CN103923214B - Anti-liver-cancer stem cell monoclonal antibody and application thereof - Google Patents
Anti-liver-cancer stem cell monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of anti-liver-cancer stem cell monoclonal antibody, CGMCC by deposit number? the mouse hybridoma cell 4P-120 of No.8800 secretes and produces, this monoclonal antibody confirms through experiment, can specific binding liver cancer cell specific recognition liver cancer tissue, its antigen identified high expression level and not expressing in cancer beside organism or low expression in the cancerous tissue of people's liver cancer, and the inverse differentiation that there is the positive differentiation to feminine gender and negative heliotropism in proliferation process; Compare the low express cell of antigen, antigen high expressing cell has stronger multiplication capacity, clonality, balling-up ability, cut transfer ability and Invasion and Metastasis ability; Therefore, the present invention is the functional monoclonal antibody that the liver cancer treatment of targeting tumor stem cells provides application prospect, and it may be used for preparing cancer treatment drug.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of monoclonal antibody.
Background technology
Liver cancer is one of common malignant tumour of China, and its case fatality rate occupies the 3rd of malignant tumour, and liver cancer patient 5 years survival rates only have 14% ~ 30%.Primary hepatocarcinoma has the advantages that morbidity is hiddenly attacked, case fatality rate is high, early diagnosis and prevent recurrence great to prognosis meaning.After monoclonal antibody technique comes out, people attempt the early diagnostic rate and the result for the treatment of that are improved liver cancer by application liver cancer-resisting monoclone antibody, but the specificity of the liver cancer-resisting monoclone antibody obtained at present is still unsatisfactory, due to the existence of Tumor Heterogeneity, its clinical application is made again to be under some influence.Therefore, be necessary to find better anti-liver cancer-specific monoclonal antibody, to improve the specificity to liver cancer diagnosis and treatment.In recent years, tumor stem cell (cancerstemcell, CSC) has become the new focus of tumor research.Due to the root that CSC is metastases, recurrence and resistance, therefore, the treatment New Policy setting up target CSC is expected to overcome the defect of existing treatment means clinically, improves the prognosis of liver cancer patient.Contriver place seminar is in earlier stage based on the similarity of tumor stem cell with embryonic stem cell, using Nanog as sorting indicia, constructing with Nanog is slow virus reporting system Lv-Nanog (the p)-GFP that promoter regulation GFP expresses, and the liver cancer cell of the Nanog positive that sorting obtains confirms to have tumor stem cell characteristic (Hepatology2012) through internal and external test.
Summary of the invention
In view of this, the object of the invention is to using the liver cancer cell of the Nanog positive as immunogen and screening antigen, the monoclonal antibody for liver-cancer stem cell is filtered out by monoclonal antibody technique, and to this monoclonal antibody for antigen study, to providing the functional monoclonal antibody of application prospect for the liver cancer treatment of targeting tumor stem cells.
After deliberation, the invention provides following technical scheme:
1. anti-liver-cancer stem cell monoclonal antibody, the mouse hybridoma cell 4P-120 being CGMCCNo.8800 by deposit number secretes and produces.
2. anti-liver-cancer stem cell monoclonal antibody is preparing the application in cancer treatment drug.
Beneficial effect of the present invention is: the present invention is using the liver cancer cell of the Nanog positive as immunogen and screening antigen, being filtered out by monoclonal antibody technique can the mouse hybridoma cell strain 4P-120 of stably excreting monoclonal antibody, monoclonal antibody of its secretion confirms through experiment, can specific binding liver cancer cell specific recognition liver cancer tissue; The antigen that this monoclonal antibody identifies high expression level and not expressing in cancer beside organism or low expression in the cancerous tissue of people's liver cancer, and the inverse differentiation that there is the positive differentiation to feminine gender and negative heliotropism in proliferation process; Compare the low express cell of antigen, antigen high expressing cell has stronger multiplication capacity, clonality, balling-up ability, cut transfer ability and Invasion and Metastasis ability; Therefore, the present invention is the functional monoclonal antibody that the liver cancer treatment of targeting tumor stem cells provides application prospect, and it may be used for preparing cancer treatment drug.
Biomaterial preservation
Mouse hybridoma cell 4P-120 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 5th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica), deposit number is CGMCCNo.8800.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe response situation of monoclonal antibody 4P-120 and different clone.
Fig. 2 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe response situation of monoclonal antibody 4P-120 and human liver cancer tissue and normal liver tissue.
Fig. 3 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression of the antigen that monoclonal antibody 4P-120 identifies in human liver cancer tissue and cancer beside organism.
Fig. 4 is mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression changing conditions of the antigen that monoclonal antibody 4P-120 identifies in proliferation process.Wherein, A, B are difference sorting 4P-120 antigen and Nanog double negative cells (P9), 4P-120 antigen list positive cell (P8), 4P-120 antigen and Nanog two positive cell (P6), these four cell colonys of 4P-120 antigen list negative cells (P7) from PLC-Nanog cell; C, D, E, F carry out to P9, P8, P7, P6 cell colony sub-elected the result that streaming returns survey; G, H, I, J are the flow cytometer detection result of after cultivating 7d, P9, P8, P7, P6 cell colony being carried out to 4P-120 antigen differentiation situation.
Fig. 5 is that 4P-120 antigen high expressing cell has stronger multiplication capacity.
Fig. 6 is that 4P-120 antigen high expressing cell has higher clonality.
Fig. 7 is that 4P-120 antigen high expressing cell has higher balling-up ability.
Fig. 8 is that 4P-120 antigen high expressing cell has higher cut transfer ability.
Fig. 9 is that 4P-120 antigen high expressing cell has higher Invasion and Metastasis ability.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.
One, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe preparation of monoclonal antibody
1, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe acquisition of hybridoma cell strain
By the PLC-Nanog cultivated
pOSnanog
negcell (being namely the liver cancer cell PLC that promoter regulation GFP expresses with Nanog) airflow classification goes out PLC-Nanog
pOScell (i.e. the PLC cell of the Nanog positive), as immunogen; Get the healthy BALB/c mouse in 4 ~ 6 week age of body weight 14 ~ 18g, in every mouse peritoneal, inject 1 × 10
6individual PLC-Nanog
pOScell carries out first immunisation, afterwards at interval of 2 weeks booster immunizations 2 times in the same way again.After final boost the 10th day, get mouse tail blood, separation of serum, adopt cell Immunofluorescent antibody (ELISA) method to detect antibody titer.
It is specific as follows that ELISA method detects antibody titer: by PLC-Nanog
pOScell is inoculated in 96 porocyte culture plates, every hole 1 × 10
4individual cell, next day takes out Tissue Culture Plate, after abandoning supernatant, PBS washes 3 times, under room temperature, 20 minutes are fixed with 4% paraformaldehyde solution, PBS rinsing 3 times, 10% foetal calf serum (FBS) is used to close 30 minutes in 37 DEG C again, abandon supernatant, different cell culture well adds the immune serum of continuous 10 times of dilutions respectively, every hole 100ul, simultaneously using the mice serum before first immunisation as negative control, PBS is as blank, 37 DEG C hatch 1 hour after abandon supernatant, PBST washes plate 5 times, add the goat anti-mouse IgG of the HRP mark diluted by 1:1000 with confining liquid again, every hole 100ul, hatch 30 minutes for 37 DEG C, discard traget antibody solution, PBST washes plate 5 times, add OPD nitrite ion again, 37 DEG C hatch 20 minutes after, by 2mol/L sulfuric acid termination reaction, measure each group of OD
490nmvalue, experimental group OD
490nmvalue is more than or equal to negative control 2.1 times of persons and is judged to the positive, to be judged to positive most highly diluted multiple as antibody titer.
Choose the mouse that antibody titer is the highest, with PLC-Nanog
pOScell carries out spleen reinforcement, after 3 days, gets its spleen and prepares splenocyte suspension, merge with murine myeloma cell SP2/0.Gained fused cell is on average assigned in 5-7 96 porocyte culture plates with the RPMI-1640 containing HAT and is cultivated, in cultivation about the 7th day, change liquid once by the RPMI-1640 full dose containing HAT, within the 10th day, collect culture supernatant, adopt ELISA method to carry out positive cell screening.
ELISA method screening positive cell is specific as follows: cultivate the 9th day, by PLC-Nanog in fused cell
pOScell is inoculated in another 96 porocyte culture plate, every hole 1 × 10
4individual cell, within 10th day, take out Tissue Culture Plate, after abandoning supernatant, PBS washes 3 times, under room temperature, 20 minutes are fixed with 4% paraformaldehyde solution, PBS rinsing 3 times, 30 minutes are closed in 37 DEG C again with 10%FBS, abandon supernatant, each hole adds different fused cell culture supernatant respectively, every hole 100ul, simultaneously using the immune serum of 1:100 dilution as positive control, using SP2/0 cells and supernatant as negative control, hatch 1 hour for 37 DEG C, abandon supernatant, PBST washes plate 5 times, add the goat anti-mouse IgG of the HRP mark diluted by 1:1000 with confining liquid again, every hole 100ul, hatch 30 minutes for 37 DEG C, discard traget antibody solution, PBST washes plate 5 times, add OPD nitrite ion again, 37 DEG C hatch 20 minutes after, by 2mol/L sulfuric acid termination reaction, measure each group of OD
490nmvalue, experimental group OD
490nmvalue is more than or equal to negative control 2.1 times of persons and is judged to the positive.
Positive porocyte is changed liquid with the RPMI-1640 containing HAT cultivate, adopt next day aforementioned ELISA method to recheck, the positive porocyte detected carries out mono-clonal screening further.
The acquisition of monoclonal hybridoma strain: adopt limiting dilution assay to carry out subclone to positive porocyte, after each subclone about the 7th day, pick out monoclonal cell hole and adopt aforementioned ELISA method to detect, retain 1-2 positive hole, repeat subclone more than three times, until all subclones are the positive, enlarged culturing is carried out to this cell strain and frozen, obtain monoclonal hybridoma strain.
The present invention merges screening through four times, obtains 64 strain monoclonal hybridoma strains altogether, confirms through qualification, and the mouse hybridoma cell of called after 4P-120 can have the monoclonal antibody of liver cancer-specific by stably excreting.
2, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe acquisition of monoclonal antibody
Choose more than body weight 20g, more than 8 week age healthy BALB/c mouse, abdominal injection by whiteruss and lanolin in mass ratio for 3:1 mixes obtained adjuvant, every 0.3ml, after 7-10 days, the mouse hybridoma cell 4P-120 PBS of cultivation or physiological saline are washed and be once injected into mouse peritoneal afterwards, mouse ascites is got after 7-10 days, carry out purifying with ProteinG post by specification, obtain mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120, measures antibody concentration ,-20 DEG C of preservations.
Two, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe specificity identification of monoclonal antibody
1, flow cytomery antibodies specific
Choose different tumor cell line: three kinds of hepatoma cell line (PLC, HU-7, T1224), two kinds of gastric carcinoma cell lines (Xn0422, SGC7901), two kinds of glioma cell line (U87, U251), three kinds of colon carcinoma cell line (SW480, SW620, HCT-116), and two kinds of normal cell systems (293, L02) the normal CIK cell (CIK-1 of and three example, CIK-2, CIK-3), culturing cell PBS is washed 1 time, add mouse hybridoma cell 4P-120 culture supernatant, hatch 30 minutes for 4 DEG C, PBS washes 3 times, add the goat anti-mouse IgG (Sigma company) that the fluorescein isothiocyanate (FITC) that dilutes by 1:500 with PBS marks again, hatch 30 minutes for 4 DEG C, PBS washes three times, after redissolving with PBS400ul, flow cytomery.
Result as shown in Figure 1, mouse hybridoma cell 4P-120 culture supernatant is all reacted in significantly high three kinds of hepatoma cell line, be low reaction to other tumor cell line, normal clone is negative reaction, the mouse-anti people liver-cancer stem cell PLC-Nanog secreted by mouse hybridoma cell 4P-120 is described
pOSmonoclonal antibody 4P-120 can specific binding liver cancer cell.
2, identified by immunofluorescence antibodies specific
Cut human liver cancer tissue and normal liver tissue respectively, fix 20 minutes with cold acetone, PBS washes 4 times, then hatches 30 minutes with 10%BSA in 37 DEG C, then adds 10ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120,4 DEG C of refrigerator overnight, PBS washes 5 times, then adds the goat anti-mouse IgG of the FITC mark diluted by 1:500 with PBS, hatch 50 minutes for 37 DEG C, then add the DAPI of 1:500 dilution, hatch 10 minutes for 37 DEG C, PBS washes 5 times, 40% glycerine mounting, and laser co-focusing detects.
Result as shown in Figure 2, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120 and human liver cancer tissue are strong positive reaction, and to be negative reaction to people's normal liver tissue, illustrate that this monoclonal antibody can specific recognition liver cancer tissue.
3, immunohistochemical methods qualification antibodies specific
Choose cancerous tissue and cancer beside organism's paraffin section of people's liver cancer respectively, bake 20 minutes under lamp, with xylene soak 2 times (each 10 minutes), use dehydrated alcohol successively again, dehydrated alcohol, 95% ethanol, 85% ethanol, 75% ethanol respectively soaks 5 minutes, use distilled water successively again, PBS respectively washes 3 times, add antigen retrieval buffers, 800W microwave heating 3 points 15 seconds, 150W microwave heating 16 minutes, distillation washing 3 times after cooling, add 3% hydrogen peroxide again, hatch 15 minutes for 37 DEG C, PBS washes 3 times, add closed serum again, hatch 15 minutes for 37 DEG C, add 5ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog again
pOSmonoclonal antibody 4P-120,4 DEG C of refrigerator overnight, PBS washes 5 times, add the goat anti-mouse IgG (Sigma company) of 1:500 dilution again, hatch 25 minutes for 37 DEG C, PBS washes 5 times, add horseradish peroxidase again, hatch 15 minutes for 37 DEG C, PBS washes 3 times, DAB dyes, tap water termination reaction, Hematorylin redyes about 10 seconds, respectively soaks 5 minutes with 75% ethanol, 85% ethanol, 95% ethanol, dehydrated alcohol, dehydrated alcohol successively, dry mounting, basis of microscopic observation.
Result as shown in Figure 3, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe antigen that monoclonal antibody 4P-120 identifies high expression level in the cancerous tissue of people's liver cancer, does not express or low expression in cancer beside organism; Statistic analysis result also shows, and the expression of the antigen that monoclonal antibody 4P-120 identifies in cancerous tissue is apparently higher than cancer beside organism.
Three, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe antigen presentation of monoclonal antibody and the functional performance of different antigen presentation amount cell detect
1, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe expression of the antigen that monoclonal antibody identifies in proliferation process
By the PLC-Nanog cultivated
pOSnanog
negcell trysinization, PBS washes 1 time, and centrifugal 3 minutes of 1000rpm, abandons supernatant, adds 50ug/ml mouse-anti people liver-cancer stem cell PLC-Nanog
pOSmonoclonal antibody 4P-120100ul, incubated at room 15 minutes, with PBS re-suspended cell 1000rpm centrifuge washing 3 times, then adds the anti-mouse IgG of AlexaFluo647 mark of 1:1000 dilution, hatch 15 minutes for 4 DEG C, go out mouse-anti people liver-cancer stem cell PLC-Nanog with selected by flow cytometry apoptosis
pOSthe antigen high expressing cell group of monoclonal antibody 4P-120 and the low express cell group of antigen, cultivate respectively with containing the DMEM substratum of 10%FBS, and by front for cultivations cell antigen expression and return after 7 days and survey result and compare.
Result as shown in Figure 4, mouse-anti people liver-cancer stem cell PLC-Nanog
pOSthe inverse differentiation of the positive differentiation to feminine gender and negative heliotropism is there is in the antigen that monoclonal antibody 4P-120 identifies in proliferation process.
2, the functional performance of the cell colony of different antigen presentation amount detects
The mouse-anti people liver-cancer stem cell PLC-Nanog get unsorted PLC cell respectively, sub-electing from PLC clone
pOSeach 1000 of the low express cell of the antigen high expressing cell of monoclonal antibody 4P-120, antigen, be inoculated in 96 porocyte culture plates, by the DMEM culture medium culturing containing 10%FBS, within 1 ~ 4 day, measure ability of cell proliferation difference afterwards with celltilterblue cell proliferation detecting kit, data detect 560nm/590nm ratio by the long microplate reader of all-wave and obtain.As shown in Figure 5,4P-120 antigen high expressing cell has the multiplication capacity being significantly better than non-sorting PLC cell, the low express cell of 4P-120 antigen to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog get unsorted PLC cell respectively, sub-electing from PLC clone
pOSeach 50 of the low express cell of the antigen high expressing cell of monoclonal antibody 4P-120, antigen, is inoculated in 24 porocyte culture plates, by the DMEM culture medium culturing containing 10%FBS, within 2 days, changes liquid once, counts and add up after 14 days to Clone formation number.As shown in Figure 6, the low express cell of the antigen compared to monoclonal antibody 4P-120,4P-120 antigen high expressing cell has higher clonality to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog get unsorted PLC cell respectively, sub-electing from PLC clone
pOSeach 20 of the low express cell of the antigen high expressing cell of monoclonal antibody 4P-120, antigen, is inoculated in 96 porocyte culture plates, by serum-free balling-up culture medium culturing, counts and add up after 14 days to balling-up number.As shown in Figure 7, compared to non-sorting cells and the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher balling-up ability to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog get unsorted PLC cell respectively, sub-electing from PLC clone
pOSthe low express cell of the antigen high expressing cell of monoclonal antibody 4P-120, antigen each 10
5individual, be inoculated in 24 porocyte culture plates, cultivate with the low serum free culture system liquid containing 2%FBS, after being paved with orifice plate, carrying out cut and taking pictures, again taking pictures after 24 hours, the width average healing state of statistics cut.As shown in Figure 8, compared to non-sorting cells and the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher cut transfer ability to result.
The mouse-anti people liver-cancer stem cell PLC-Nanog get unsorted PLC cell respectively, sub-electing from PLC clone
pOSthe low express cell of the antigen high expressing cell of monoclonal antibody 4P-120, antigen each 10
5individual, be inoculated in 8umtranswell cell, little indoor are 200ul plasma-free DMEM medium, for 1300ul is containing the DMEM substratum of 10%FBS in lower floor 24 orifice plate, dye after 24 hours to porate cells number, statistics porate cells number.As shown in Figure 9, compared to the low express cell of 4P-120 antigen, 4P-120 antigen high expressing cell has higher Invasion and Metastasis ability to result.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (2)
1. anti-liver-cancer stem cell monoclonal antibody, is characterized in that, the mouse hybridoma cell 4P-120 being CGMCCNo.8800 by deposit number secretes and produces.
2. anti-liver-cancer stem cell monoclonal antibody as claimed in claim 1 is preparing the application in cancer treatment drug.
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| CN106366190B (en) * | 2015-07-22 | 2020-01-10 | 中国医学科学院肿瘤医院 | Monoclonal antibody for resisting human liver cancer stem cells |
| CN105132445B (en) * | 2015-10-23 | 2018-10-02 | 马健颖 | A kind of receptor protein of specially recognizing tumor cells, T lymphocytes and NK cells |
| CN107474139B (en) * | 2017-06-23 | 2021-04-27 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody targeting human tumor stem cells and application thereof |
| EP3650469A4 (en) * | 2017-06-23 | 2021-03-24 | Suzhou Bojuhua Biomedical Technology Co. Ltd | Monoclonal antibody of targeted human tumor stem cells and application of monoclonal antibody |
| CN107163146B (en) * | 2017-06-23 | 2021-04-23 | 苏州博聚华生物医药科技有限公司 | Monoclonal antibody targeting human tumor stem cells and application thereof |
| CN111363034B (en) * | 2018-12-25 | 2023-04-25 | 上海市第十人民医院 | Antibody of NANOG Ser68, inhibitor and application |
| CN111303290B (en) * | 2020-05-11 | 2020-12-01 | 绍兴百奥尼科技有限公司 | Antibody specifically binding to liver cancer stem cells and application of antibody in preparation of liver cancer detection kit |
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| CN1940062A (en) * | 2005-09-28 | 2007-04-04 | 上海市肿瘤研究所 | Liver-cancer stem cell, its separation and use |
| WO2012176175A1 (en) * | 2011-06-24 | 2012-12-27 | Universite De Geneve | New uses of nanog inhibitors and related methods |
| CN102892879A (en) * | 2010-05-13 | 2013-01-23 | 加州大学董事会 | Method and composition for inducing human pluripotent stem cells |
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| CN1940062A (en) * | 2005-09-28 | 2007-04-04 | 上海市肿瘤研究所 | Liver-cancer stem cell, its separation and use |
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| WO2012176175A1 (en) * | 2011-06-24 | 2012-12-27 | Universite De Geneve | New uses of nanog inhibitors and related methods |
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