CN102634487A - GPC3 (glypican-3) monoclonal antibody hybridoma strain 8G6, and preparation method and application thereof - Google Patents
GPC3 (glypican-3) monoclonal antibody hybridoma strain 8G6, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a GPC3 (glypican-3) monoclonal antibody hybridoma strain 8G6, and a preparation method and application thereof, which belong to the technical field of cell engineering. The preservation code of the GPC3 monoclonal antibody hybridoma strain 8G6 is CGMCC (china general microbiological culture collection center) No.5427. The preparation method of the GPC3 monoclonal antibody hybridoma strain 8G6 includes: using GPC3 protein as immunogen to immunize a mouse; and fusing mouse splenocytes having serum titer more than 1:104 with SP2/0 myeloma cells, using an HATRPMI-1640 medium to screen fusion cells, and finally obtaining the hybridoma strain 8G6 by ELISA (enzyme-linked immunosorbent assay) and repeated limiting dilution. The GPC3 monoclonal antibody hybridoma strain 8G6 is high in yield of secretory antibodies, and is easy to survive, and the secreted monoclonal antibodies are high in titer, sensitive in reaction, easy to detect, low in production cost and widely applicable to detection of GPC3 protein expression.
Description
Technical field
The present invention relates to the cell engineering field, be specifically related to GPC3 monoclonal antibody hybridoma cell strain 8G6.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma; HCC) be one of modal malignant tumour in the world, the serious harm human life is healthy, has report to show; In all diseases, the hepatocellular carcinoma sickness rate occupies the 5th and case fatality rate is in the 3rd.According to estimates, there are the 1000000 newfound HCC patients of surpassing in the whole world every year, and about 1,000,000 people die of illness.Because HCC early symptom and sign is not obvious or lack of specific; Cause the early diagnostic rate of HCC lower; The patient seeks medical advice and is middle and advanced stage often, the meeting of forfeiture treatment machine (research shows, but early metaphase HCC operative treatment; 5 years survival rates of small liver cancer excision postoperative are nearly 62.9%, and big 5 years survival rates of liver cancer postoperative then are merely 34.6%).Simultaneously, because HCC is all little responsive to radiation and chemotherapy, prognosis is totally relatively poor, and surgical operation comprises that liver transplantation is still the unique method of treatment recovery from illness liver cancer.Yet long-term survival rate was lower after high recurrence caused performing the operation with the high rate of transform, and result of treatment is undesirable.When treatment does not at present still have the better healing strategy,, be the effective way that improves survival rate, reduces case fatality rate to high risk population's effective screening and the early diagnosis of HCC.And to the early discovery and the diagnosis of small liver cancer, the tool advantage of biomarker.The most classical and putative in diagnosing cancer of liver at present is ALPHA-FP AFP, but its positive detection rate also is merely 30 ~ 60%, and specificity is not enough, can not satisfy clinical early diagnosis to the AFP negative HCC.The pathogenesis of liver cancer is a complex process multifactor, that multistep is rapid, thus so far still none specific parameters can use clinically, but generation, recurrence and transfer through the multiple index early discovery of joint-detection liver cancer, thereby carry out early treatment.Therefore, seek efficient diagnosis and treatment target, become emphasis, difficult point and the direction of HCC research.
(glypican-3 is a film property Suleparoid polysaccharide protein GPC3), at the fetus liver expressed in abundance to Monophosphoinositideproteoglycans proteoglycans-3; Do not express at adult human liver; When liver cancer takes place, usually activating, is an important substance in the HCC incidence and development again, and usefulness Northern results of hybridization such as Li Shenjing demonstration GPC3 gene is not expressed in normal liver tissue; And be high expression level in liver cancer tissue; And be proportionate with tumour size and liver cancer pathological grading, prompting GPC3 gene plays an important role in liver cancer genesis and development, and is relevant with the lesion degree of liver cancer.This this gene of explanation might be the hepatocellular carcinoma tissue-specific marker gene, can be used as the reference index of clinical early diagnosis, treatment and the judgement grade malignancy of liver cancer, has certain potential applicability in clinical practice.GPC3 is specificity overexpression in tumor tissues not only, simultaneously also can be to be detected in peripheral blood, and usefulness ELISA methods such as Capurro record the C end fragment that has GPC3 in 53% liver cancer patient blood serum.And be none positive in 53 the normal healthy controls group in sample size, only an example is positive in sample size is 20 liver cirrhosis patient control group, the higher specificity of prompting GPC3.The C2 end antibody that usefulness such as Nakatsura are different is also obtained similar result.The variation of its amount again with the existence of HCC focus whether, the severity close association (can reflect perioperative state and result of treatment, responsive) of the state of an illness than AFP.Especially GPC3 is specific expressed in the AFP negative HCC, and joint-detection AFP and GPC3 can significantly improve diagnostic detection rate and the accuracy of HCC, so are considered to a kind of novel liver cancer marker that has potential.Simultaneously, research shows that also GPC3 has higher immunogenicity, in the incidence and development of HCC, plays important effect, might become the novel targets of hepatoma-targeting treatment.
The antibody product that can detect GPG3 at present is the product of more external companies such as BioMosaics, costs an arm and a leg, and has limited the application of this albumen as clinical target checking and functional study aspect thereof.
Summary of the invention
The objective of the invention is to provides GPC3 (human phosphatidyl-inositol proteoglycan 3) monoclonal antibody hybridoma cell strain 8G6 to above-mentioned deficiency of the prior art.
Another object of the present invention provides the preparation method of above-mentioned GPC3 monoclonal antibody hybridoma cell strain 8G6.
Another purpose of the present invention provides the application of above-mentioned GPC3 monoclonal antibody hybridoma cell strain 8G6.
The present invention realizes above-mentioned purpose through following technical scheme:
GPC3 monoclonal antibody hybridoma cell strain 8G6; Classification called after glypican-3 (GPC3) monoclonal antibody hybridoma cell strain 8G6; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 04th, 2011; Deposit number is CGMCC No.5427, the preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the present invention of this cell strain abbreviate GPC3 monoclonal antibody hybridoma cell strain 8G6 as.
By above-mentioned GPC3 monoclonal antibody hybridoma cell strain 8G6 excretory monoclonal antibody.
The preparation method of above-mentioned GPC3 monoclonal antibody hybridoma cell strain 8G6, step is following: with GPC3 albumen is the immunogen immune BALB/c mouse, gets serum titer at 1:10
4Above mouse boosting cell and SP2/0 myeloma cell are merged; With the HAT RPMI-1640 screening of medium fused cell that contains the 20wt% calf serum; Encapsulate elisa plate with GPC3 albumen, indirect elisa method screening fused cell culture supernatant liquid, the highest hybridoma cell strain of selecting to tire carries out subcloning; Be cloned into 100% cell positive rate repeatedly with limiting dilution assay, obtain GPC3 monoclonal antibody hybridoma cell strain 8G6 at last.
In the aforesaid method; Described GPC3 albumen is preferably recombinant protein, and concrete preparation method is: with sequence shown in SEQ ID NO:1 ~ 2 is the upstream and downstream primer, is template with the cDNA of Huh7 (can purchase the Shanghai cell bank in the Chinese Academy of Sciences) cell total rna reverse transcription; PCR method amplification GPC3 gene, amplified production is used
EcoThe R I with
SalI is carried out double digestion; Enzyme is cut product and is connected with the carrier PET-11b (can purchase the company in Merck Chemicals) of the same double digestion of process, obtains recombinant vectors GPC3/PET-11b, transformed into escherichia coli; The IPTG abduction delivering, the purified GPC3 recombinant protein that obtains of expression product.
In the aforesaid method, mouse boosting cell and SP2/0 myeloma cell are merged preferred usefulness 50 wt % PEG-4000.
The application of GPC3 monoclonal antibody hybridoma cell strain 8G6 excretory monoclonal antibody of the present invention in detecting the GPC3 protein expression.The technical fields such as immunohistochemistry detection that if can be used for ELISA, time-resolved fluoroimmunoassay, chemoluminescence, western-blot, immunofluorescence and tissue.
As a kind of application method, hybridoma cell strain 8G6 excretory monoclonal antibody of the present invention can be prepared into a kind of GPC3 immunity detection reagent.
Compared with prior art, the present invention has following beneficial effect:
GPC3 monoclonal antibody hybridoma cell strain 8G6 secretory antibody output of the present invention is high; And be prone to survival, the secreted monoclonal antibody height of tiring, being quick on the draw is easy to detect; Can be widely used in the detection of GPC3 protein expression; In the hepatocellular carcinoma early detection, have outstanding advantage, and reduced the production cost of GPC3 antibody, help clinical target checking and the functional study thereof of GPC3.
Description of drawings
Fig. 1. GPC3 N end group is because of being GPC3 72-369 bp fragment amplification product electrophoresis result and recombinant vectors GPC3/PET-11b double digestion qualification result; Wherein 1 is the PCR result of GPC3 gene fragment; 2 is marker; DL2000,3 is GPC3/PET-11b double digestion result, 4 is the GPC3/PET-11b recombinant plasmid.
SDS-PAGE electrophoresis (12%) result of Fig. 2 .GPC3 recombinant protein IPTG abduction delivering, m:marker wherein, 1 induce with 3:GPC3/PET-11b/Rosetta before; 2 with after 4:GPC3/PET-11b/Rosetta1 induces.
Fig. 3. SDS-PAGE electrophoresis (12%) result after GPC3 recombinant protein thalline is ultrasonic.1, the ultrasonic supernatant of 3:GPC3/PET-11b/Rosetta; 2, the 4:GPC3/PET-11b/Rosetta1 ultrasound precipitation.
Fig. 4. SDS-PAGE electrophoresis (12%) result of GPC3 recombinant protein purification.M:marker wherein; 1,2: purifying protein.
Fig. 5. monoclonal antibody 8G6 of the present invention is to the result and the results of comparison under the common light microscopic (200 times of microscope magnifications) of liver cancer cell Huh7 immunofluorescence dyeing.
Embodiment
Further explain the present invention below in conjunction with specific embodiment; Should understand; These embodiment only are used to explain the present invention; And protection scope of the present invention is not carried out any type of restriction, under the prerequisite that does not deviate from technical scheme of the present invention, any change that those of ordinary skills that the present invention did are realized easily all will fall within the claim protection domain of the present invention.
1. the structure of GPC3/PET-11b recombinant vectors
According to people GPC3mRNA (NM_001164619) sequence that provides among the GenBank; Remove the terminal signal peptide of N; 72-369 base to sequence designs a pair of primer, upstream primer: 5'TAATGAATTCATGCAGCCCCCGCCGCC 3' (SEQ ID NO:1) downstream primer: 5'AGCGGTCGACTCACTTGGCATGGCGAACAACAA 3' (SEQ ID NO:2).Introduce respectively at the 5' of two primers end
EcoThe R I with
SalThe I restriction enzyme site; With conventional PCR method with the cDNA of Huh7 (available from Chinese Academy of Sciences's Shanghai cell bank) cell total rna reverse transcription be template transfer GPC3 N end group because of; Agarose gel electrophoresis shows, successfully obtained GPC3 N end group because of, fragment length with expect consistent (Fig. 1).
Prokaryotic expression carrier PET-11b (available from Merck Chemicals company) and the GPC3 gene fragment behind the sepharose purifying are used
EcoThe R I with
SalThe I double digestion is handled, and enzyme is cut the purified back of product and connected, and obtains recombinant plasmid GPC3/PET-11b, and recombinant plasmid is identified (Fig. 1) through double digestion, send company's order-checking with recombinant plasmid simultaneously, and sequencing result shows that the GPC3 fragment and the implementation sequence of reorganization is in full accord.
2. GPC3 Recombinant Protein Expression
With recombinant plasmid GPC3/PET-11b transformed into escherichia coli, the recombinant expressed bacterium of GPC3/PET-11b is cultivated in the LB substratum that contains 100 μ g/mL ammonia joint penicillium mould, when A600 reaches 0.6 left and right sides; The IPTG that adds final concentration 1 mmol/L induces 3 h in 37 ℃, and the bacterium liquid after inducing is in 4 ℃, centrifugal 10 min of 8000 rpm; Collect thalline, binding buffer liquid (Binding Buffer) washs once, and wet bacterium deposition is resuspended with binding buffer liquid; Place ice bath, centrifugal 20 min of 12000 rpm after the carrying out ultrasonic bacteria breaking go up cleer and peaceful precipitation and do not carry out the SDS-PAGE electrophoresis; The result shows; The fusion protein molecule amount of expressing is about 25 KDa (see Fig. 22 and 4), is inclusion body expression (Fig. 3), and this fusion rotein is the GPC3 recombinant protein.
3. the purifying of GPC3 recombinant protein is with quantitative
Bacterium after collection is induced, PBS washs once, and wet bacterium deposition is washed with the binding buffer liquid vibration of 8mol/L urea, centrifugal 20 min of 12000 rpm, supernatant is a protein sample to be purified.
His6-Ni Superflow Resin filler (available from clonetech company) is loaded in the purification column; Purification column is connected with Akta purifying appearance; With level pad (method according to product description provides is prepared) 5 column volumes of balance; Protein sample to be purified is with appearance on the speed of 1. 5 mL/min, and last appearance back is washed till baseline with level pad, with albumen elution buffer wash-out; Collect protein peak, elutriant is 4 ℃ of gradient dialysis in the binding buffer liquid that contains 6 mol, 4 mol, 2 mol, 1 mol, 0 mol urea.The SDS-PAGE electrophoretic analysis shows that the GPC3 recombinant protein has single band (see figure 4) behind the His affinity purification about molecular weight 25 kDa, conforms to desired value, and it is 0.12 mg/mL that the Bradford method is measured protein concentration.
1. the foundation of hybridoma cell strain
(1) mouse immune
The GPC3 recombinant protein of getting purifying mixes with 250 μ L Bentonite adjuvants; Amount subcutaneous abdomen multi-point injection BALB/c mouse with 100 μ g/500 μ L; Three weeks began from immunity for the third time with the amount subcutaneous abdomen multi-point injection BALB/c mouse of 100 μ g/500 μ L at interval, and second all tail veins after each immunity are gathered mouse blood; The indirect ELISA method detects serum titer and reaches the preparation fusion of the above back of 1:50000; Merged preceding 3 days, the tail vein injection booster immunization once, antigen dose 100 μ g.
(2) immune serum titration
Adopting indirect elisa method to measure immune serum tires.Get 30 μ gGPC3 recombinant proteins and be dissolved in 10 mL, 0.05 M pH9.6 carbonate buffer solution, encapsulate little 96 orifice plates of PS, 100 μ L/ holes, 4 ℃ are spent the night.Next day; Use PBS (containing 0.1% (V/V) Tween-20) to wash plate three times; Contain 10% NBCS confining liquid, 200 μ L/ holes with 10 mM PBS, 37 ℃ of sealing 1 h use PBS (containing 0.1% (V/V) Tween-20) to wash plate three times; Mouse was in immune back 15 days for the third time tail vein bloods, and the mouse immune serum is with containing 2% NBCS, 10 mM PBS with 10
-1～10
-8Doubly dilution adds 96 orifice plates, 37 ℃ of 1 h in 100 μ L/ holes; After PBS (containing 0.1% (V/V) Tween-20) washes plate three times, add 1:10000 doubly dilute the horseradish peroxidase-labeled goat anti-mouse igg (Sigma, INC.); 37 ℃ of 1 h in 100 μ L/ holes, the same wash plate after, TMB colour developing; 100 μ L/ holes, room temperature lucifuge 20 min add 50 μ L/ hole 2M H
2SO
2Termination reaction is surveyed 450 nm absorption values,, must compare>=2.1 with measured value and control value and positively judge tiring of immune serum as negative control with mice serum before the immunity.
(3) preparation of hybridoma
Get serum titer greater than 1:10
4Mouse, merged preceding 3 days, get recombinant antigen and isopyknic PBS mixing after, carry out booster immunization with the amount abdominal injection BALB/c mouse to be merged of every 100 μ g/500 μ L.The aseptic mouse spleen of getting is processed the mixed of the murine myeloma cell strain SP20 of splenocyte suspension and logarithmic phase by 10:1, centrifugal 5 min of 500 * g room temperature; Abandon supernatant, flick the centrifuge tube bottom, make deposition loose with finger; Centrifuge tube places 37 ℃ of water-baths, will be at 50% polyoxyethylene glycol (PEG, the MW4000 of 37 ℃ of water bath heat preservations; Sigma company) with in the one after another drop of adding centrifuge tube of dropper, shakes centrifuge tube while dripping, drip off in 1 min; Leave standstill 2 min after dripping off, serum-free 1640 substratum 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 10 mL that whenever added 37 ℃ of preheatings at a distance from 1 minute stop the effect of polyoxyethylene glycol, centrifugal 5 min of cell mixture 500 * g room temperature; Abandon supernatant, add HAT nutrient solution (xanthoglobulin (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma company)) re-suspended cell gently; Cell is divided to 96 orifice plates every hole 200 μ L.Cultivate after three days, observation of cell merges situation, changes half HAT nutrient solution; The continuous a few days; Until there being the clone to form, changing HT nutrient solution (xanthoglobulin (H) and thymidine (T) (HT, Sigma company)) and cultivated three days; Through the indirect ELISA method filter out can with the hybridoma (ratio with negative serum A 450 is judged to the positive greater than 2.1) of GPC3 recombinant protein reaction, change 1640 perfect mediums.
(4) hybridoma of the anti-people GPC3 monoclonal antibody of screening secretion
Indirect elisa method screening cells and supernatant; The selection high positive clone hybridization oncocyte of tiring carries out subcloning; And,, obtain 13 strains of stably excreting resisting GPC 3 cell strain of monoclonal antibody at last until to 100% cell positive rate with the continuous cloning of limiting dilution assay 2 ~ 3 times.Use time-resolved method and match antibody screening, hybridoma cell strain 8G6 antibody response fluorescent value is the highest, positive rate after the cloning is reached 100% cell amplification and cultivates the back liquid nitrogen cryopreservation.
(5) preparation of ascites and purifying
With hybridoma cell strain 8G6 with 1 * 10
6/ amount is only injected the whiteruss BALB/c female mice abdominal cavity in pretreated 8 ~ 10 ages in week, and breeding observing extracts ascites when mouse web portion expands after 6 ~ 7 days.Gather mouse ascites after 7 ~ 10 days, 12000 rpm, centrifugal 10 min collect supernatant; Carry out purifying with ProteinGharose (available from GE company): the ProteinG filler is loaded in the purification column; Purification column is connected with Akta purifying appearance, with level pad (provide to specifications method preparation) 5 column volumes of balance, ascites to be purified with after 10 times of the level pad dilutions with appearance on the 1.5 mL/min speed; Last appearance back is washed till baseline with level pad; With elution buffer wash-out antibody, collect the antibody peak, the antibody of wash-out is used Tris-HCl (pH9.0) neutralization immediately.The Bradford method is measured AC.Antibody purification is in-20 ℃ of preservations.
(6) CHARACTERISTICS IDENTIFICATION of monoclonal antibody
The identified by immunofluorescence of antibody: GPC3 expresses male liver cancer cell Huh7 (available from Chinese Academy of Sciences's Shanghai cell bank) after cultivation for some time; Collecting cell after the trysinization is laid on grow overnight on the sheet glass, the PBS washed cell; Use the Paraformaldehyde 96 fixed cell of precooling; Add the resisting GPC 3 monoclonal antibody 8G6 (1:10 dilution) of hybridoma cell strain 8G6 preparation respectively, hatch 1h for 37 ℃, with the negative contrast of PBS.1:1000 added fluorescent mark sheep anti mouse two anti-(Sigma companies) after PBS developed a film, and hatched 1h for 37 ℃, and after PBS developed a film, fluorescent microscope is observed down, all observes green fluorescence through the painted cell of resisting GPC 3 monoclonal antibody 8G6, and was as shown in Figure 5.The result proves that the resisting GPC 3 monoclonal antibody 8G6 of hybridoma 8G6 preparation can discern natural people GPC3 albumen.
SEQUENCE?LISTING
< 110>Nanfang Medical Univ
< 120>GPC3 monoclonal antibody hybridoma cell strain 8G6
<130>
<160> 2
<170> PatentIn?version?3.3
<210> 1
<211> 27
<212> DNA
< 213>artificial sequence
<400> 1
taatgaattc?atgcagcccc?cgccgcc 27
<210> 2
<211> 33
<212> DNA
< 213>artificial sequence
<400> 2
agcggtcgac?tcacttggca?tggcgaacaa?caa 33
Claims (7)
1.GPC3 monoclonal antibody hybridoma cell strain 8G6 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 04th, 2011, deposit number is CGMCC No.5427.
2. the said GPC3 monoclonal antibody hybridoma cell of claim 1 strain 8G6 excretory monoclonal antibody.
3. the preparation method of the said GPC3 monoclonal antibody hybridoma cell of claim 1 strain 8G6, it is characterized in that step is following: with GPC3 albumen is the immunogen immune BALB/c mouse, gets serum titer at 1:10
4Above mouse boosting cell and SP2/0 myeloma cell are merged; With the HAT RPMI-1640 screening of medium fused cell that contains the 20wt% calf serum; Encapsulate elisa plate with GPC3 albumen, indirect elisa method screening fused cell culture supernatant liquid, the highest hybridoma cell strain of selecting to tire carries out subcloning; Limiting dilution assay is cloned into 100% cell positive rate repeatedly, obtains GPC3 monoclonal antibody hybridoma cell strain 8G6 at last.
4. according to the preparation method of the said GPC3 monoclonal antibody hybridoma cell of claim 3 strain 8G6; It is characterized in that said GPC3 albumen is recombinant protein; Concrete preparation method is: with sequence shown in SEQ ID NO:1 ~ 2 is the upstream and downstream primer; CDNA with the reverse transcription of Huh7 cell total rna is a template, PCR method amplification GPC3 gene, and amplified production is used
EcoThe R I with
SalI is carried out double digestion, and enzyme is cut product and is connected with the carrier PET-11b of the same double digestion of process, obtains recombinant vectors GPC3/PET-11b, transformed into escherichia coli, IPTG abduction delivering, the purified GPC3 recombinant protein that obtains of expression product.
5. according to the preparation method of the said GPC3 monoclonal antibody hybridoma cell of claim 3 strain 8G6, it is characterized in that mouse boosting cell and SP2/0 myeloma cell merge with 50 wt % PEG-4000.
6. the application of the said GPC3 monoclonal antibody hybridoma cell of claim 2 strain 8G6 excretory monoclonal antibody in detecting the GPC3 protein expression.
7. a GPC3 immunity detection reagent is characterized in that comprising the said GPC3 monoclonal antibody hybridoma cell of claim 2 strain 8G6 excretory monoclonal antibody.
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| CN111040999A (en) * | 2019-12-31 | 2020-04-21 | 南京拂晓生物科技有限公司 | Recombinant CHO cell strain for stably expressing GPC3 and application thereof |
| CN111175520A (en) * | 2020-01-15 | 2020-05-19 | 河北省科学院生物研究所 | Glyphican-3 double-antibody sandwich method detection kit and detection method |
| CN111378628A (en) * | 2020-04-08 | 2020-07-07 | 扬州大学 | Hybridoma cell strain secreting mycobacterium tuberculosis ESAT6 protein specific antibody, antibody and application thereof |
| CN112608907A (en) * | 2020-12-18 | 2021-04-06 | 十堰市太和医院（湖北医药学院附属医院） | Phosphatidylinositolglycan 3 monoclonal antibody, hybridoma cell strain and application |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104610441A (en) * | 2015-03-04 | 2015-05-13 | 首都医科大学 | Artificial hapten used for preparing glypican-3 (GPC3) monoclonal antibody, preparation method and obtained monoclonal antibody |
| CN104610441B (en) * | 2015-03-04 | 2018-02-13 | 首都医科大学 | For preparing artificial semiantigen, preparation method and the monoclonal antibody of acquisition of glypican-3 (GPC3) monoclonal antibody |
| CN111040999A (en) * | 2019-12-31 | 2020-04-21 | 南京拂晓生物科技有限公司 | Recombinant CHO cell strain for stably expressing GPC3 and application thereof |
| CN111040999B (en) * | 2019-12-31 | 2022-05-06 | 南京拂晓生物科技有限公司 | Recombinant CHO cell strain for stably expressing GPC3 and application thereof |
| CN111175520A (en) * | 2020-01-15 | 2020-05-19 | 河北省科学院生物研究所 | Glyphican-3 double-antibody sandwich method detection kit and detection method |
| CN111378628A (en) * | 2020-04-08 | 2020-07-07 | 扬州大学 | Hybridoma cell strain secreting mycobacterium tuberculosis ESAT6 protein specific antibody, antibody and application thereof |
| CN111378628B (en) * | 2020-04-08 | 2021-07-23 | 扬州大学 | Hybridoma cell strain secreting mycobacterium tuberculosis ESAT6 protein specific antibody, antibody and application thereof |
| CN112608907A (en) * | 2020-12-18 | 2021-04-06 | 十堰市太和医院（湖北医药学院附属医院） | Phosphatidylinositolglycan 3 monoclonal antibody, hybridoma cell strain and application |
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