CN108341876A - Anti-human CEACAM5 monoclonal antibodies and its preparation method and application - Google Patents
Anti-human CEACAM5 monoclonal antibodies and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3007—Carcino-embryonic Antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention discloses a kind of anti-human CEACAM5 monoclonal antibodies and its preparation method and application, which is CCTCC NO by preserving number:The mouse hybridoma cell system of C2016129 generates.The preparation method is：S1：Use the whole-cell protein that gastric carcinoma cells crack as immunogen immune mouse；S2：The splenocyte that mouse is immunized obtained in step S1 is merged with murine myeloma cell；S3：After cell clone occurs in cell fusion in step S2, the hybridoma cell line of stably excreting antibody is selected；S4：The anti-human CEACAM5 monoclonal antibodies are made from the hybridoma cell line of stably excreting antibody described in step S3.The monoclonal antibody can be used for preparing the composition for the treatment of malignant tumour.Anti-human CEACAM5 monoclonal antibodies provided by the invention such as can be widely applied to flow cytometry, immunohistochemical staining, immunoprecipitate at all kinds of routine operations.
Description
Technical field
The present invention relates to antibody technique fields, and in particular to a kind of anti-human CEACAM5 monoclonal antibodies and preparation method thereof
And application.
Background technology
Malignant tumour is to endanger the major disease of human health, still in research and is explored at present for its treatment means
Stage.After traditional operative treatment, radiotherapy, chemotherapy and immunization therapy, to combine genetic engineering and protein engineering
The emerging research field for just becoming treatment tumour for the targeting antibodies drug therapy of representative, is ground by preclinical medicine and clinical medicine
Study carefully the extensive concern of field researcher.Most important one kind is that the special monoclonal of tumour antigen is anti-wherein in targeted molecular
Body (mAb).
Antibody monomer molecule is to pass through two sulphur of interchain by two identical heavy chains (H chains) and two identical light chains (L chains)
Four peptide chain structure made of key connection.H chains and L chains include aminoterminal (N) and c-terminus (C), close to the variable region (areas V) of N-terminal
It is made of hypervariable region/complementary determining region (HVR/CDR) and skeleton area (FR)；It is constant region (areas C) close to C-terminal.Heavy chain variable region
(VH) and the protein folding of light chain variable region (VL) formation is antigen-binding site, and CDR/HVR therein is antibody and antigen
Determine that the reaction after antigen-antibody identification is caused at the position that base complementation combines, the areas C.Antibody can be divided into people according to the areas FR/C difference
Source, mouse source etc. have immunogenicity, easily cause the immune response of human body when murine antibody uses in human body, these are immune
Reaction can cause the hypersensitivity of removing and immune complex mediation to murine antibody.In order to overcome murine antibody
Defect needs chimeric antibody, single-chain antibody or the humanized antibody of the specificity for building high-affinity.
The incidence of gastric cancer is occupied in China first of malignant tumor of digestive tract, occupies second in the world, year death toll about
The 24% of mortality of malignant tumors number is accounted for, five year survival rate is only about 27%.The diagnosis discovery of patients with gastric cancer often reaches an advanced stage,
Therefore poor prognosis.Effective target therapeutic agent is had no for gastric cancer.
Carcinomebryonic antigen (carcinoembryonic antigen, CEA) is a kind of embrane-associated protein, generally in fetus liver sausage
The tissue expressions such as pancreas.Normal secretions enter enteron aisle, and content is seldom in serum.It is horizontal in serum to increase when cell carcinogenesis, to pancreas
Cancer, colon cancer early diagnosis have Auxiliary Significance, there is certain reference value to the diffusion of tumour, curative effect, recurrence and prognosis.4th
Army medical university Fan awards for penetrating judgment finds that glycosylated CEA is very high to the Sensitivity Specificity of a variety of digestive system cancers.Clinical research
Show to glycosylate positive rates of the CEA in stomach organization 80% or more, in colon cancer tissue positive rate 40% or more,
In gastric precancerous lesion positive rate 30% or more, positive rate is 18% or more in esophageal cancer tissue.Clinical trial results simultaneously
It has been shown that, in 28 postoperative serum of patients with gastric cancer the positive rate of glycosylation CEA than it is preoperative it is apparent under, prompt glycosylation CEA and gastric cancer
There is substantial connection (Gadler et al., Int J Cancer 25 (1) for morbidity:91-4,1980).Carcinomebryonic antigen is related thin
Born of the same parents' adhesion molecule 5 (carcinoembryonic antigen-related cell adhesion molecule 5,
CEACAM5) contactin, the high expression in a variety of epithelial tumours such as gastric cancer, and it is close with angiogenesis
Correlation can effectively activate the breeding of tumour cell, inhibit apoptosis of tumor cells.Therefore, using the mAb of CEACAM5 or
Genetic engineering antibody blocks the signal transduction of CEACAM5 to be expected to provide new strategy for the treatment of a variety of epithelial tumours such as gastric cancer
And means.But although generation and development that CEACAM5 participates in a variety of epithelial tumours such as gastric cancer are had proven at present, especially
The function of CEACAM5 is related to the glycosylation of its height, there is no the glycosylation modified antibody for this albumen, serious shadow at present
The application in terms of diagnosis and treatment of the molecule in the tumours such as gastric cancer is rung.
Invention content
The technical problem to be solved by the present invention is to lack in the prior art for the anti-of glycosylation CEACAM5 to overcome
Body and then lead to not provide a kind of anti-human using the molecule this problem in the diagnosis and treatment of the tumours such as gastric cancer
CEACAM5 monoclonal antibodies and its preparation method and application.Anti-human CEACAM5 monoclonal antibodies provided by the invention can be special
Property there is higher affinity with glycosylated CEACAM5 antigen bindings and with glycosylated CEACAM5, be that gastric cancer etc. is a variety of
The treatment and diagnosis of epithelial tumour provide the strategy and means for being effectively targeted to treatment.
Term used herein " antibody " refers to the heterologous four glycan albumen of about 150KDa, by two identical light chains
(L) it is formed with two identical heavy chains (H).Every light chain is connected by a covalent disulfide bonds with heavy chain, and different antibodies are same
Disulfide bond number between the heavy chain of kind of type is different, the intrachain disulfide bond at each heavy chain and light chain also regular interval.Each heavy chain
One end have variable region (VH), be followed by multiple constant regions.One section of every light chain has variable region (VL), and the other end has constant
Area；The constant region of light chain is opposite with the first of heavy chain constant region, and the variable region of light chain is opposite with the variable region of heavy chain.Special
Amino acid residue forms interface between light chain and the variable region of heavy chain.Light chain and heavy chain variable region also contain there are three hypervariable region,
Also become complementary determining region or CDR.CDR is mainly responsible for be combined with the epitope of antigen.The CDR of every chain be commonly referred to as CDR1,
CDR2 and CDR3, the serial number since N-terminal, and usually identified by the chain where the specific CDR.With different spies
Anisotropic antibody, that is, have the antibody of different binding sites that there is different CDR to different antigen.Native heavy and light chain
Variable region in respectively contain four areas FR, they generally be in b- folding configurations, be connected by three CDR of formation connection ring,
Part b- foldable structures can be formed in some cases.CDR in daily chain by the areas FR be tightly against instrument and with it is another
The antigen-binding site that the CDR of chain together forms antibody [is shown in Kabat etc., NIH Publ.NO.91-3242 volume I, 647-669
Page (1991)].Constant region does not participate in the combination of antibody and antigen directly, but they show different effector functions.
Term used herein " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform group, i.e.,
The single antibody for including in the group is identical, in addition to minority is there may be the mutation naturally occurred.Monoclonal antibody Gao Te
The opposite sex is directed to single antigen site.And (typically there is the difference for different determinants from conventional polyclonal antibody preparation
Antibody) it is different, each monoclonal antibody will not be polluted by other immunoglobulins for the single determinant on antigen.Modification
Language " monoclonal " illustrates the specificity of antibody, is obtained from substantially uniform antibody population, is not construed as needing to appoint
What specific process produces antibody.
Term used herein " affinity " refers to affinity of the antibody to antigen.In one embodiment, affine
Power is measured by the dissociation rate of antigen/antibody.
The present invention solves above-mentioned technical problem by the following technical programs：
First aspect present invention provides a kind of anti-human CEACAM5 monoclonal antibodies, is CCTCC NO by preserving number:
The mouse hybridoma cell system of C2016129 generates.
Preferably, the immunoglobulin of the anti-human CEACAM5 monoclonal antibodies is IgG1 subclass, κ type light chains.
In the present invention, the mouse hybridoma cell system has been deposited in Chinese Typical Representative culture guarantor on June 23rd, 2016
Tibetan center (CCTCC), preservation address：Wuhan City, Hubei Province Wuchang District Bayi Road 299, postcode：430072, deposit number is：
CCTCC NO:C2016129, culture title are hybridoma cell strain MG7.
In a specific embodiment, anti-human CEACAM5 monoclonal antibodies of the present invention are CCTCC by preserving number
NO:The mouse hybridoma cell system of C2016129 generates, and immunoglobulin is IgG1 subclass, κ type light chains.
Second aspect of the present invention provides a kind of composition including aforementioned anti-human CEACAM5 monoclonal antibodies.
Preferably, the composition can also include pharmaceutically acceptable carrier.
In general, the content of aforementioned anti-human CEACAM5 monoclonal antibodies is the content of this field routine in the composition,
Such as mass percent 0.001~99.99%.
Third aspect present invention provides a kind of hybridoma cell line generating aforementioned anti-human CEACAM5 monoclonal antibodies,
Its preserving number is CCTCC NO:C2016129.
Fourth aspect present invention provides the preparation method of aforementioned anti-human CEACAM5 monoclonal antibodies, includes the following steps：
S1：Use the whole-cell protein that gastric carcinoma cells crack as immunogen immune mouse；
S2：The splenocyte for the mouse being immunized in step S1 is merged with murine myeloma cell；
S3：After cell clone occurs in cell fusion in step S2, the hybridoma cell line of stably excreting antibody is selected；
S4：Aforementioned anti-human CEACAM5 monoclonals are made from the hybridoma cell line of stably excreting antibody described in step S3
Antibody.
Preferably, in step S1, the gastric carcinoma cells can be the cell of this field routine, as immortalized
Human gastric cancer cell line or the stomach cancer cell detached from Patients with Gastric Cancer.Described be immunized can be the operation of this field routine, compared with
It is 3 times immune goodly, is spaced 4 weeks between second for the first time, is spaced 2 weeks between second and third time.
Preferably, in step S2, the murine myeloma cell can be the small of the routine that antibody art uses
Rat bone marrow tumour cell, such as SP2/0 cells.The fusion can be that the cell fusion of this field routine operates, as polyethylene glycol is situated between
The cell fusion led.
Preferably, in step S3, described select can be the operation of this field routine, to thin as described in appearance
Born of the same parents, which clone, carries out indirect ELISA detection, the cell of positive colony is carried out cloning using limiting dilution assay, until obtaining can
The hybridoma cell line of stably excreting antibody.
Preferably, it in step S4, is prepared from the hybridoma cell line of the stably excreting antibody aforementioned anti-human
The method of CEACAM monoclonal antibodies can be the operation of this field routine, such as mouse ascites method.
Fifth aspect present invention provides aforementioned anti-human CEACAM5 monoclonal antibodies in the combination for preparing treatment malignant tumour
Application in object.
Preferably, the malignant tumour is one or more in gastric cancer, cancer of pancreas, colon cancer and cancer of the esophagus；More
Goodly, the malignant tumour is gastric cancer.
The composition can be the composition of this field routine, preferably, being pharmaceutical composition；More preferably, the medicine
Compositions include aforementioned anti-human CEACAM5 monoclonal antibodies and pharmaceutically acceptable carrier.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The beneficial effects of the present invention are：Anti-human CEACAM5 monoclonal antibodies provided by the invention can specifically with
Glycosylated people CEACAM5 antigen bindings, and there is higher affinity and antigentic specificity with glycosylated CEACAM5,
All kinds of routine operations such as it can be widely applied to flow cytometry, immunohistochemical staining, immunoprecipitate, being on gastric cancer etc. is a variety of
The treatment and diagnosis of skin tumour provide the strategy and means of effective targeted therapy.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1, mouse anti human CEACAM5 monoclonal antibodies MG7-Ab and different cell pyrolysis liquids and people's liver lysate Western are miscellaneous
Knot fruit；
Fig. 2, mouse anti human CEACAM5 monoclonal antibody MG7-Ab and KATOIII cell pyrolysis liquid Immunoprecipitation studies；
Fig. 3, the anti-human CEACAM5 monoclonal antibodies of commercialization and mouse anti human CEACAM5 monoclonal antibodies MG7-Ab and KATOIII of the present invention
Cell pyrolysis liquid co-immunoprecipitation result；
Fig. 4, the anti-human CEACAM5 monoclonal antibodies of commercialization and mouse anti human CEACAM5 monoclonal antibodies MG7-Ab of the present invention and external source mistake
Express the HEK293T cell pyrolysis liquid Western results of hybridization of CEACAM5；
Fig. 5, by after the expression of CEACAM5 intracellular KATOIII shRNA silences, monoclonal antibody MG7-Ab is anti-with commercialization
Result after body Western hybridization；
The cellular immunofluorescence detection streaming cell detection results of Fig. 6, mouse anti human CEACAM5 monoclonal antibodies；
The immune group of people CEACAM5 in Fig. 7, mouse anti human CEACAM5 monoclonal antibodies MG7-Ab detection stomach organization paraffin sections
Change result；
In Fig. 8, the anti-human CEACAM5 monoclonal antibodies of commercialization and mouse anti human CEACAM5 monoclonal antibody MG7-Ab and HEK293T cells
The glycosylation of overexpression and deglycosylated people CEACAM5 albumen Western results of hybridization.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
The present inventor after extensive and in-depth study, has found to go to be immunized with the cracking obtained total protein of gastric carcinoma cells
After mouse, the monoclonal antibody of a collection of gastric cancer specificity can be obtained, wherein being named as tissue and serum of the antibody in gastric cancer of MG7-Ab
In expression average level it is very high, after detected by proteomic techniques, find this antibody identification antigen be people
CEACAM5.On this basis, the Dan Ke of the anti-human CEACAM5 of purifying has further been made using hybridoma technology by inventor
Grand antibody, it is thus identified that the DNA sequence dna and corresponding protein sequence and packet of the monoclonal antibody heavy variable region and light chain variable region
It containing CDR sequence inside, and finds that the antibody is not only stronger with antigen affinity, but also can specifically identify glycosylated
CEACAM5 provides support, so as to complete the present invention for the humanized recombinant antibodies of the monoclonal antibody.
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
Product specification selects.
SP2/0, SGC7091, KATOIII, MCF7, HEK293T and MKN45 used in the present invention are purchased from the Chinese Academy of Sciences
Extra large cell bank.
The monoclonal antibody that anti-human CEACAM5 is commercialized is purchased from Abcam companies of the U.S., and article No. ab4451, it is mouse that animal, which is immunized,
(Mus musculus)。
MG7-Ab refers to mouse anti human CEACAM5 monoclonal antibodies of the present invention in following embodiment, and it is anti-that Anti-CEA refers to commercialization
People's CEACAM5 monoclonal antibodies.The mouse hybridoma cell system for generating the monoclonal antibody MG7-Ab is deposited in China on June 23rd, 2016
Type Tissue Collection (CCTCC), preservation address：Wuhan City, Hubei Province Wuchang District Bayi Road 299, postcode：430072,
Deposit number is：CCTCC NO:C2016129, culture title are hybridoma cell strain MG7.
1 the present embodiment of embodiment is related to the preparation of mouse anti human CEACAM5 monoclonal antibodies
The preparation method reference of mAb《Cell and molecular immunology experimental technique》First edition Jin Bai springs The Fourth Military Medical University goes out
Version society 2002, the method that P9-P17 is recorded, steps are as follows：
(1) gastric carcinoma cells MKN-46-9 is cracked, the whole-cell protein of lysate is extracted；
(2) whole-cell protein that step (1) obtains is used to press the dose immunization BALB/c mouses of 50 μ g/ only 3 times；Exempt from for the first time
Epidemic disease, using Freund's complete adjuvant, the 2nd time immune to use incomplete Freund's adjuvant, is spaced 4 weeks, is subcutaneous multi-point injection, and the 3rd
It is secondary to be immunized as intraperitoneal injection, with the 2nd immunization interval 2 weeks；
(3) it takes a blood sample within 7-10 days after the completion of step (2) is immune, potency is detected with indirect elisa method；3 days abdomens before cell fusion
Chamber injecting immune original carries out booster immunization；Booster immunization takes the mouse myeloma of mouse boosting cell and exponential phase thin after 3 days
Born of the same parents SP2/0 is counted, and prepares immune spleen cell suspension, and SP2/0 cells and splenocyte are pressed 1:5 ratio mixing carries out cell and melts
It closes, fusion reagent is polyethylene glycol.Containing feeder cells, (normal BALB/c mouse abdominal cavity macrophage is thin for cell suspension addition after fusion
Born of the same parents) 96 orifice plates, 37 DEG C, 5%CO2Incubator culture；
(4) it after cell clone appearance, is detected with indirect elisa method, select has atopy for CEACAM5
Positive cell clone carries out cloning using limiting dilution assay, is 100% for standard with continuous 2 time cloning Positive rate,
Until obtaining the hybridoma cell line for capableing of stably excreting antibody；
(5) hybridoma cell line for taking the stably excreting antibody of step (4) acquisition, is prepared by mouse ascites preparation method and is wrapped
Ascites containing monoclonal antibody；
(6) pure using QFF anion exchange chromatographies by 45% saturated ammonium sulphate of ascites made from step (5)
After change, the antibody of SDS-PAGE purification Identifications.
Obtained antibody belongs to IgG1 subclass by measuring, and κ type light chains reach 95% by purity after purification.It will be obtained
The antibody obtained is named as MG7-Ab.
2 the present embodiment of embodiment is related to the immune-blotting method of mouse anti human CEACAM5 monoclonal antibodies
Using western blotting method detection mouse anti human CEACAM5 monoclonal antibodies and gastric carcinoma cell lines SGC7901,
KATOIII cell pyrolysis liquids and non-gastric carcinoma cell lines MCF7, HEK293T, AD293, A2780, N38 cell pyrolysis liquid and people's liver
CEACAM5 binding abilities in Tissue lysates, method reference《Cell and molecular immunology experimental technique》First edition gold Bai Quan
Publishing house of The Fourth Military Medical University 2002, the method that P53-P55 is recorded, using mouse anti human CEACAM5 monoclonal antibodies as primary antibody,
The goat anti-mouse antibody of HRP labels is secondary antibody, Alpha Innotech FluorChem FC2 imaging systems (Alpha
Innotech, USA) analyze the result that Western hybridizes.
As shown in Figure 1, mouse anti human CEACAM5 monoclonal antibodies MG7-Ab of the present invention can be split with gastric carcinoma cell lines KATOIII
It solving liquid and generates reaction, shape generates band at about 180KD, and with the lysate of non-stomach cancer cell without generating item at 180KD
Band, it is that an idiocrasys of the MG7-Ab in gastric carcinoma cell lines corresponds to antigen to prompt the 180KD albumen.
3 the present embodiment of embodiment is related to the identification of mouse anti human CEACAM5 monoclonal antibody combination antigens
The antibody MG7-Ab antigens combined are identified using the method for immunoprecipitating (IP), the step of IP is as follows：1)
1ml IP lysis buffers (Pierce IP Lysis are added to 90% fusion in 10cm culture plates routine culture KATOIII cells
Buffer, Prod#87788, the cOmplete Tablets of Roche containing protease inhibitors Mini, EDTA-free), it splits on ice
Solution centrifugation in 30 minutes, 13,000 × g, 4 DEG C takes supernatant after ten minutes.2) a small amount of lysate is taken in case Western blot analysis,
Remaining lysate adds protein A/G sepharose 4Bs (20mg/ml) to carry out pre cleaning, and 4 DEG C of rotations are incubated 2 hours.3)1000×
G, 4 DEG C of centrifugations take supernatant after 1 minute, this is the cell IP lysates after pre cleaning.4) by 40mg protein A/G agaroses
Pearl is put into clean three times with PBS in 1ml Ep pipes after be resuspended with 1mlPBS, 10 μ g primary antibodies or control antibodies, 4 DEG C of rotations are added and incubate
It educates 2 hours.5) 1000 × g, 4 DEG C centrifugation 1 minute after abandon supernatant.This is the protein A/G sepharose 4Bs for combining primary antibody.6)
Cell IP lysates after pre cleaning are added to the protein A/G sepharose 4Bs for combining primary antibody, often pipe 1ml.4 DEG C of rotations are incubated
It educates 12 hours.8) 1000 × g, 4 DEG C of centrifugations abandon supernatant after 1 minute, at the uniform velocity rotate Qingxu 3 times, every time 5 points with 4 DEG C of IP lysates
Clock, then cleaned 2 times with PBS solution.9) after immune precipitation, 1000 × g, 4 DEG C centrifugation 1 minute after abandon supernatant, by agarose
Pearl centrifuges to tube bottom, and supernatant is carefully sucked, and 2 × SDS sample-loading buffers (DTT containing 20mM) of 30 μ l, 100 DEG C of heating 5 are added
Minute.It is cooled to room temperature.10) 40,000 × g are centrifuged 20 minutes, and supernatant is IP products.
The antigen of above-mentioned enrichment combined with MG7-Ab is analyzed by mass spectrometry, SDS- is run before IP product mass spectra loadings
PAGE, steps are as follows for Gel Treatment：
I. film dosim
(1) by the protein band parallel divisional of each swimming lane at 3~4 pieces of blob of viscoses, each blob of viscose is chopped into about 1mm × 1mm
Small blob of viscose, be fitted into the EP pipes of 1.5mL；
(2) blob of viscose decolourizes：200-400 μ L 25mM NH are added4HCO3/ 30%ACN, be vortexed concussion 10min, and centrifugation sucks
Liquid is repeated twice；
(3) it is dehydrated：Add 100 μ l, 100% acetonitriles, be vortexed 10~15min of concussion.Vacuum is drained after sucking liquid；
(4) it restores：100 μ l 20mMDTT, 56 DEG C of incubation 1h are added to suck DTT solution；
(5) it closes：50 μ l 50mM iodoacetamides (indoacetamide), incubation at room temperature 20min alkylations are added to suck liquid
Body；
(6) it is dehydrated：Add 200 μ l, 100% acetonitriles, be vortexed 10~15min of concussion.Liquid is sucked, vacuum drains about 25min；
(7) it digests：Add about 50 μ l 10ng/ μ l trypsin/10mM NH4HCO3To cover blob of viscose, 4 DEG C of placement 30-
60min.Again plus 20-30 μ l 10mM NH4HCO3Blob of viscose after covering expansion.37 DEG C digest overnight.
(8) peptide fragment recycles：Add 150 μ l 60%ACN/0.1%TFA, ultrasonic 15min.In transfer liquid to new EP pipes,
Repeat elution twice.
(9) 12000rpm centrifuges 10min, collects supernatant, is put into vacuum drying instrument and drains liquid, -20 DEG C of storages.
II. desalination：
(1) 100% acetonitrile, 200 μ l neutral solutions activation Millipore C18ZipTip are added.After centrifuging 100g × 1min
It discards supernatant.
(2) reversed-phase liquid chromatography mobile phase A solution is added, for balancing Millipore C18ZipTip,.Centrifuge 100g
It is discarded supernatant after × 1min.Step experiment is repeated 3 times.
(3) it is added and waits for desalted sample, discarded supernatant after centrifuging 100g × 1min.
(4) reversed-phase liquid chromatography mobile phase A solution washing sample.It is discarded supernatant after centrifugation 100g × 1min.The step is tested
It is repeated 3 times.
(5) 70% acetonitrile, 200 μ l are added, centrifuge 100g × 1min, efflux is purification of samples.
(6) sample Speed Vac are centrifuged into vacuum drying to dry powder-shaped.
(7) add 12 2% acetonitriles of μ l/98%H2O/0.1%TFA sample dissolutions, upper LC-MS analyze sample.
As a result see Fig. 2, compared with negative control cell system HEK293T, exist in KATOIII cell pyrolysis liquids and MG7-Ab
The albumen for the 180KD being combined, and can be obviously enriched with by MG7 antibody.To immunoprecipitating being tied with MG7-Ab phases of being enriched with
The molecular weight of conjunction is analyzed by mass spectrometry for the antigen of 180KD shows that the antigen is CEACAM5.
4 the present embodiment of embodiment is related to immunoprecipitating for mouse anti human CEACAM5 monoclonal antibodies and hybridizes inspection with Western
It surveys
Further MG7-Ab is immunoprecipitated with gastric carcinoma cell lines SGC7901, MKN45 and KATOIII cell respectively
Detection；With the expression for the HEK293T and CEACAM5 for being overexpressed people CEACAM5 by the KATOIII cells of RNAi silences to MG7-Ab
Western hybridization checks are carried out, using the commercial antibody of anti-human CEACAM5 as positive control.The step of IP, is the same as embodiment 3
In it is recorded；The step of Western hybridizes is as follows：1) cell line is carried out on ice with the IP lysates containing protease inhibitors
Albumen is extracted in cracking after 15 minutes, Bradford methods are quantitative, SDS sample-loading buffers are added, boiling sample 5min makes its change
Property；2) SDS-PAGE electrophoresis is carried out, constant current, 25mA, albumen applied sample amount is 30 μ g；3) semidry method carries out electric transferring film, will pass through electricity
On protein delivery to nitrocellulose filter (NC) after swimming separation, constant pressure, 20V, 30 minutes；4) 5% skimmed milk power closes 3 points
Clock；5) milk is discarded, the diluted primary antibody of 2.5% skim milk is added dropwise, 4 DEG C of overnight incubations or room temperature are incubated for 3 hours；6)1×
TBST washes film 3 times, each 5min, adds the secondary antibody of corresponding HRP labels, is incubated at room temperature 1h；7) 1 × TBST washes film 3 times, every time
10min drops evenly ECL luminescent solutions in the darkroom of Western blot imagers and carries out colour developing capture imaging.
As a result as seen in figures 3-5.Made from the anti-human CEACAM5 monoclonal antibodies and the embodiment of the present invention 1 of Fig. 3 display of commodity
Monoclonal antibody MG-Ab can be enriched with the albumen that gastric carcinoma cell lines KATOIII middle-molecular-weihydroxyethyls are 180KD；Fig. 4 shows, in itself not table
The anti-human CEACAM5 monoclonal antibodies and the present invention being commercialized after up to the CEACAM5 of overexpression external source in the HEK293T cells of CEACAM5
Monoclonal antibody MG-Ab made from embodiment 1 can be combined the band for generating and hybridizing with the CEACAM5 of the external source of overexpression；Fig. 5 is aobvious
Show, after the expression of CEACAM5 intracellular KATOIII shRNA silences, monoclonal antibody MG7-Ab and commercial antibody Western
It is generated without signal after hybridization.
The above results in conjunction with the embodiments 3 Mass Spectrometric Identification the result shows that, monoclonal antibody MG7-Ab can be with people CEACAM5 specificity
Ground combines, and is a kind of mouse anti human CEACAM5 monoclonal antibodies.
5 the present embodiment of embodiment is related to the titration of mouse anti human CEACAM5 monoclonal antibodies
Indirect elisa method measures ascites and the after purification relative affinity of mAb before purification in embodiment 1.Envelope antigen
For glycosylated people CEACAM antigens, i.e. CEACAM5, sample to be tested is the ascites being serially diluted and mAb, detection are anti-after purification
Body is sheep anti mouse-HRP enzymic-labelled antibodies, and substrate uses ABTS.
Testing result shows that the titer of ascites containing monoclonal antibody screened in embodiment 1 is 1 × 10-7, far above reaching 1
×10-5The mean level that can be used.
6 the present embodiment of embodiment is related to the cellular immunofluorescence detection of mouse anti human CEACAM5 monoclonal antibodies
Using indirect IF staining method combination Flow cytometry monoclonal antibody MG7-Ab and human gastric cancer cell line
The binding ability of the natural CEACAM5 molecules of KATOIII, operating procedure reference《Cell and molecular immunology experimental technique》The first edition
Publishing house of Jin Bai springs The Fourth Military Medical University 2002, the method that P78-80 is recorded, with mouse anti human made from the embodiment of the present invention 1
CEACAM5 monoclonal antibodies are primary antibody, using the goat anti-mouse antibody of FITC labels as secondary antibody, carry out flow cytometry analysis.
For testing result as shown in fig. 6, compared with the Isotype control for being directly added into immunofluorescence secondary antibody, mouse of the invention is anti-
People CEACAM5 monoclonal antibodies and the gastric carcinoma cell lines KATOIII binding abilities of high expression people CEACAM5 are remarkably reinforced, and illustrate the present invention
Mouse anti human CEACAM5 monoclonal antibodies can be combined specifically with the CEACAM5 molecules on the surfaces gastric carcinoma cell lines KATOIII.
7 the present embodiment of embodiment is related to the Immunohistochemical detection of mouse anti human CEACAM5 monoclonal antibodies
Using mouse anti human made from embodiment 1 as primary antibody, gastric cancer group is identified with immunohistochemical method detection CEACAM5
Knit the ability of people CEACAM5 in paraffin section.The operating procedure reference of immunohistochemical staining《Pathological technique》The first edition
(People's Health Publisher, beam hero), P367-372.
Testing result as shown in fig. 7, CEACAM5 molecules in stomach organization in heterogeneous expression, Fig. 7 a, 7b, 7c and 7d
Expression intensity be divided into from weak to strong-,+, ++, +++.Heterogeneity of the CEACAM5 molecules in stomach organization is expressed as further
The state of an illness, the course of disease and the relationship of prognosis for studying the molecule and gastric cancer provide theoretical foundation.
8 the present embodiment of embodiment is related to specific detection of the mouse anti human CEACAM5 monoclonal antibodies to mannosylated antigen
The external source CEACAM5 being overexpressed in 293T cells is handled with n-glycosylase F (PNGase F), every 20 μ g cells are complete
1 μ l of 500U/ μ l PNGaseF are added in albumen, 37 DEG C are incubated 1 hour, and the mouse anti human CEACAM5 made from embodiment 1 is mono-
Clonal antibody carries out Western hybridization checks respectively with commercial antibody as primary antibody, and Western steps are the same as aforementioned.
The results are shown in Figure 8, the anti-human CEACAM5 monoclonal antibodies of commercialization and glycosylated and deglycosylated people
CEACAM5 can be combined and be generated band, and mouse anti human CEACAM5 monoclonal antibodies of the present invention and deglycosylated people
CEACAM5 is not combined, and is only combined with glycosylated people CEACAM5 and generates band.Therefore mouse anti human CEACAM5 Dan Ke of the present invention
Grand antibody can specifically identify glycosylated people CEACAM5.
In conclusion only presently preferred embodiments of the present invention, is not used for limiting the scope of implementation of the present invention, it is all according to
The equivalent changes and modifications carried out by shape, construction, feature and spirit described in scope of the invention as claimed should all be included in this
In the right of invention.
Claims (5)
1. a kind of anti-human CEACAM5 monoclonal antibodies, which is characterized in that by preserving number be CCTCC NO:The mouse of C2016129
Hybridoma cell line generates.
2. anti-human CEACAM5 monoclonal antibodies as described in claim 1, which is characterized in that the anti-human CEACAM5 monoclonals
The immunoglobulin of antibody is IgG1 subclass, κ type light chains.
3. a kind of anti-human CEACAM5 monoclonal antibodies as claimed in claim 1 or 2 are in the composition for preparing treatment malignant tumour
In application.
4. a kind of composition containing anti-human CEACAM5 monoclonal antibodies as claimed in claim 1 or 2.
5. a kind of preserving number is CCTCC NO:The hybridoma cell line of C2016129.
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