CN109111519A - Anti- S100P protein monoclonal antibody and application thereof - Google Patents
Anti- S100P protein monoclonal antibody and application thereof Download PDFInfo
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- CN109111519A CN109111519A CN201811015870.0A CN201811015870A CN109111519A CN 109111519 A CN109111519 A CN 109111519A CN 201811015870 A CN201811015870 A CN 201811015870A CN 109111519 A CN109111519 A CN 109111519A
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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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.15595), and the monoclonal antibody Umab24 that thus hybridoma cell strain generates.The invention further relates to monoclonal antibody Umab24 to prepare the application in the immune detection tool for detecting S100P albumen, application of the immunohistochemical kit and monoclonal antibody Umab24 of the Umab24 containing monoclonal antibody in the kit that preparation is used for marked tumor.Monoclonal antibody of the present invention can in conjunction with S100P protein-specific, and with other intracellular albumen no cross reactions, significantly improve specificity, the accuracy and reliability of the detection of S100P protein immunization.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to can specific bond S100P albumen monoclonal antibody Umab24,
Generate the cell strain of the monoclonal antibody and using the diagnostic method of the antibody and purposes.
Background technique
S100 protein family has now been found that about 25 members, belongs to EF-hand superfamily molecule, protein structure letter
It is single, but participate in adjusting many cellular processes, such as adjust cell growth and movement, cell cycle progression and differentiation.S100 albumen is fixed
Positioned at the cytoplasm and/or nucleus of various kinds of cell, the immune response of various organization types, while and and neuron are mainly mediated
It develops related.Studies have shown that the expression of S100 abnormal protein is significant related to cancer progression.
S100P is the S100 family member containing 2 EF-hand calcium binding motifs, encodes 95 amino acid.Transcriptional activation
Afterwards, it expresses and increases in the kinds of tumor cells such as lung cancer, breast cancer, oophoroma and cancer of pancreas.The unusual high levels of S100P are recognized
For the generation and transfer that will lead to tumour.Immunohistochemistry (IHC) Pathological experiment is clinically commonly used at present detects tumour cell
The expression situation of middle albumen, however the core of IHC experiment is the monoclonal antibody of binding proteins specific, the superiority and inferiority of performance is straight
It connects and decides the sensitivity and specificity entirely detected.Therefore, it is higher for S100P albumen to develop a kind of binding specificity
Monoclonal antibody to IHC detection S100P expression have great importance.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of monoclonals of the higher S100P albumen of binding specificity to resist
Body, and its preparing the application in the immune detection tool for detecting S100P albumen.
The present invention provides a kind of hybridoma cell strains, and it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers (referred to as CGMCC), the deposit date is on April 26th, 2018, deposit number was CGMCC No.15595.
The present invention also provides a kind of monoclonal antibody Umab24 for specifically binding S100P albumen, by above-mentioned hybridoma
Cell strain generates.
Monoclonal antibody of the present invention the preparation method is as follows:
(1) building of recombinant expression carrier: according to S100P ORF nucleotide sequence, (S100P ORF nucleotide sequence is such as
Shown in SEQ ID NO.1,288bp；S100P amino acid sequence is as shown in SEQ ID NO.2)
Design primer PCR amplification S100P ORF 1bp to 288bp bit sequence, gene two sides introduce restricted respectively
Restriction enzyme site SgfI and MluI are inserted into expression vector pCMV6-Entry, construct S100P amino acid sequence the 1st to the 95th
Recombinant expression plasmid pCMV6-rS100P；Upstream amplification primer sequence, SEQ ID NO.3:CACGCGATCGCATGACGGAA
CTAGAGACAGCC downstream amplification primer sequence SEQ ID NO.4:ACCGACGCGT TTTGAGTCCTGCCTTCTCAAAG
(2) S100P recombinant expression plasmid the expression and purification of S100P recombinant protein: is converted into HEK293T cell, cracking
Centrifugation obtains supernatant, and DDK affinity chromatography column purification obtains the S100P recombinant protein of purifying；
(3) screening and preparation of monoclonal antibody: being immunized Balb/c mouse using the S100P recombinant protein of above-mentioned purifying,
Mouse spleen cells are taken to be merged with SP2/0 cell, limiting dilution assay obtains monoclonal, and ELISA method screens positive hybridoma
Cell obtains the hybridoma cell strain that can secrete anti-S100P specific antibody, is named as Umab24, subtype identification IgG1；It is logical
It crosses collection ascites and prepares antibody, S100P monoclonal antibody Umab24 is obtained by affinity chromatography column purification.Pass through respectively
Western Blot, immunofluorescence experiment and 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 10,000 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 Umab24 is hybridized with said chip and determines positive signal site by the present invention, as the result is shown
Monoclonal antibody Umab24 of the present invention specifically binds S100P albumen, and with other albumen no cross reactions.
The present invention also provides monoclonal antibody Umab24 in preparing the immune detection tool for detecting S100P 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 Umab24 can detect the expression situation of S100P 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
State the tumour being proliferated and the expression of S100P is closely related that tumour specifically refers to tumour cell, including but not limited to cancer of pancreas, lung
Cancer, breast cancer and oophoroma.
Compared with prior art, the present invention provides a kind of hybridoma cell strain (deposit number CGMCCNo.15595),
And the monoclonal antibody Umab24 that thus hybridoma cell strain generates.The present invention also provides monoclonal antibody Umab24 to make
The application being ready for use in the immune detection tool of detection S100P albumen, the immunohistochemical kit of the Umab24 containing monoclonal antibody,
And application of the monoclonal antibody Umab24 in the kit that preparation is used for marked tumor.Monoclonal antibody of the present invention can
In conjunction with S100P protein-specific, and with other intracellular albumen no cross reactions, significantly improve the inspection of S100P protein immunization
Specificity, the accuracy and reliability of survey, it is true to reflect S100P protein expression level in tumour cell, can be applied to cancer of pancreas,
The expression of S100P in the associated tumor tissues such as lung cancer, breast cancer and oophoroma.
Preservation information
The classification naming of hybridoma cell strain Umab24 for preservation are as follows: the hybridoma of S100P mouse monoclonal antibody is thin
Born of the same parents' strain；
Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center；
Depositary institution's abbreviation: CGMCC；
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；
Preservation date: on April 26th, 2018；
Deposit number: CGMCC No.15595.
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 that embodiment 2 recombinates S100P albumen Western blot testing result figure, with anti-DDK antibody test weight
Expression of the group S100P albumen in HEK293T cell, wherein left side swimming lane is the HEK293T cell pyrolysis liquid for transfecting empty carrier
It is the inspection of antigen for the HEK293T cell pyrolysis liquid that the testing result of antigen, right lanes are transfection pCMV6-rS100P plasmid
Survey result；
Fig. 3 shows that embodiment 2 recombinates S100P protein SDS-PAGE result figure, is recombinated with anti-DDK affinity chromatography column purification
S100P albumen, albumen after purification pass through SDS-PAGE glue electricity arteries and veins, coomassie brilliant blue staining；
Fig. 4 show embodiment 3 with monoclonal antibody Umab24 identify complete S100P (Full length S100P,
S100P-FL) the Western blot testing result figure of albumen.Swimming lane 1 is the cell pyrolysis liquid for transfecting pCMV6-Entry；Swimming lane
2 be the cell pyrolysis liquid for transfecting pCMV6-S100P-FL；
Fig. 5 shows the Hela cell that embodiment 3 identifies that wild type Hela cell and S100P are knocked out with monoclonal antibody Umab24
The Western blot testing result figure of endogenous S100P albumen in lysate, PCNA are compareed as internal reference.WT:Wild type
Hela；KO:S100P knockout Hela.
Fig. 6 shows that 4 formalin of embodiment is fixed, (primary antibody is that S100P is mono- to the bladder cancer ImmunohistochemistryResults Results figure of paraffin embedding
Clonal antibody Umab24)；
Fig. 7 show 4 formalin of embodiment fix, human colon carcinoma ImmunohistochemistryResults Results figure (the primary antibody S100P of paraffin embedding
Monoclonal antibody Umab24)；
Fig. 8 shows that 4 formalin of embodiment is fixed, (primary antibody is that S100P is mono- to the Human plactnta ImmunohistochemistryResults Results figure of paraffin embedding
Clonal antibody Umab24)；
Fig. 9 shows that 4 formalin of embodiment is fixed, (primary antibody is that S100P is mono- to the human liver cancer ImmunohistochemistryResults Results figure of paraffin embedding
Clonal antibody Umab24)；
Figure 10 show 4 formalin of embodiment fix, human gastric cancer ImmunohistochemistryResults Results figure (the primary antibody S100P of paraffin embedding
Monoclonal antibody Umab24)；
Figure 11 show 4 formalin of embodiment fix, people's spleen ImmunohistochemistryResults Results figure (primary antibody S100P of paraffin embedding
Monoclonal antibody Umab24)；
Figure 12 shows that (upper left corner is phalloidine to 5 human cervical carcinoma cell Hela immunofluorescence dyeing result figure of embodiment
The microfilament of phalloidin label, upper right corner primary antibody are S100P monoclonal antibody Umab24, and the lower left corner is DAPI core dye, bottom right
Angle is overlay chart)；
Figure 13 shows that (primary antibody is S100P monoclonal antibody Umab24,1:100 to embodiment 6OriGene 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, S100P recombinant expression plasmid
With the plasmid RC201533 (ORF288bp containing S100P) obtained from Biotechnology Co., Ltd of Aureal Dongyuan County of the U.S.
For template, designs two primers and introduce restriction enzyme site SgfI and MluI respectively, clone insertion expression vector pCMV6-Entry,
Establish S100P recombinant expression plasmid.Cloning site design is as shown in Figure 1.
The expression and purification of embodiment 2, S100P 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 S100P 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 WB identification recombination S100P's
Expression, result figure 2.
3, DDK affinity chromatography column purification: with 0.45 μM, the lysate supernatant after 33mm pvdf membrane filter filter centrifugation is simultaneously
It is transferred to 15mL pipe, the Beads 1mL mixed is added, is put into after sealing in 360 degree of vortex mixers, is combined 2 hours in 4 DEG C；It takes out
15mL pipe, lysate is poured into BIO-RAD chromatographic column, and catch and penetrate liquid, and liquid sampling WB detection is penetrated after drop is most；With cracking
Buffer rinses column material 1-2 times, is rinsed Beads 3 times with TBST after drop is most, is washed after dripping to the greatest extent with 0.1M Glycine pH3.5 again
De-, 200 μ L, drop are not collected to the greatest extent for the first time, second and third each 500 μ L, the 4th 250 μ L are collected to a 1.5mL centrifuge tube
In, and it is rapidly added NaH2PO4(pH=11.0) it is neutralized to pH7.0 or so, glycerol is added to final concentration of 10% in every pipe,
Tween-80 to final concentration of 0.1%.Recombination S100P albumen after purification is identified with SDS-PAGE, sees Fig. 3.
By Fig. 2 result as it can be seen that in 12kD after WB detection in the HEK293T cell pyrolysis liquid of transfection pCMV6-rS100P plasmid
There is apparent specific band at place, is consistent substantially with the theoretical molecular weight 10.2kD of polypeptide.Show to recombinate S100P albumen spy in cell
Different expression.
Theory point by Fig. 3 result as it can be seen that the albumen of purifying has apparent specific band in PAGE glue 10kD, with polypeptide
Son amount 10.2kD is consistent substantially.Show to have obtained the preferable S100P recombinant protein of purity.
The preparation and screening of embodiment 3, S100P monoclonal antibody
The S100P protein fragments of the purifying generated according to standard method recombination (tie up tonneau China in Beijing to Balb/c mouse
Experimental animal Technology Co., Ltd.) it is immunized.The specific method is as follows:
1, animal immune: purified S100P antigen is emulsified with complete Freund's adjuvant, immune using subcutaneous injection method
6-8 week old Balb/c mouse, immunizing dose are 30 μ g/, and progress is immune for the second time after two weeks at interval, with incomplete Freund's adjuvant
Emulsification, immunizing dose are 30 μ g/.It is immune that tail blood is taken to measure serum titer with ELISA method gradient dilution afterwards twice；According to result
Determine whether booster immunization, chooses the highest mouse of antibody titer and carry out cell fusion.
2, cell fusion: myeloma cell uses the sp2/0 in the source Balb/c, and logarithmic growth phase is in when fusion；It takes
Immune mouse spleen, is made lymphocyte single cell suspension；Myeloma cell is mixed with mouse spleen lymphocyte with 1:5-1:10,
37 DEG C 50%PEG (PH 8.0) 1mL is added dropwise, incomplete culture medium terminate liquid and remaining terminate liquid is added, after supernatant is abandoned in centrifugation
HAT culture medium be added suspend and mix, MC constant volume to 50mL is dispensed into 3.5cm culture dish, is put in wet box, be placed in 37 DEG C,
5%CO2It is cultivated in constant incubator.
3, screen and clone: fusion selected cell clone in 7-10 days, carried out ELISA using S100P purification of recombinant proteins
Test.Mark cell strain number.Limiting dilution is carried out to positive hole cell, 5-6 days measurement ELISA values after each limiting dilution are chosen
The higher monoclonal hole of OD280 positive value is taken to carry out limiting dilution, hardened fruit is the positive entirely until ELISA measures 96 orifice plates.It chooses
The monoclonal singling for taking positive value high.It is Umab24 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 Umab24 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 Umab24 can be good at identifying S100P full-length proteins；By Fig. 5 result as it can be seen that Umab24
Identify the endogenous S100P albumen in Hela cell, after Hela cell-specific knocks out S100P gene, this is thin for Umab24 nonrecognition
Intrinsic protein in born of the same parents, molecular weight is consistent with expected molecular size range, shows that monoclonal antibody Umab24 can specifically Western
Blot detects complete S100P albumen.
Embodiment 4, the immunohistochemistry that monoclonal antibody Umab24 is primary antibody detect
(1), experimental method:
1, take the bladder cancer that formalin is fixed, human colon carcinoma, Human plactnta, human liver cancer, human gastric cancer and people's spleen tissue into
Row paraffin embedding is sliced using Finesse histotome, and tissue thickness is 6 μm.
2, dewaxing and aquation: analyzing pure 3 × 10min of dimethylbenzene, dehydrated alcohol 3 × 10min, 95% ethyl alcohol 5min, and 85%
Ethyl alcohol 5min, 75% ethyl alcohol 5min, deionized water impregnate 3min × 3 time.
3, antigen retrieval buffers (0.01M, pH6.0 sodium citrate buffer) pressure cooker hot high pressure is added and repairs 3min, to height
When pressure pot temperature is down to about 90 DEG C, pressure cooker is opened, takes out sample, then cooled to room temperature.Deionized water impregnates 3min
× 3 times.
4, using 3% hydrogen peroxide deactivation tissue endogenous peroxydase, it is stored at room temperature 10min.Deionized water is impregnated
5min × 3 time.
5, confining liquid (+5% Normal Goat Serum of PBS+5% skimmed milk power) is added, 37 DEG C of incubation 60min.
6, confining liquid is removed, is not rinsed, is added S100P monoclonal antibody (Umab24), thinner ratio: 1:100 or 1:200 uses 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, is washed every time
5min.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. 6-11.
(2), experimental result:
By Fig. 6-11 result as it can be seen that in bladder cancer, human colon carcinoma, Human plactnta, human liver cancer, human gastric cancer and people's spleen tissue can
See specific cell matter or nuclear targeting.As a result consistent with S100P positioning in the cell and tissue expression specificity, table
Bright monoclonal antibody Umab24 can be used for the level of Immunohistochemical detection S100P albumen.
Embodiment 5, the Immunofluorescence test that monoclonal antibody Umab24 is primary antibody
1), experimental method:
1, cell spreads version: taking the human cervical carcinoma cell Hela of in vitro culture to tile into 24 orifice plates, places incubator culture
24 hours.
2, culture medium is removed, washed once with the PBS of preheating, cell monolayer culture is made.
3, fixed: 4% paraformaldehyde (PBS preparation) room temperature fixes 10 minutes.
4, fixer is removed, PBS is washed 3 times.
5, it penetrates: penetrating punching 5 minutes with 0.1%Triton-X-100 (PBS preparation) at room temperature, PBS is washed 3 times.
6, confining liquid (PBS+2%BSA) is added, 37 DEG C are incubated for 60 minutes.
7, S100P monoclonal antibody (Umab24) is added, thinner ratio: 1:50 is diluted using confining liquid, and 37 DEG C are incubated for 60 points
Clock.PBS is washed 3 times.
8, Alexa is added- 488AffiniPure Goat Anti-Mouse IgG (H+L), thinner ratio: 1:
500, it is diluted using confining liquid, room temperature is protected from light incubation 60 minutes.PBS is washed 3 times.
9, Alexa is added594-Phalloidin (phalloidine) marks microfilament.
10, DAPI (HH3342) redyes nucleus 3 minutes, microscopy.
As a result as shown in figure 12, visible specific cell nuclear staining, shows monoclonal in human cervical carcinoma cell Hela
Antibody Umab24 can be used for the level of Immunofluorescence test S100P albumen.
The specific detection of embodiment 6, monoclonal antibody Umab24
Lysate, every hatching egg are overexpressed comprising 10,000 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 Umab24 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 Umab24 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 Umab24 can specifically identify S100P albumen, table on OriGene protein chip
The specificity of bright monoclonal antibody Umab24 is preferably.
<110>Wuxi Origene Bio-tech Co., Ltd.
<120>anti-S100P protein monoclonal antibody and application thereof
<210> 1
<211>288
<212> DNA
<213>artificial sequence
<400> 1
1 ATGACGGAAC TAGAGACAGC CATGGGCATG ATCATAGACG TCTTTTCCCG ATATTCGGGC
61 AGCGAGGGCA GCACGCAGAC CCTGACCAAG GGGGAGCTCA AGGTGCTGAT GGAGAAGGAG
121 CTACCAGGCT TCCTGCAGAG TGGAAAAGAC AAGGATGCCG TGGATAAATT GCTCAAGGAC
181 CTGGACGCCA ATGGAGATGC CCAGGTGGAC TTCAGTGAGT TCATCGTGTT CGTGGCTGCA
241 ATCACGTCTG CCTGTCACAA GTACTTTGAG AAGGCAGGAC TCAAATGA
//
<210> 2
<211>95
<212> PRT
<213>artificial sequence
<400> 2
ORIGIN
1 MTELETAMGM IIDVFSRYSG SEGSTQTLTK GELKVLMEKE LPGFLQSGKD KDAVDKLLKD
61 LDANGDAQVD LDANGDAQVD ITSACHKYFE KAGLK
//
Claims (7)
1. a kind of monoclonal antibody, which is characterized in that it specifically binds with S100 calbindin P (S100P)；The Dan Ke
Grand antibody is generated by hybridoma cell strain Umab24, and deposit number is CGMCC No.15595.
2. a kind of hybridoma cell strain, deposit number is CGMCC No.15595.
3. monoclonal antibody as described in claim 1 is in preparation for detecting the application in S100P 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 that cancer of pancreas, lung cancer, breast cancer are related to oophoroma etc.
Tumour.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116496394A (en) * | 2022-01-26 | 2023-07-28 | 东莞市朋志生物科技有限公司 | Antibodies against S100 protein, reagents and kits for detecting S100 protein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103460045A (en) * | 2011-04-05 | 2013-12-18 | 奥林巴斯株式会社 | Pancreas test method, and pancreas test kit |
| CN106188288A (en) * | 2015-05-05 | 2016-12-07 | 北京傲锐东源生物科技有限公司 | Anti-S100P protein monoclonal antibody and application thereof |
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2018
- 2018-10-16 CN CN201811015870.0A patent/CN109111519A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103460045A (en) * | 2011-04-05 | 2013-12-18 | 奥林巴斯株式会社 | Pancreas test method, and pancreas test kit |
| CN106188288A (en) * | 2015-05-05 | 2016-12-07 | 北京傲锐东源生物科技有限公司 | Anti-S100P protein monoclonal antibody and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116496394A (en) * | 2022-01-26 | 2023-07-28 | 东莞市朋志生物科技有限公司 | Antibodies against S100 protein, reagents and kits for detecting S100 protein |
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Application publication date: 20190101 |