CN1752104B - Monoclonal antibody of synapse protein gamma and its application - Google Patents
Monoclonal antibody of synapse protein gamma and its application Download PDFInfo
- Publication number
- CN1752104B CN1752104B CN 200410078299 CN200410078299A CN1752104B CN 1752104 B CN1752104 B CN 1752104B CN 200410078299 CN200410078299 CN 200410078299 CN 200410078299 A CN200410078299 A CN 200410078299A CN 1752104 B CN1752104 B CN 1752104B
- Authority
- CN
- China
- Prior art keywords
- sncg
- antibody
- test kit
- monoclonal antibody
- horseradish peroxidase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A monoclonal antibody against to SNCG, the hybridoma cell line (CGMCC 1196) for secreting it, and the reagent kit for testing the SNCG level of specimen are disclosed. Its advantages are high specificity and sensitivity. It can be used to diagnose breast cancer.
Description
Technical field
The present invention relates to field of immunology, relate to particularly at the monoclonal antibody of SNCG (synapse protein gamma) and the hybridoma cell line of this antibody of secretion.The invention still further relates to the application of described antibody in test sample SNCG level, and test kit and this antibody may use in oncotherapy of utilizing described antibody test sample SNCG level.
Background of invention
Mammary cancer is human common a kind of malignant tumour.Though the treatment level to mammary cancer improves constantly in recent years, significantly do not improve patient's overall lifetime.Major cause is that mammary cancer promptly shifts in early days, and this transfer is difficult to discover clinically, thereby influence is to the rational therapy of breast carcinoma of early stage.Therefore, searching and detection and Metastasis in Breast Cancer clinical detection index in close relations for the treatment level that improves mammary cancer, to prolong survival time of patients significant.
Synapse protein gamma (synuclein γ) is called for short SNCG, belongs to the synuclein family member, and this family is the small molecules neuroprotein family with high conservative.Except that SNCG, known this family also comprises synuclein α and synuclein β at present.Synuclein α is main relevant with some nerve degenerative diseases, for example alzheimer's disease (AD) and Parkinson's disease (PD).Synuclein β and breast cancer related does not express in the normal galactophore tissue, and breast cancer tissue expresses.And most of ovarian cancers are obviously expressed at least a synuclein albumen.
Main and the mammary cancer of the expression of discovered in recent years SNCG in close relations.Ji etc. confirm that by immunohistochemical methods and hybridization in situ experiment the expression of SNCG has following characteristics: do not detect SNCG in (1) normal galactophore tissue or the mammary gland benign lesion tissue; (2) part is expressed SNCG in breast ductal carcinoma in situ; (3) express SNCG in III phase of extensively soaking into or IV primary breast cancer camber.Ji etc. detect human breast cancer cell strain SKBR-3 by Northern blot again, MCF-7, H3396, MDA-MB-231, T47D, H3922, H3914, ZR-75-1, discovery is except that H3396, all the other cell strains are all expressed SNCG mRNA, especially with the highest (the Ji H of expression of infitrating ductal carcinoma cell strain H3922, Liu YE, Jia T, Wang M, Liu J, Xiao G, Joseph B K, Shi YE.Identification of a breastcancer-specific gene, BCSG1, by direct differential cDNA sequencing.Cancer Res.1997; 57:759-764.).These find all to point out the expression of SNCG relevant with transfer with the infiltration of mammary cancer.Confirmed at present that it is that the patient with breast cancer is with nodus lymphoideus transferring rate and the not good relatively independent index of prognosis that crossing of SNCG expressed.
The SNCG high expression level is not only a kind of phenomenon of mammary cancer, and can promote the infiltration and the transfer of mammary cancer.Matrix metalloproteinase (MMP) is a class and the closely-related proteolytic ferment of tumor-infiltrated transfer, comprise MMP1, MMP2, MMP3, MMP7, MMP9 and MMP10 etc., Jia etc. find the expression of crossing expression obvious promote MMP2 and MMP9 of SNCG in breast cancer cell.Microtubule bindin (MAPs) participates in the assembling of microtubule and increases the stability of microtubule, think that now the cytoskeleton reorganization of breast cancer cell can cause cell to have bigger aggressive, SNCG is by regulating the structure of cytoskeleton and dynamically assembling with the MAPs effect, strengthen mobility (the Jia T of breast cancer cell, Liu YE, Liu J, Shi E.Stimulation of breast cancer invasion and metastasis by breastcancer-specific synuclein γ (SNCG) .Cancer Res.1999; 59:742-7).In addition, SNCG obviously suppresses tumor growth supressor OM (oncostatin M).
Therefore, the detection that SNCG is expressed may become the important clinical index of early diagnosing mammary cancer and estimating prognosis.
Immuning tissue's (cell) chemistry (abbreviation immunohistochemical methods) is on the basis that keeps tissue and original form of cell and structure, antibody and its reaction with specific antigen, method by spike a technical skill that antigen antibody reaction is manifested then. because this method can not only show intuitively whether certain specific antigen exists in inspected tissue or cell, the more important thing is the position and the relative content that can show intuitively that antigen exists in tissue or cell. so immunohistochemical methods is widely used in medical research field.
But the domestic and international at present research about SNCG all is to utilize the polyclonal antibody of SNCG to carry out, and does not see the report of SNCG monoclonal antibody.The poor specificity of polyclonal antibody, colour developing background height when being used for immunohistochemical methods, quality is wayward, is difficult to promote in clinical diagnosis.And have the false-positive shortcoming of easy contaminated generation with the genetic expression of PCR method detection SNCG, can not accurately reflect proteinic real standard simultaneously, thereby greatly limit the application of SNCG detection in clinical.Monoclonal antibody being widely used in clinical detection can be used in several different methods such as immunohistochemical methods, ELISA, has high specificity, advantage that susceptibility is high.
Under native state, the conformation of antigenic determinant keeps better, the easy and antibodies of antigen.Therefore, be in the test experience of native state at antigens such as ELISA, immunoprecipitation, frozen sections, not high relatively to the requirement of monoclonal antibody.But in clinical the most frequently used paraffin section immunohistochemical methods detects, tissue is after formalin fixed, paraffin embedding and dehydration of alcohol etc. are handled, the space conformation that makes antigenic determinant owing to protein denaturation is by partial destruction, and therefore specific recognition and the binding ability to monoclonal antibody requires high.Same reason needs higher antibody titer in the paraffin section immunohistochemical methods.Simultaneously, consider, also require corresponding hybridoma to have the ability of stronger secrete monoclonal antibody from the economy of preparation antibody.
The present clinical SNCG monoclonal antibody that satisfies above-mentioned requirements that presses for, and can secrete the tire hybridoma of said monoclonal antibody of height, so that predict guiding clinical treatment in early stage possibility and the patient's prognosis of just pathology being shifted that mammary cancer is found.
The invention summary
The invention provides a kind of monoclonal antibody at SNCG, or can specificity in conjunction with the bioactive fragment of SNCG.Described monoclonal antibody by be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC
1196Hybridoma secretion.
The present invention also provides a kind of hybridoma cell line of the SNCG of secretion monoclonal antibody, its on July 22nd, 2004 with preserving number CGMCC
1196Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (Zhong Guan-cun, Haidian District, BeiJing, China city north one No. 13).
The invention still further relates to the application of described monoclonal antibody in test sample SNCG level.The invention provides the test kit of SNCG level in a kind of test sample, it contains described monoclonal antibody, bonded second antibody with it, chromogenic substrate or fluorescence/radio-labeled that can be detected, and suitable damping fluid.In a specific embodiments of the present invention, be marked with vitamin H on the second antibody of described test kit, and further contain Streptavidin in conjunction with horseradish peroxidase/alkaline phosphatase, chromogenic substrate is DAB/AP-Red.In another specific embodiments of the present invention, directly indicate horseradish peroxidase/alkaline phosphatase on the second antibody of described test kit.The present invention also provides a kind of ELISA test kit of test sample SNCG level, comprise microtiter plate with described monoclonal antibody or active fragments bag quilt, the SNCG standard antigen, the SNCG polyclonal antibody of purifying, be marked with vitamin H can with SNCG polyclonal antibody bonded second antibody, the Streptavidin of horseradish peroxidase/alkali phosphatase enzyme mark, the corresponding chromogenic substrate of marker enzyme, and suitable damping fluid.In another specific embodiments of the present invention, directly indicate horseradish peroxidase/alkaline phosphatase on the second antibody in the above-mentioned ELISA test kit, and do not contain the Streptavidin of horseradish peroxidase/alkali phosphatase enzyme mark.
The present invention also provides a kind of diagnostic method of tumors, it is characterized by with SNCG protein expression level in the monoclonal antibody vitro detection tissue of the present invention. in a specific embodiments of the present invention, may carry out immunohistochemical staining for the tissue of tumour to what take from the patient with monoclonal antibody of the present invention.
The present invention also provides a kind of monitoring method of oncotherapy, comprises the dynamic change that detects SNCG protein level in the tumour patient sample with monoclonal antibody of the present invention.The present invention also provides a kind of tumor treatment method, comprise need the treatment the patient treatment significant quantity the SNCG monoclonal antibody or from this antibody can with SNCG specificity bonded bioactive fragment.Correspondingly, the present invention relates to the application in the preparation antitumor drug of SNCG monoclonal antibody of the present invention or its bioactive fragment.
Detailed Description Of The Invention
The inventor obtains the SNCG albumen of purifying by prokaryotic expression, and obtains the monoclonal antibody of SNCG and the hybridoma cell line of secreting this antibody by hybridoma technology.
The present invention is a template with the cDNA of SNCG gene, from initiator codon design sense primer, from terminator codon design antisense primer, with the cDNA fragment of PCR method amplification SNCG gene.The pcr amplification product directed cloning to prokaryotic expression carrier pGEX4T-1, is made up protokaryon recombinant plasmid pGEX4T-1-SNCG, and at expression in escherichia coli.Select the mono-clonal positive bacteria, a large amount of abduction delivering SNCG albumen and purifying.
Protein induced expression condition well known in the art is: temperature between 28～30 ℃, the concentration of inductor IPTG is that 0.1～0.5mmol/L, induction time are 6～8 hours, uses the e. coli jm109 bacterial strain.It is generally acknowledged that under these conditions the abduction delivering level of alien gene is higher, and be difficult for forming inclusion body.But under these conditions, SNCG does not express.In the present invention, the contriver explores the abduction delivering condition of SNCG, uses e. coli strain bl21, and temperature is 37 ℃, and the concentration of inductor IPTG is 1mmol/L, and induction time is 2～3 hours.With this understanding, the expression amount of SNCG is the highest, and expressed proteins mainly is a secreted protein.
B lymphocyte hybridoma technology (Kohler G with reference to Kohler and Milstein foundation, Milstein C, Continuous cultures of fused cells secreting antibody ofpredefined specificity.Nature.1975Aug 7; 256 (5517): 495-7.), the present invention's SNCG protein immunization BALB/c mouse of purifying.The splenocyte and the myeloma cell SP2/0 of immune mouse are merged the preparation hybridoma.With ELISA method screening positive cell clone.The inventor has obtained many strains positive cell clone through test of many times.The monoclonal antibody that these positive colonies are secreted is carried out Analysis and Identification, find a strain of hybridoma strain, its excretory SNCG monoclonal antibody 4E9 is positive in the immunohistochemical methods test.Through identifying that the subclass of described monoclonal antibody is IgG1.This hybridoma cell strain on July 22nd, 2004 with preserving number CGMCC
1196Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (address: Zhong Guan-cun, Haidian District, BeiJing, China city north one No. 13).
The invention further relates to the application of described SNCG monoclonal antibody in test sample SNCG level.Used detection method comprises all immunization methods that can use antibody, for example immunohistochemical methods, ELISA, immunoprecipitation, immunofluorescence, radioimmunoassay etc.
The invention provides the test kit of SNCG level in a kind of test sample, it contains described monoclonal antibody, bonded second antibody with it, chromogenic substrate or fluorescence/radio-labeled that can be detected, and suitable damping fluid.
In specific embodiments of the present invention, adopt the immunohistochemical methods method to detect the expression of tissue or cell SNCG.Used kit comprises described SNCG monoclonal antibody, the antibody of the anti-mouse IgG of the antibody of biotin labeled goat anti-mouse IgG or rabbit, Streptavidin in conjunction with horseradish peroxidase/alkaline phosphatase, the corresponding chromogenic substrate DAB/AP-red of enzyme, and the damping fluid that is applicable to immunohistochemical methods.
The cell climbing sheet of preparation breast cancer cell line mcf-7 and SKBR-3, behind the fixed cell, drip the hybridoma supernatant liquor of secretion 4E9 antibody, incubation. wash creep plate with damping fluid, drip the antibody (second antibody) of biotin labeled goat anti-mouse IgG then, incubation. wash creep plate with damping fluid, drip Streptavidin then in conjunction with horseradish peroxidase/alkaline phosphatase, incubation. perhaps directly be marked with horseradish peroxidase/alkaline phosphatase on the second antibody, and no longer drip Streptavidin in conjunction with horseradish peroxidase/alkaline phosphatase. use DAB (diamino benzidine then, benzidine)/AP-red (alkalinephosphatase-red, alkaline phosphatase is red, claim Fast Red again, red soon) carry out color reaction.If cell expressing SNCG, then the color reaction result for cytoplasm presents pale brown look/redness, otherwise do not develop the color.
In another embodiment of the invention, the sample that is used to detect is a breast cancer tissue.Tissue is made the section of 3～5 μ m through formalin fixed, dehydration of alcohol, paraffin embedding.The method that experimental technique is expressed with above-mentioned detection cell strain SNCG.
The present invention also provides a kind of test kit with ELISA method test sample SNCG level, is suitable for detecting the SNCG level in blood or the tissue juice.
In a specific embodiments of the present invention, use the ELISA method to detect the level of SNCG in the serum.Used kit comprises the microtiter plate with described SNCG monoclonal antibody bag quilt, the SNCG standard antigen, the SNCG polyclonal antibody (first antibody) of purifying, be marked with vitamin H and can with SNCG polyclonal antibody bonded antibody (second antibody), the Streptavidin of horseradish peroxidase/alkali phosphatase enzyme mark, the corresponding chromogenic substrate OPD or the BCIP/NBT of marker enzyme, and suitable damping fluid.
Add the standard antigen solution of different concns in each hole that microtiter plate is used for doing the standard antigen curve successively, in sample well, add the test serum sample, incubation for some time so that SNCG albumen fully combine with the SNCG monoclonal antibody.The washing microtiter plate adds first antibody, and incubation for some time is so that the SNCG polyclonal antibody fully combines with SNCG albumen on being fixed in microtiter plate.The washing microtiter plate adds second antibody, and incubation for some time is so that second antibody fully combines with SNCG polyclonal antibody on being fixed in microtiter plate.The washing microtiter plate, the Streptavidin of adding horseradish peroxidase/alkali phosphatase enzyme mark, incubation for some time is so that Streptavidin fully combines with vitamin H.The washing microtiter plate adds OPD, the colour developing of room temperature lucifuge.Read the OD492 value of each reacting hole with microplate reader.The drawing standard curve, the concentration of SNCG in the calculating testing sample.
The present invention also provides a kind of diagnostic method of tumors, it is characterized by with SNCG protein expression level in the monoclonal antibody vitro detection tissue of the present invention.In a specific embodiments of the present invention, may carry out immunohistochemical staining with monoclonal antibody of the present invention for the tissue of tumour to what take from the patient as mentioned above, examine under a microscope the tissue staining result and determine whether to express SNCG protein, thereby whether be that tumour is made diagnosis organizing.By judging that the SNCG positive cell accounts for the ratio of tumour cell and what (powers of positive signal) of expression can further be assisted stage of tumor.For example in mammary cancer, the part cancer cells is expressed SNCG in the breast ductal carcinoma in situ, and staining power is medium; And most of cancer cells is expressed SNCG in III phase of extensively soaking into or the IV primary breast cancer, and coloration result is generally strong positive.
The present invention also provides a kind of monitoring method of oncotherapy, comprises the dynamic change with monoclonal antibody of the present invention SNCG protein level in vitro detection tumour patient sample.In a specific embodiment of the present invention, as mentioned above, the serum of taking from tumour patient is carried out conventional ELISA experiment with the microtiter plate of SNCG monoclonal antibody bag quilt of the present invention, detect the proteinic level of SNCG in the serum.If result of treatment is good, then with before the treatment compares SNCG protein level among the patients serum and reduce or be difficult to detect; If the state of an illness occurs repeatedly, then the proteinic level of SNCG increases in the serum.
As confirming in the following drawings and Examples, SNCG monoclonal antibody of the present invention can with the SNCG specificity, efficient combination. therefore, give tumour patient monoclonal antibody of the present invention or this antibody can with SNCG protein-specific bonded bioactive fragment, the activity that may suppress SNCG in the tumour cell, suppress tumor-infiltrated thereby play, the effect of shifting. therefore, the present invention also provides a kind of method of treatment animal (comprising the people) tumour, the monoclonal antibody of the present invention or its bioactive fragment that comprise the treatment of animals significant quantity that needs treatment. " tumour " used herein is not particularly limited, as long as relevant, but be preferably mammary cancer with expressing excessively of SNCG.
Correspondingly, the present invention relates to the application in the medicine of preparation antitumor drug, particularly mammary cancer of monoclonal antibody of the present invention or its bioactive fragment.The preparation method of described medicine can be any method well known to those skilled in the art, for example monoclonal antibody of the present invention or its bioactive fragment is mixed with appropriate excipients.Described vehicle is the vehicle that this area routine is used for the antibody drug preparation, as water, salt solution, buffer reagent etc.The route of administration of described medicine can be the conventional route of administration of any antibody drug, for example, injects in intravenous injection, subcutaneous injection, the tumour etc.
Further illustrate the present invention below in conjunction with the drawings and specific embodiments, but scope of the present invention is not subjected to the restriction of these drawings and Examples.
The accompanying drawing summary
Fig. 1: the 12%SDS-PAGE electrophoresis is identified the purity of 4E9 antibody.Wherein the first road electrophoresis is 2 μ g antibody, and second road is 5 μ g antibody, and the swimming lane that is labeled as M is the protein molecular weight standard of 66KD.The electrophoresis direction is for from top to bottom.The fast band (following band) of electrophoretic velocity is the less monoclonal antibody light chain of molecular weight, and the slow band (top band) of electrophoretic velocity is the bigger monoclonal antibody heavy chain of molecular weight.The result shows, the antibody purity height that the positive hybridoma cell strain that filters out is secreted does not have assorted band.
Fig. 2: with wrapping by the microtiter plate of GST-SNCG recombinant protein tiring with ELISA methods analyst 4E9 antibody.With wrapping by the negative contrast of the microtiter plate of GST-VEGF.The antibody purification 4E9 that concentration is respectively 0.5,1,2,5,10 μ g/ml is added in the microtiter plate.Ordinate zou is OD
492The absorbancy numerical value at place, X-coordinate is the 4E9 antibody concentration.The result shows that 5 μ g/ml are best working concentrations of 4E9 antibody, and with other albumen such as GST-VEGF no cross reaction.
Fig. 3: detect the expression of SNCG among the breast carcinoma cell strain SKBR-3 with immunocytochemistry.The result shows that the cytoplasm colour developing of SKBR-3 is pale brown look, illustrates SKBR-3 cell strain expression SNCG.
Fig. 4 A: detect the expression of SNCG in the mammary gland benign lesion with the immunohistochemical methods method, the result shows and does not express SNCG in the mammary gland benign lesion.
Fig. 4 B: with the expression of SNCG in the immunohistochemical methods method detection III primary breast cancer pathology, the result shows that the expression of SNCG in the III primary breast cancer tissue is strong.
Embodiment
1, the antigenic preparation of SNCG
Design 5 ' of SNCG gene cDNA fragment respectively and rectify adopted primer 5 '-GACGGATCCATGGATGTTTTCAAGAAGGC-3 ' and 3 ' end antisense primer 5 '-ATCCTCGAGCTAGTCTCCCCCACTCTG-3 ' (primer is given birth to worker bio-engineering corporation by Shanghai and synthesized), and introduce BamH I and Xho I restriction enzyme site respectively in the primer two ends.With PCR method amplification SNCG cDNA fragment, product length is 393bp.
PCR product and pGEM-T Easy carrier (available from Promega company) are connected to form pGEM-T Easy-SNCG plasmid.PGEM-T Easy-SNCG plasmid is transformed into intestinal bacteria JM-109 bacterial strain to increase.PGEM-T Easy-SNCG plasmid with BamH I and Xho I digest amplification, again with BamH I and Xho I digestion prokaryotic expression carrier pGEX4T-1, the SNCG gene cDNA fragment that above-mentioned digestion is obtained inserts among the prokaryotic expression carrier pGEX4T-1, the SD sequence downstream that forms pGEX4T-1-SNCG. prokaryotic expression carrier pGEX4T-1 is exactly glutathione sulfydryl transferase (GST) gene, clone's external source SNCG gene then links to each other with gst gene. when genetic expression, expression product is the syzygy GST-SNCG of GST and SNCG gene product, to make things convenient for the purifying of SNCG.
With pGEX4T-1-SNCG transformed into escherichia coli BL21 bacterial strain, the BL21 bacterial strain of selecting positive colony carries out a small amount of abduction delivering of GST-SNCG recombinant protein.Getting in a small amount, abduction delivering is accredited as a large amount of abduction deliverings that the male bacterial strain carries out the GST-SNCG recombinant protein.With the glutathione S epharose 4B that can adsorb gst fusion protein (available from HiTrap company) purifying GST-SNCG recombinant protein, and reclaim, identify.
2, SNCG MONOCLONAL ANTIBODIES SPECIFIC FOR
With the BALB/c mouse in ages in GST-SNCG recombinant protein immunity 6～8 week of purifying, 6 week the back reinforced immunologicals.Mouse boosting cell that immunity is good and myeloma cell SP2/0 merge.Hybridoma with the anti-SNCG monoclonal antibody of ELISA method screening secretion.With limiting dilution assay the male hybridoma is carried out repeatedly subclone again.Choose strong positive clone's enlarged culturing, build strain, the anti-SNCG monoclonal antibody called after 4E9 that it is secreted, and this hybridoma cell strain is the 4E9 cell strain.This hybridoma cell strain on July 22nd, 2004 with preserving number CGMCC
1196Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (address: Zhong Guan-cun, Haidian District, BeiJing, China city north one No. 13).With the 4E9 cell inoculation in 10 age in week BALB/C mice the abdominal cavity in, form to ascites and to put to death mouse when increasing the special bulge of belly and get ascites, it includes a large amount of 4E9 antibody.
Subclass with conventional ELISA method evaluation 4E9 antibody is confirmed that it is IgG1, therefore selects for use high salt Pro.A Sepharose-4B post to carry out purifying, and identifies its purity with the 12%SDS-PAGE electrophoresis.The results are shown in Figure 10, show antibody purity height, do not have assorted band.
Embodiment 3 measures tiring of 4E9 antibody
With the 4E9 antibody of PBS damping fluid dialysis purifying, measure the concentration of antibody at the 280nm place with ultraviolet spectrophotometer.
1, in the microtiter plate of GST-SNCG albumen bag quilt, adds the 4E9 antibody that concentration is 0.5,1,2,5,10 μ g/ml respectively.And the 4E9 antibody of same concentration is added in the microtiter plate of GST-VEGF albumen bag quilt as negative control.Room temperature incubation 1 hour.
2,0.05%Tween-20/PBS washing reaction hole is 3 times, PBS washing reaction hole 2 times.
3, the antibody that adds the sheep anti-mouse igg of horseradish peroxidase-labeled, room temperature incubation 1 hour.
4,0.05%Tween-20/PBS washing reaction hole is 3 times, PBS washing reaction hole 2 times.
5, add horseradish peroxidase substrate OPD in reacting hole, color development at room temperature added stop buffer (12.5%H after 30 minutes
2SO
4).Measure OD with microplate reader
492The absorbance at place.The results are shown in Figure 2, show that 5 μ g/ml are best working concentrations of 4E9 antibody, and with the GST-VEGF no cross reaction.
Embodiment 4 immunohistochemical methodss detect the expression of SNCG among the breast carcinoma cell strain SKBR-3
1, ordinary method prepares the cell climbing sheet of breast carcinoma cell strain SKBR-3, and cold acetone/methyl alcohol (volume ratio 1: 1) is fixing.
2, creep plate is soaked in 3%H
2O
2(PBS dilution) interior 10 minutes is to remove endogenous peroxydase.
3, PBS washing creep plate is 2 times.Use 1%BSA (PBS dilution) to seal 30 minutes down then at 37 ℃.Drip the 4E9 antibody of suitable concentration, reacted 1 hour down at 37 ℃.
4, PBS washing creep plate is 2 times, drips antibody working fluid (the DAKO ENVISION of the sheep anti-mouse igg of horseradish peroxidase-labeled
TM+ System HRP Mouse), reacted 1 hour down at 37 ℃.
5, wash creep plate 2 times with PBS, colour developing is 20 minutes in the solution of horseradish peroxidase substrate DAB.
6, use the PBS color development stopping, cover on slide glass creep plate is counter, microscopically is observed, and is positive to occur the brown yellow granule shape in the cytoplasm.The results are shown in Figure 3, show that the SKBR-3 cytoplasm is pale brown look, SKBR-3 cell strain expression SNCG is described.
1, chooses the III primary breast cancer and mammary gland benign lesion tissue carries out conventional formalin fixed, dehydration of alcohol, paraffin embedding, be cut into 4 μ m slabs.60 ℃ of bakings 4 hours, so that section is attached on the slide glass securely.
2, with dimethylbenzene dewaxing treatment is carried out in section, gradient alcohol makes the section aquation, uses PBS washing slice 2 times.
3, section is soaked in 3%H
2O
2(PBS dilution) interior 10 minutes is to remove endogenous peroxydase.Use the PBS washing slice then 3 times.
4, section is soaked in the 0.1M sodium citrate buffer of pH6.0, boiled 20 minutes, carry out antigen retrieval at 92 ℃～100 ℃.After slice naturally cools to room temperature,, use the PBS washing slice again 2 times with deionized water wash section 2 times.
5,5% lowlenthal serum (PBS dilution) is dripped in section,, carry out the antigen sealing 37 ℃ of following incubations 30 minutes.
6, in section, drip the 4E9 antibody of suitable concentration, spend the night under 4 ℃.Use the PBS washing slice then 3 times.
7, in section, drip antibody working fluid (the DAKO ENVISION of the sheep anti-mouse igg of horseradish peroxidase-labeled
TM+ System HRP Mouse), 37 ℃ of following incubations 1 hour.
8, use PBS washing slice 2 times, section is soaked in the solution of horseradish peroxidase substrate DAB developed the color 20 minutes.
9, use the PBS color development stopping, section is redyed with phenodin.With 1% hydrochloride alcohol differentiation section, gradient alcohol dehydration, dimethylbenzene is transparent, then cover glass is covered on tissue slice, and microscopically is observed.The results are shown in Figure 4, show in the mammary gland benign lesion and do not express SNCG (Fig. 4 A), and the high expression level (Fig. 4 B) of SNCG is arranged in the III primary breast cancer tissue.
Embodiment 6SNCG expresses the correlation analysis with the mammary cancer clinical pathologic characteristic
The inventor carries out the paraffin section immunohistochemical analysis to the 113 routine clinical breast lesion tissues of collecting, comprising 47 routine mammary gland benign lesions and 66 routine mammary cancer pathologies, 1 example is non-infiltration mammary cancer (intraductal carcinoma) in the mammary cancer pathology, and 65 examples are infiltrative breast carcinoma.As a result, in the 47 routine mammary gland benign lesion tissues, 1 routine weak expression SNCG is arranged.And have 58 examples to express SNCG in the 66 routine breast cancer tissues.Through Pearson X
2Check, the expression of SNCG in mammary gland benign lesion and breast cancer tissue has significant difference (X
2=77.50, P＜0.001).
Whether relevant in order further to analyze the expression of SNCG in mammary cancer with the mammary cancer clinical stages, the inventor has compiled the clinical data of 66 routine mammary cancer cases, comprise patient age, tumour size, TNM by stages, nodus lymphoideus transferring rate, have or not vascular cancer embolus etc., and relation is between the two analyzed.Because there are 3 routine cases not have TNM by stages, thus TNM analyzed by stages the time, in this 3 example is not calculated.Statistic analysis result sees Table 1, shows that expression and the nodus lymphoideus transferring rate of SNCG has certain dependency (X
2=3.913, P＜0.05), tangible dependency (X is arranged by stages with TNM
2=24.77, P＜0.001), and do not have obviously relevant (X with having or not the vascular cancer embolus
2=3.57, P＞0.05).In the III phase or IV primary breast cancer of extensively soaking into, SNCG highly expresses.
This result shows that the expression of SNCG in the breast cancer tissue and patient's clinical stages is closely related, and clinical stages has determined treatment plan and patient's prognosis.Therefore, further specify the detection SNCG of breast cancer tissue and be expressed in decision treatment plan and the importance of predicting in patient's prognosis.
Claims (8)
1. proteinic monoclonal antibody of SNCG, it is CGMCC by preserving number
1196Hybridoma secretion.
2. preserving number is CGMCC
1196The hybridoma cell line of secretion SNCG monoclonal antibody.
3. test kit with immunological method test sample SNCG level, it contains the antibody of claim 1.
4. the test kit of claim 3 wherein also contains second antibody, Color Appearance System or fluorescence or radio-labeled that can be detected, and suitable damping fluid.
5. the test kit of claim 4 wherein is marked with vitamin H on the second antibody, and further contains the Streptavidin in conjunction with horseradish peroxidase, and its chromogenic substrate is DAB; The Streptavidin that perhaps contains the combined alkali acid phosphatase, its chromogenic substrate are AP-Red.
6. the test kit of claim 4 wherein directly indicates horseradish peroxidase on the second antibody, and its chromogenic substrate is DAB; Perhaps directly indicate alkaline phosphatase, its chromogenic substrate is AP-Red.
7. the test kit of claim 3, it is the ELISA test kit, comprise microtiter plate with the described antibody sandwich of claim 1, the SNCG standard antigen, the SNCG polyclonal antibody of purifying, be marked with vitamin H can with SNCG polyclonal antibody bonded second antibody, the Streptavidin of horseradish peroxidase or alkali phosphatase enzyme mark, the corresponding chromogenic substrate of marker enzyme, and suitable damping fluid.
8. the test kit of claim 7 wherein directly indicates horseradish peroxidase or alkaline phosphatase on the second antibody, and does not contain the Streptavidin of horseradish peroxidase or alkali phosphatase enzyme mark.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410078299 CN1752104B (en) | 2004-09-21 | 2004-09-21 | Monoclonal antibody of synapse protein gamma and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410078299 CN1752104B (en) | 2004-09-21 | 2004-09-21 | Monoclonal antibody of synapse protein gamma and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1752104A CN1752104A (en) | 2006-03-29 |
| CN1752104B true CN1752104B (en) | 2010-05-05 |
Family
ID=36679116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410078299 Expired - Fee Related CN1752104B (en) | 2004-09-21 | 2004-09-21 | Monoclonal antibody of synapse protein gamma and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1752104B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570575B (en) * | 2008-04-30 | 2012-02-29 | 北京市肿瘤防治研究所 | Sncg monoclonal antibody and application thereof |
| CN102586407A (en) * | 2011-01-12 | 2012-07-18 | 上海交通大学医学院附属新华医院 | Application of synuclein gamma (SNCG) gene in diagnosis and hormone therapy of breast cancer |
| CN111435137A (en) * | 2019-01-14 | 2020-07-21 | 深圳先进技术研究院 | Quantitative detection kit for DcR3 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002075317A2 (en) * | 2001-03-15 | 2002-09-26 | Novartis Ag | Screening for drugs against disorders associated with schizophrenia |
| WO2003069332A2 (en) * | 2002-02-14 | 2003-08-21 | The University Of Lancaster | Detection and/or monitoring of synuclein-related diseases |
-
2004
- 2004-09-21 CN CN 200410078299 patent/CN1752104B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002075317A2 (en) * | 2001-03-15 | 2002-09-26 | Novartis Ag | Screening for drugs against disorders associated with schizophrenia |
| WO2003069332A2 (en) * | 2002-02-14 | 2003-08-21 | The University Of Lancaster | Detection and/or monitoring of synuclein-related diseases |
Non-Patent Citations (2)
| Title |
|---|
| Pang-hsien Tu et al.Glial Cvtodasmic Inclusions in White MatterOligodendrocytesof Multiple System Atrophy Brains ContainInsoluble α-Synuclein.Annals of Neurology44.1998,44415-422. * |
| Tongli Jia et al.Stimulation of Breast Cancer Invasion and Metastasis by Synuclein γ.CANCER RESEARCH59.1999,59742-747. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1752104A (en) | 2006-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101570575B (en) | Sncg monoclonal antibody and application thereof | |
| KR20120041175A (en) | Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers | |
| CN101949935A (en) | HE4 (Human Epididymis Protein) monoclonal and polyclonal antibody preparation and development of corresponding diagnostic reagent kit | |
| Rodriguez-Pineiro et al. | Secreted clusterin in colon tumor cell models and its potentialas diagnostic marker for colorectal cancer | |
| CN106706912A (en) | Marker for diagnosis of inflammation-associated HCC and application thereof | |
| CN102782500B (en) | Gastric cancer marker, and method for detecting gastric cancer | |
| CN103045541A (en) | Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same | |
| CN103765220B (en) | The detection mark of colorectal cancer or cancer of the esophagus and inspection method | |
| CN103923212A (en) | EHD2 antibody and application of EHD2 antibody to preparation of immunohistochemical detection reagent for breast cancer | |
| Dai et al. | Analysis of the clinical significance of DCLK1+ colorectal cancer using novel monoclonal antibodies against DCLK1 | |
| KR101777259B1 (en) | Specific monoclonal antibody to EN2 protein or composition comprising the same for diagnosis of prostate cancer | |
| CN1752104B (en) | Monoclonal antibody of synapse protein gamma and its application | |
| CN102782499B (en) | Gastric cancer marker, and method for detecting gastric cancer | |
| US20060063214A1 (en) | Methods and compositions for diagnosing neoplastic disease | |
| CN104364374A (en) | Method for detecting cancer, and antibody capable of recognizing pancreas-specific ribonuclease 1 | |
| CN101514230B (en) | Monoclone antibody of ARD1 and application thereof | |
| CN100504393C (en) | Use of specificly bound reagent of protein MASP in preparing diagnosis reagent | |
| CN103180455A (en) | Yang shiaw-der | |
| ES2285469T3 (en) | USE OF THE TYPE II RETINOIC ACID TRANSPORTER CELL PROTEIN (CRABP II) AS A BREAST CANCER MARKER. | |
| Zhang et al. | Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis | |
| AU2015215008B2 (en) | Composition and method for detecting malignant neoplastic disease | |
| JP2014115186A (en) | Method for detecting stomach cancer, lung cancer, and/or esophagus cancer | |
| CN103842503A (en) | Antibody for detecting epithelial ovarian cancer marker and method for diagnosing epithelial ovarian cancer | |
| CN104945506A (en) | Immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment | |
| CN104945496A (en) | Polypeptide and application thereof in preparing and purifying EHD2-specific antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100505 Termination date: 20120921 |