CN106501518A - A kind of SABC antibody reagent of detection EGFRv III and detection method - Google Patents
A kind of SABC antibody reagent of detection EGFRv III and detection method Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of SABC antibody reagent of detection EGFRv III, high have the advantages that high specificity, susceptibility, including anti-III monoclonal antibody of EGFR mutant and antibody diluent；The antibody diluent is produced by doctor's moral Bioisystech Co., Ltd, and its commodity article No. is AR1016.The invention also discloses a kind of ImmunohistochemistryMethods Methods of detection EGFRv III：Using the SABC antibody reagent of detection EGFRv III, the expression that the EGFRv III in sample tissue to be detected is detected by ImmunohistochemistryMethods Methods, testing result can be used as one of diagnosis basis of clinician, diagnosis for clinical tumor plays Index for diagnosis, helps diagnosis, classification, Index for diagnosis and the curative effect evaluation of clinical tumor.
Description
Technical field
The present invention relates to a kind of SABC antibody reagent of detection EGFRv III and detection method, belong to tumor-marker and face
Bed application and research field.
Background technology
EGF-R ELISA (epidermal growth factor receptor, EGFR) molecular weight is 170kD,
Belong to the Eb r B families of receptor tyrosine kinase, be made up of extracellular region, transmembrane region and intracellular region, its normal activation can mediate thin
The growth of born of the same parents, breed, break up, sticking and the activity such as mobile.Many epithelial origin tumours are increased with activity of EGFR or exception has
Close.EGFR overexpression generally in mankind's difference tumour, and the overexpression of EGFR is related to the grade malignancy of some tumours.Closely
Year research finds that, during the occurrence and development of tumour, the expression of EGFR albumen is not very stable, the expansion of gene usually occurs
Increase and reset, cause gene mutation to cause the antigenic phenotype of tumor cell surface that modulation occurs.Now research finds that EGFR has 3 kinds
Extracellular type mutant, i.e. EGFRv I, EGFRv II and EGFRv III, the EGFR signal paths after the receptor binding being mutated with these are led to
Often abnormal activation, causes abnormal cell proliferation.Wherein EGFRv III is most commonly seen mutant form in human tumor.
EGFRv III is compared with EGFR complete structures, and III molecular weight of EGFRv is 130kD, in 26 extrons the 2nd～7 outer
Aobvious son is deleted, and causes 801 base pair deletions, and which deletes end by new codon glycine (GGT) connection, translates into
After protein, 6～273 amino acid of extracellular region is substituted by a glycine residue.Lose the EGFRv of extracellular ligand binding site
III can in the case of without ligand binding constitutively activated EGFR-TK, produce autophosphorylation, induce downstream signal
Transduction pathway, causes cascade reaction, affects the biological behaviour of tumour cell.
So far, EGFRv III is not yet detected in the normal tissue, is considered the specific expressed EGFR of malignant tumour and is dashed forward
Variant, in glioma (positive rate 30～50%), lung cancer (positive rate 39%), breast cancer (positive rate 20～36%), ovary
Cancer (positive rate 75%), incidence cancer (positive rate 42%), colon cancer (positive rate 34%), prostate (positive rate 100%), liver
Cancer (positive rate 36.8%), cancer of the stomach (positive rate 35.2%)) etc. be found.EGFRv III only occurs in tumour cell, and
Normal tissue cell is not expressed, so it is a kind of good tumour specific antigen, this make EGFRv III become one swollen well
The target of knurl treatment.EGFRv III also has certain relation with tumor patient prognosis, and the expression of tumor tissues EGFRv III is to sentence
The independent indication of disconnected prognosis, EGFRv III express high person's poor prognosis.
How accurately and rapidly to detect each tumor tissues EGFRv III expression be target immunization therapy key
Factor.At present, the method that the expression of detection EGFRv III is mainly adopted has direct sequencing, real-time quantitative PCR method and tissue
The methods such as Immunohistochemical Method.These methods are each advantageous, but there is complex operation, time-consuming, cost is relatively large, poor specificity
Etc. weak point, still there is improved necessity.
Content of the invention
For above-mentioned prior art, the invention provides a kind of SABC antibody reagent of detection EGFRv III, Yi Jijian
Survey method.Detect that expression of the EGFRv III in tumour, the positive staining positioning of III albumen of EGFRv mainly exist using Immunohistochemical Method
On cell membrane and cytoplasm, brown yellow granule is shown as；As a result sxemiquantitative integration method is adopted, is existed by 2 diagosis persons per a section
Interpretation in the case of blind state, this result can be that the diagnosis prognosis of clinical tumor is sentenced as one of diagnosis basis of clinician
Disconnected effect, helps diagnosis, classification, Index for diagnosis and the curative effect evaluation of clinical tumor.
The present invention is achieved by the following technical solutions：
A kind of SABC antibody reagent of detection EGFRv III, including anti-III monoclonal antibody of EGFR mutant, (antibody is former
Liquid) and antibody diluent；Anti- III monoclonal antibody of EGFR mutant, its preparation method are documented in document
Oncotarget.2016 Dec 22.doi:In 10.18632/oncotarget.14088.；The antibody diluent, mainly into
It is divided into Tris buffer solutions (pH 7.2), and containing NaN3 and protein, is produced by doctor's moral Bioisystech Co., Ltd, its commodity
Article No. is AR1016, trade name antibody diluent.
Further, anti-III monoclonal antibody of EGFR mutant (the antibody stoste) with the volume ratio of antibody diluent is
1:1～5000, preferably 1:1000～1:4000.
A kind of method (Immunohistochemical Method) of detection EGFRv III, be：Resisted using the SABC of above-mentioned detection EGFRv III
Body reagent, detects sample tissue to be detected by ImmunohistochemistryMethods Methods (tumor tissues for performing the operation or puncture are obtained after process)
In EGFRv III expression, testing result can be the diagnosis of clinical tumor as one of diagnosis basis of clinician
Play Index for diagnosis, help diagnosis, classification, Index for diagnosis and the curative effect evaluation of clinical tumor (to be embodied as treatment side
Case need to also be referring again to the clinical manifestation situation of patient).
Further, expression value of the testing result for EGFRv III, is obtained by the methods of marking of following SABC
Go out：The expression value of EGFRv III=cell membrane staining power score value × cell membrane dyeing score value+cytoplasm staining power score value ×
Cytoplasm dyes score value.The cell membrane staining power score value is the score value of staining power on cell membrane, the cytoplasm dye
Intensity of colour score value is the score value of staining power on cytoplasm；The score value marking of the staining power is according to being：It is unstained=0,
Weak dyeing=1, moderate stain=2, strong dyeing=3.The cell membrane dyes the marking foundation of score value：Cell membrane staining cell
0%～5% corresponding score value of number is 0；6%～25% corresponding score value of cell membrane staining cell number is 1；Cell membrane staining cell
26%～50% corresponding score value of number is 2；51%～100% corresponding score value of cell membrane staining cell number is 3.The cytoplasm
The marking of score value is according to being：It is 0 for 0%～5% corresponding score value of cytoplasm staining cell number；Cytoplasm staining cell number 6%
～25% corresponding score value is 1；26%～50% corresponding score value of cytoplasm staining cell number is 2；Cytoplasm staining cell number
51%～100% corresponding score value is 3.When the expression value of EGFRv III is more than 4 points, it is judged as that " EGFRv III is expressed as sun
Property ", when the expression value of EGFRv III is less than 4 timesharing, be judged as " EGFRv III is expressed as feminine gender " (be unstained, weak dyeing, medium dye
Color, the judgement of strong dyeing, the judgement of cell membrane staining cell number is normal experiment skill that those skilled in the art are grasped
Can).
Anti- III monoclonal antibody of EGFR mutant is to develop (present applicant by Department Of Medicine, Peking University
Collaboration, participates in developing；Anti- III monoclonal antibody of EGFR mutant, related content have been published an article, its document
Information is：Oncotarget.2016Dec 22.doi:10.18632/oncotarget.14088., molecular weight is 150KD, is
IgG2a hypotypes.The antibody is the mouse monoclonal antibody of III albumen of anti-human EGFRv, can with histocyte on III antigens of EGFRv
Specific binding, and recognize and combine with enzyme mark goat anti-mouse immunoglobulin polymer, by immunohistochemical staining, make
Occur yellow or brownish discoloration in histotomy on antigen site, using EGFRv III in light microscope observable tissue
Expression.
The present invention has obtained best results through experiment screening on the basis of anti-III monoclonal antibody of EGFR mutant
Antibody diluent (is not meant to which can be directly as antibody reagent work after developing anti-III monoclonal antibody of EGFR mutant
Applied as liquid, suitable antibody diluent is also the key of the SABC product of high-quality), have developed EGFRv III and exempt from
Epidemic disease group antibody reagent working solution, itself and Wild type EGFR no cross reaction are high have the advantages that high specificity, susceptibility, are
The targeted therapy of EGFRv III provides good premise and basis.
Description of the drawings
Fig. 1：Antibody is combined into SABC testing result schematic diagram after working solution, wherein, a with different antibodies dilution：
Doctor's moral (AR1016)；b：Beijing Zhong Shan Golden Bridge (ZLI-9028)；c：Foochow steps new (ABD-0030)；d：Denmark Dako
(K8007).The word of figure central lower is：F98EGFRvⅢ.
Fig. 2：The SABC section of glioma, wherein, 1：Astrocytoma gradeⅡ；2：Oligodendroglioma II
Level；3：III grade of human anaplastic astrocytoma；4：Between III grade of denaturation oligodendroglioma；5：Glioblastoma.
Fig. 3：III expressions of EGFRv in the glioma of different malignant grades, wherein, A：The expression of EGFRv III and tissue
Learn type；B：The expression of EGFRv III is classified with WHO.
Fig. 4：EGFRv III and ATRX, MIB1, IDH1R132, are mutated the correlation of expression of the P53 in glioma.
Fig. 5：The western-blot figures of III monoclonal antibodies of anti-EGFRv, wherein, band from left to right is represented respectively
F98、F98EGFR、F98EGFRvIII、U251、U251EGFRvIII.
Fig. 6：EGFR and III RT-PCR of EGFRv runs glue and sequencing analysis, wherein, 1:F98；2:F98EGFR；3:F98EGFRvIII；
4:U251；5:U251EGFRvIII.
Fig. 7：The immunofluorescence experiment result figure of the rabbit source polyclonal antibody of existing targeting EGFR v III.
Fig. 8：The immunofluorescence experiment result figure of the monoclonal antibody of anti-EGFRv III.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
Involved instrument, reagent, material etc. in following embodiments, unless otherwise noted, are in prior art existing
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental technique in following embodiments, inspection
Survey method etc., unless otherwise noted, is existing normal experiment method, detection method etc. in prior art.
The screening of 1 antibody diluent of embodiment
Immunohistochemical assay step：Tissue is soaked 10 minutes after 60 DEG C of baking boxs are roasting 40 minutes in dimethylbenzene, so as to
Paraffin is removed, is then passed through to soak 2 minutes in 100% ethanol twice, is then soaked 2 minutes two in 95% ethanol solution
Secondary, soak 2 minutes in 85% ethanol solution and soak 2 minutes in 75% ethanol solution, carry out aquation, finally with steaming
Distilled water is washed 2 minutes, altogether 5 times.
In pressure cooker membranous antigen is added to repair liquid (225ml distilled waters:4.5ml citric acid:20.5ml trisodium citrates),
Pressure cooker is uncapped and on electromagnetic oven is heated to seething with excitement tissue antigen recovery liquid, section is all submerged in reparation liquid, is covered
Pressure cooker is naturally cooled to from fire after 2 points of high pressure and take out after room temperature section by pot cover, and PBS solution is cleaned 5 times, every time 2 minutes.
Liquid (30%H is blocked by fresh configuration peroxidase2O2:Distilled water=1:9) 37 DEG C of baking ovens are incubated 30 minutes, with eliminating
The activity of endogenous peroxidase enzyme.PBS solution is cleaned 5 times, every time 2 minutes.Carefully get rid of and wipe away and be excessive on and around tissue
Liquid, in the tissue regions of SABC pen delineation plus 37 DEG C of 100ul5% lowlenthal serums are incubated 30 minutes, non-to close
Specific antigen.
Do not wash, an anti-working solution is added dropwise, 37 DEG C are incubated 1～2 hour or 4 DEG C overnight.Phosphate buffer (PBS) rinses 2
Minute, totally 5 times.Two anti-working solution Dako K5007 of appropriate horseradish peroxidase-labeled are added dropwise, 37 DEG C are incubated 30 minutes.Phosphoric acid
Salt buffer (PBS) is rinsed 2 minutes, 5 times totally.Through chromogenic reagent 1～2 minute, the in good time color development stopping of Microscopic observation, haematoxylin
Dye core 1 minute, after running water rinses unnecessary haematoxylin, then through dehydration, transparent, mounting, basis of microscopic observation are simultaneously made film.
The one anti-working solution is by one anti-(prepared by embodiment 7,5.3 μ g/ μ l of concentration) and antibody diluent composition, the two body
Product compares 1:2000.
Used one anti-be by Department Of Medicine, Peking University develop (present applicant collaboration participates in developing
Exploitation；Anti- III monoclonal antibody of EGFR mutant, related content have been published an article, and its documentation ＆ info is：
Oncotarget.2016Dec 22.doi:10.18632/oncotarget.14088., molecular weight are 150KD, are that IgG2a is sub-
Type.
The present embodiment has investigated four kinds of antibody diluents, respectively：Beijing Zhong Shan Golden Bridge (ZLI-9028), Foochow step new
(ABD-0030), doctor's moral (AR1016), Denmark Dako (K8007).
Microscope film making result as shown in figure 1, as seen from the figure, the antibody working solution that combined by doctor's moral (AR1016), the back of the body
Scape is clean, and dyeing is clear, high specificity, and susceptibility is high, and the antibody working solution of other three kinds of antibody diluent combinations manifests
Go out cross reaction.Therefore, doctor's moral (AR1016) is the antibody diluent for being best suitable for anti-III monoclonal antibody of EGFR mutant.
F98EGFRvⅢIt is positive with the negative brown rat brain glioblastoma cell system of III protein expressions of EGFRv respectively with F98 cells,
It is purchased from American Type Culture collection warehousing (ATCC).
Embodiment 2 is obtained by the method for SABC and is available for the pathology piece for reading
First the tumor specimen that operation or puncture are obtained is fixed in 10% formalin, then with gradient concentration
Ethanol is fully dehydrated, and the tissue after dehydration treats that wax stone coagulates again through dimethylbenzene is transparent and the saturating wax of paraffin afterwards with FFPE
Gu after, it is cut into the slice, thin piece of 3 μ m-thicks.Immunohistochemical assay step can be executed by embodiment 1.
The expression value of the EGFRv III in computation organization's pathology piece.
The expression value of EGFRv III=cell membrane staining power score value × cell membrane dyeing score value+cytoplasm staining power point
Value × cytoplasm dyes score value.The cell membrane staining power score value is the score value of staining power on cell membrane, the cell
Matter staining power score value is the score value of staining power on cytoplasm；The score value marking of the staining power is according to being：It is unstained
=0, weak dyeing=1, moderate stain=2, strong dyeing=3.The cell membrane dyes the marking foundation of score value：Cell membrane is dyeed
0%～5% corresponding score value of cell number is 0；6%～25% corresponding score value of cell membrane staining cell number is 1；Cell membrane is dyeed
26%～50% corresponding score value of cell number is 2；51%～100% corresponding score value of cell membrane staining cell number is 3.Described thin
The marking of kytoplasm score value is according to being：It is 0 for 0%～5% corresponding score value of cytoplasm staining cell number；Cytoplasm staining cell number
6%～25% corresponding score value is 1；26%～50% corresponding score value of cytoplasm staining cell number is 2；Cytoplasm staining cell
51%～100% corresponding score value of number is 3.When the expression value of EGFRv III is more than 4 points, it is judged as that " EGFRv III is expressed as
Positive ", when the expression value of EGFRv III is less than 4 timesharing, it is judged as " EGFRv III is expressed as feminine gender ".
It is computed, in the present embodiment, the expression value of EGFRv III is >=4, determines that patient EGFRv III is expressed as the positive.This
Bright EGFRv III is positioned clearly, used in EGFRv III only detects positive expression, and the present invention on cell membrane and cytoplasm
III antibody titers of anti-EGFRv are high, high with sensitivity, the characteristics of specific good.
The diagnostic value of detection method is set up to further establish this research, is clinically verified.
The determination of 3 diagnostic criteria of embodiment
Glioma cells in tissue sample 99 of three hospital of Bo Nao sections of Beijing from 2011～2016 surgery excisions is collected, its
The middle male sex 53, women 46,17～69 years old age, the median age 43 years old.Any treatment such as non-row chemicotherapy before operation in patients,
The FFPE pathological tissue specimen that Postoperative pathological inspection is made a definite diagnosis, wherein astrocytoma II level, human anaplastic astrocytoma
III grade of III grade, oligodendroglioma II levels and a denaturation oligodendroglioma are equal 20,19 (tissues of glioblastoma
Section is as shown in Figure 2).By SABC conventional method, using the antibody working solution in embodiment 1, (antibody diluent is doctor
Moral (AR1016)) detection patient tumors sample EGFRv III, as a result as shown in table 1, Fig. 3, each pathology is calculated according to formula
The expression value (see embodiment 2) of EGFRv III in piece.
With the expression value of EGFRv III 4 points and 4 points determined above be expressed as the positive as patient EGFRv III, as EGFRv III
Expression value less than 4 (not comprising 4), represent that the EGFRv III of patient is expressed as feminine gender.According to table 1 and Fig. 3：99 brain glue
Matter knurl EGFR positive rate is (54) 54%, and wherein star II levels and II level positive rates of dashing forward less account for 40% (16/40).III grade of star and
III grade of positive rate of dashing forward less accounts for 55% (22/40).Glue blastoma positive rate accounts for 80% (16/20).Similar, patient expresses
EGFRv III accounts for the 34 (34%) of sum, and wherein star II levels and II level positive rates of dashing forward less accounts for 22.5% (9/40).III grade of star and
III grade of positive rate of dashing forward less accounts for 30% (12/40).Glue blastoma positive rate accounts for 65% (13/20).As can be seen here, the high expression of EGFR
In patient, III positive expressions of EGFRv account for 62.96%.And the positive coloring sites of EGFR and EGFRv III are predominantly located at cell membrane and thin
Endochylema.The positive rate of the malignant grade of patient's glioma and EGFR and EGFRv III is proportionate (both P<0.01).
In order to illustrate clinical meanings of the EGFRv III in human glioma, we compare EGFRv III and express and clinical disease
Reason feature, including the age, sex, histological type, WHO ranks, TP53 are mutated, and MIB1 is expressed, IDH1R132 mutation status and
ATRX expresses (table 2).The expression of EGFRv III and histological type (P<0.001, Fig. 3 A) and WHO classification (P=0.001, Fig. 3 B) be in
Positive correlation.Additionally, the unfavorable prognosis that TP53 mutation status, ATRX expression and MIB1 expression are reported as patients with gliomas refers to
Mark.Our result also shows the expression of EGFRv III and these index positive correlations (all P<0.05, table 32, Fig. 4).IDH1R132
It is common in rudimentary glioma and Secondary cases spongioblast to be mutated, and the reagent with the mutation has longer depositing
Live time.The invention demonstrates that all gliomas are primary tumor and IDH1R132 sports feminine gender.EGFRv III expression with
IDH1R132 mutation status negative correlation (P=0.031).These results show that III trends of EGFRv are aggressiveness human nerve's colloids
The unfavorable factor of knurl.
The table expression of 1 EGFR and EGFRv III in glioma
2 Patients with gliomas EGFRv of table III expresses the correlation with clinical pathologic characteristic
The western-blot identifications of 4 anti-III monoclonal antibody of EGFR mutant of embodiment
Extract each total protein of cell, Bradford standard measures.After loading, after 10%SDS-PAGE electrophoresis, electricity is walked around and is moved to
On pvdf membrane.Skimmed milk power under room temperature with 5% is added dropwise corresponding one and resists 4 DEG C overnight after closing 2h, wash film, horseradish peroxidating is added dropwise
Two anti-room temperatures 2h of thing enzyme mark, carry out chemiluminescence detection after rinsing, image.Can recognize simultaneously used in experimentation
The EGFR antibody of III albumen of EGFR and EGFRv and only III antibody of EGFRv of identification III albumen of EGFRv.This experiment purpose is used for reflecting
Determine clone F98, F98EGFR、F98EGFRvⅢ, U251 and U251EGFRvⅢIn III albumen of EGFRv stable expression.
As a result as shown in figure 5, the monoclonal antibody western-blot qualification result of the present invention, by western-blot
Identification, monoclonal antibody specificity ground with III protein combinations of EGFRv, and with Wild type EGFR no cross reaction.Wherein, M is egg
White molecular weight standard (kDa).F98EGFRvⅢ、F98EGFRWith tri- kinds of cells of F98 be respectively III protein expressions of EGFRv positive, negative and
Negative brown rat brain glioblastoma cell system, is purchased from American Type Culture collection warehousing (ATCC).U251 is expression wild type
EGFR is positive, U251EGFRvⅢIt is the human glioma cell line for expressing III protein positives of EGFRv, is purchased from Beijing Union Medical College
Cell resource center, surely turns U251EGFRvⅢCell.
5 reverse transcription-polymerase chain reaction of embodiment (RT-PCR) and DNA sequencing analysis
The expression of wild-typ EGFR and EGFRv III can be more preferably verified in RT-PCR reactions.Total-RNA is carried from cell
Rear reverse transcription is taken for cDNA, PCR-system expands purpose fragment.III primer sequence of EGFR and EGFRv for RT-PCR reactions
For same sequence：(Forward) 5'-CTT CGG GGA GCA GCG ATG CGA C-3' and (Reverse) 5'-ACC AAT
ACC TAT TCC GTT ACA C-3'.Pcr amplification reaction condition is as follows：95℃1min；95℃15s、60℃15s、72℃
30s, 72 DEG C of 7min, 4 DEG C of ∞, totally 40 circulations.Gapdh is used as internal reference simultaneously.Question response terminates rear 2% agarose gel electrophoresis
After detection, the further sequence verification of PCR primer.Each PCR primer carries out two-way sequencing, compares through sequencer map, judges corresponding sample
This whether producer mutation, and to exclude be the situation of gene pleiomorphism.
As a result as shown in fig. 6, the cementing fruit of the RT-PCR races of the present invention shows：F98EGFREGFR is detected with U251 cells
Gene expression (1044bp), F98npEGFRvⅢCell detection is to III gene expressions of EGFRv (243bp), and U251EGFRvⅢCell is simultaneously
Expression EGFR (1044bp) and EGFRv III (243bp) gene.Normal F98 clones are not detected by any gene expression.Sequencer map
Spectrum can also clearly observe the mutational site of mutant egf Rv III.
The immunofluorescence of the monoclonal antibody of 6 anti-EGFRv III of embodiment and the rabbit source polyclonal antibody of existing EGFRv III
Experiment
The immunofluorescence experiment result of the rabbit source polyclonal antibody of existing targeting EGFR v III is as shown in fig. 7, in the present invention
The immunofluorescence experiment result of the monoclonal antibody of anti-EGFRv III is as shown in Figure 8.The rabbit source of the targeting EGFR v III on market is more
The immunofluorescence experiment of clonal antibody, specific poor, the targeting EGFR v of the rabbit source polyclonal antibody of targeting EGFR v III
III rabbit source polyclonal antibody, also can be in conjunction with wild type F98EGFR in addition to can be in conjunction with EGFRv III.The rabbit of targeting EGFR v III
There is cross reaction with wild type F98EGFR in source polyclonal antibody.The monoclonal antibody of the anti-EGFRv III of the present invention has good
Target specificity, which is combined with EGFRv III, and wild type F98EGFR of getting along well is combined, and the monoclonal of the anti-EGFRv III resists
Body and wild type F98EGFR no cross reaction.Therefore, the specificity of the monoclonal antibody of the anti-EGFRv III used in the present invention
Good, can be very good specific to be combined with EGFRv III.
The preparation of 7 anti-III monoclonal antibody of EGFR mutant of embodiment (is documented in document Oncotarget.2016Dec
22.doi:10.18632/oncotarget.14088. in)
(1) foundation of hybridoma cell line
Step one, experiment material prepare：
1st, immunogene：With EGFRv III as target, three amino acid polypeptides are prepared by the way of chemical synthesis, wherein
The sequence of three amino acid polypeptides is respectively LEEKKGNYVVTDHC；EKKGNYVVTDHC；KKGNYVVTDHC.Every amino acid
The purity requirement of polypeptide is more than 90%, and three polypeptides are coupled with KLH and are prepared into immunogene.
2nd, culture medium：DMEM culture mediums are purchased from Hyclone companies；It is public that HAT, HT Selective agar medium, norphytane are purchased from sigma
Department.
3rd, animal used as test：Three Balb/c mouse are numbered respectively and are：1#, 2#, 3#, are 8-12 week old, female, SPF levels
Animal is cultivated.
4th, other materials：Freund's complete adjuvant, freund 's incomplete adjuvant are purchased from Sigma companies；PEG4000 is purchased from Fluka
Company；HRP- goat anti-mouse IgG antibodies are purchased from Jackson Immune companies；Remaining reagent is domestic pure analysis pure product.
Step 2, animal immune
1) fundamental immunity：Antigen is mixed with Freund's complete adjuvant equal-volume and fully emulsified, branch hypodermic injection, per only
Balb/c mouse per injections amount is 80-120 μ g, preferably 100 μ g.
2) booster immunization：Booster immunization is using antigen and the emulsion of freund 's incomplete adjuvant.3 before cell fusion is carried out
My god, through normal saline solution of the lumbar injection containing 100-200ug antigens, the preferably normal saline solution of 150ug antigens.
3) tail blood examination is surveyed：The tail blood for taking immune mouse carries out indirect elisa method detection.The operating procedure of indirect elisa method
As follows：The wrapper sheet of three polypeptide 100ng is respectively taken, is resisted as one using the tail blood of the mouse after three immunity and is incubated, every afterwards
Hole adds 1:100 μ l of 2000HRP- goat anti-mouse IgGs, finally determine 450nm OD values.
As a result：Using the tail blood of 2# immune mouses as an anti-450nm OD readings to all of three polypeptides reactives as most
High.Therefore the splenocyte from 2# immune mouses carries out the preparation of hybridoma.
Step 3, the preparation of hybridoma
The splenocyte and SP2/0 cells for collecting mouse according to a conventional method presses 20:1 to 5：1 ratio is with 400-600g/L's
PEG4000 is merged.The splenocyte of mouse and the ratio of SP2/0 cells, preferably 10:1, PEG4000 preferably concentration
For 500g/L.Culture is selected with HAT nutrient solutions, 10～15 days after fusion, supernatant is taken anti-using indirect elisa method screening secretion
Former hybridoma cell strain.Gained positive colony hybridoma cell strain is subcloned using limiting dilution assay.Indirect ELISA
The operating procedure of method is as follows：The wrapper sheet of three polypeptide 100ngs is respectively taken, with immune mouse tail blood 1:2000 used as positive control, nothing
The culture medium supernatant Normal Mouse Serum of clonal growth adds 1 as negative control per hole:2000HRP- goat anti-mouse IgGs
100 μ l, finally determine 450nm OD values.All OD450Value is more than more than 2 times persons of negative control, you can preliminary judgement is positive gram
Grand.
Step 4, the foundation of hybridoma cell line
Repeat step 2, carries out 2 cell fusions, screens through 4 subclones and indirect ELISA, obtains 4 plants stable point
Secrete the hybridoma cell line of the monoclonal antibody of anti-EGFR mutant III (EGFRv III), four plants of monoclonal antibody hybridoma cells
The numbering difference of system：4G1、4D12、7C7、1G8.
Step 5, the bioactivity for applying above-mentioned hybridoma cell line gained monoclonal antibody
1) cell culture supernatant titration：Indirect elisa method detects that above-mentioned Hybridoma Cell Culture supernatant potency is：
1：50000～1：100000.Wherein most strong with the signal of 4G1.
2) mouse ascites titration：Indirect elisa method detects that titer of ascites prepared by above-mentioned hybridoma is：1：
500000～1：1000000.Wherein most strong with the signal of 4G1.
Step 6, the Secondary Culture of hybridoma cell line
Above-mentioned hybridoma is tied up to and is proceeded to cultivate in the DMEM culture mediums containing 10% hyclone, is passed on,
After cultivating for 10 generations, hybridoma cell line remains able to well-grown, stably passes on, and nutrient solution supernatant potency still can reach
1:More than 10000.Gained hybridoma cell line of the present invention stably can be passed on, and can continue, the list of the anti-EGFRv III of stably excreting
Clonal antibody.
After obtaining the hybridoma of the monoclonal antibody for producing required, a part of hybridoma is preserved.Preservation side
Method is as follows：Prepare material, cell：Take the logarithm the cell in growth period；(dimethyl sulfoxide (DMSO) can be damaged 10% dimethyl sulfoxide (DMSO) protection liquid
Filter, and destroyed by high pressure, so can not filter or autoclave sterilization.Itself it is exactly drugs, aseptic)：Contain 10% diformazan
Base sulfoxide, 20% inactivated fetal bovine serum, 70%RPMI-1640 liquid；20%FBS-1640 nutrient solutions：100U/ml containing penicillin, chain
100 μ g/ml of mycin；2ml ampullas of sterilizing etc..
2nd, method of operating, (1) remove the old nutrient solution in Tissue Culture Flask, add 10%FBS-1640 liquid, make cell
Suspend.(2) 1000rpm/min centrifugations 10min, removes supernatant.Cell precipitation makes suspension with 10% dimethyl sulfoxide (DMSO) protection liquid, makes
Into 1.0 × 107Cell/ml.(3) sample, platform expects blue dyeing, and living cell counting should be more than 95%.(4) will be thin with syringe
Born of the same parents dispense ampulla, every bottle of 0.5ml～1.0ml, sealing by fusing ampulla.(5) 4 DEG C × 2h is put.(6) liquid nitrogen container gaseous parts (- 70 DEG C) are put
15h.(7) liquid nitrogen part is proceeded to.
(2) preparation of the monoclonal antibody of anti-EGFRv III
Select BALB/c mouse of growing up, intraperitoneal inoculation norphytane, every mouse 0.5ml.7-10 days pneumoretroperitoneum inoculation numberings
For the hybridoma of 4G1, every mouse 1 × 106～2 × 106Individual.Treat that belly substantially expands, touch after 5 days in interval
When, skin has tension, you can gather ascites with No. 9 syringe needles.
Ascites is centrifuged (13000rpm/min 30 minutes), cell component and other sediments is removed, is collected supernatant.
Purified with Protein G-Sepharose CL-4B, PBS of the upper prop liquid for 20mM, column chromatography eluent is：pH
The glycine buffer of=2.7,20mM, obtains the monoclonal antibody stoste (5.3 μ g/ μ l of concentration) of anti-EGFRv III.
Attached：Part abbreviation, English and Key Term definition：
EGFR：epidermal growth factor receptor.
EGFRvⅢ：epidermal growth factor receptor variantⅢ.
PBS：Phosphate buffer.
Term " SABC " refers to the principle combined by applied immunology general principle, i.e. antigen with antibody specificity,
The chromogenic reagent of labelled antibody is made by chemical reaction to determine histocyte endoantigen, which is carried out positioning, sxemiquantitative is ground
Study carefully.
Term " transfer " refers to tumour cell and invades lymphatic vessel, blood vessel or other organs from original site, is formed at original
The tumour of region tumors same type is sent out, this process is referred to as shifting.
Term " puncture " refers to be exactly by this apparatus of hollow needle, to the suspicious lesions of organ (such as：Lump, thicken
Region, calcification etc.) puncture take out portion of tissue checked.
Term：" tumour " refers to body under various factors effect, and some cell of local organization is in gene level
On lose the normal regulation grown by which, cause its clonal abnormality hyperplasia and the abnormality that formed.Educational circles is typically by tumour
It is divided into benign and malignant two big class.
Term " patient " is referred to through radical tumorectomy or the patient of partial eradication tumorectomy.
Term " tumor specimen " is referred to by performing the operation or puncturing the breast tumor tissues for obtaining, and the tissue then carries out solid
Fixed, dehydration, transparent with waxdip, FFPE and the tumor specimen tissue for SABC detection is obtained through paraffin section again.
Claims (5)
1. a kind of SABC antibody reagent of detection EGFRv III, it is characterised in that：Anti- including anti-III monoclonal of EGFR mutant
Body and antibody diluent；The antibody diluent is produced by doctor's moral Bioisystech Co., Ltd, and its commodity article No. is AR1016,
Trade name antibody diluent.
2. the SABC antibody reagent of detection EGFRv III according to claim 1, it is characterised in that：The anti-EGFR
III monoclonal antibody of mutant is 1 with the volume ratio of antibody diluent:1～5000.
3. the SABC antibody reagent of detection EGFRv III according to claim 2, it is characterised in that：The anti-EGFR
III monoclonal antibody of mutant is 1 with the volume ratio of antibody diluent:1000～4000.
4. a kind of ImmunohistochemistryMethods Methods of detection EGFRv III, it is characterised in that：Using any one of claim 1～4
The SABC antibody reagent of detection EGFRv III, detects the EGFRv's III in sample tissue to be detected by ImmunohistochemistryMethods Methods
Expression, obtains testing result.
5. ImmunohistochemistryMethods Methods of detection EGFRv III according to claim 4, it is characterised in that：The testing result is
The expression value of EGFRv III, is drawn by the methods of marking of following SABC：The expression value of EGFRv III=cell membrane dye
Intensity of colour score value × cell membrane dyeing score value+cytoplasm staining power score value × cytoplasm dyeing score value；
The cell membrane staining power score value is the score value of staining power on cell membrane；
The cytoplasm staining power score value is the score value of staining power on cytoplasm；
The score value marking of the staining power is according to being：It is unstained=0, weak dyeing=1, moderate stain=2, strong dyeing=3；
The cell membrane dyes the marking foundation of score value：0%～5% corresponding score value of cell membrane staining cell number is 0；Cell
6%～25% corresponding score value of film staining cell number is 1；26%～50% corresponding score value of cell membrane staining cell number is 2；Carefully
51%～100% corresponding score value of after birth staining cell number is 3；
The marking of the cytoplasm score value is according to being：It is 0 for 0%～5% corresponding score value of cytoplasm staining cell number；Cytoplasm
6%～25% corresponding score value of staining cell number is 1；26%～50% corresponding score value of cytoplasm staining cell number is 2；Cell
51%～100% corresponding score value of matter staining cell number is 3；
When the expression value of EGFRv III is more than 4 points, it is judged as " EGFRv III is expressed as the positive ", when the expression value of EGFRv III
Less than 4 timesharing, it is judged as " EGFRv III is expressed as feminine gender ".
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