CN101633693A - Monoclonal antibody for resisting GPC3 - Google Patents
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Abstract
The invention discloses a monoclonal antibody of GPC3, which is generated by the secretion of a human GPC3 resistant monoclonal antibody hybrid tumor cell strain with the preservation number of CGMCC No.3232. The monoclonal antibody can be used for an immunohistochemical test of hepatocellular carcinoma.
Description
Technical field
The invention belongs to field of biomedicine technology.Specifically, the present invention relates to monoclonal antibody and a kind of test kit that is used for the detection of hepatocellular carcinoma immunohistochemical methods of a kind of anti-glypican albumen 3 (GPC3).
Background technology
Primary hepatocarcinoma (HCC) is one of modal malignant tumour, and worldwide its sickness rate is in rising trend at present.The annual new cases 50 of global in recent years liver cancer～600,000 wherein about 50% occur in China.Because concealment of its morbidity, atypical symptoms is difficult to early diagnosis, and most of liver cancer has been middle and advanced stage when finding, has not only missed the best period of treatment, and more owing to lack special therapeutic modality and high recurrent, its prognosis is not good.At present, liver cancer occupies second of mortality of malignant tumors in China.
At present, diagnostic imaging, pathological diagnosis and chemical diagnosis are three big pillars of diagnosing tumor.The pathological diagnosis of liver cancer is the basis of understanding the cause of disease, mechanism, hepatic pathology variation, type, patient's clinical treatment and the Practical Research of hepatopathy for the clinicist.The tissue-derived complexity of liver primary tumor, various optimum, malignant tumours surpass 100 kinds, and there are certain difficulty in pathological diagnosis and differential diagnosis.Hepatocellular carcinoma (HCC) is a difficult problem that often runs in the surgery pathological diagnosis with the differential diagnosis of stones in intrahepatic bile duct cancer (ICC) and adenocarcinoma metastatic (MAC), although most of cases can be made correct diagnosis by clinical characters, conventional H E dyeing, immunohistochemical methods has very important value when atypical symptoms and pathology are uncertain.
Immunohistochemistry (Immuno-histochemistry, immunohistochemical methods) technology is indispensable, a most important supplementary means in the hepatic pathology diagnosis.Yet alternative liver cell specific antibody kind is few in the immunohistochemical experiment.The widely used diagnosis of hepatoma mark of clinical pathology mainly is Hep Par-1, CD34, pCEA, AFP etc. at present, but its specificity and susceptibility have certain limitation, bring very big inconvenience [Wee A..ApplImmunohistochem Mol Morphol.2006 for the pathological diagnosis and the differential diagnosis of liver cancer; 14:266-272].Therefore need new tumor markers, set up many indexs joint-detection system, remedy the deficiency of single index.
Glypican albumen 3 (GPC3) assignment of genes gene mapping is in human chromosome Xq26, and its cDNA sequence total length is 2263bp, is made of the Suleparoid proteinoid glycan that coding is made up of 580 amino acid 8 exons.The molecular weight of GPC3 is 60-70kDa, and (Glycosyl-phosphatidylinositol, GPI) anchor is connected in cytolemma, belongs to proteoglycan family by glycosyl-phosphatidyl inositol.
Existing experimental study shows, GPC3 mRNA and GPC3 albumen specificity overexpression in liver cancer, with the generation development of liver cancer very substantial connection [Zhu ZW, Friess H, Wang L are arranged, Abou-Shady M, Zimmermann A, Lander AD, Korc M, Kleeff J, B ü chler MW.Gut 2001; 48:558-64].Existing research and bibliographical information all show, the generation development of GPC3 and liver cancer has substantial connection, not only liver cancer higher recall rate arranged in early days, and along with progression of HCC, its recall rate also increases thereupon.By hepatocarcinoma patient is studied, we detect has GPC3 to express in 76.7% (23/30) liver cancer tissue, and wherein there is the weak positive expression of GPC3 in 13.3% (4/30) cancer beside organism; And GPC3 expresses negative patient in the other 7 routine liver cancer tissues, and the GPC3 of its cancer beside organism also expresses feminine gender.Importantly, in the liver cancer tissue of Serum AFP positive patients, have 88% to express GPC3mRNA, and can find still that in patient's liver cancer of Serum AFP feminine gender 73% is the GPC3mRNA expression positive.In diameter＜3cm liver cancer, the GPC3mRNA positive expression rate is 77%, and this is significantly higher than AFPmRNA expression rate 41%[Zhou Xueping in serum afp rate of rise 43% and the liver cancer, Wang Hongyang, Chinese surgical magazine, 1999; 37 (3): 171-173].In adenoma of liver, cholangiocellular carcinoma, metastatic liver cancer and 12 kinds of common solid tumors and 21 non-liver cancer clone, all not detecting GPC3 expresses, show that GPC3 is the gene of specifically expressing in the liver cancer, has higher recall rate in the liver cancer patient of AFP feminine gender.Therefore, GPC3 is a new liver cancer marker, the very potential detection that is used for liver cancer.
Summary of the invention
Based on above-mentioned discovery, primary and foremost purpose of the present invention is to provide a kind of monoclonal antibody that can be used for the resisting GPC 3 of hepatocellular carcinoma immunohistochemical methods detection.
Another object of the present invention is to provide a kind of test kit that the hepatocellular carcinoma immunohistochemical methods detects that is used for.
For achieving the above object, the present invention adopts following technique means:
A kind of monoclonal antibody of resisting GPC 3, it is the anti-people GPC3 monoclonal antibody hybridoma cell strain secretion generation of CGMCC No.3232 by deposit number.
Said monoclonal antibody prepares according to following steps: inoculation hybridoma CGMCC No.3232 in the mouse body, produce ascites antibody, and take out ascites and carry out purifying, obtain the monoclonal antibody of resisting GPC 3; Perhaps vitro culture hybridoma CGMCCNo.3232, the separation and purification nutrient solution obtains the monoclonal antibody of resisting GPC 3.
A kind of test kit that is used to implement above-mentioned application, it comprises:
(1) monoclonal antibody of above-mentioned resisting GPC 3; And
(2) when GPC3 is incorporated into described monoclonal antibody, can be incorporated into the traget antibody of GPC3.
Studies show that, monoclonal antibody of the present invention is the immunoglobulin (Ig) of IgG type, can be incorporated into GPC3 albumen specifically, therefore it can be used for the detection of hepatocellular carcinoma immunohistochemical methods, sensitivity, specificity and the accuracy rate of diagnosing cancer of liver can be effectively improved, and prognosis evaluation, result of treatment assessment and the course of disease monitoring of liver cancer can be extended to.
Description of drawings
Fig. 1 is the SAS-PAGE electrophoresis photo of the GPC3 fusion rotein of intestinal bacteria XL1 Blue expression.Wherein left side and intermediate strap are the GPC3 fusion rotein behind the purifying; Right side band (M) is molecular weight standard thing Marker.
Fig. 2 is the qualification result of monoclonal antibody Ig class and subclass.We obtain a large amount of hybridoma supernatants by the cell in vitro cultured method, (HyCult biotechnology bv MouseMonoclonal Antibody Isotyping Kit P/N:HL2010) carries out Ig class and subgroup identification to adopt mouse monoclonal antibody Ig class and subclass detection kit.The result shows (see figure 2), and 6 kinds of monoclonal antibody Z1C15 heavy chains are the IgG2b type, and light chain is the κ chain.
Fig. 3 is the detection photo that carries out Western Blot experiment behind 293T cell transient transfection GPC3 and the empty carrier plasmid 48h.The left side is GPC3 albumen (a 293T cell transient transfection GPC3 plasmid lysate), and the right side is blank (a 293T cell transient transfection empty carrier pcDNA3.1A plasmid lysate).Experiment condition is: the sex change sample carries out the SDS-PAGE protein electrophoresis; By electroporation albumen is transferred to nitrocellulose filter (Schleicher and Schcell company); 5% skim-milk sealing 1h, TBST gives a baby a bath on the third day after its birth inferior, each 5min; Z1C15 one is anti-for 5%BSA dilution resisting GPC 3 monoclonal antibody, hatches 2h, and TBST gives a baby a bath on the third day after its birth inferior, each 5min; 1h is hatched in 5% skim-milk dilution goat-anti mouse, two anti-(Hua Shun companies), and TBST gives a baby a bath on the third day after its birth inferior, each 5-10min; Chemoluminescence method (ECL) develops.
Fig. 4 is the detection photo that Huh7, PLC, SMMC7721, Hep3B, L02 and Hela cell pyrolysis liquid is carried out Western Blot experiment.Experiment condition is: the sex change sample carries out the SDS-PAGE protein electrophoresis; By electroporation albumen is transferred to nitrocellulose filter (Schleicher and Schcell company); 5% skim-milk sealing 1h, TBST gives a baby a bath on the third day after its birth inferior, each 5min; Z1C15 one is anti-for 5%BSA dilution resisting GPC 3 monoclonal antibody, hatches 2h, and TBST gives a baby a bath on the third day after its birth inferior, each 5min; 1h is hatched in 5% skim-milk dilution goat-anti mouse, two anti-(Hua Shun companies), and TBST gives a baby a bath on the third day after its birth inferior, each 5-10min; Chemoluminescence method (ECL) develops.
The microbial preservation explanation
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC);
Preservation address: Da Tun road, Beijing Institute of Microorganism, Academia Sinica;
Preservation date: on August 13rd, 2009;
Deposit number: CGMCC No.3232;
Classification name: anti-people GPC3 monoclonal antibody hybridoma cell strain;
The biomaterial of ginseng certificate: Z1C15.
Embodiment
The design philosophy that the invention provides the monoclonal antibody of resisting GPC 3 is to prepare monoclonal antibody according to the method that may further comprise the steps:
A) abduction delivering people GPC3 albumen or its fragment in intestinal bacteria obtain the GPC3 fusion rotein;
B) be the immunogen immune mouse with purified GPC3 fusion rotein;
C) get the splenocyte of immunized mice, merge with murine myeloma cell;
D) filter out GPC3 albumen test positive cells clone, as the hybridoma cell strain of secretion resisting GPC 3 protein monoclonal antibody; And
E) produce ascites antibody by inoculation hybridoma in the mouse body, take out ascites and carry out purifying, obtain the monoclonal antibody of resisting GPC 3; Perhaps cultivate separation and purification, the monoclonal antibody of acquisition resisting GPC 3 by cell in vitro.
In embodiments of the present invention, people GPC3 albumen or its fragment can obtain in several ways, such as clone its encoding gene by molecular biology method, in prokaryotic cell prokaryocyte such as intestinal bacteria etc., eukaryotic cell such as yeast, insect cell, vegetable cell or mammalian cell such as CHO etc., express then, obtain through purifying.
For example, for passing through escherichia expression system, people GPC3 albumen or its segmental preparation method can be: with people GPC3 albumen or its fragment coding gene is template, design suitable primer, increase with polymerase chain reaction (PCR), through restriction enzyme such as EcoRI, after the XhoI enzyme is cut, enzyme is cut product to be connected such as the T carrier with prokaryotic expression carrier, change in the intestinal bacteria again and cultivate, extraction contains the recombinant expression plasmid of people GPC3 albumen or its fragment coding gene, change abduction delivering in the intestinal bacteria then over to, can obtain the GPC3 fusion rotein through purifying.
The hybridoma cell strain that is used to secrete the monoclonal antibody of resisting GPC 3 can adopt the method preparation of present technique field routine.Such as, the splenocyte of the mice immunized of learning from else's experience or rat merges such as the SP2/0 cell with mouse or rat myeloma cell, can obtain hybridoma after screening.
The acquisition of monoclonal antibody can be adopted the method for present technique field routine.A kind of method is, by Mammals such as the mouse body in the inoculation hybridoma produce ascites antibody, take out ascites and carry out purifying and obtain.Another kind method is to obtain by the vitro culture hybridoma.
The present invention is that the prepared monoclonal antibody of Z1C15 is a kind of immunoglobulin (Ig) by mouse hybridoma cell, but its specificity is incorporated into the GPC3 albumen of GPC3 albumen, especially solubility.
Through identifying that the prepared immunoglobulin (Ig) of the present invention is the monoclonal antibody of IgG type.Antibody ascites tires＞and 105.
Hepatocellular carcinoma immunologic combined detection reagent kit of the present invention comprises at least:
(1) monoclonal antibody of resisting GPC 3; And
(2) when GPC3 is incorporated into described monoclonal antibody, can be incorporated into the traget antibody of GPC3.
Preferred mentioned reagent box also can comprise:
(3) by the standard of the solution composition that contains known quantity GPC3; And
(4) be used to the antibody labeling thing that detects, it can combine with the monoclonal antibody of resisting GPC 3 and form conjugate.
More have select test kit also can further comprise in subordinate's article one of at least: (5) carry instrument, its spatial division is for accommodating the restriceted envelope of one or more containers, 96 orifice plates or lath, this container for example is medicine bottle, test tube and analogue, and every sample container all contains an independent component that is used for the inventive method; (6) auxiliary reagent, such as, developer, enzyme inhibitors, damping fluid, stablizer, thinner, washing reagent and analogue; (7) specification sheets, it can write on bottle, test tube and the analogue, perhaps writes on the independent paper, perhaps outside or inner at container; Also can be multimedia form, such as CD, compact disk, video recording or the like.
Preferred antibody can be fixed on the solid phase carrier, forms capture antibody.
Undoubtedly, monoclonal antibody of the present invention and related kit can be applied to prognosis evaluation, result of treatment assessment and the course of disease monitoring of liver cancer.
Used herein term " monoclonal antibody " is identical with the implication of " monoclonal antibody ", and the two is interchangeable; Term " Z1C15 monoclonal antibody " is identical with the implication of " monoclonal antibody of resisting GPC 3 ", and the two is interchangeable; Term " Z1C15 hybridoma " is identical with " hybridoma Z1C15 cell " or the implication of " Z1C15 cell ", is used interchangeably.
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only to be used to the present invention is described and be not used in the scope of the present invention that limits.
The expression of people GPC3 protein fragments
1.1GPC3-N end group is because of segmental amplification
From people's placenta cDNA, cloned the GPC3-N end group of 981bp because of fragment, as amplification template with PCR method.
Design following PCR primer:
Primer 1:
5’-CGGC?GAA?TTC?AA?GCC?ACC?TGT?CAC?CAA?GTC-3’
Primer 2:
5’-CGC?CTC?GAG?TCA?AAA?TCT?ATA?TTG?GCG?TTG-3’
The PCR reaction system:
The KOD enzyme, 1 μ l; KOD cushions night, 5 μ l; The MgCl of 25mmol/L
2, 3 μ l; The P1 of 10 μ mol/L, 1 μ l; The P2 of 10 μ mol/L, 1 μ l; DNTP, 5 μ l; Template, 0.5 μ l; DdH
2O, 34.5 μ l; Totally 50 μ l.
Pre-sex change 4min under 94 ℃; 94 ℃ of sex change 30sec then, 57 ℃ of annealing 30sec, 68 ℃ of chain extension 60sec, 30 circulations.
Reclaim the PCR product of test kit (Qiagen Gel Extraction Kit) purifying gained with gel.With reference to " molecular cloning " J. Sa nurse Brooker, D.W. Russell (U.S.) writes, Huang Peitang etc. translate, Science Press, Beijing, 2002, the method of describing in the 68th page, connect in T carrier (Invitrogen company), with EcoRI and two kinds of restriction enzymes of XhoI (Promega company) digestion purpose fragment and empty carrier pPROEX HTb (Invitrogen company), with Qiagen GelExtraction Kit respectively glue reclaim two fragments.Under 22 ℃, connect purpose fragment and prokaryotic expression pPROEX HTb carrier (Invitrogen company), 10～12 hours with T4DNA ligase enzyme (Invitrogen company).
With the carrier transformed into escherichia coli XL1 Blue (Invitrogen company) that obtains, amplification, with reference to " molecular cloning " J. Sa nurse Brooker, D.W. Russell (U.S.) writes, and Huang Peitang etc. translate, Science Press, Beijing, the method of describing in 2002, the 27 pages extracts plasmid and identifies with corresponding restriction enzyme, proves conclusively by dna sequencing.
The segmental sequencing result of the purpose of cloning be SEQ ID NO 1.The GPC3-N end group is because of the 242nd to 1222 dna sequence dna among its behaviour placenta cDNA.
1.2 abduction delivering GPC3 fusion rotein
With reference to " molecular cloning " J. Sa nurse Brooker, D.W. Russell (U.S.) writes, and Huang Peitang etc. translate, Science Press, and Beijing, the method for describing in 2002, the 97 pages is passed through CaCl with the above-mentioned plasmid that obtains
2Method changes among the intestinal bacteria XL1Blue (Invitrogen company), cultivates 5 hours down in 37 ℃ in the LB substratum.Behind the IPTG abduction delivering, with Ni-NTA agarose affinity chromatography method purifying GPC3 fusion rotein.
1.3SAS-PAGE electrophoretic analysis
For the GPC3 fusion rotein behind the purifying, carry out 10%SDS-PAGE and detect.The result as shown in Figure 1.
As seen from Figure 1, the molecular weight of GPC3 fusion rotein is about 32.5kDa.
Embodiment 2
The preparation of Z1C15 monoclonal antibody and purifying
2.1 with GPC3 fusion protein immunization mouse
With immune 5-6 week female Bab/c mouse in age (The 2nd Army Medical College Experimental Animal Center) behind the GPC3 albumen of purifying among the embodiment 1 and good fortune formula adjuvant (CFA) mixing fully, intracutaneous multi-point injection, 100 μ g/ are only.4 week back booster immunization intraperitoneal injections for the first time, 100 μ g/ only.6 week back booster immunization intraperitoneal injections for the second time, 100 μ g/ only.Immunity is got immunity back mouse tail hematometry the 8th week respectively and is tired, and uses GPC3 antigen 3 μ g/ml bag quilt, the 10%FCS sealing, serum dilution back is added, use goat anti-mouse igg mark HRP as two antibody, the OPD colour developing uses microplate reader (Bio-RAD 550 types) at OD
492Following reading.
2.2 the hybridoma cell strain of secretion resisting GPC 3 protein antibodies is set up, screening
In immune mouse, prepare murine myeloma cell SP2/0 cell.Preceding 3 days of cytogamy, booster immunization, the direct abdominal injection of antigen, 100 μ g/ are only.
Get mouse boosting cell,, after carrying out fusion reaction under the PEG1500 effect, implant 96 well culture plates respectively, 37 ℃, 5%CO with SP2/0 cell proportion 1: 10
2Cultivate under the condition.Cultivate after 14 days, mirror detects down and grows the clone cell hole for merging positive hole, calculates total fusion rate＞95%.Select the supernatant in monoclonal cell hole to detect under the mirror as far as possible; Picking OD
492The porocyte of value＞0.8 carries out limited subclone, the final cell clone that obtains GPC3 albumen test positive rate＞99%, and as the hybridoma cell strain of secretion resisting GPC 3 protein monoclonal antibody, it is a hybridoma Z1C15 cell.
With the positive hybridoma Z1C15 cell clone cultivation that obtains, cultivate through the cloning of 2-3 wheel, obtain stable can produce height the tire hybridoma cell clone of monoclonal antibody and frozen guarantor's kind.
Submit this hybridoma to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, deposit number is CGMCC No.3232.
2.3Z1C15 the preparation of monoclonal antibody and purifying
Contain the ascites preparation of antibody: after 6-8 age in week, female Bab/c mouse was handled 7-10 days with paraffin oil, get hybridoma CGMCC No.3232 with 2 * 10
6Individual cell/only carry out abdominal injection.From mouse peritoneal, obtain the ascites that is rich in antibody after 7-14 days, measure ascites to tire＞10
5
The purifying of Z1C15 monoclonal antibody: adopt protein G affinity chromatography.At first prepare protein G affinity column, behind PBS balance pillar, get ascites and cross post, clean pillar with PBS then, approach zero to the OD value, with the glycine hydrochloride eluant solution of 50nmol/L, collect elutriant, measure the OD value of each collection tube, keep the elutriant of peak region, elutriant is collected after dialysing.
The evaluation of Z1C15 monoclonal antibody
3.1Z1C15 cells in vitro is cultivated
With the Z1C15 hybridoma CGMCC No.3232 of preparation among the embodiment 2, carry out cell in vitro and cultivate (DMEM, 10%FBS substratum, 37 ℃, 5%CO
2Cultivate in the incubator), obtain a large amount of hybridoma supernatants.
3.2 the monoclonal antibody type is identified
(HyCult biotechnology bv MouseMonoclonal Antibody Isotyping Kit P/N:HL2010) carries out Ig class and subgroup identification, and qualification result is seen Fig. 2 to adopt mouse monoclonal antibody Ig class and subclass detection kit.Studies show that the monoclonal antibody heavy chain is the IgG2b type, light chain is the κ chain.
3.3 monoclonal antibody is measured the proteic affinity of GPC3
Can the evaluation monoclonal antibody discern the GPC3 albumen of external source.With reference to " molecular cloning " J. Sa nurse Brooker, D.W. Russell (U.S.) writes, Huang Peitang etc. translate, Science Press, Beijing, 2002, the method of describing in the 1282nd page, with GPC3 protein coding gene and empty carrier plasmid to 293T cell (Shanghai cell institute of the Chinese Academy of Sciences) transient transfection 48h after, collecting cell carries out Western Blot experiment.Detected result as shown in Figure 3.
The result shows that yet visible molecular weight GPC3 core protein and glycosylated protein band but do not occur in the lysate of 293T cell transfecting empty carrier plasmid pcDNA3.1A in the 293T cell transient transfection GPC3 plasmid lysate.As seen, the monoclonal anti physical efficiency identifies endogenous GPC3 albumen under the native state.GPC3 is represented among the figure be 293T cell transient transfection GPC3 plasmid lysate, EF represented be the lysate of 293T cell transfecting empty carrier plasmid pcDNA3.1A.
Simultaneously, we have collected Huh7, PLC, and SMMC7721, Hep3B, L02 and Hela cell pyrolysis liquid carry out WesternBlot and detect, and detected result is as shown in Figure 4.
The result shows: the albumen of an also visible about 60kDa of molecular weight in hepatoma cell line HuH7, the Hep3B lysate, hepatoma cell line SMMC7721, normal liver cell be L02 and non-liver cancer clone Hela band then do not appear.The instruction book clonal antibody can detect GPC3 albumen in liver cancer cell, all can not detect GPC3 albumen in normal liver cell and non-liver cancer cell, and this shows that monoclonal antibody can be specifically in conjunction with endogenous GPC3 albumen.
Embodiment 4
Monoclonal antibody with resisting GPC 3 detects liver cancer
Hospital of liver and gall surgical department takes 114 routine 22-73 year cancer experimental subjects (94 routine hepatocellular carcinomas at random in the Orient, 20 routine cholangiocellular carcinomas) hepatic tissue, 19 routine normal liver tissues, 42 routine liver innocent tumours (focal hyperplasia 22 examples of liver, adenoma of liver 20 examples) sample, use the Z1C15 monoclonal antibody of preparation among the embodiment 2, reference literature [Fromowitz FB, Viola MV, Chao S, Oravez S, Mishriki Y, Finkel G, Grimson R, Lundy J.Hum Pathol.1987 (18): 1268-1275] the middle method of describing, detect GPC3 protein expression level in the tissue by immunohistochemical method.
Studies show that, normal liver tissue is not expressed GPC3 albumen, all do not express GPC3 albumen in the benign tumor tissue (adenoma of liver, the focal hyperplasia of liver), all do not express GPC3 albumen in the Hepatocholangiocarcinoma tissue, the GPC3 positive rate reaches 79.8% in the hepatocellular carcinoma tissue, the pathology immunohistochemical methods that this explanation resisting GPC 3 monoclonal antibody can be used for hepatocellular carcinoma detects, and has good specificity and susceptibility.
More than with embodiment technical scheme of the present invention is set forth, the present invention undoubtedly can extend to diagnosis, prognosis evaluation, result of treatment monitoring and the PD monitoring of liver cancer, this is conspicuous for a person skilled in the art.Therefore, under thought of the present invention, various changes or modification that those skilled in the art can make the present invention on this basis should belong to scope of the present invention equally.
Sequence table
＜110〉Second Military Medical University, PLA
＜120〉a kind of monoclonal antibody of resisting GPC 3
<130>061588JN
<141>2009-08-21
<160>1
<170>PatentIn?version?3.3
<210>1
<211>981
<212>DNA
＜213〉among people's placenta cDNA the GPC3-N end group because of the 242-1222 position
<220>
<221>gene
<222>(1)..(981)
<400>1
gccacctgtc?accaagtccg?ctccttcttc?cagagactgc?agcccggact?caagtgggtg 60
ccagaaactc?ccgtgccagg?atcagatttg?caagtatgtc?tccctaaggg?cccaacatgc 120
tgctcaagaa?agatggaaga?aaaataccaa?ctaacagcac?gattgaacat?ggaacagctg 180
cttcagtctg?caagtatgga?gctcaagttc?ttaattattc?agaatgctgc?ggttttccaa 240
gaggcctttg?aaattgttgt?tcgccatgcc?aagaactaca?ccaatgccat?gttcaagaac 300
aactacccaa?gcctgactcc?acaagctttt?gagtttgtgg?gtgaattttt?cacagatgtg 360
tctctctaca?tcttgggttc?tgacatcaat?gtagatgaca?tggtcaatga?attgtttgac 420
agcctgtttc?cagtcatcta?tacccagcta?atgaacccag?gcctgcctga?ttcagccttg 480
gacatcaatg?agtgcctccg?aggagcaaga?cgtgacctga?aagtatttgg?gaatttcccc 540
aagcttatta?tgacccaggt?ttccaagtca?ctgcaagtca?ctaggatctt?ccttcaggct 600
ctgaatcttg?gaattgaagt?gatcaacaca?actgatcacc?tgaagttcag?taaggactgt 660
ggccgaatgc?tcaccagaat?gtggtactgc?tcttactgcc?agggactgat?gatggttaaa 720
ccctgtggcg?gttactgcaa?tgtggtcatg?caaggctgta?tggcaggtgt?ggtggagatt 780
gacaagtact?ggagagaata?cattctgtcc?cttgaagaac?ttgtgaatgg?catgtacaga 840
atctatgaca?tggagaacgt?actgcttggt?ctcttttcaa?caatccatga?ttctatccag 900
tatgtccaga?agaatgcagg?aaagctgacc?accactattg?gcaagttatg?tgcccattct 960
caacaacgcc?aatatagatt?t 981
Claims (6)
1. the monoclonal antibody of a resisting GPC 3, it is that the anti-people GPC3 monoclonal antibody hybridoma cell strain secretion of CGMCC No.3232 produces by deposit number.
2. monoclonal antibody as claimed in claim 1 is characterized in that it prepares according to following steps:
Inoculation hybridoma CGMCC No.3232 produces ascites antibody in the mouse body, takes out ascites and carries out purifying, obtains the monoclonal antibody of resisting GPC 3; Perhaps
Vitro culture hybridoma CGMCC No.3232, separation and purification nutrient solution, the monoclonal antibody of acquisition resisting GPC 3.
3. monoclonal antibody as claimed in claim 1 or 2 is characterized in that, it is an immunoglobulin (Ig).
4. monoclonal antibody as claimed in claim 3 is characterized in that, it is the IgG type.
5. one kind is used for the test kit that the hepatocellular carcinoma immunohistochemical methods detects, and it comprises:
(1) as each described monoclonal antibody in the claim 1～4; And
(2) when GPC3 is incorporated into described monoclonal antibody, can be incorporated into the traget antibody of GPC3.
6. test kit as claimed in claim 5 is characterized in that, also comprises:
(3) by the standard of the solution composition that contains known quantity GPC3; And
(4) be used to the antibody labeling thing that detects, it can combine with the monoclonal antibody of resisting GPC 3 and form conjugate.
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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 |
| CN106397593A (en) * | 2015-08-03 | 2017-02-15 | 科济生物医药（上海）有限公司 | An anti-glypican-3 antibody and an application thereof |
| CN106589128A (en) * | 2015-10-20 | 2017-04-26 | 天津医科大学 | Preparation and applications of liver cancer marker monoclonal antibodies |
| CN106674350A (en) * | 2015-11-09 | 2017-05-17 | 天津医科大学 | Preparation of single chain antibody of human liver cancer marker and application thereof |
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