Abstract:
An EHD2 antibody and an application thereof in the preparation of an immunohistochemical detection reagent for breast cancer. The antibody can be used for specifically recognizing a human EHD2 protein, and the amino acid sequence on a recognition site is: 503-SEQ ID NO:1-543.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the biotechnology field, and more particularly it relates to a significant correlation between the expression plus subcellular localization of EHD2 and the survival of patients with breast cancer, which can be used as a molecular functional marker for malignancy diagnosis of the disease and prognosis anticipation of breast cancer. 
       BACKGROUND OF THE INVENTION 
       [0002]    Breast cancer is one of the most common malignant tumors in women, one million women develop this disease worldwide every year. Since the late 1970s, the incidence rate of breast cancer has ranked first in tumors of female in China. North America and northern Europe used to be high incidence areas of breast cancer, but now the incidence rate has grown from 17 per 100, 000 of 5 years ago to 52 per 100, 000 of last year in China, showing a trend of rapid escalation, moreover, the age of onset is getting much younger than before, with the youngest one being only 14 years old. Factors that trigger breast cancer could be various, in addition to genetic factors there exist other factors such as environmental aspects, mental elements and extensive working pressure. Furthermore, a city whose economy is more developed, the incidence rate will be more higher than that in rural areas. Nowadays, most countries in the world have put considerable energy and material into investigating the occurrence and development of this disease, so as to adopt positive precautions. 
         [0003]    At present, discrimination of malignant degree of tumors is mainly based upon conventional morphological observation performed on tumor tissues and tumor cells at the histopathological level in clinical practice, which has a stronger ability to discriminate differentiation degrees and aggressive properties of cancer cells, with an accuracy of 50%-75%. However, on one hand, this interpretation based on morphology is affected by human factors, such as experience of pathologists, on the other hand, it lacks molecular evidences at a deeper level associated with individual patient&#39;s etiological factors, therefore, a combination with current molecular technologies is very necessary to increase the accuracy and specificity of judgement, especially to provide a theoretical foundation for individualized treatment pathways and protocol selection. 
         [0004]    Although the occurrence of tumor is related to multiple factors, the ultimate root comes mainly from abnormal changes at the genetic level, including amplification, mutation, deletion and translocation, etc. which might lead to abnormal changes of multiple functional proteins needed to maintain the normal physiological state of cells in expression levels, biological activities, and even subcellular distributions, and in case such changes endow cells with an ability to proliferate uncontrollably and metastasize, and these resultant cells cannot be reversed or killed by endogenous and exogenous protective mechanisms of a human body, tumor will appear. Therefore, the premise for a deep understanding of the malignant nature of tumor and an effective control of the disease is to clarify the abnormal characteristics of these functional proteins, which requires immunohistochemical means. 
         [0005]    Using basic principles of antigen-antibody interactions in immunology, through specific recognition of antibodies to protein molecule antigens in the body and color development of secondary antibodies with chemical labelling (fluorophore, enzyme, metal ion, isotope and etc.), immunohistochemical techniques are employed to detect the expression quantity and subcellular localization of the target antigen (polypeptide and protein) in tissues and cells. In recent years, with the development of immunohistochemical techniques and the successful research and development of various specific antibodies, more importantly with the continuously deep understanding of molecular mechanisms underlying tumors, the application value of immunohistochemistry in tumor diagnosis and identification has been generally recognized. For example, estrogen receptor (ER), progesterone receptor (PR) and Her2 of the epidermal growth factor receptor family have been widely used in the clinically molecular pathological diagnosis of breast cancer, which plays an active role of guidance in pathological classification and clinical medication of breast cancer. These achievements are inseparable from the advance in basic tumor research, it is expected that new molecular markers will be further revealed and incorporated into clinical application, thus continuously improving the diagnosis and treatment level of tumors. EHD2 gene and its coded protein may be such new molecular markers. 
         [0006]    EHD2 is one of the EHD proteins(Epsin homology [EH]-domain-containing proteins) that are members of a new category of membrane trafficking regulatory proteins, it contains a highly conserved EH domain which is a fragment of about 100 amino acid residues and was first discovered in EGFR Kinase substrate Eps15 (Epidermal Growth Factor Receptor Pathway Substrate Clone 15). EHD2 is involved in the regulation of multiple steps in cellular membrane transport, including internalization, transition between early endosomes and late endosomes and regulation of a cycling pathway. The regulation of various steps on membranes and membrane proteins, including digestive degradation and recycling for regeneration, is an indispensable important part in the whole mechanism of cellular membrane trafficking and plays a key role in maintaining the homeostasis of signal transduction and material transport in a variety of cellular processes, whereas its imbalance will lead to improper signal responses, cell functional disorders and probably in turn cause diseases. 
         [0007]    Chinese literature “ Effects of EHD 2 Interference on Proliferation and Migration of Immortalized Breast Epithelial Cells HBL100” (WANG Hongyu, et al.  Chinese Journal of Clinical Oncology,  2011, 38(11)) disclosed the a lower expression level of EHD2 gene in breast carcinoma, suggesting that EHD2 might be inhibitory to breast tumor progression. However, it is found from a further immunohistochemical study that although the overall expression quantity of EHD2 shows a downward trend, this trend mainly exists in nuclei; on the contrary, the expression in cytoplasms has an increasing trend. Moreover, the expression level of EHD2 in nuclei is closely related to the survival condition of patients. Therefore, immunohistochemical detection of EHD2 will more objectively and accurately reflect an alienated nature of tumors and individual patient&#39;s clinical prognosis conditions. 
         [0008]    There are over ten existing commercial antibodies to EHD2, which are provided by mainstream antibody reagent companies such as Santa Cruz and Abcam, but all these antibodies belong to reagents for basic research only, and there is no evidence in suggesting that these antibodies, like ours, have qualified specificity and can be used for immunohistochemical detection, especially at present there is still no other antibodies to EHD2 that are verified by means of immunohistochemistry on a large number of tissue samples and there are no results would showing that abnormal subcellular localization detection of EHD2 can achieve the goals of prognosis anticipation of malignant degree of tumors and survival condition of patients. Compared with the antibody in the present application, they have the following defects: 
         [0009]    1. According to common knowledge in the art, as for antibodies which can be used for immunohistochemistry (IHC), clear identification and indication will be provided in their user manuals. As indicated in instructions for these commercial antibodies, most of them cannot be used for immunohistochemical (IHC) detection. Although it is indicated in some instructions that individual antibodies can be used for immunohistochemistry (IHC), it is found that it is not the case after careful studies. One is an EHD2 antibody authorized to be sold by Nanjing SenBeiJia Biological Technology Co., Ltd., but the antibody is indicated only for membrane localization detection. The other is an antibody with the item No. 11440-1-AP from Proteintech, but the antibody has some problems in specificity: first, a main signal band location obtained from immunoblotting detection is significantly smaller than 60 KD, whereas a signal at the right location of 70 KD is weak, suggesting that it is able to detect EHD2, but also detect more other unknown molecules; second, it can be seen from their immunohistochemical pictures of a reaction of the antibody to lung cancer tissues published by the company that staining signals obviously belong to a nonspecific coloration of matrix components. 
         [0010]    2. All these antibodies are reagents used for research, are not tested or verified by means of a large sample of clinical cases, and have no key capacity to detect a nuclear localization of EHD2, therefore, they have no precondition and basis of “achieving the goals of prognosis anticipation of malignant degree of tumors and survival condition of patients by the abnormal subcellular localization detection of EHD2”. 
         [0011]    3. A strict specificity in immunoblotting for all these antibodies is not verified, especially the verification of cross-reactions with EHD1, EHD3 and EHD4 proteins homologous to EHD2 protein in immunoblotting experiments, which is very crucial to immunohistochemical detection methods of EHD2, due to the high homology between them (&gt;70%). 
         [0012]    Chinese literature “ Down - regulation of EHD 2  Enhanced Transformed Growth of Breast Epithelial Cell ” (TIAN Gang, et al.  Journal of Modern Laboratory Medicine,  2012, 27(1): 49-51) disclosed a self-made antibody to EHD2, which is prepared by using our method for preparing the antibody, but without antigen purification, and can be used for immunoblotting detection, but is not able to satisfy high specificity demands in immunohistochemical detection. 
       SUMMARY OF THE INVENTION 
       [0013]    It is found from the immunohistochemical studies that EHD2 is primarily distributed in a cell nucleus in normal epithelium cells, and expressed in cytoplasm and membrane at a low level. However, in breast cancer tissues, the localization of EHD2 changes abnormally, and its nuclear distribution drops significantly, whereas the cytoplasmic and membranous distribution shows a trend of escalation. Analytical results of clinical factors show that the nuclear distribution conditions of EHD2 are closely related to a prognosis of patients, that is, the lower the nuclear expression is, the worse the survival status of patients will be. These discoveries show that the malignant degree of breast cancer is closely associated with extents of abnormal changes in the expression location and level of EHD2 protein in cancer tissues and cells, and immunohistochemical techniques are the only reliable means to detect the extents of such expression disorders. Therefore, a problem to be solved in the prior art is to provide a method for detecting the expression conditions of expressed products of EHD2 gene at different locations inside cancer cells, especially in nuclei. 
         [0014]    In order to solve the above-mentioned problem, the present invention provides the following technical means and solutions: 
         [0015]    An antibody specific to a human EHD2 protein, characterized in that the antibody is able to specifically recognize a human EHD2 protein, and an amino acid sequence on a recognition site is: 503-SEQ ID NO:1-543. 
         [0016]    Use of EHD2 gene and its coded protein in diagnosis and prognosis anticipation of breast cancer by an immunohistochemical method, wherein an internationally universal sequence number for the EHD2 gene in GeneBank is: NM_014601, an internationally universal sequence number for the protein coded by the EHD2 gene in GeneBank is: NP_055416. 
         [0017]    The use of EHD2 gene and its coded protein in diagnosis and prognosis anticipation of breast cancer by the immunohistochemical method, characterized by using the aforementioned antibody. 
         [0018]    Use of EHD2 gene and its coded protein in the preparation of a reagent for diagnosis and prognosis anticipation of breast cancer by an immunohistochemical method, wherein an internationally universal sequence number for the EHD2 gene in GeneBank is: NM_014601. 
         [0019]    The use of EHD2 gene and its coded protein in the preparation of a reagent for diagnosis and prognosis anticipation of breast cancer by an immunohistochemical method, characterized by using the aforementioned antibody. 
         [0020]    A polypeptide, characterized in that an amino acid sequence for the polypeptide is SEQ ID NO: 1. 
         [0021]    Preferably, the polypeptide undergoes a modification with the aforementioned amino acid sequence as the core sequence, wherein the modification is achieved by connecting a cysteine to the N-terminal of the polypeptide. 
         [0022]    Use of the polypeptide in the preparation of the aforementioned antibody. Preferably, the polypeptide is used as an antigen to prepare an antibody to EHD2 by an immunization against animals. 
         [0023]    The use of the polypeptide in the preparation of the antibody specific to the EHD2 protein. Preferably, the use is to perform antigen purification of the antibody. 
         [0024]    An immunohistochemical reagent for diagnosis and prognosis judgment of breast cancer, characterized by using the aforementioned antibody specific to the human EHD2 protein as primary antibody or core antibody. 
         [0025]    In the Specification of the present invention, the following terms will be used: 
         [0026]    “Protein encoded by EHD2 gene” means “EHD2 protein”. It should be appreciated for those skilled in the art that when “expression of EHD2” is mentioned in the Specification, it means “expression of EHD2 gene and its coded protein”. 
         [0027]    “Core sequence” refers to the an amino acid sequence from position 503 to 543 in human EHD2 protein (the protein number in GeneBank is NP_055416), after chemical synthesis or recombinant expression, the resultant polypeptide fragment corresponding to the core sequence, with or without modification, can be used for immunization to produce the antibody specific to EHD2 protein. 
         [0028]    “Modification” means that the polypeptide fragment corresponding to the above-mentioned core sequence undergoes amino acid introduction, chemical group coupling or medium purification adopting common methods such as a chemical process and recombinant DNA technique, for the purpose of being used for immunization to produce the antibody or antigen purification of the antibody. 
         [0029]    It is found from the study that expression distribution of expressed products of EHD2 gene shows a trend of disorder in expression distribution if mammary epithelial cells become cancerous, and its extent of disorder, especially the expression condition in nuclei, is closely related to the survival condition of patients, and the detection using immunohistochemical techniques and artificial interpretation of results from the above detection are the only reliable means to learn the extent of expression disorder of this gene at different locations inside cells. However, most of the existing antibodies to EHD2 cannot be used for immunohistochemical detection, and there is no immunoblotting evidence showing that antibodies alleged to be able to be used for immunohistochemical detection have a qualified specificity, especially cross-reactions with homologous proteins of EHD2 protein cannot be excluded, in addition, it is found from the practical application that they may only react to the EHD2 protein in the cell membrane, while cannot recognize a valuable EHD2 protein in nuclei, or what they detect are just nonspecific signals from interstitial substance. Moreover, all the existing antibodies are not tested or statistically analyzed on the basis of a large sample size at the tissular level to show that they have the properties of recognizing the nuclear expression localization of EHD2 and the function of being used for prognosis anticipation. We provide an antibody specific to EHD2 with antigen purification and a use thereof in the preparation of an immunohistochemical detection reagent. The EHD2 antibody provided in the present invention has qualified specificity, it can specifically recognize the EHD2 protein, while cannot recognize other highly homologous proteins through testing using the immunoblotting method, and can quantitatively determine the EHD2 protein using the immunoblotting method, moreover, it can be prepared into an immunohistochemical reagent directly used for judging the expression and localization conditions of EHD2 in tissue cells, especially for monitoring the expression and localization conditions of EHD2 in the cell nucleus, thus better used for diagnosis and prognosis judgment of breast cancer. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0030]      FIG. 1  shows an immunoblotting photo obtained from Example 1 of the specificity detection; 
           [0031]      FIG. 2  shows an immunohistochemical photo of normal tissue cells in Example 1 of an immunohistochemical detection; 
           [0032]      FIG. 3  shows an immunohistochemical photo of breast cancer cells in Example 1 of an immunohistochemical detection; 
           [0033]      FIG. 4  shows an immunohistochemical photo of breast cancer cells in Example 1 of an immunohistochemical detection; 
           [0034]      FIG. 5  shows an immunohistochemical photo of breast cancer cells in Example 1 of an immunohistochemical detection; 
           [0035]      FIG. 6  shows a progression-free survival curve of a total of 260 samples in Example 1 of an immunohistochemical detection 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0036]    The present invention will now be detailedly described by way of examples and with reference to the accompanying drawings. 
       Example 1 of an Antibody Preparation 
     Preparation of an Antibody Specific to EHD2 
       [0037]    (1) Sources of Experimental Materials 
         [0038]    New England white rabbits were purchased from ChinaPeptides Co., Ltd.; The polypeptide of 
         [0039]    CDEEFALASHLIEAKLEGHGLPANLPRRLVPPSKRRHKGSAE (the polypeptide was obtained by modification with another polypeptide having an amino acid sequence of SEQ ID NO: 1 as a core sequence, and the modification method was connecting a cysteine to its N-terminal.) was custom-made by Tianjin Saier Biotechnology Co., Ltd. and was coupled with KLH. 
         [0040]    CNBr-Activated gel beads, Freund&#39;s complete adjuvant and Freund&#39;s incomplete adjuvant were purchased from Invitrogen. 
         [0041]    (2) Animal Immunization 
         [0042]    Three four-month-old New Zealand white rabbits were taken for the study, 100 pg of an antigen polypeptide was dissolved in 0.2 ml of 0.1 M PBS (pH 7.2), the obtained solution was thoroughly mixed with an equal volume of Freund&#39;s complete adjuvant, which was multi-point injected into the abdominal subcutaneous region of each rabbit. On day 15 and day 29 after the initial immunization, a booster immunization is given by using 100 μg of the polypeptide/0.2 ml of PBS thoroughly mixed with an equal volume of Freund&#39;s incomplete adjuvant, respectively. 
         [0043]    (3) Preparation of an Antiserum Against EHD2 
         [0044]    Blood was collected from a carotid artery one week after the last immunization, then kept still at 37° C. for 3 hours and centrifuged to get the serum. 
         [0045]    (4) Preparation of Antigen Gel Beads 
         [0046]    CNBr-Activated gel beads were soaked in 1 mM HCl for 30 min, and washed with a coupling buffer (containing 0.1 M of NaHCO 3  with pH=8.3, and 0.5 M of NaCl), then a reaction system was mixed according to a proportion of adding 1 ml of gel to per 1 mg of the polypeptide. After coupling at 4° C. overnight, the system was soaked in 1 M ethanolamine for 3 hours, then washed in a cross manner with a washing liquid 1 (containing 50 mM Tris, 1 M NaCI, pH 8.0) and a washing liquid 2 (containing 50 mM glycine, 1 M NaCl, pH 3.5) for a total of 8 times, followed by washing once with PBS. 
         [0047]    (5) Purification of EHD2 Antibody 
         [0048]    The serum was mixed with the above-mentioned antigen gel beads according to a volume proportion of 20:1 to get a mixed system, then an equal volume of PBS was added to the system, followed by mixing uniformly and one hour later taking centrifugation, the gel beads were washed with PBS and the antibody coupled to the gel beads was eluted with sodium citrate solution with pH=3, then the pH value was adjusted to 6.5-7.5, the a purified EHD2 antibody was finally obtained. 
       Example 1 of a Detection 
     Specificity Detection of EHD2 Antibody Using an Immunoblotting Method 
       [0049]    (1) Sources of Experimental Materials 
         [0050]    293T cells were purchased from American Type Culture Collection (ATCC); culture medium RPMI 1940, BSA, HRP-labeled Anti-Rabbit Secondary Antibodies, Lipo2000 Transfection Reagent, RIPA lysis buffer, BCA protein concentration assay reagent, and ECL chemiluminescence assay reagent were all purchased from Invitrogen; EHD1, EHD2, EHD3 and EHD4 expression plasm ids were self-made. 
         [0051]    (2) Cell Culture 
         [0052]    293T cells were cultured in the culture medium RPMI 1940, and were grown by adherent culture at 37° C. with 5% CO2; when the cells were passaged, firstly the culture medium was discarded, then the cells were washed twice with phosphate buffered saline (PBS), after that, 0.05% trypsin was added for digestion, 2 minutes later, the culture medium was added to stop the digestion. The cells were kept in good condition, and passed one generation every two days. At the time of transfection, the plasmids that express EHD1, EHD2, EHD3 and EHD4 and the transfection reagents were added, respectively, two days later, the cells were collected for immunoblotting experiments. 
         [0053]    (3) Immunoblotting Method 
         [0054]    Different cells in sufficient quantities were reserved in centrifuge tubes, after centrifugation the cells were lysed in RIPA lysis buffer, followed by boiling and another centrifugation so as to obtain samples. After the samples were made, their protein concentrations were determined by the BCA assay reagent. For each sample, 80pg of total protein was taken for SDS-PAGE electrophoresis. When the electrophoresis was completed, the proteins in the gels were electrotransfered onto PVDF membranes, followed by blocking with 5% milk at room temperature for 1 hour. After washing, the membranes were incubated with a primary antibody (the antibody prepared in Example 1 was diluted 1/2000 in PBS containing 5% BSA) at room temperature for 1 hour. Next, the membranes were incubated with Anti-Rabbit Secondary Antibodies diluted 1/5000 at room temperature for 1 hour. Finally, the membranes were detected with the chemiluminescence assay reagent and the detection results were shown in  FIG. 3 . It can be seen from the figure that the antibody specific to EHD2 provided in the present invention is able to specifically recognize the EHD2 protein, while not able to recognize other homologous EHD proteins. 
         [0055]    (4) Results 
         [0056]    The antibody against EHD2 has the ability to specifically and immunologically recognize the EHD2 protein, whereas has no cross-reactions with other homologous proteins. The detection results were shown in  FIG. 1 , in  FIG. 1 , sample Ve is the a empty expression vector, no signal; sample No.1 represents overexpression of EHD1 protein, no signal; sample No.2 represents overexpression of EHD2 protein, there appears a main band at the location corresponding to a molecular weight of 70 KD, the signal is clear and there is no other obvious background bands; sample No.3 represents overexpression of EHD3 protein, no signal; and sample No.4 represents overexpression of EHD4 protein, no signal. In conclusion, the antibody provided in this example can produce the intensive signal at the right location of 70 KD, whereas cannot produce signals at other locations, these results show that the antibody provided in the present invention has perfect specificity. 
       Example 1 of an Immunohistochemical Detection 
     Immunohistochemical Detection of EHD2 
       [0057]    (1) Sources of Experimental Materials: 
         [0058]    Breast cancer slices were obtained from the a tumor tissue bank of Tianjin Cancer Hospital, with routine conventional dewaxing. The total number of sample is 260. Diluent of the primary antibody, Horseradish Peroxidase (HRP)-labeled Universal Secondary Antibody, Diaminobenzidine (DAB) substrate, and Substrate Diluent Solution were purchased from ZSGB-BIO Co. Ltd. 
         [0059]    (2) Preparation Conditions for Immunohistochemical Detection Reagents and Detection Methods for EHD2 Expression and Localization in Tissue Samples: 
         [0060]    Main steps for the method are as follows: tissue slices were dewaxed until the wax had been replaced with water, then performed antigen retrieval and blockage of endogenous peroxidases. The antibody prepared from Example 1 was used as the primary antibody, and dropped inside at 1/200 dilution, incubated at 4° C. overnight. Samples were washed with buffer three times, 5 min for each time. The HRP-labeled universal secondary antibody was dropped inside for incubation at room temperature for 30 min. Samples were washed again with buffer three times, 5 min for each time. DAB staining, restaining, dehydration, and mounting were performed for staining observation under a microscope. 
         [0061]    (3) Results: 
         [0062]    EHD2 expression in epithelial nuclei of normal tissues was positive, and the expression in the cytoplasm and membrane was weakly positive. In cancer tissues, EHD2 expression produced a disorder and the expression in nuclei tended to be weak. Typical immunohistochemical photos were shown in  FIG. 2 - FIG. 5 ,  FIG. 2  was an immunohistochemical photo for normal tissues, showing that EHD2 was expressed in epithelial nuclei of normal cells,  FIG. 3 - FIG. 5  were immunohistochemical photos for different breast cancer tissue samples, in the breast cancer tissue sample as shown in  FIG. 3 , EHD2 was expressed both in cancer nuclei and cytoplasm, in the breast cancer tissue sample as shown in  FIG. 4 , EHD2 was not expressed in cancer nuclei, whereas was intensively expressed in the cytoplasm; in the breast cancer tissue sample as shown in  FIG. 4 , an overall loss of EHD2 expression was found in breast cancer cells, showing that the expression of EHD2 in cancer cells produced a disorder and the expression in nuclei was weakened. A progression-free survival curve of a total of 260 samples was shown in  FIG. 6 , and legends for the figure were as follows: 0 represented negative expression in nuclei; 1 represented positive expression in nuclei; censored represented death. The horizontal axis displayed progression-free survival months, and the vertical axis displayed a percentage of progression-free survival cases. It can be seen from the figure that the prognosis of the cases with negative expression in nuclei is apparently poorer than that of cases with positive expression. 
         [0063]    The above experimental results indicate that the immunohistochemical detection method adopting the antibody provided in the present invention as the core reagent has an ability to favorably detect the expression quantity and localization of EHD2 in breast cancer tissue cells, so as to directly interpret the localization and expression conditions of EHD2 in nuclei of cancer cells in order to anticipate malignant degree of breast cancer and survival prospects of patients.