CN104945506A - Immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment - Google Patents

Abstract

The invention relates to an immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment, which is characterized in that a human-EHD2-protein-specific antibody is used as a primary antibody. The antibody can specifically recognize the human EHD2 protein, and the amino acid sequence of the recognition site is 503-SEQ ID NO:1-543.

Description

A kind of immunohistochemical methods reagent for breast cancer diagnosis and Index for diagnosis

Technical field

The present invention relates to biological technical field, specifically the expression of EHD2 and the existence of Subcellular Localization and patient with breast cancer have significant correlation, can as a molecular indexes of mammary cancer grade of malignancy diagnosis and prognosis anticipation.

Background technology

Mammary cancer is women's common cancer.The whole world has 1,000,000 women ill every year, and from late 1970s, the sickness rate of China's mammary cancer occupies the first place of female tumor always.North America, Northern Europe are former is the district occurred frequently of mammary cancer, the sickness rate of present China is also increased to 52/100000ths of last year from 100,000 before 5 years/17, in quick ascendant trend, and age of onset also more and more rejuvenation, what the age was minimum only has 14 years old.The factor causing mammary cancer is many-sided, except foreign genetic element also has the factors such as environmental factors, mental element and operating pressure are too large.And the city sickness rate that economy reaches all the more is more higher than rural area.The current countries in the world larger energy of input and material resources study generation, the development of this disease, to take positive preventive measures.

Clinically the grade of malignancy of tumour is differentiated that mainly being also based on histopathology level carries out routine morphological observation to tumor tissues and tumour cell at present, it has stronger resolving ability to the differentiation degree of cancer cells and infringement characteristic, and accuracy rate can reach 50%-75%.But, thisly to affect by human factors such as Pathology Doctors ' experiences on the one hand based on morphologic deciphering, lack the deeper molecule foundation relevant to individual patient pathogenic factors on the other hand, therefore need badly and combine with molecular approach, could improve the accuracy and specificity that judge, especially the selection of individualized treatment path and scheme provides theoretical foundation.

Although the generation of tumour is relevant to many factors; but final root mainly comes from the ANOMALOUS VARIATIONS of gene level; comprise the amplification of gene, sudden change, disappearance, dystopy etc.; the multiple functional proteins maintained needed for cell normal physiological condition are caused to change in the height, the power of biochemical activity, even subcellular structure distribution etc. of expression level; when this change imparts cell unlimited multiplication capacity and transfer ability; when can not be reversed by inside and outside protection mechanism or kill, just create tumour.Therefore, be the mutation situation of these functional protein molecules clear and definite to the heightened awareness of malignancy of tumor essence and the prerequisite of effectively control, this needs by immunohistochemical means.

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, metal ion, isotropic substance etc.) is used for test set and knits expression amount 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, immunohistochemical methods receives general accreditation at diagnosing tumor and the using value in discriminating.Such as the Her2 of estrogen receptor ER, progesterone receptor PR and Epidermal Growth Factor Receptor Family has been widely used in the clinical molecular pathological diagnosis of mammary cancer, and it serves positive directive function to the pathological classification of mammary cancer and clinical application.These achievements be unable to do without the progress of tumour fundamental research, and expect that new molecular indexes is disclosed further and includes clinical application in, improve constantly tumor diagnosis and treatment level.Namely EHD2 gene and proteins encoded thereof may be this new molecular indexes.

EHD2 (Epsin homology (EH)-domain-containing proteins) belongs to the one of EHD albumen, it is a class new membrane transhipment modulin, EH structural domain containing high conservative, this EH structural domain is a fragment containing 100 amino-acid residues of having an appointment, and is previously found in the kinase substrate Eps15 (Epdemal Growth Factor Receptor PathwaySubstrate Clone15) of EGFR.Each step control of EHD2 participation after birth transhipment, comprises the transition of interior turn and forward and backward inclusion body and regulates and controls circulation path; Each road of film and membranin regulates, and comprises digestion degraded and cyclic regeneration.It is the requisite important step of whole after birth transporting mechanism, to safeguarding that in various cellular activity the running balance of material transferring intracellular signaling plays a part key, its imbalance will cause signal response improper, cell functional disorders, and possible and then diseases induced.

China document " propagation of EHD2 disturbing influence immortalization mammary epithelial cell and migration " (Wang Hongyu etc., China's clinical tumor, 38th volume o. 11th in 2011) dysregulated gene expression that discloses EHD2 in breast tumor declines, and points out this gene may be a new mammary gland cancer suppressor gene.But we find in further immunohistochemical study, although EHD2 presents downward trend on overall expression amount, this trend is mainly in core; On the contrary, the expression in cytoplasm but has the trend of rising.And the Survival of the expression level of EHD2 in core and patient is closely related.Therefore carry out immunohistochemical methods to EHD2 to detect and reflect the alienation essence of tumour and the clinical prognosis situation of individual patients by more objective and accurate.

Existing commercialization EHD2 antibody has more than ten to plant, thered is provided by main flow antibody reagent company Santa Cruz 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 immunohistochemical methods and detect, particularly at present still verify without any other EHD2 antibody through the immunohistochemical methods of a large amount of tissue sample and demonstrate can by reaching the object of the grade of malignancy of tumour and survival of patients situation being carried out to prognosis anticipation to the ubcellular exception detection and localization 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 immunohistochemical methods (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 immunohistochemical methods (IHC) and detect.Although individual antibody is indicated can be used for immunohistochemical methods (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 immunohistochemical methods figure of cancerous lung tissue.

2, these antibody are research reagent, not through the test of large sample clinical case or checking, there is no the crucial nuclear location detectivity to EHD2, therefore just there is no precondition and the basis of " by the abnormal detection and localization of the ubcellular of EHD2 being reached to the object of the grade of malignancy of tumour and survival of patients situation being carried out to prognosis anticipation " yet.

3, strict immunoblotting 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 mammary 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 immunohistochemical methods detection can not be met.

Summary of the invention

We find in immunohistochemical study, and in normal epithelium cell, EHD2 is mainly distributed in nucleus, also have a small amount of expression in cytoplasm and film simultaneously.But in breast cancer tissue, mutation occurs for the location of EHD2, core distribution significantly declines, and slurry and film distribute the trend but occurring raising.Clinical factor analytical results shows, core distribution situation and patient's prognosis of EHD2 are closely related, and the Survival of the more weak then patient of nuclear expression is poorer.These grade of malignancy finding to show mammary cancer and the expression position of EHD2 albumen and the degree close association of expression level generation mutation in cancerous tissue cell, and immunohistochemistry technique is unique reliable means of the disorderly degree of this expression of detection.Therefore the method providing the expression of a kind of expression product of the EHD2 of detection gene in the inner different positions of cancer cells particularly core is the problem needing in prior art to solve.

In order to solve the problem, the invention provides following technique means and scheme:

An antibody special to people EHD2 albumen, it is characterized in that described antibody capable specific recognition people EHD2 albumen, the aminoacid sequence of described recognition site is: 503-SEQ ID NO:1-543.

Gene EHD2 and the application of proteins encoded in the anticipation of mammary cancer immunohistochemical method diagnosis and prognosis thereof, the international sequence numbering of GeneBank of described gene EHD2 is: NM_014601, and the international sequence numbering of GeneBank of described gene EHD2 proteins encoded is: NP_055416.

Described gene EHD2 and the application of proteins encoded in the anticipation of mammary cancer immunohistochemical method diagnosis and prognosis thereof, is characterized in that have employed afore mentioned antibodies.

Gene EHD2 and the application of proteins encoded in preparation mammary cancer immunohistochemical method diagnosis and prognosis anticipation reagent thereof, the international sequence numbering of GeneBank of described gene EHD2 is: NM_014601.

Described gene EHD2 and the application of proteins encoded in preparation mammary cancer immunohistochemical method diagnosis and prognosis anticipation reagent thereof, is characterized in that have employed afore mentioned antibodies.

One peptide species, is characterized in that described polypeptid acid sequence is SEQ ID NO:1.

Described polypeptide, preferably with aforementioned polypeptid acid sequence for core sequence is modified it, described in be modified to and hold connection halfcystine at polypeptide N.

Described polypeptide is preparing the application in afore mentioned antibodies.Described application preferably carries out immunity to prepare EHD2 antibody as antigen to animal.

The application of described polypeptide in the antibody that preparation is special to EHD2.Described application is preferably antagonist and carries out antigen purification.

A kind of for breast cancer diagnosis and Index for diagnosis immunohistochemical methods reagent, it is characterized in that adopting the aforementioned antibody special to people EHD2 albumen as primary antibodie or core antibody.

The term adopted in specification sheets of the present invention:

" proteins encoded of gene EHD2 " i.e. " EHD2 albumen ".It will be understood by a person skilled in the art that when mentioning " expression of EHD2 " in specification sheets, namely referring to " expression of gene EHD2 and proteins encoded thereof ".

" core sequence " is the 503 to the 543 amino acids sequence of people EHD2 albumen (genebank albumen NP_055416), polypeptide fragment corresponding to this core sequence after chemosynthesis or recombinant expressed after through or not modifiedly can be used for immunity to produce the antibody special to EHD2.

" modification " for polypeptide fragment corresponding to above-mentioned core sequence chemically or the ordinary method such as recombinant DNA carry out interpolations amino acid, conjugation chemistry group or purification media, object carries out antigen purification for immunity generation antibody or antagonist.

We find under study for action, expression product expression and distribution when mammary epithelial cell canceration of gene EHD2 gets muddled trend, the Survival close association of its disorderly degree especially nuclear expression situation and patient, and to adopt immunohistochemistry technique to carry out detecting and carry out artificial interpretation to result be understand unique reliable means that this gene different positions in cell expresses disorderly degree.But existing EHD2 antibody, how can not be used for immunohistochemical methods to detect, and claim that the antibody that can be used in immunohistochemical methods detection does not have immunoblotting evidence and shows qualified specificity, particularly can not get rid of the cross reactivity of the homologous protein to EHD2, and find in actual applications or only can produce reaction and EHD2 albumen in important core can not be identified by the EHD2 albumen in cell membrane, or the non-specific signals just to interstitial components detected.And existing antibody is all less than detecting and statistical study display has the characteristic that identifies that EHD2 nuclear expression is located and for the effect of prognosis anticipation organizing level to carry out large sample.We provide a kind of antibody through antigen purification special to EHD2 and preparing the application in Immunohistochemical detection reagent.EHD2 antibody provided by the invention has qualified specificity, can specific recognition EHD2 albumen and other albumen of very high homology can not be identified through western blotting method test, western blotting method can be adopted to carry out the detection of EHD2 protein quantification, immunohistochemical methods reagent can also be prepared into, be directly used in the expression and positioning scenarios that judge EHD2 in histocyte, especially may be used for the expression and localization situation of monitoring EHD2 in nucleus, thus can better judge for the diagnosis and prognosis of mammary cancer.

Accompanying drawing explanation

Fig. 1 is the western blot figure obtained in specific detection embodiment 1;

Fig. 2 is the immunohistochemical methods figure that immunohistochemical methods detects normal tissue cell in embodiment 1;

Fig. 3 is the immunohistochemical methods figure that immunohistochemical methods detects breast cancer cell in embodiment 1;

Fig. 4 is the immunohistochemical methods figure that immunohistochemical methods detects breast cancer cell in embodiment 1;

Fig. 5 is the immunohistochemical methods figure that immunohistochemical methods detects breast cancer cell in embodiment 1;

Fig. 6 is the Progression free survival graphic representation that immunohistochemical methods detects 260 routine samples in embodiment 1.

Embodiment:

Below in conjunction with drawings and Examples, the present invention will be described in detail.

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;

This polypeptide of peptide C DEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE(is that the polypeptide of SEQ ID NO:1 carries out modifying obtaining for core sequence with aminoacid sequence, and its modifying method is hold connection halfcystine at N.) to be customized by Tianjin Purcell Biological Technology Co and with KLH coupling;

The gel beads that CNBr activates, Freund's complete adjuvant and Freund's incomplete adjuvant are purchased from Invitrogen company.

(2) animal immune

Get 4 monthly age new zealand white rabbit 3,100ug antigenic peptide is dissolved in 0.2ml0.1M PBS (pH7.2), fully mixes with equal-volume 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 Freund's incomplete adjuvant respectively.

(3) the sero-fast preparation of EHD2

After immunity terminates, one week carotid artery 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 is soaked 30 minutes in 1mM HCl, with coupling buffer (containing 0.1M NaHCO3pH8.3, with 0.5M NaCl) cleaning, add the ratio hybrid reaction system of 1ml gel in 1mg polypeptide, 4 degree of couplings are spent the night, 1M thanomin soaks after 3 hours and contains 50mM Tris with scavenging solution 1(, 1M NaCl, pH8.0) and scavenging solution 2(containing 50mM glycine, 1M NaCl, pH3.5) intersection cleaning eight times, PBS cleans one 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 immunoblotting to carry out specific detection to EHD2 antibody

(1) experiment material source:

293T cell is purchased from American Type Culture Collection (ATCC) company of the U.S.; RPMI1940 nutrient solution, BSA, HRP mark anti-rabbit two are anti-, Lipo2000 transfection reagent, RIPA lysate, BCA protein concentration detection reagent, ECL chemiluminescence detection reagent etc. are purchased from Invitrogen company; EHD1, EHD2, EHD3, EHD4 expression plasmid is self-control.

(2) cell cultures

293T cell cultures in RPMI1940 nutrient solution, 37 DEG C, 5%CO2 cultivates adherent growth; First discard nutrient solution during passage, then use phosphate buffered saline buffer (phosphate buffered saline, PBS) to wash twice, add 0.05% tryptic digestion 2 minutes, add substratum 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 lysing cell, 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, detected result is shown in Fig. 3.Therefrom can find out, the antibody special to EHD2 provided by the invention can specific recognition EHD2 albumen, and can not identify other EHD albumen of homology.

(4) result: anti-EHD2 antibody has the specific immune recognition capability to EHD2 albumen, and does not produce cross reaction to other homologous protein.Fig. 1 is shown in by detected result picture, 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.

Immunohistochemical methods detects embodiment 1:EHD2 Immunohistochemical detection

(1) experiment material source:

Tianjin Tumour Hospital's tumor tissues sample storehouse is taken from mammary cancer section, and conventional dewaxing, sample size is 260.General two anti-, diaminobenzidine (DAB) substrate, substrate dilution etc. that primary antibodie diluent, horseradish peroxidase (HRP) mark are purchased from Zhong Shan Golden Bridge.

(2) in immunohistochemical methods detection reagent assembly condition and sample tissue, EHD2 expresses the detection method with location:

Method key step is: tissue slice 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:

In healthy tissues, EHD2 expresses the positive in epithelial nucleus, slurry or the weak positive of film expression.In cancerous tissue, EHD2 expresses and gets muddled, and nuclear expression tends to become weak.Typical Western group figure is shown in Fig. 2 ~ Fig. 5, Fig. 2 is the immunohistochemical methods figure of healthy tissues, demonstrate normal cell EHD2 at epithelial cell nuclear expression, Fig. 3 ~ Fig. 5 is the immunohistochemical methods figure of different breast cancer tissues sample, in the sample of breast cancer tissue shown in Fig. 3, EHD2 all has expression in cancer cells core with slurry, in the sample of breast cancer tissue shown in Fig. 4, EHD2 does not express in cancer cells core, strongly expressed in slurry; In the sample of breast cancer tissue shown in Fig. 4, EHD2 expresses overall disappearance in breast cancer cell, shows that EHD2 expresses in cancer cells and to get muddled and nuclear expression weakens.The Progression free survival graphic representation of 260 routine samples is shown in Fig. 6, this figure legend: 0 is that nuclear expression is negative; 1 is that nuclear expression is positive, and censored represents dead.X-coordinate is Progression free survival moon number, and ordinate is Progression free survival case percentage ratio.As can be seen from the figure its prognosis of case of nuclear expression feminine gender is obviously compared with the difference of the nuclear expression positive.

Above experimental result shows, adopt and the invention provides the method for immunohistochemical detection that antibody is core, the expression amount of EHD2 in breast cancer tissue's cell can be detected well and express position, being convenient to location in cancerous tissue nucleus of from immunohistochemical methods figure direct interpretation EHD2 and expression with anticipation mammary cancer grade of malignancy and survival of patients prospect.

Claims (1)

1. the immunohistochemical methods reagent for breast cancer diagnosis and Index for diagnosis, it is characterized in that adopting a kind of antibody special to people EHD2 albumen as primary antibodie or core antibody, described antibody capable specific recognition people EHD2 albumen, the aminoacid sequence of described recognition site is 503-SEQ ID NO:1-543.