CN105467133A - Application of EHD2 antibody in preparation of ovarian cancer immunohistochemical detection reagent - Google Patents
Application of EHD2 antibody in preparation of ovarian cancer immunohistochemical detection reagent Download PDFInfo
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- CN105467133A CN105467133A CN201510995383.5A CN201510995383A CN105467133A CN 105467133 A CN105467133 A CN 105467133A CN 201510995383 A CN201510995383 A CN 201510995383A CN 105467133 A CN105467133 A CN 105467133A
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Abstract
The invention belongs to the technical field of biologics, and particularly relates to application of an EHD2 antibody in preparation of an ovarian cancer immunohistochemical detection reagent. The application is characterized by comprising preparation of a specific EHD2 antibody and preparation of the ovarian cancer immunohistochemical detection reagent, wherein the preparation of the specific EHD2 antibody is characterized in that the GeneBank international sequence number of a gene EHD2 is: NM_014601, the GeneBank international sequence number of a protein encoded by the gene EHD2 is: NP_055416, the specific EHD2 antibody is an antibody specific to the EHD2 protein of a human body and can specifically identify the EHD2 protein of the human body, and the amino acid sequence of an identification site is: 503-SEQ ID NO: 1-543; the preparation of the ovarian cancer immunohistochemical detection reagent is characterized in that the ovarian cancer immunohistochemical detection reagent is used for detecting the expression level and the location of the EHD2 in an ovarian cancer tissue, and the antibody specific to the EHD2 protein of the human is used as a primary antibody or a core antibody.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the application of a kind of EHD2 antibody in preparation oophoroma SABC detection reagent.
Background technology
Malignant tumour is common disease and the frequently-occurring disease of serious harm human life and health, and be cause one of disabled and early dead principal disease, in 35 ~ 59 years old age group, malignant tumour occupies the cause of the death first.Data shows, and China's malignant tumour year morbidity number of cases is 2,000,000, death about 1,500,000, and with 3% speed increase and in rejuvenation trend, occupy mortality ratio in various diseases.
Oophoroma is a kind of malignant tumour of ovarian neoplasm, and refer to the malignant tumour of growth on ovary, wherein 90% ~ 95% is the cancer of ovarian primary, and other 5% ~ 10% is that the metastasis of cancer at former of other position is to ovary.The incidence of disease of oophoroma occupies the 3rd of gynecologic malignant tumor lower than cervical carcinoma and carcinoma of endometrium, but mortality ratio exceedes cervical carcinoma and carcinoma of endometrium sum, is in gynecological cancer first place, is the maximum illness of serious threat WomanHealth.Although chemotherapy occupies very important status in the treatment of malignant tumour, in clinical practice, result is often not fully up to expectations.At present, the Tumor Heterogeneity problem comprising oophoroma earns widespread respect, even if tumour in clinical manifestation or on traditional pathological roughly the same, the molecule root of its morbidity is also not quite similar, this just requires more in depth to probe into tumour from molecular pathology level, improves the precision of diagnosis and treatment.At present, utilizing immunohistochemical method to carry out histology to crucial tumor correlated albumen is the important step being related to the accurate diagnosis and treatment of tumour.
Immunohistochemistry technology's applied immunology antigen-antibody reaction ultimate principle, by the specific recognition of anti-body to body internal protein molecular antigen, and the colour developing etc. of the second antibody of chemical labeling (fluorescein, enzyme, metallic ion, isotope etc.) is used for test set and knits expression and the Subcellular Localization thereof of cell internal object antigen (peptide and protein).In recent years, along with the development of immunohistochemistry technology and the successful research and development of each strain specific antibodies, deepening continuously more importantly to tumor cells mechanism understanding, SABC receives general accreditation at the molecular diagnosis of tumour with the using value in discriminating.
The application's early-stage Study finds that the expression of EHD2 albumen is relevant to the grade malignancy of oophoroma, and the EHD2 specific antibody of independent research can be used for the SABC detection of oophoroma.EHD2 (Epsinhomology (EH)-domain-containingprotein2) is the one of EHD albumen, is a class new membrane transhipment modulin, the EH domain containing high conservative.Participate in the multistep regulation and control of the after birth transhipments such as acceptor endocytosis, it is the requisite important step of whole after birth transporting mechanism, molecule and the key effect such as matter transportation and intracellular signaling is played in various kinds of cell, its imbalance will cause cell signal to reply improper and cell functional disorders, and possible and then diseases induced.Therefore carry out SABC to EHD2 to detect and reflect the alienation essence of tumour and the clinical characteristics of individual patients by more objective and accurate.At present, the SABC reagent prepared by EHD2 specific antibody has no report, and the application of EHD2 antibody in preparation oophoroma SABC detection reagent also beyond example.
For solving the problem, the invention discloses the application of a kind of EHD2 specific antibody in preparation oophoroma SABC detection reagent.
Although existing commercialization EHD2 antibody has more than ten to plant, thered is provided by main flow antibody reagent company SantaCruz and Abcam etc., but all belong to fundamental research reagent, have no basis and show that these antibody equally with our antibody can have qualified specificity and can be used for the detection of oophoroma SABC, particularly at present still verifying without any other EHD2 antibody through the SABC of a large amount of tissue specimen and demonstrate can by detecting the object reaching and Ovarian Cancer degree and survival of patients situation are carried out to prognosis anticipation to the histocyte expression of EHD2.They have following defect compared with present patent application antibody:
1, according to general knowledge known in this field, the antibody that can be used in SABC (IHC) can be made and clearly be identified and indicate in its operation instruction, by these antibody explanation shown in, great majority all can not be used for SABC (IHC) and detect.Although individual antibody is indicated can be used for SABC (IHC), carefully study discovery really not so.One is the EHD2 antibody of Nanjing Sen Beijia corporate agent, but has indicated and can only detect film location.Another is the article No. of Proteintech company is the antibody of 11440-1-AP, but the specificity of this antibody existing problems: the main signal position that first immune-blotting method obtains is significantly less than 60kD, and the normal position signal of 70kD is very weak, although illustrate and can EHD2 be detected, what detect is other unknown molecular more; Secondly this antibody delivered from company is the non-specific painted of obvious matrix components to visible stain signal the SABC figure of ovarian cancer tissue.
2, these antibody are research reagent, not through the test of large sample clinical case or checking, do not have the crucial celluar localization detectability to EHD2.
3, strict Western blotting specificity verification is not all passed through, particularly verify with the immunoblot experiment of the cross reaction of homologous protein EHD1, EHD3, EHD4 of EHD2 albumen, and these checkings are even more important concerning the method for immunohistochemical detection of EHD2, because have very high homology (>70%) between them.China's document " EHD2 lowers the research promoting that galactophore epithelial cell transforms " (Tian Gang etc., modern medicine inspection magazine, 27th volume the 1st phase in 2012,49-51) disclose one by its homemade EHD2 antibody, this antibody be adopt we not through the antibody of antigen purification, may be used for immune-blotting method, but the high degree of specificity requirement of SABC detection can not be met.
The application's early-stage Study finds that the expression of EHD2 albumen produces mutation with being positioned in oophoroma, and the SABC that the EHD2 specific antibody of independent research can be used for oophoroma detects.EHD2 (Epsinhomology (EH)-domain-containingprotein2) is the one of EHD albumen, is a class new membrane transhipment modulin, the EH domain containing high conservative.Participate in the multistep regulation and control of the after birth transhipments such as acceptor endocytosis, it is the requisite important step of whole after birth transporting mechanism, molecule and the key effect such as matter transportation and intracellular signaling is played in various kinds of cell, its imbalance will cause cell signal to reply improper and cell functional disorders, and possible and then diseases induced.Therefore carry out SABC to EHD2 to detect and reflect the alienation essence of tumour and the clinical characteristics of individual patients by more objective and accurate.At present, the SABC reagent prepared by EHD2 specific antibody has no report, and the application of EHD2 antibody in preparation oophoroma SABC detection reagent also beyond example.
For solving the problem, the invention discloses the application of a kind of EHD2 specific antibody in preparation oophoroma SABC detection reagent.
Summary of the invention
For solve in background technology mention being mainly also based on histopathology level to the differentiation of tumour clinically carries out routine morphological observation to tumor tissues and tumour cell at present, affect larger by human factors such as Pathology Doctors ' experiences, lack the problem of the deeper molecule foundation relevant to individual patient pathogenic factors, the present invention discloses the application of EHD2 specific antibody in preparation oophoroma SABC detection reagent.
The technical matters that the present invention solves is achieved through the following technical solutions:
EHD2 antibody detects the application in reagent in preparation oophoroma SABC, it is characterized in that, comprises the preparation of EHD2 specific antibody and the preparation of oophoroma SABC detection reagent.
The preparation of described EHD2 specific antibody, comprising:
The international sequence numbering of GeneBank of gene EHD2 is: NM_014601, and the international sequence numbering of GeneBank of described gene EHD2 encoding proteins is: NP_055416.
Described EHD2 specific antibody is a kind of antibody special to people EHD2 albumen, and described antibody capable specific recognition people EHD2 albumen, the amino acid sequence of described recognition site is: 503-SEQIDNO:1-543.
One peptide species, its amino acid sequence is SEQIDNO:1, with described polypeptid acid sequence for core sequence is modified it, described in be modified to and hold connection halfcystine at polypeptide N; Described polypeptide after modification is purified carries out immunity to prepare EHD2 antibody and to carry out antigen purification as antigen to animal afterwards.
Described oophoroma SABC detects the preparation of reagent, comprising:
Oophoroma SABC detects reagent, for detecting the expression of EHD2 in ovarian cancer tissue and location, is adopt the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.
Beneficial effect
The invention discloses the application of EHD2 antibody in preparation oophoroma SABC detection reagent, the multistep of transporting due to EHD2 albumen participation acceptor and after birth regulates and controls, and in various cell, maintenance matter transportation and intracellular signaling etc. are played a crucial role, closely related with the generation of the disease such as tumour.Therefore carrying out SABC to EHD2 to detect more objective and accurate from the alienation essence of molecular level reflection tumour and the clinical characters of individual patients, meets accurate medical principle and the trend of tumour.
EHD2 antibody disclosed by the invention detects the application in reagent in preparation oophoroma SABC, using to the special antibody of people EHD2 albumen as primary antibodie or core antibody, preparation oophoroma SABC detects reagent, and the reagent specificity prepared is good, highly sensitive, colour rendering is good.
Accompanying drawing explanation
Fig. 1 is western blot figure;
Fig. 2 is SABC figure under the ovarian cancer tissue's 200 times of mirrors in EHD3 slurry under weak expression;
Fig. 3 is SABC figure under the ovarian cancer tissue's 200 times of mirrors in EHD3 slurry under high expressed;
Fig. 4 is ovarian cancer patients survival analysis figure.
Embodiment
Antibody prepares embodiment 1: to the preparation of EHD2 specific antibody
1) experiment material source
New England's White Rabbit is purchased from Shanghai and shines by force biology, polypeptide
CDEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE customized by Tianjin Purcell Biological Technology Co and with KLH coupling, CNBr activation gel beads, FuShi Freund's complete adjuvant and FuShi Freund's incomplete adjuvant are purchased from Invitrigen company.
2) animal immune
Get 4 monthly age new zealand white rabbit 3,100ug antigen polypeptide is dissolved in 0.2ml0.1MPBS (pH7.2), fully mix with equal-volume FuShi Freund's complete adjuvant, subcutaneous abdomen multi-point injection.First time the immune rear 15th and 29 days, fully mix rear booster immunization with 100ug polypeptide/0.2mlPBS with equal-volume FuShi Freund's incomplete adjuvant respectively.
3) the sero-fast preparation of EHD2
After immunity terminates, one week arteria carotis gets blood, and 37 DEG C leave standstill centrifuging and taking serum after 3 hours.
4) antigen gel beads preparation
The gel beads of CNBr activation soaks 30 minutes in 1mMHCl, with coupling buffer (containing 0.1MNaHCO3pH8.3, and 0.5MNaCl) cleaning, add the ratio hybrid reaction system of 1ml gel in 1mg polypeptide, 4 degree of couplings are spent the night, 1M monoethanolamine soaked after 3 hours uses cleaning fluid 1 (containing 50mMTris, 1MNaCl, pH8.0) and cleaning fluid 2 (containing 50mM glycocoll, 1MNaCl, pH3.5) intersection cleaning eight times, PBS cleans 1 time.
5) purifying of EHD2 antibody
Serum and above-mentioned antigen gel beads mixing by volume for 20:1, add and mix the rear isopyknic PBS of system, mixing centrifugal after 1 hour, gel beads is cleaned with PBS, be associated in the antibody in gel beads with pH3 sodium citrate solution wash-out, adjust pH to 6.5-7.5, obtain the EHD2 antibody after purifying.
Detect embodiment 1: adopt Western blot to carry out specific detection to EHD2 antibody
1) experiment material source:
293T cell is purchased from U.S. AmericanTypeCultureCollection (ATCC) company, RPMI1940 nutrient solution, BSA, HRP mark anti-rabbit two are anti-, Lipo2000 transfection reagent, RIPA lysate, BCA protein concentration detect reagent, ECL chemiluminescence detection reagent etc. and be purchased from Invitrogen company, and EHD1, EHD2, EHD3, EHD4 expression plasmid is self-control.
2) cell chulture
293T cell chulture in RPMI1940 nutrient solution, 37 DEG C, 5%CO
2cultivate adherent growth; First discard nutrient solution during passage, then use phosphate buffer (phosphatebufferedsaline, PBS) to wash twice, add 0.05% Trypsin Induced 2 minutes, add nutrient culture media and stop digestion.Cell maintains a good state, and within two days, passes a generation.Add the plasmid and transfection reagent of expressing EHD1, EHD2, EHD3, EHD4 during transfection respectively, collect cell after 2 days and do immunoblot experiment.
3) western blotting method
Various cell is reserved sufficient amount in centrifuge tube, centrifugal rear RIPA lysate cell lysis, boils sample, more centrifugal.After obtained sample, BCA detection reagent is used to carry out the mensuration of protein concentration.Each sample is got 80ug Tot Prot respectively and is carried out SDS-PAGE electrophoresis.After electrophoresis terminates, forward on pvdf membrane by glue setting egg(s) white appliances, room temperature 5% milk closes 1 hour.After washing, with primary antibodie (antibody prepared in embodiment 1 adds PBS and 5%BSA with 1:2000) incubated at room 1 hour.The anti-incubated at room of the anti-rabbit two of then diluting with 1:5000 1 hour.Finally detect with chemiluminescence detection reagent.
4) result: above-mentioned antibody has the specificity of height to EHD2 albumen, shows as and only just has signal for EHD2, and can not identify other EHD albumen of very high homology.Testing result is shown in Fig. 1.In figure: sample Ve is empty expression vector, no signal; Sample 1 process LAN EHD1 albumen, no signal; Sample 2 process LAN EHD2 albumen, be a master tape in corresponding molecular weight 70kd position, signal is clear, not other obvious background band; Sample 3 process LAN EHD3 albumen, no signal; Sample 4 process LAN EHD4 albumen, no signal.To sum up, the antibody that the present embodiment provides has strong signal at 70kd place, normal position, and in the equal no signal in other positions, result shows that antibody provided by the invention has good specificity.
SABC detects embodiment 1:EHD2 Immunohistochemical detection
1) experiment material source:
Tianjin Tumour Hospital's tumor tissues sample storehouse is taken from oophoroma section, conventional dewaxing.General two anti-, diaminobenzidine (DAB) substrate, substrate dilution etc. that primary antibodie dilution, horseradish peroxidase (HRP) mark are purchased from Zhong Shan Golden Bridge.
2) SABC detects EHD2 in reagent assembly condition and sample tissue and expresses the detection method with location:
Method key step is: histotomy dewaxing is to water, and antigen retrieval, Inner source peroxidase blocks, and drips embodiment (1) antibody for preparing as primary antibodie, 4 DEG C of overnight incubation using 1:200.3 each 5min washed by damping fluid.Drip general HRP ELIAS secondary antibody, at room temperature hatch 30 minutes.3 each 5min washed by damping fluid.DAB develops the color, and redyes, basis of microscopic observation staining conditions after dehydration mounting.
3) result:
Painted in various degree in slurry of EHD2 in ovarian cancer patients histocyte, in slurry, the weak positive is shown in Fig. 2, paintedly by force in slurry sees Fig. 3.Complex clinical prognosis data, finds that the survival region of weak positive patient in slurry is significantly better than strong positive patient (Fig. 4) in slurry
Above experimental result shows, adopt and the invention provides the method for immunohistochemical detection that antibody is core, the expression of EHD2 in ovarian cancer tissue's cell can be detected well and express position, be convenient to location in cancerous tissue cell of from SABC figure direct interpretation EHD2 and expression, and differentiate the heterogeneous and survival region of the individuation of oophoroma on this basis.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature, and not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement etc., all should be included within protection scope of the present invention.
Claims (3)
1.EHD2 antibody detects the application in reagent in preparation oophoroma SABC, it is characterized in that, comprises the preparation of EHD2 specific antibody and the preparation of oophoroma SABC detection reagent;
The preparation of described EHD2 specific antibody, the international sequence numbering of GeneBank comprising gene EHD2 is: NM_014601, and the international sequence numbering of GeneBank of described gene EHD2 encoding proteins is: NP_055416; Described EHD2 specific antibody is a kind of antibody special to people EHD2 albumen, described antibody capable specific recognition people EHD2 albumen, and the amino acid sequence of described recognition site is: 503-SEQIDNO:1-543;
Described oophoroma SABC detects the preparation of reagent, comprises oophoroma SABC and detects reagent, for detecting the expression of EHD2 in ovarian cancer tissue and location, is adopt the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.
2. EHD2 antibody according to claim 1 detects the application in reagent in preparation oophoroma SABC, it is characterized in that, containing a peptide species, its amino acid sequence is SEQIDNO:1, with described polypeptid acid sequence for core sequence is modified it, described in be modified to and hold connection halfcystine at polypeptide N; Described polypeptide after modification is purified carries out immunity to prepare EHD2 antibody and to carry out antigen purification as antigen to animal afterwards.
3. EHD2 antibody according to claim 1 detects the application in reagent in preparation oophoroma SABC, it is characterized in that, the flushing after the preparation of described EHD2 specific antibody also comprises development step, redye, dry and sealing.
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