recombinant C D10 after purification is identified with SDS-PAGE, is seen
Fig. 2.
From Fig. 2 results, purified by His affinity columns, obtain the CD10 recombinant proteins of high-purity.
Embodiment 3：The preparation screening of anti-CD10 monoclonal antibody hybridoma cells and its monoclonal antibody
Total length CD10 recombinant proteins rCD10/411-750aa (the following letters of the purifying produced according to standard method with restructuring
Referred to as CD10 antigens) it is used to B6/C57 mouse (Beijing Vital River Experimental Animals Technology Co., Ltd.) are immunized.Specifically
Method is as follows：
1st, animal immune：Purified CD10 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, and ELISA is carried out using the CD10 recombinant proteins of purifying
Test.Mark cell line number.Positive hole cell is carried out to determine ELISA values within 5-6 days after limiting dilution, each limiting dilution, chosen
The monoclonal hole for taking the positive values of OD280 higher carries out limiting dilution, until it is the positive that ELISA, which determines the complete hardened fruit of 96 orifice plates,.Choose
The monoclonal singling for taking positive value high.Its correspondence fusion plate cell line is UMAB235.
4th, the preparation and purification of cell conditioned medium monoclonal antibody：The DMEM culture mediums containing 15% serum are used to train cell line UMAB235
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 UMAB235 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：CD10 monoclonal antibodies UMAB235 for primary antibody protein blot (Western-Blot, hereinafter referred to as
WB) detect
(1), experimental method：
1st, cell is prepared：Prepare K562, HL-60, Ramos and Raji cells (are bought from ATCC), are incubated at containing 5%
CO237 DEG C of cell culture incubators in.
2nd, cell pyrolysis liquid is prepared：By above-mentioned cell dissociation, horizontal centrifuge 800-1000rpm/5min centrifugations, PBS washes 2
Secondary, cell precipitation is managed with PBS constant volumes to 400ul-EP, adds cell pyrolysis liquid, after ice bath is cracked 20 minutes, 12000rpm centrifugations
10 minutes, collect supernatant addition 5X Loading Buffer and be diluted to 1X as cell pyrolysis liquid, -80 DEG C of storages are standby.
3rd, SDS-PAGE electrophoresis：Constant pressure 180V 50min bromjophenol blues are to gel bottom.
4th, transferring film：Nc films and filter paper are soaked with transfering buffering liquid in advance, sandwich formats are placed in wet film instrument of walking around, order
For：Electrode (-)-sponge-filter paper-gel-NC film-filter paper-electrodes (+), must ensure glue in lower film upper, i.e., gel is close
Negative pole.Constant current 600mA, 45 minutes.
5th, dye：With the beginning of spring red S dyeing 2min, cleaned, showed to marker bands with distilled water, mark is carried out with ball pen
Note.Distilled water flushing film.
6th, close：37 DEG C are placed in confining liquid (TBST for containing 5% defatted milk) submergence NC films to be incubated 1 hour.
7th, primary antibody is incubated：NC films are placed in PR mouse monoclonal antibodies UMAB235 (antibody is diluted into 500 times with confining liquid), 37 DEG C
After incubation 1 hour, then with 15mLTBST wash film 15min*3 times.(TBST consumptions can adjust according to container size)
8th, secondary antibody is incubated：NC films are placed in sheep anti-mouse igg secondary antibody (Jackson, the Catlog No.115- of HRP marks
035-146；1：5000) in, after room temperature shaker is incubated 1 hour, TBST washes film 5 times, 15min/ times.
9th, substrate exposes：Luminol and hydrogen peroxide 1：X-ray film in (1ml/ films), pressure is added on NC films after 1 mixing to expose
Light, film then developed and be fixed and (can estimate gorgeous time in darkroom according to the fluorescence on film).
10th, according to feminine gender, positive, blank control carries out interpretation of result and judgement, sees Fig. 3.
(2), experimental result：
From Fig. 3 results, CD10 mouse monoclonal antibodies UMAB235 (1:500) it can detect in K562, Ramos and Raji cells
CD10 albumen, molecular size range is consistent with expection, and then without correspondingly sized band, table in HL-60 and Jurkat cell
Bright UMAB235 can detect the specific expressed of CD10 albumen.
Embodiment 5：The monoclonal antibody that UMAB235 secretions are produced detects for the SABC of primary antibody
(1), experimental method：
1st, clear cell carcinoma of kidney, Bukitt lymthomas and follicular lymphoma, breast cancer, normal tonsillotome and kidney are taken respectively
Tissue block carries out FFPE, is cut into slices using SAKURA histotomes, 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, pH9.0 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, CD10 monoclonal antibodies (UMAB235 secretions are produced), thinner ratio 1 is added:600, 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 PV8000 in polymer HRP staining kits is added dropwise, and (purchase is limited from Beijing Zhong Shan Golden Bridge biotechnology
Company, article No. PV-8000), 37 DEG C are incubated 15 minutes.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, CD10 albumen is in specific cell film expression in the tumour cell of renal clear cell carcinoma,
Tumour cell as indicated in the figures by an arrow.
From Fig. 5 results, CD10 albumen is in specific cell film and cell on the lymphocyte in lymphoma tissue
Matter is expressed, as indicated in the figures by an arrow lymphoma cell.
From Fig. 6 results, CD10 albumen is in specific cell film and cytoplasm in the musculoepithelia cell of breast cancer tissue
Express, as indicated in the figures by an arrow musculoepithelia cell.
From Fig. 7 results, CD10 albumen is in specific cell film table on the lymphocyte in normal tonsil
Reach, as indicated in the figures by an arrow lymphocyte.
From Fig. 8 results, CD10 albumen is thin in specificity on the renal tubule and messangial cell in normal kidney tissue
After birth and cytoplasmic expression, as indicated in the figures by an arrow histocyte.
As a result show that the monoclonal antibody that UMAB235 of the present invention is produced being capable of specific recognition clear cell carcinoma of kidney, lymph
CD10 albumen in tumor tissue, breast cancer, normal tonsillotome and nephridial tissue.
Embodiment 6：The specific proteins chip detection for the monoclonal antibody that UMAB235 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 UMAB235 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 UMAB235 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. 9 is seen.
Fig. 9 results show that the CD10 albumen on protein chip secretes the monoclonal antibody specificity produced with UMAB235
With reference to, and with other albumen without any binding signal, show that monoclonal antibody UMAB235 of the present invention can specific recognition CD10 eggs
In vain, and with 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- CD10 protein monoclonal antibodies and application thereof
<210> 1
<211> 2250
<212> DNA
<213>Artificial sequence
<400>
1ATGGGCAAGTCAGAAAGTCAGATGGATATAACTGATATCAACACTCCAAAGCCAAAGAAGAAACAGCGAT
GGACTCCACTGGAGATCAGCCTCTCGGTCCTTGTCCTGCTCCTCACCATCATAGCTGTGACAATGATCGC
ACTCTATGCAACCTACGATGATGGTATTTGCAAGTCATCAGACTGCATAAAATCAGCTGCTCGACTGATC
CAAAACATGGATGCCACCACTGAGCCTTGTACAGACTTTTTCAAATATGCTTGCGGAGGCTGGTTGAAAC
GTAATGTCATTCCCGAGACCAGCTCCCGTTACGGCAACTTTGACATTTTAAGAGATGAACTAGAAGTCGT
TTTGAAAGATGTCCTTCAAGAACCCAAAACTGAAGATATAGTAGCAGTGCAGAAAGCAAAAGCATTGTAC
AGGTCTTGTATAAATGAATCTGCTATTGATAGCAGAGGTGGAGAACCTCTACTCAAACTGTTACCAGACA
TATATGGGTGGCCAGTAGCAACAGAAAACTGGGAGCAAAAATATGGTGCTTCTTGGACAGCTGAAAAAGC
TATTGCACAACTGAATTCTAAATATGGGAAAAAAGTCCTTATTAATTTGTTTGTTGGCACTGATGATAAG
AATTCTGTGAATCATGTAATTCATATTGACCAACCTCGACTTGGCCTCCCTTCTAGAGATTACTATGAAT
GCACTGGAATCTATAAAGAGGCTTGTACAGCATATGTGGATTTTATGATTTCTGTGGCCAGATTGATTCG
TCAGGAAGAAAGATTGCCCATCGATGAAAACCAGCTTGCTTTGGAAATGAATAAAGTTATGGAATTGGAA
AAAGAAATTGCCAATGCTACGGCTAAACCTGAAGATCGAAATGATCCAATGCTTCTGTATAACAAGATGA
CATTGGCCCAGATCCAAAATAACTTTTCACTAGAGATCAATGGGAAGCCATTCAGCTGGTTGAATTTCAC
AAATGAAATCATGTCAACTGTGAATATTAGTATTACAAATGAGGAAGATGTGGTTGTTTATGCTCCAGAA
TATTTAACCAAACTTAAGCCCATTCTTACCAAATATTCTGCCAGAGATCTTCAAAATTTAATGTCCTGGA
GATTCATAATGGATCTTGTAAGCAGCCTCAGCCGAACCTACAAGGAGTCCAGAAATGCTTTCCGCAAGGC
CCTTTATGGTACAACCTCAGAAACAGCAACTTGGAGACGTTGTGCAAACTATGTCAATGGGAATATGGAA
AATGCTGTGGGGAGGCTTTATGTGGAAGCAGCATTTGCTGGAGAGAGTAAACATGTGGTCGAGGATTTGA
TTGCACAGATCCGAGAAGTTTTTATTCAGACTTTAGATGACCTCACTTGGATGGATGCCGAGACAAAAAA
GAGAGCTGAAGAAAAGGCCTTAGCAATTAAAGAAAGGATCGGCTATCCTGATGACATTGTTTCAAATGAT
AACAAACTGAATAATGAGTACCTCGAGTTGAACTACAAAGAAGATGAATACTTCGAGAACATAATTCAAA
ATTTGAAATTCAGCCAAAGTAAACAACTGAAGAAGCTCCGAGAAAAGGTGGACAAAGATGAGTGGATAAG
TGGAGCAGCTGTAGTCAATGCATTTTACTCTTCAGGAAGAAATCAGATAGTCTTCCCAGCCGGCATTCTG
CAGCCCCCCTTCTTTAGTGCCCAGCAGTCCAACTCATTGAACTATGGGGGCATCGGCATGGTCATAGGAC
ACGAAATCACCCATGGCTTCGATGACAATGGCAGAAACTTTAACAAAGATGGAGACCTCGTTGACTGGTG
GACTCAACAGTCTGCAAGTAACTTTAAGGAGCAATCCCAGTGCATGGTGTATCAGTATGGAAACTTTTCC
TGGGACCTGGCAGGTGGACAGCACCTTAATGGAATTAATACACTGGGAGAAAACATTGCTGATAATGGAG
GTCTTGGTCAAGCATACAGAGCCTATCAGAATTATATTAAAAAGAATGGCGAAGAAAAATTACTTCCTGG
ACTTGACCTAAATCACAAACAACTATTTTTCTTGAACTTTGCACAGGTGTGGTGTGGAACCTATAGGCCA
GAGTATGCGGTTAACTCCATTAAAACAGATGTGCACAGTCCAGGCAATTTCAGGATTATTGGGACTTTGC
AGAACTCTGCAGAGTTTTCAGAAGCCTTTCACTGCCGCAAGAATTCATACATGAATCCAGAAAAGAAGTG
CCGGGTTTGG2250
<210> 1
<211> 750
<212> PRT
<213>Artificial sequence
<400>
1MetGlyLysSerGluSerGlnMetAspIleThrAspIleAsnThrProLysProLysLysLysGlnArgTrp
ThrProLeuGluIleSerLeuSerValLeuValLeuLeuLeuThrIleIleAlaValThrMetIleAlaLeuTyrAl
aThrTyrAspAspGlyIleCysLysSerSerAspCysIleLysSerAlaAlaArgLeuIleGlnAsnMetAspAlaT
hrThrGluProCysThrAspPhePheLysTyrAlaCysGlyGlyTrpLeuLysArgAsnValIleProGluThrSer
SerArgTyrGlyAsnPheAspIleLeuArgAspGluLeuGluValValLeuLysAspValLeuGlnGluProLysTh
rGluAspIleValAlaValGlnLysAlaLysAlaLeuTyrArgSerCysIleAsnGluSerAlaIleAspSerArgG
lyGlyGluProLeuLeuLysLeuLeuProAspIleTyrGlyTrpProValAlaThrGluAsnTrpGluGlnLysTyr
GlyAlaSerTrpThrAlaGluLysAlaIleAlaGlnLeuAsnSerLysTyrGlyLysLysValLeuIleAsnLeuPh
eValGlyThrAspAspLysAsnSerValAsnHisValIleHisIleAspGlnProArgLeuGlyLeuProSerArgA
spTyrTyrGluCysThrGlyIleTyrLysGluAlaCysThrAlaTyrValAspPheMetIleSerValAlaArgLeu
IleArgGlnGluGluArgLeuProIleAspGluAsnGlnLeuAlaLeuGluMetAsnLysValMetGluLeuGluLy
sGluIleAlaAsnAlaThrAlaLysProGluAspArgAsnAspProMetLeuLeuTyrAsnLysMetThrLeuAlaG
lnIleGlnAsnAsnPheSerLeuGluIleAsnGlyLysProPheSerTrpLeuAsnPheThrAsnGluIleMetSer
ThrValAsnIleSerIleThrAsnGluGluAspValValValTyrAlaProGluTyrLeuThrLysLeuLysProIl
eLeuThrLysTyrSerAlaArgAspLeuGlnAsnLeuMetSerTrpArgPheIleMetAspLeuValSerSerLeuS
erArgThrTyrLysGluSerArgAsnAlaPheArgLysAlaLeuTyrGlyThrThrSerGluThrAlaThrTrpArg
ArgCysAlaAsnTyrValAsnGlyAsnMetGluAsnAlaValGlyArgLeuTyrValGluAlaAlaPheAlaGlyGl
uSerLysHisValValGluAspLeuIleAlaGlnIleArgGluValPheIleGlnThrLeuAspAspLeuThrTrpM
etAspAlaGluThrLysLysArgAlaGluGluLysAlaLeuAlaIleLysGluArgIleGlyTyrProAspAspIle
ValSerAsnAspAsnLysLeuAsnAsnGluTyrLeuGluLeuAsnTyrLysGluAspGluTyrPheGluAsnIleIl
eGlnAsnLeuLysPheSerGlnSerLysGlnLeuLysLysLeuArgGluLysValAspLysAspGluTrpIleSerG
lyAlaAlaValValAsnAlaPheTyrSerSerGlyArgAsnGlnIleValPheProAlaGlyIleLeuGlnProPro
PhePheSerAlaGlnGlnSerAsnSerLeuAsnTyrGlyGlyIleGlyMetValIleGlyHisGluIleThrHisGl
yPheAspAspAsnGlyArgAsnPheAsnLysAspGlyAspLeuValAspTrpTrpThrGlnGlnSerAlaSerAsnP
heLysGluGlnSerGlnCysMetValTyrGlnTyrGlyAsnPheSerTrpAspLeuAlaGlyGlyGlnHisLeuAsn
GlyIleAsnThrLeuGlyGluAsnIleAlaAspAsnGlyGlyLeuGlyGlnAlaTyrArgAlaTyrGlnAsnTyrIl
eLysLysAsnGlyGluGluLysLeuLeuProGlyLeuAspLeuAsnHisLysGlnLeuPhePheLeuAsnPheAlaG
lnValTrpCysGlyThrTyrArgProGluTyrAlaValAsnSerIleLysThrAspValHisSerProGlyAsnPhe
ArgIleIleGlyThrLeuGlnAsnSerAlaGluPheSerGluAlaPheHisCysArgLysAsnSerTyrMetAsnPr
oGluLysLysCysArgValTrp750
Claims (8)
1. anti-CD10 protein monoclonal antibodies hybridoma, it is characterised in that deposit number is CGMCC NO：13286.
2. deposit number is CGMCC NO：Application of 13286 hybridoma in anti-CD10 protein monoclonal antibodies are prepared.
3. anti-CD10 protein monoclonal antibodies, it is characterised in that by deposit number be CGMCC NO：13286 hybridoma
Secretion is produced.
4. deposit number is CGMCC NO：The anti-CD10 protein monoclonal antibodies that 13286 hybridoma secretion is produced are in system
Application in the immune detection product of standby detection CD10 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：13286 hybridization
The anti-CD10 protein monoclonal antibodies that oncocyte secretion is produced.
7. deposit number is CGMCC NO：The anti-CD10 protein monoclonal antibodies that 13286 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, clear cell carcinoma of kidney and breast
Gland cancer.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611224291.8A CN107058238A (en) | 2016-12-27 | 2016-12-27 | Anti- CD10 protein monoclonal antibodies and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611224291.8A CN107058238A (en) | 2016-12-27 | 2016-12-27 | Anti- CD10 protein monoclonal antibodies and application thereof |
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| CN107058238A true CN107058238A (en) | 2017-08-18 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116731181A (en) * | 2023-07-17 | 2023-09-12 | 武汉爱博泰克生物科技有限公司 | Anti-human CD10 protein rabbit monoclonal antibody and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1645292A1 (en) * | 1999-05-07 | 2006-04-12 | Genentech, Inc. | Treatment of autoimmune diseases with antagonists which bind to B cell surface markers |
| WO2006121159A1 (en) * | 2005-05-12 | 2006-11-16 | Kyowa Hakko Kogyo Co., Ltd. | Humanized cdr-grafted antibody specifically reacting with cd10 and antibody fragment of the same |
| JP2007129903A (en) * | 2003-10-08 | 2007-05-31 | Kyowa Hakko Kogyo Co Ltd | Antibody composition specifically binding to cd10 |
| CN103880960A (en) * | 2014-04-02 | 2014-06-25 | 福州迈新生物技术开发有限公司 | Monoclonal antibody for secreting anti-CD10 molecule and application thereof |
-
2016
- 2016-12-27 CN CN201611224291.8A patent/CN107058238A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1645292A1 (en) * | 1999-05-07 | 2006-04-12 | Genentech, Inc. | Treatment of autoimmune diseases with antagonists which bind to B cell surface markers |
| JP2007129903A (en) * | 2003-10-08 | 2007-05-31 | Kyowa Hakko Kogyo Co Ltd | Antibody composition specifically binding to cd10 |
| WO2006121159A1 (en) * | 2005-05-12 | 2006-11-16 | Kyowa Hakko Kogyo Co., Ltd. | Humanized cdr-grafted antibody specifically reacting with cd10 and antibody fragment of the same |
| CN103880960A (en) * | 2014-04-02 | 2014-06-25 | 福州迈新生物技术开发有限公司 | Monoclonal antibody for secreting anti-CD10 molecule and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116731181A (en) * | 2023-07-17 | 2023-09-12 | 武汉爱博泰克生物科技有限公司 | Anti-human CD10 protein rabbit monoclonal antibody and application thereof |
| CN116731181B (en) * | 2023-07-17 | 2024-01-12 | 武汉爱博泰克生物科技有限公司 | Anti-human CD10 protein rabbit monoclonal antibody and application thereof |
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Application publication date: 20170818 |