WO2013104104A1 - 诊断和预示乳腺癌的标志物 - Google Patents

诊断和预示乳腺癌的标志物 Download PDF

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WO2013104104A1
WO2013104104A1 PCT/CN2012/070151 CN2012070151W WO2013104104A1 WO 2013104104 A1 WO2013104104 A1 WO 2013104104A1 CN 2012070151 W CN2012070151 W CN 2012070151W WO 2013104104 A1 WO2013104104 A1 WO 2013104104A1
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seq
cystatin
cst4
kit
protein
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PCT/CN2012/070151
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English (en)
French (fr)
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王弢
渠香云
陈菲
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苏州工业园区为真生物医药科技有限公司
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Priority to US14/371,099 priority Critical patent/US20150160221A1/en
Priority to JP2014550608A priority patent/JP6192123B2/ja
Priority to CN201280066460.3A priority patent/CN104136630B/zh
Priority to PCT/CN2012/070151 priority patent/WO2013104104A1/zh
Priority to GB1414104.8A priority patent/GB2513771B/en
Publication of WO2013104104A1 publication Critical patent/WO2013104104A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin

Definitions

  • the present invention relates to the fields of biotechnology and medicine, and more particularly, to a breast cancer marker and its use, a reagent or kit for diagnosis, dynamic detection, and prognosis of breast cancer, and a method for using the same.
  • WHO World Health Organization
  • breast cancer has risen to the top of female malignancies.
  • the incidence of breast cancer in Shanghai has risen from 17.7/100,000 to 70/100,000, ranking first in all kinds of female cancers. Its incidence has jumped from the second place in female malignant tumors to the first place, with a high mortality rate. 40% or more.
  • the development of breast cancer diagnostic products with high sensitivity and specificity is one of the keys to improve the early detection rate of breast cancer and improve the prognosis of patients.
  • the natural inhibitory protein of cathepsin the Cystatin gene family
  • the Cystatin family of proteins reversibly binds to cysteine proteases, preventing excessive activity of cathepsins.
  • Cystatin C is the strongest inhibitor of protein by cathepsin B, but there is a different level of expression of cystatin C in ovarian cancer and head and neck cancer.
  • cystatin A a member of the cystatin family
  • cystatin B is significantly reduced at the mRNA level
  • cystatin F also known as leukocystatin or CMAP
  • cystatin is not positively related to the development of tumors, because in gliomas, the low expression of cystatin C means the late stage of the disease, the patient's survival is shorter, and it is prone to recurrence, the conclusion is in mRNA and Protein levels have been verified.
  • CST4 also known as cystatin S
  • cystatin S is a member of the cystatin protease family (cystatin), which contains 141 amino acids and is found in a variety of body fluids and secretions such as tears, saliva, serum, and plasma.
  • cystatin protease family cystatin protease family
  • One object of the present invention is to provide an epitope of CST4 gene, CST4 gene mRNA, cDNA of CST4 gene cleavage, amplicon corresponding to CST4-specific primer, CYSTATIN S protein encoded by CST4 gene, and CYSTATIN S protein.
  • a new application of peptides which provides new ideas for the diagnosis of breast cancer.
  • CST4 gene, CST4 gene mRNA, CST4 gene cleavage cDNA, CST4 specific primer corresponding amplicon, CST4 gene-encoded CYSTATIN S protein and CYSTATIN S protein epitope peptide in the preparation of diagnosis and predictive breast cancer For use in the marker, the nucleotide sequence of the CST4 gene is set forth in SEQ ID No: 42.
  • the probe of the CST4 gene, the mRNA of the CST4 gene, or the cDNA of the CST4 gene cleavage is as shown in SEQ ID No: 3.
  • the specific primer of the amplicon has an upstream primer such as SEQ ID No: 1, 4, 6, 8, 10, 12, 14, 16, 18, 20; ID No: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, the upstream primer SEQ ID No: 1 is paired with the downstream primer such as SEQ ID No: 2; upstream primer SEQ ID No: 4 Paired with a downstream primer such as SEQ ID No: 5; upstream primer SEQ ID No: 6 paired with a downstream primer such as SEQ ID No: 7; upstream primer SEQ ID No: 8 paired with a downstream primer such as SEQ ID No: 9; upstream primer SEQ ID No: 10 is paired with a downstream primer such as SEQ ID No: 11; upstream primer SEQ ID No: 12 is paired with a downstream primer such as SEQ ID No: 13; upstream primer SEQ ID No: 14 is paired with a downstream primer such as SEQ ID No: 15.
  • upstream primer SEQ ID No: 1 is paired with the downstream primer such as SEQ ID No: 2; upstream primer SEQ
  • the upstream primer SEQ ID No: 16 is paired with the downstream primer such as SEQ ID No: 17; the upstream primer SEQ ID No: 18 is paired with the downstream primer such as SEQ ID No: 19; the upstream primer SEQ ID No: 20 and the downstream primer such as SEQ ID No: 21 pairing.
  • the amino acid sequence of the epitope peptide of the CYSTATIN S protein is shown as SEQ ID No: 50.
  • the diagnosis and prediction are preferably: The diagnosis and prediction are specifically: metastasis, micrometastasis, pTNM staging, dynamic detection during treatment, and prognosis of breast cancer.
  • a second object of the invention is to provide several capture agents that bind specifically to breast cancer markers.
  • the technical solution of the present invention is: A capture agent for a breast cancer marker, wherein the capture agent is a capture agent for the diagnosis and prediction of a breast cancer marker, wherein the breast cancer marker is CST4 gene, CST4 gene mRNA, CST4 gene cleavage cDNA, CST4
  • the nucleotide sequence of the primer in the 3) is shown in SEQ ID No: 1-2.
  • the nucleotide sequence of the probe in the 2) is shown in SEQ ID No: 3.
  • the nucleotide sequence of the amplicon in the 3) is shown in SEQ ID No: 43.
  • the capture agent is a specific antibody to the CYSTATIN S protein or the epitope peptide recognizing the CYSTATIN S protein.
  • amino acid sequence of the CYSTATIN S epitope peptide is shown as SEQ ID No: 50.
  • the third object of the present invention is to provide a new application of a capture agent and a kit prepared according to the application principle and a method for using the same; the application and the kit provide a new idea for detecting breast cancer, and the accuracy thereof is high; the method is simple in operation , suitable for clinical large-scale use.
  • a diagnostic kit containing the capture agent is provided.
  • the diagnostic kit is specifically:
  • the upstream primer is as shown in SEQ ID No: 1, the downstream primer SEQ ID No: 2; and the internal reference primer, the upstream primer of the internal reference primer is SEQ ID No: 30, the downstream primer is shown as SEQ ID No: 31; or
  • CST4 mRNA quantitative detection kit based on ligase chain reaction, which comprises 4 probes, the nucleotide sequences of which are shown in the nucleotide sequences of SEQ ID Nos: 33-36, respectively;
  • a quantitative detection kit for CST4 mRNA based on isothermal chain displacement amplification which contains primers and probes, The primers are shown in SEQ ID Nos: 37-40, and the probes are shown in SEQ ID No: 41.
  • the diagnostic kit may preferably be:
  • a double-antibody sandwich Elisa kit comprising a solid phase carrier, the capture agent immobilized on a solid support, the biotin-labeled capture agent, and a chromogenic substrate; the capture immobilized on a solid support
  • the agent is a specific monoclonal antibody
  • the biotin-labeled capture agent is a polyclonal antibody
  • a Western blotting kit comprising a solid phase carrier, a capture agent, an enzyme-labeled secondary antibody, and a chromogenic substrate, the capture agent comprising a specific monoclonal antibody, and the biotin-labeled capture agent is a specific polyclonal antibody;
  • a competitive ELISA kit comprising a solid support, an antigen immobilized on a solid support; a biotin-labeled capture reagent and a chromogenic substrate; a specific monoclonal antibody; the biotin-labeled capture agent is more specific anti.
  • the diagnostic kit also includes a positive control, a negative control or/and a blank control.
  • the specific monoclonal antibody is a murine monoclonal antibody, an anti-CYSTATIN S protein, the solid phase carrier is an ELISA plate, and the biotin-labeled specific polyclonal antibody is added. Bioantibody-labeled rabbit anti- CYSTATIN S protein polyclonal antibody.
  • the kit is an ELISA diagnostic kit based on a double-anti-sandwich method
  • the solid phase carrier is an ELISA plate
  • the capture agent immobilized on the solid phase carrier is a mouse anti-CYSTATIN S protein monoclonal antibody (R&D, MAB1296 (5ug/ml)
  • the biotin-labeled capture agent is a polyclonal antibody against rabbit anti-CYSTATIN S protein (1: 1000)
  • the chromogenic substrate is alkaline phosphatase; or the kit is based on the competitive Elisa method.
  • the solid phase carrier is a ELISA plate
  • the concentration of the CYSTATIN S protein is 5 ug/ml
  • the specific monoclonal antibody is a mouse anti-CYSTATIN S protein monoclonal antibody (R&D, MAB 1296, 1 : 2000) 3% ⁇
  • the enzyme-labeled secondary antibody is alkaline phosphatase-labeled goat anti-mouse IgG
  • the enzyme standard secondary antibody titer of 1: 2000 (Jackson, 1: 2000, dissolved in TBS 0.3% In BSA)
  • the chromogenic substrate is an alkaline phosphatase substrate
  • the volume ratio of the CYSTATIN S protein, the enzyme-labeled secondary antibody, and the chromogenic substrate is 1:2;
  • the kit is an immunoblotting-based diagnostic kit
  • the solid phase carrier is a nitrocellulose membrane
  • the capture agent is CYSTATIN S mouse monoclonal antibody (1: 1000)
  • the enzyme-labeled secondary antibody is conjugated to a peroxide.
  • Enzyme (Jackson) goat anti-rabbit IgG, chromogenic substrate is a commercial TMB solution (Kirkkeard and Perry Laboratories Inc. (Gaithersburg; MD)) "TMB Peroxidase Substrate" solution Cat. No. 50-76-01).
  • Corning ELISA plate wells were covered with CYSTATIN S (Abnova, cat. No H00001472-P01) (5 ug/ml) and blocked with 3% BSA. Serum samples containing anti-CYSTATIN S murine mAb (R&D, cat. No MAB 1296) (1:2000) and 8 dilutions were incubated overnight at 4 degrees. It was then added to the covered ELISA plate and incubated for 1 hour at 37 degrees.
  • a fourth object of the present invention is to provide an in vitro diagnostic method and an in vitro diagnostic kit for breast cancer, which are simple in operation, high in specificity and sensitivity.
  • the diagnostic kit to detect or predict breast cancer using the diagnostic kit to detect the content or expression level of the breast cancer marker in the sample to be tested, and comparing the obtained content or expression level with a normal person Determining whether the sample to be tested is positive; or directly determining whether the content or expression level exceeds a threshold, and when the threshold is exceeded, determining to be positive; the threshold is by comparing a normal person with a flag in a body fluid or tissue of a breast cancer patient
  • the content or expression level of the substance is statistically obtained;
  • the sample to be tested is any one or more of blood, urine, bone marrow, breast cancer cell line or breast cancer and adjacent surgical tissue and lymph nodes.
  • the threshold for the CYSTATIN S protein is at 3. 434 ng/ml.
  • kits for diagnosing and predicting breast cancer including a solid phase carrier, the capture agent immobilized on a solid phase carrier, the biotin-labeled capture agent, and a coloring substrate; the capturing agent immobilized on the solid phase carrier is a specific monoclonal antibody, and the biotin-labeled capturing agent is a specific polyclonal antibody;
  • the kit is for detecting the level of CYSTATIN S protein
  • the kit includes a solid phase carrier, a CYSTATIN S protein coated with a solid phase carrier, a CYSTATIN S-specific murine monoclonal antibody, an enzyme-labeled secondary antibody, and a chromogenic bottom.
  • the kit is for detecting CYSTATIN S protein levels, including a solid phase carrier, a capture agent, an enzyme-labeled secondary antibody, and a chromogenic substrate, the capture agent includes a specific monoclonal antibody, and the biotin-labeled capture agent is specific. Multi-antibody.
  • the kit is an ELISA diagnostic kit based on a double-anti-sandwich method, wherein the solid phase carrier is an ELISA plate, and the capture agent immobilized on the solid phase carrier is a mouse anti-CYSTATIN S protein monoclonal antibody.
  • the biotin-labeled capture agent is a polyclonal antibody against rabbit anti-CYSTATIN S protein with a titer of 1:1000, and the chromogenic substrate is alkaline phosphatase;
  • the kit is a diagnostic kit based on the competitive Elisa method, wherein the solid phase carrier is an ELISA plate, the concentration of the CYSTATIN S protein is 5 ug/ml, and the specific monoclonal antibody is a mouse antibody.
  • a CYSTATIN S protein monoclonal antibody having a potency of 1:2000, the enzyme-labeled secondary antibody being an alkaline phosphatase-labeled goat anti-mouse IgG having a titer of 1:2000.
  • the chromogenic substrate is an alkaline phosphatase substrate, and the volume ratio of the CYSTATIN S protein, the enzyme-labeled secondary antibody, and the chromogenic substrate is 1:2;
  • the kit is a diagnostic kit based on immunoblotting, characterized in that the solid phase carrier is a nitrocellulose
  • the membrane is a CYSTATIN S murine monoclonal antibody with a titer of 1:1000, a goat anti-rabbit IgG conjugated to peroxidase, and a chromogenic substrate of TMB solution.
  • the main advantages of the present invention are as follows: 1) It is found for the first time that CST4 mRNA and protein expression levels are closely related to breast cancer diagnosis, dynamic monitoring and prognosis, and the sample size is verified and the results are accurate. This correlation provides a new approach to breast cancer diagnosis, dynamic monitoring, and prognosis. 2) Developed reagents or kits suitable for breast cancer diagnosis, dynamic monitoring, and prognosis. The detection sensitivity is good.
  • Figure 1 shows the recombinant plasmid map of CST4 and Pmdl8-T
  • Figure 2 shows the expression of CST4 in normal human tissues (chip), including tonsil, posterior pituitary, thyroid, salivary gland, skeletal muscle, bone marrow, peripheral blood, lung, stomach, liver, heart, and red blood cells and platelets. , kidney, adrenal gland, intestine, colon, pancreas spleen, bladder, prostate, ovary, uterus, placenta, testis and breast cancer cell line HCC1937, SK-BR-3, MCF-7, human normal breast cell line Hs578Bst;
  • Figure 3 shows the difference in expression levels of CST124; CST1; CST2; CST4 in 20 pairs of breast cancer and adjacent tissues by dye method real-time quantitative PCR;
  • Figure 4 shows the expression distribution of CST4 in 100 cases of breast cancer and adjacent tissues by real-time PCR absolute quantification
  • Figure 5 shows the expression distribution of CST4 in 40 cases of breast cancer and 40 cases of biopsy specimens by Real-time PCR absolute quantification method
  • Figure 6 shows the expression distribution of CST4 in 30 pathologically positive lymph nodes and 30 pathologically negative lymph nodes in real breast cancer by absolute-time PCR.
  • Figure 7 is a comparison of the accuracy and cytological detection of peripheral blood free breast cancer cells by Real-time PCR absolute quantification
  • Figure 8 is a comparison of the accuracy and cytological detection of breast cancer bone marrow metastasis by Real-time PCR absolute quantification
  • Figure 9 is a graph showing the difference between the amount of CST4 in the cell-free RNA of breast cancer patients and the inflammation/normal person.
  • A is the amount of CST4 in the plasma Cell-free RNA of breast cancer patients (50 cases) by Real-time PCR absolute quantification method. Differences from inflammation (30 cases)/normal (30 cases); B is the ROC curve obtained by Real-time PCR absolute quantification, distinguishing breast cancer/inflammation/normal sensitivity and specificity;
  • Figure 10 shows the difference between CST4 in plasma Cell-free RNA and inflammation (30 cases)/normal (30 cases) in breast cancer patients (50 cases) by ligase chain reaction (LCR) method.
  • LCR ligase chain reaction
  • Figure 11 shows the difference between CST4 in plasma Cell-free RNA and inflammation (30 cases)/normal (30 cases) in breast cancer patients (50 cases) by reverse transcription strand displacement amplification (RTSDA). ;
  • Figure 12 shows the difference in expression of CST4 in urine samples from 30 cases of breast cancer, 20 cases of mastitis, and 20 cases of normal humans by Nucleic Acid based Amplificatin (NASBA).
  • NASBA Nucleic Acid based Amplificatin
  • Figure 13 shows the differential expression of CST4 in 80 cases of breast cancer (pTNM stage, 30 cases of ⁇ + ⁇ , 50 cases of III+IV) by different transcriptional-mediated amplification (TMA).
  • Figure 14 shows the expression of CYSTATIN S in breast cancer cell culture supernatants and normal human serum.
  • Figure 15 shows the expression of CYSTATIN S in breast cancer cell lines and normal human serum.
  • Figure 16 shows the expression of CYSTATIN S in serum of 20 normal and 30 breast cancer patients by monoclonal antibody competition ELISA.
  • Figure 17 shows the sensitivity and specificity of the ELISA method for detecting breast cancer CYSTATIN S protein and CEA.
  • Figure 18 shows a median array of breast cancer patients who were higher than CYSTATIN S protein expression levels after treatment and a disease-free survival curve below the median array.
  • the specimens used in the examples were obtained after signing the informed consent form with the patient and were obtained according to the hospital's prescribed procedures, which were obtained from Beijing Friendship Hospital.
  • samples such as lymph nodes obtained during surgery should be immediately extracted or stored in liquid nitrogen or RNAlater (Ambion products).
  • Peripheral blood, bone marrow or urine samples were first centrifuged at 4000 rpm, 4 ° C, 20 minutes, supernatant, 13000 rpm, 4 ° C, centrifugation for 10 minutes, the supernatant and precipitate were separated, RNA was immediately extracted or placed at -20 ° Store at C to -80 ° C.
  • Real-time PCR of TaqMan hydrolysis probes The above samples were carried out using a commercial nucleic acid extraction method.
  • a common, non-limiting example is the phenol/chloroform extraction method, such as the extraction of total RNA by the Trizol method of Invitrogen. And carry out RNA quality identification, related methods can be described by reference books such as "Molecular Biology Experiment”.
  • the reverse transcription process of mRNA was carried out using a commercial reverse transcription kit and according to the manufacturer's instructions, and the resulting cDNA was diluted to the required working concentration.
  • the specific primers used were optimized, and the upstream and downstream primers of CST4 were designed on exon 1.
  • a recombinant plasmid containing the CST4 amplicon was prepared, the desired plasmid was commercialized Pegm-TX promega) (Fig. 1), and the upstream and downstream primers were designed on exons 1, 3.
  • CST4 amplification was performed using Real-time PCR based on TaqMan hydrolysis probe.
  • Upstream primer gctctcaccctcctctccctg ( SEQ ID No: 1 )
  • Probe 5'-fam-ctccagctttgtgctctgcctctg-tamra-3' (SEQ ID No: 3)
  • the amplification length was 142 bp.
  • Upstream primer tgcctcgggctctcaccctcctct ( SEQ ID No : 22 )
  • the experimental group, the positive control group, and the negative control group were amplified together with the recombinant plasmid standard.
  • the standard curve is based on the concentration gradient of the recombinant plasmid and the corresponding CP (cross point) after amplification.
  • the copy number of the experimental group, the positive control group, and the negative control group was given according to the standard curve.
  • Dye-based real-time quantitative PCR Sample-treated Real-time PCR with TaqMan hydrolysis probe, CST1, 2, 4 (primers capable of simultaneously amplifying CST1, CST2, CST4) Primer, upstream primer: agtcccagcccaacttgga (SEQ ID No: 24 ), downstream bow I: gggaacttcgtagatctggaaaga (SEQ ID No: 25); CST4 upstream bow
  • RNA template can be amplified 2 ( 912 ), and the reaction product is placed in a fluorescence detector to detect the fluorescence intensity (U), thereby reacting the initial template amount.
  • the human tissue specimens used were collected from the Beijing Friendship Hospital in addition to the normal breast tissue, and other organizations were purchased from commercial organizations.
  • the Affymetrix nucleotide chip HG_U95Av was used to compare the expression of CST4 mRNA in each normal tissue. The procedure was as follows. The relative expression value of this experiment is a normalized signal value normalized by the housekeeping gene ⁇ -actin fluorescence value.
  • CST4, CST1, CST2, and CST4 mRNA expression levels were compared in 20 pairs of C1-C20 breast cancer tissues and adjacent tissues. mRNA expression in breast cancer and adjacent tissues is quite different, second only to CST1. The results are shown in Figure 3. All the above specimens were confirmed by pathology, real-time quantitative PCR was performed by dye method, positive samples were amplified normally, and negative samples were not amplified.
  • the kit contains:
  • CST4 bow I including:
  • Upstream primer agtacaacaa ggccaccgaa gat (SEQ ID No: 4)
  • Downstream bow I agaagcaaga aggaaggagg gag (SEQ ID No: 5)
  • Upstream bow I tacaacaagg ccaccgaaga tga (SEQ ID No: 6)
  • Downstream bow I agaagcaaga aggaaggagg gag (SEQ ID No: 7)
  • Upstream primer tgctactcct gatggctacc ctg (SEQ ID No: 8)
  • Downstream bow I gtggccttgt tgtactcgct gat (SEQ ID No: 9)
  • Upstream bow I agtacaacaa ggccaccgaa gat (SEQ ID No: 10)
  • Upstream primer tgctactcct gatggctacc ctg ( SEQ ID No: 12 )
  • Upstream primer tgctactcct gatggctacc ctg ( SEQ ID No: 14 )
  • Upstream primer tgggattatc ctattctcct ccttg (SEQ ID No: 16)
  • Downstream primer ctccagcttt gtgctctgcc tct ( SEQ ID No: 17)
  • Upstream primer tgctactcct gatggctacc ctg ( SEQ ID No: 18 )
  • Downstream primer ctcatcttcg gtggccttgt tgt (SEQ ID No: 19)
  • Upstream primer tacagtgggt gggagtgggt ggt ( SEQ ID No: 20)
  • Upstream primer aagatcattgctcctcctg (SEQ ID No: 30)
  • Downstream primer cgtcatactcctgcttgc ( SEQ ID No: 31 )
  • kit containing:
  • Primers, probes including:
  • Upstream primer gctctcaccctcctctccctg ( SEQ ID No: 1 )
  • RNA samples were extracted and reverse-transcribed cDNA samples were obtained after pathological verification.
  • Real-time PCR absolute quantitative method was used to identify the expression difference of CST4 in breast cancer and adjacent tissues. The number of test samples was 100 pairs. Linearity, amplification efficiency meets the requirements, positive samples are positive, negative samples are negative, and no template control is not amplified.
  • the sample of the needle biopsy is quite different from the sample taken by the surgery, mainly in the thin tissue of the sampled tissue.
  • the proportion of cells varies greatly, and sometimes it may only occupy a small part of the whole organization, and some may not even. Therefore, the inventors statistically compared the expression of CST4 in 40 specimens of breast cancer and 40 specimens of mastitis in a biopsy specimen.
  • the median number of copies of the cancer specimen was about 9.15 times the median copy number of the specimen, if at 113.795
  • the line at the copy can separate the cancer from the inflammation, which can provide a reference for the diagnosis of breast cancer by needle biopsy.
  • the results are shown in Fig. 5.
  • the method also uses Real-time PCR absolute quantification method.
  • the linearity of the standard curve and the amplification efficiency meet the requirements.
  • the positive samples are positive, the negative samples are negative, and the template control is not amplified.
  • the pathological evidence obtained during the operation was 30 lymph nodes with positive metastasis of breast cancer, and the size of metastases was different.
  • Pathologically-negative lymph nodes were mainly obtained from patients with early-stage breast cancer because the lymph nodes with negative pathological diagnosis but with micrometastasis were minimized to avoid experimental errors.
  • the experiment uses Real-time PCR detection method.
  • the specific materials and processes are the same as those in Example 2.
  • the linearity of the standard curve and the amplification efficiency meet the requirements.
  • the positive samples are positive, the negative samples are negative, and the template control is not amplified.
  • CST4 is highly expressed in metastatic positive lymph nodes and only in negative lymph nodes.
  • the positive lymph node expression of CST4 mRNA was 8.458 times higher than that in the negative lymph node. If the line is drawn from the 120.66 copy, it is basically possible to distinguish between positive and negative lymph nodes in breast cancer.
  • Two cases of pathologically negative lymph nodes detected CST4 weakly positive lymph nodes, which were observed by pathologists for detailed observation and identification of micrometastases. Therefore, the degree of expression of CST4 mRNA is not only 100% distinguishable from cytological detection results, but also can detect lymph nodes with micrometastases that are not detectable by cytology, and has higher sensitivity than cytological detection.
  • RNA was extracted from peripheral blood nucleated cells of red blood cells and platelets, and the expression level of CST4 mRNA was quantitatively detected by Real-time PCR. The expression of CST4 mRNA was determined by comparison with the expression of mastitis patients and normal humans. The presence of breast cancer cells is aligned with cytological detection.
  • the results of Figure 7 show that the detection method of the present invention 100% confirms the positive result of cytological identification, and at the same time, can detect partial metastasis cases in the case of negative cytological identification, indicating that the method has higher sensitivity than cytological detection.
  • the presence of micrometastases that are not detectable by cytology can be detected.
  • the bone marrow of breast cancer patients obtained by puncture and other methods were quantitatively detected by Real-time PCR.
  • the expression of CST4 mRNA was determined by comparison with normal bone marrow.
  • the presence or absence of metastasis and micrometastasis was confirmed by bone marrow. Compare.
  • kits were used to extract Cell-free RNA from plasma. Real-time PCR was used to detect breast cancer patients (50 cases). The amount of CST4 in plasma Cell-free RNA was associated with inflammation (30 cases), and normal subjects (30 cases). difference.
  • Figure 9-A shows that the median copy number of CST4 in cancer is about 8.87 times that of inflammation, and the normal 25.62, at the copy number of 71.218, can separate cancer from inflammation/normality.
  • ROC curve in Figure 9-B shows that the method for diagnosing breast cancer based on CST4 expression has higher sensitivity and specificity (area under the curve of 0.987), so CST4 can be used as a specific molecular marker for breast cancer diagnosis in non-invasive plasma samples. .
  • LCR Ligase chain reaction
  • the CST4 mRNA quantitative detection kit based on Ligase chain reation (LCR) contains the kit containing:
  • the median of relative light units (RLU) of CST4 in cancer was 10.881 times of inflammation and 35.286 times of normal, and the line was plotted at a relative fluorescence intensity of 17.458 RLU, which could be cancerous.
  • the inflammation/normality is separated.
  • RTSDA Reverse transcription strand displacement amplification
  • thermophilic strand displacement amplification tSDA
  • the kit contains: CST4 B 1 bow I substance: cccggcctctgtgtaccctgcta (SEQ ID No: 37)
  • kits were used to extract Cell-free RNA from plasma, and rtSDA was used to detect differences in CST4 levels in plasma cell-free RNA between patients with breast cancer (50 patients) and inflammation (30 patients) and normal subjects (30 patients).
  • the results are shown in Figure 11.
  • the median relative fluorescence intensity (RLU) of CST4 in cancer is 34.58 times that of inflammation, 35.89 times normal, and is plotted at a relative fluorescence intensity of 24.095 RLU, which serves as a threshold for cancer. It is separate from the inflammation/normal.
  • the kit contains:
  • CST4 bow I, probe including:
  • Upstream primer aattctaatacgactcactataggg-gctctcaccctcctctcctg (SEQ ID No: 32)
  • Avian myeloid leukemia virus (AMV) reverse transcriptase ribonucleotide (NTP), deoxyribonucleotide (dNTP), RNA fluorescent dye (Ribo-Green fluorescent dye).
  • AMV Avian myeloid leukemia virus
  • NTP ribonucleotide
  • dNTP deoxyribonucleotide
  • RNA fluorescent dye Ribo-Green fluorescent dye
  • kits were used to extract Cell-free RNA from urine, and Nucleic Acid based Amplificatin (NASBA) was used to detect CST4 and inflammation in urine-derived Cell-free RNA (30 cases). 20 cases), the difference between normal people (20 cases).
  • NASBA Nucleic Acid based Amplificatin
  • the median fluorescence intensity of CST4 in cancer is about 15.86 times that of inflammation, about 38.35 times that of normal, and the line number is 30.92, which can distinguish cancer from inflammation/normality.
  • Example 4 CST4 was used as a CST4 mRNA quantitative detection kit for transcriptional-mediated amplification (TMA) in pTNM staging, dynamic monitoring during treatment, and prognosis.
  • TMA transcriptional-mediated amplification
  • kit containing:
  • Primers, probes including:
  • Upstream primer aattctaatacgactcactataggg-gctctcaccctcctctcctg (SEQ ID No: 32)
  • Downstream primer tatcctattctcctccttgg (SEQ ID No: 2)
  • reagents are reagents other than primers and probes in the Gen-probe TMA assay.
  • CST4 can be used as an indicator of dynamic monitoring during the treatment of breast cancer patients.
  • Table 1 shows the amount of CST4 in the blood of breast cancer patients during chemoradiotherapy by real-time quantitative PCR.
  • Patient CST4 expression ⁇ l(copy) Imaging (cm)
  • Table 2 shows the amount of CST4 in the blood of breast cancer patients after real-time quantitative PCR or 1 month, 3 months, and 1 year after radiotherapy and chemotherapy. Table 2 shows the amount of CST4 in the blood of breast cancer patients during follow-up by real-time quantitative PCR.
  • the present invention relates to antibodies, kits, assays, and methods of use thereof for non-invasive labeling for diagnosis and monitoring of breast diseases and evaluation of therapeutic effects.
  • the CYSTATIN S recombinant protein of the present invention was purchased from Abnova (concentration 0.06 ug/ul, article number H00001472-P01); the CYSTATIN S mouse monoclonal antibody was purchased from R&D (potency 1:2000, article number MAB 1296); CYSTATIN S rabbit polyclonal The antibody was purchased from Abeam (potency 1:800, article number ab58515).
  • the present invention provides an assay for diagnosing the condition of a breast in a sample of a subject and whether it is metastasized or relapsed; in addition, an assay for evaluating whether the treatment is effective according to a sample of the subject is also provided.
  • the assay detects at least one selected protein in a patient sample, preferably a CYSTATIN S protein, more preferably the assay is to quantify or at least semi-quantitatively detect CYSTATIN S levels in a patient sample.
  • any type of reporter can be used to detect a protein, it is preferred that the reporter be a CYSTATIN S specific antibody or a fragment thereof.
  • the methods, assays and kits of the invention can be used to find early A person who is asymptomatic or asymptomatic.
  • the assay optionally and preferably utilizes at least one, and preferably a plurality of antibodies or fragments thereof that specifically bind to at least one CYSTATIN S epitope peptide for detecting the presence of an immune response to CYSTATIN S in a subject sample.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the monoclonal antibody binds to SEQ ID NO: 50, and/or can be obtained from any one of the sequences. More preferably, the amount of CYSTATIN S is determined.
  • the present invention also provides a method for quantifying the level of CYSTATIN S in a sample by quantifying the level of an immune response to CYSTATIN S in the sample using at least one of the above antibodies.
  • the optional and preferred subject is human and the detected immune response is characterized by antibodies and human polypeptides.
  • the immune response can optionally be detected by using any suitable assay, including but not limited to ELISA (enzyme-linked immunosorbent assay) or immunoblotting such as Western blot or a combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting such as Western blot or a combination thereof.
  • a competitive ELISA and a double-antibody sandwich ELISA are utilized.
  • the diagnosis or monitoring of the condition of the breast, the monitoring or monitoring of the effect of the treatment of the breast disease is effected by the quantification of the detected immune response, which represents the amount of the labeled polypeptide (CYSTATIN S ) in the sample.
  • the invention also relates to a test kit for detecting a labeled protein in a sample.
  • the test kit comprises at least one antibody or fragment thereof of the invention, which is capable of interacting with the labeled polypeptide present in the fluid being examined ( CYSTATIN S) reaction, and comprising at least one reporter component capable of detecting a complex consisting of an antibody or fragment thereof and a marker protein.
  • the antibody may optionally be a polyclonal or monoclonal antibody.
  • the detection kit, the reporter component can be an antibody, an antibody or fragment thereof used in the kit, and displaying a label.
  • the reporter component is preferably a suitable IgG antibody or a suitable IgM antibody.
  • the label is optionally and preferably an enzyme capable of catalyzing a color reaction, such as a peroxidase, and more preferably covalently bound to a second antibody.
  • the label can be a fluorescent moiety, or a colorimetric antibody.
  • the test kit is an ELISA test kit.
  • the ELISA assay kit is a competitive ELISA kit and a double-antibody sandwich ELISA kit as described in the Examples section below.
  • the antibody is a monoclonal antibody.
  • the protein used to produce the anti-CYSTATIN S antibody is coupled to a microtiter plate (also referred to herein as a matrix) used as an exemplary solid phase material, and the pre-incubated sample is subsequently perfused into the plate and not previously present in the serum.
  • the epitope-bound antibody binds to the protein on the plate.
  • the reporter component consists of the appropriate immunoglobulin, especially It is an anti-IgG antibody and/or an anti-IgM antibody that detects antibodies that bind to the plate and is coupled to the enzyme and/or fluorescent label shown by the catalysis.
  • the detection kit is an immunoblot, also called a Western blot (Western blot in the detection kit of the characteristic, the protein in the sample utilizes an electrophoresis gel, such as a polyacrylamide gel Transfer to a solid substrate (for example, a nitrocellulose membrane). Transfer can be carried out, for example, by electrotransfer.
  • An immunoreaction occurs between the protein present on the substrate and an antibody against the protein. Preferably, a monoclonal antibody or a fragment thereof is utilized. The reaction can then be detected by a suitable method, such as an antibody using an enzyme-labeled and/or fluorescently labeled anti-antibody.
  • the detection kit is for use in a flow assay.
  • an antibody or a fragment thereof is bound to a column, and a sample to be tested is perfused through the column.
  • the antibody may be polyclonal or monoclonal as described above, and preferably the monoclonal antibody used.
  • the above specific antibodies mentioned in the present invention are included.
  • the sample to be tested is poured into the column and flows through the column.
  • the protein bound to the column flows through or through the competitive antibody. Washing is carried out by changing the buffer conditions.
  • they are washed at different times.
  • a test kit for determining the level of CYSTATIN S taken from a sample of a subject is provided, wherein The dual indicator indicates whether the CYSTATIN S level is at a normal level or above a normal level.
  • the sample taken from the subject is a fluid sample.
  • the test kit preferably includes a container containing the sample fluid, a specific antibody or fragment thereof, and an indicator.
  • the antibody is monoclonal, and more preferably a monoclonal antibody as described above for the present invention.
  • the kit is further selected to include a solution and a buffer required for the assay, and optionally a print containing instructions for performing the assay and interpreting the results.
  • the kit can be used by those skilled in the art and can be used in any physical location, including but Not limited to hospitals, clinics and private homes.
  • kits of the present invention may be treated as a breast discomfort, and the test includes screening the subject to detect breast cancer.
  • the subject against which the kit of the invention is directed may be a mastitis patient, and the test includes screening the subject to detect breast cancer.
  • the subject against which the kit of the present invention is directed may be a family of breast cancer, and the test includes sieving the subject Check to detect breast cancer.
  • Samples contemplated by the present invention include, but are not limited to, serum samples, plasma samples, urine samples, or blood samples. Blood samples may include whole blood samples or blood fraction samples.
  • the antibodies known in the present invention can be used for the evaluation of breast disease diagnosis or whether or not breast cancer metastasizes.
  • Some methods well known to those skilled in the art can be used to apply this guidance, such as using the antibody kit conjugated to a detectable moiety (e.g., a fluorescent moiety) as described herein for assessing serum, plasma, urine samples obtained from a test subject.
  • a detectable moiety e.g., a fluorescent moiety
  • CYSTATIN S recombinant protein purchased from Abnova (concentration 0. 06ug/ul, article number H00001472-P01);
  • CYSTATIN S rabbit polyclonal antibody purchased from Abeam (valence 1 : 800, part number ab58515 ); CYSTATIN S mouse monoclonal antibody was purchased from R&D (potage 1: 2000, article number MAB 1296)
  • Immunoprecipitation of cell supernatant was performed directly by adding 2 mM PMSF, Protein A-Sepharose and anti-CYSTATIN S antibody, and then rotated overnight at 4 degrees.
  • a serum sample 200 ul was immunoprecipitated in a similar manner using an anti-Cystatin antibody conjugated to Protein A-Sepharose by dimethl pimel imidate.
  • the immunoprecipitate was washed as described in an earlier study, and the immunoprecipitate was treated with N-glycosidase F and SDS_PAGE.
  • SDS_PAGE was performed on 15% polyacrylamide (Lae l i gel ) unless otherwise stated. The gel was analyzed by fluorescence autoradiography using 20% 2,5-diphenyloxazole and quantified by densitometry as previously described.
  • Protein electroporation and immunoblotting The protein spots were transferred to a nitrocellulose membrane. Blocking was carried out for 2 hours at room temperature with 5% skim milk powder and 0.1% Brij-35 in PBS. After blocking, they were incubated overnight with CYSTATIN S rabbit polyclonal antibody at 4 degrees. The blot was washed three times in PBS containing 0.1% Brij-35 and incubated with 0.227 ul of goat anti-rabbit IgG conjugated to peroxidase (Jack) for 1 hour at 37 °C. 0. l%Brij-35 wash in PBS Dilute 4 times and wash once with PBS, then use commercial TMB solution (Kirkegaard and Perry Laboratories Inc.
  • Alkaline phosphatase substrate (KPL, lOOul per L, Blue Phos solution cat. No 508805 Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) was added, and 0D was quantified at 405 nm using an ELISA reader.
  • biotin-labeled rabbit anti-CYSTATIN S protein polyclonal antibody was added (1: 1000). (Biotin was labeled and purified before the experiment)), incubated at 37 degrees for 1 hour.
  • Alkaline phosphatase substrate KPL, lOOul per L, Blue Phos solution cat. No 508805 Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) was added, and 0D was quantified at 405 nm using an ELISA reader.
  • CST4 mRNA is abnormally highly expressed in breast cancer, and CYSTATIN S is a secreted protein distributed in various body fluids and secretions.
  • CYSTATIN S is a secreted protein distributed in various body fluids and secretions.
  • the breast cancer cell line HCC1973 with high mRNA expression as shown in Fig. 14, the cell supernatant of lane 1_2, the normal human serum of one donor (S1, lanes 3-4), and subjected to 15% SDS_PAGE.
  • the protein was then transferred to a nitrocellulose membrane and the blot was reacted with an anti-CYSTATIN S antibody followed by reaction with goat anti-rabbit peroxidase.
  • Band detection was performed using a TMB membrane peroxidase substrate (3, 3', 5, 5'-tetramethylbenzidine). All methods are basically like the above materials Performed as described in the Methods section.
  • the method is the same as "1", and the result is shown in Fig. 15.
  • the lower part is the band of the ⁇ -actin protein of the internal reference.
  • Lanes 3, 4 represent normal serum samples with only very light bands; lanes 1, 2 represent a more pronounced band in serum samples from breast cancer patients.
  • CYSTATIN S peptide 5 ⁇ g/ml CYSTATIN S peptide was added to the ELISA plate at 4 degrees overnight; normal human serum (20 cases), breast cancer patient serum (30 cases) and anti-CYSTATIN S monoclonal antibody (1: 2000, soluble in TBS (0.3% BSA in 154 mM NaCl in 10 mM Tris-HCl in pH 7.5) was pre-incubated with 4 degrees overnight. The sample incubated with the monoclonal antibody was then added to an ELISA plate coated with CYSTATIN S peptide and incubated for 1 hour at room temperature. Subsequently, it was incubated with 0.
  • the median serum CYSTATIN S concentration was 1. 35 ng / ml
  • the median serum CYSTATIN S concentration of breast cancer patients was 2. 95 ng / ml, at 3. 105 ng / ml
  • the scribing can basically distinguish cancer from normal, so it can be used as a reference value for clinical diagnosis of breast cancer.
  • the CYSTATIN S protein detection method is the same as "3", and the CEA detection commercialization kit (Germany DRG, item number EIA5071) is operated according to its instructions.
  • the results are shown in Figure 17 and Table 3.
  • the area under the CYSTATIN S curve is 0.832, which is greater than CEA. That is, the ELISA method for detecting CYSTATIN S in serum/plasma is superior to the detection of CEA in terms of sensitivity and specificity.
  • Table 3 Area under the curve
  • Variables tested CYSTATIN S , CEA has at least one variation between the actual positive group and the actual negative group.
  • This example provides a number of non-limiting, illustrative embodiments of the assays, kits, and methods of use thereof.
  • Methods for detecting breast disease include: immobilizing the soluble protein CYSTATIN S antigen to a support, washing with R&D antibody (MAB1296), and adding a labeled secondary antibody (alkaline phosphatase-labeled goat anti-rabbit IgG, Biyuntian, Cat. No. A0239), washing and directly or indirectly detecting and/or measuring the label, which reflects the amount of the protein of interest bound to the antibody.
  • R&D antibody MAB1296
  • a labeled secondary antibody alkaline phosphatase-labeled goat anti-rabbit IgG, Biyuntian, Cat. No. A0239
  • supports include, but are not limited to, latex particles, cellulosic materials such as cellulose sheets, plastic assay plates and particles.
  • the antigens used may optionally be immobilized on the support, e.g., by covalent bonding or physical adsorption.
  • the sample to be tested is human serum or the like.
  • the surface of the optional and preferred support is "closed” by pre-incubation with bovine serum albumin (BSA) or the like prior to sample addition to at least reduce the likelihood of other antibodies non-specifically binding the support in the sample.
  • BSA bovine serum albumin
  • the support is then washed with a suitable buffer, such as a phosphate buffer containing a surfactant.
  • a non-limiting example of a labeled second antibody is a labeled anti-mouse polyclonal antibody.
  • Effective labels include, but are not limited to, various types of enzymes such as alkaline phosphatase, luciferase, peroxidase, ⁇ -galactosidase, and the like, as well as various fluorescent compounds such as fluorescein and the like.
  • Compounds such as biotin, avidin, streptavidin, digitoxin, etc. can be inserted between the antibody and the label.
  • labeled as an enzyme When labeled as an enzyme, its presence can optionally be detected and/or measured by the addition of a substrate and detecting and/or measuring luminescence or color development that occurs as a result of the catalytic action of the enzyme and/or by measuring changes in light absorption.
  • labeled as a fluorescent compound it can optionally be detected and/or measured by illuminating the reaction system with ultraviolet light and detecting and/or measuring the emitted fluorescence.
  • a sensitizer can be used if necessary.
  • the agent for detecting and/or measuring the marker protein CYSTATIN S using an antibody of the invention that binds to at least one epitope of the marker (SEQ ID NO: 50) preferably comprises an antibody or fragment thereof, a necessary amount of a second antibody or matrix (if needed) And, optionally, one or more "support” reagents necessary for the action of the previously described reagents.
  • These agents are optionally and preferably provided in a kit.
  • the above reagents can be used as an agent for diagnosing breast cancer, staging breast cancer, evaluating whether to metastasize, and evaluating the therapeutic effect.
  • the kit is preferably characterized by a specific antibody or fragment thereof.
  • the kit is further characterized by a reporter component that is capable of detecting the presence of a marker protein in a subject sample.
  • the reporter component is a suitable second antibody, optionally and preferably also comprising a label that detects the second antibody, if desired.
  • the kit may also optionally and preferably be pre-incubated with one or more buffers, such as a "blocking" buffer that is preincubated with a substrate to block non-specific reactions with the matrix and/or proteins immobilized thereon; Or a plurality of buffers for the sample, the second antibody, the matrix, and/or for washing the substrate between incubation with the previously described reagents.
  • buffers such as a "blocking" buffer that is preincubated with a substrate to block non-specific reactions with the matrix and/or proteins immobilized thereon; Or a plurality of buffers for the sample, the second antibody, the matrix, and/or for washing the substrate between incuba
  • the kit is provided for use in a competition assay wherein the control protein binds to a solid phase material.
  • the antibody or fragment thereof is pre-incubated with the serum sample and subsequently applied to the plate.
  • the binding of the antibody to the plate is detected by the reporter component, whereby one skilled in the art can determine whether the antibody binds to an epitope from the serum, and preferably determines the amount of antibody that binds to an epitope in the serum, and thereby determines the labeled protein in the sample. the amount.
  • the kit provides for a double-antibody sandwich assay in which an anti-CYSTATIN S antibody binds to a solid phase material, and then the standard CYSTATIN S and the treated test sample serum are separately added to the plate, and the anti-reporter The binding of a CYSTATIN S polyclonal antibody to a plate, whereby one of skill in the art can determine whether the antibody binds to an epitope from the serum, and preferably determines the amount of antibody that binds to an epitope in the serum, and thereby determines the amount of labeled protein in the sample. .
  • the type of reagent and/or kit, and/or the need for other types of equipment other than the kit and/or other types of equipment combined with the kit, each of which depends on the use of the kit The type of assay.
  • non-limiting examples of the above assays include ELISA, Western blot or flow cytometry.
  • An exemplary method for performing an ELISA assay is as described above. Western blots are often more accurate than ELISA but require more time and/or equipment for operation.
  • Serum Whole blood specimens should be placed at room temperature for 2 hours or at 4 °C overnight, centrifuged at 1000 ⁇ g for 20 minutes, the supernatant can be taken for testing, or the specimen can be placed at -20 °C or -80 °C. Storage, but should avoid repeated freezing and thawing; Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge at 1000xg for 2 minutes at 2-8°C for 15 minutes within 30 minutes after collection, or place the specimen at -20°C or -80°. C save, but should avoid repeated freezing and thawing.
  • the kit contains the following: 1) Enzyme-linked plates, coated. 2) Standard: Concentration of lOOng/ml CYSTATIN S, first diluted to 10 ng/ml CYSTATIN S with 1% BSA in PBST, and then diluted to 10 ng/ml CYSTATIN S, 5 ng/ Ml CYSTATIN S , 2. 5 ng/ml CYSTATIN S , Ing/mL CYSTATIN S, 0. 5 ng/ml CYSTATIN S, 0. 25 ng/ml of CYSTATIN S , sample dilution directly as standard concentration 0 ng/ml CYSTATIN S, prepared within 15 minutes before use.
  • CYSTATIN S standard Take 0.5 ml (not less than 0.5 ml) 8 ng/ml CYSTATIN S The above standard is added to an Eppendorf tube containing 0.5 ml of the sample dilution, and mix well. The rest of the concentration is deduced by analogy. 3) 3% BSA in PBST blocking solution.
  • the specific steps include:
  • Blocking Add 200 ul of PBS containing 3% BSA to each well, 37 degrees lh or 4 degrees overnight. Discard the solution in the well and wash it 3 times with washing buffer for 3 minutes each time.
  • Biotin-labeled rabbit anti-human CYSTATIN S polyclonal antibody lOOul (diluted with 1% BSA in PBST 1:200 before use) was added to the well, and the plate was coated with a membrane and reacted at 37 ° C for 60 minutes.
  • the experimental method was the same as the above "7", pathologically confirmed breast cancer T stage 20 patients, N stage patients 30, M stage 30, the results are shown in Table 4, as the disease progresses, CYSTATIN S protein expression level also Increase, suggest that CYSTATIN S protein expression levels can be used for breast cancer pTNM staging
  • CYSTATIN S protein expression levels are used to assess whether breast cancer metastasizes
  • the experimental method was the same as the above "7", 20 patients with pathologically confirmed breast cancer were not metastasized, and 30 patients with metastatic disease were shown in Table 5.
  • the expression level of CYSTATIN S protein in metastatic patients was higher than that in non-metastatic patients, suggesting that CYSTATIN S protein expression level is available. Indicates whether breast cancer has metastasized.
  • CYSTATIN S protein expression level is used to evaluate the efficacy of adjuvant endocrine therapy combined with chemotherapy in breast cancer.
  • the above-mentioned “7" the study included 2885 patients with N0-1 breast cancer, 4 cycles of AC (multiple ratio) Star + cyclophosphamide) or AT (doxorubicin + paclitaxel) regimen chemotherapy, and then according to the hormone receptor (HR) status to choose whether to assist endocrine therapy, the follow-up time was 76 months.
  • the expression level of CYSTATIN S protein was detected at the end of the treatment period.
  • the effective data was 776 cases, and the median expression of CYSTATIN S protein was 3.96 ng/ml.
  • the results are shown in Figure 19. Patients above this median had a disease-free survival (DFS) of 49% (PFS), and patients below this median had a disease-free survival of 64%.
  • DFS disease-free survival
  • PFS disease-free survival

Abstract

提供了CST4基因、CST4基因的mRNA、CST4基因剪切子的cDNA、CST4特异性引物对应的扩增子、CST4基因编码的CYSTATIN S蛋白和CYSTATIN S蛋白的表位肽在制备诊断和预示乳腺癌标志物中的应用。其可应用于乳腺癌的诊断、动态监测及预后判断。

Description

诊断和预示乳腺癌的标志物
技术领域 本发明属于生物技术和医学领域， 更具体的， 本发明涉及一种乳腺癌标志物及其用 途， 乳腺癌诊断、 动态检测以及预后判断的试剂或试剂盒及其使用方法。 背景技术 据世界卫生组织 （WHO) 统计， 全世界每年约有 120 万妇女发生乳腺癌。 我国每年 有超过 40 万人罹患乳腺癌， 发病率逐年上升。 在北京、 上海等大城市， 乳腺癌已经升至 女性恶性肿瘤的第一位。 30年来， 上海乳腺癌发病率已从 17.7/10万上升至 70/10万， 居 各类女性肿瘤发病之首， 其发病率已由女性恶性肿瘤中的第 2 位跃至首位， 死亡率高达 40%以上。
乳腺癌早期检出率和总体治愈率不断提高， 但 WHO2008年报告每年约有 54万乳腺 癌患者死亡， 国内致死率每年还在以 3%的速度增长。 乳腺癌的防治仍存在大量亟待解决 的问题， 包括如何更早期发现、 及时干预， 如何实现疗效评估和监测， 如何对术后患者做 到实时准确的复发监控。
发展具有高灵敏度、 特异性强的乳腺癌诊断产品， 是提高乳腺癌早期检出率、 改善患 者预后的关键之一。 在肿瘤发生发展过程中， 作为组织蛋白酶的天然抑制蛋白 Cystatin基 因家族与肿瘤的发生发展关系密切。 在组织和体液中， Cystatin家族蛋白可逆性地结合半 胱氨酸蛋白酶， 防止组织蛋白酶的活性过度。 Cystatin C 是组织蛋白酶 B 最强的抑制蛋 白， 但是在卵巢癌和头颈部癌症中， cystatin C 的表达有不同水平的上升。 而在非小细胞 肺癌中， stefin A ( cystatin 家族成员） 的表达水平有所增加； 在脑膜瘤中， stefin B 在 mRNA水平上有明显降低； cystatin F (亦称为 leukocystatin或 CMAP) 在多种肿瘤中的表 达水平都有显著增加。 研究结果显示， cystatin 的几个家族成员在不同肿瘤中的表达水平 有所上升， 很可能是由于在肿瘤发生发展过程中需要组织蛋白酶的参与， 先诱导其表达量 增加， 再激活机体本身的应激机制， 导致 cystatin 的表达量上升以抑制过盛的蛋白酶活 性。 但 cystatin的表达量并不与肿瘤的发展成正相关的关系， 因为在胶质瘤中， cystatin C 的低表达意味着疾病的晚期， 病人的生存期较短， 且易于复发， 该结论在 mRNA 和蛋白 水平上都得到了验证。
本发明证实 cystatin 家族成员 CST4 及其剪切子的表达水平与乳腺肿瘤关系密切。 CST4, 又名 cystatin S， 是半胱氨酸蛋白酶抑制剂 （cystatin) 家族成员， 含 141 个氨基 酸， 分布于多种体液和分泌物中， 如眼泪、 唾液、 血清、 血浆等。 发明内容 本发明的目的之一在于提供 CST4基因、 CST4基因的 mRNA、 CST4基因剪切 子的 cDNA、 CST4特异性引物对应的扩增子、 CST4基因编码的 CYSTATIN S蛋白 和 CYSTATIN S蛋白的表位肽的新应用， 该应用为乳腺癌的诊断提供了新思路。
为实现上述目的， 本发明的技术方案为：
1. CST4 基因、 CST4 基因的 mRNA、 CST4 基因剪切子的 cDNA、 CST4 特异性引物对应的扩增子、 CST4基因编码的 CYSTATIN S蛋白和 CYSTATIN S蛋白 的表位肽在制备诊断和预示乳腺癌标志物中的应用， 所述 CST4 基因的核苷酸序列 如 SEQ ID No: 42所示。
优选的， CST4 基因、 CST4 基因的 mRNA、 CST4 基因剪切子的 cDNA 的 探针如核苷酸序列如 SEQ ID No: 3所示。
优选的， 所述扩增子的特异性引物， 其上游引物如 SEQ ID No: 1， 4,6,8,10,12,14,16,18,20 ； 所 示 ， 下 游 弓 I 物 如 SEQ ID No ： 2, 5,7,9,11,13,15,17,19, 21 所示， 上游引物 SEQ ID No: 1 与下游引物如 SEQ ID No: 2配对； 上游引物 SEQ ID No: 4与下游引物如 SEQ ID No： 5配对； 上游引物 SEQ ID No: 6与下游引物如 SEQ ID No: 7配对； 上游引物 SEQ ID No: 8与下游引物如 SEQ ID No: 9配对； 上游引物 SEQ ID No: 10与下 游引物如 SEQ ID No: 11配对； 上游引物 SEQ ID No: 12与下游引物如 SEQ ID No: 13配对； 上游引物 SEQ ID No: 14与下游引物如 SEQ ID No： 15配 对； 上游引物 SEQ ID No: 16与下游引物如 SEQ ID No: 17配对； 上游引物 SEQ ID No: 18与下游引物如 SEQ ID No: 19配对； 上游引物 SEQ ID No: 20与下游引物如 SEQ ID No: 21配对。
优选的， 所述 CYSTATIN S蛋白的表位肽的氨基酸序列如 SEQ ID No: 50所示。 所述诊断和预示优选为： 所述诊断和预示具体为： 乳腺癌的转移、 微转 移、 pTNM分期、 治疗过程中的动态检测及预后判断。
本发明的目的之二在于提供若干种捕获剂， 其能和乳腺癌标志物特异性的结 合。
为实现上述目的， 本发明的技术方案为： 乳腺癌标志物的捕获剂， 所述捕获剂为所述诊断和预示乳腺癌标志物的捕获 剂， 所述乳腺癌标志物为 CST4 基因、 CST4 基因的 mRNA、 CST4 基因剪切子 的 cDNA、 CST4特异性引物对应的扩增子、 CST4基因编码的 CYSTATIN S蛋白 和 CYSTATIN S蛋白的表位肽。
所述 3) 中的所述引物的核苷酸序列如 SEQ ID No : 1 -2所示。
所述 2) 中的所述探针的核苷酸序列如 SEQ ID No : 3所示。
所述 3) 中的所述扩增子的核苷酸序列如 SEQ ID No : 43所示。
所述捕获剂为所述 CYSTATIN S 蛋白或所述识别 CYSTATIN S 蛋白的表位肽的特异性抗 体。
所述 CYSTATIN S表位肽的氨基酸序列如 SEQ ID No : 50所示。
本发明的目的之三在于提供捕获剂的新应用及根据该应用原理制备的试剂盒及其使用方 法； 该应用和试剂盒为检测乳腺癌提供了新思路， 其准确性高； 该方法操作简单， 适用于临 床大规模使用。
为实现上述目的， 本发明的技术方案为：
所述捕获剂在制备乳腺癌检测试剂或试剂盒中的应用。
含有所述的捕获剂的诊断试剂盒。
所述诊断试剂盒具体为：
1 ) 为基于 Taqman 水解探针法的 CST4 mRNA 实时定量检测试剂盒， 其上游引物如 SEQ ID No:l， 其下游引物 SEQ ID No:2所示， 所述探针的核苷酸序列如 SEQ ID No:3所 示； 或
2 ) 为基于染料法的 CST4 mRNA 实时定量检测试剂盒， 其上游引物如 SEQ ID No:l， 其下游引物 SEQ ID No:2所示； 及以内参引物， 所述内参引物的上游引物如 SEQ ID No: 30所示， 下游引物如 SEQ ID No: 31所示； 或
3) 为基于核酸序列扩增法的 CST4 mRNA定量检测试剂盒或基于转录介导的扩增的 CST4 mRNA定量检测试剂盒， 两者均含有 CST4引物和探针， 所述 CST4的上游引物如 SEQ ID No:32所示， 所述下游引物如 SEQ ID No:2所示， 所述探针如 SEQ ID NO: 3所 示； 或
4) 为基于连接酶链反应的 CST4 mRNA定量检测试剂盒， 其含有 4条探针， 所述探 针的核苷酸序列分别如 SEQ ID No:33-36的核苷酸序列所示； 或
5) 为基于适温链置换扩增的 CST4 mRNA 定量检测试剂盒， 其含有引物和探针， 所 述引物如 SEQ ID No:37-40所示， 所述探针如 SEQ ID No:41所示。
所述诊断试剂盒可优选为：
1) 双抗夹心 Elisa试剂盒， 包括固相载体、 固定在固相载体上的所述捕获剂、 生物素 标记的所述捕获剂及显色底物； 固定在固相载体上的所述捕获剂为特异性单抗， 生物素标 记的所述捕获剂为多抗；
或 2) 蛋白印迹试剂盒 包括固相载体、 捕获剂、 酶标二抗及显色底物， 捕获剂包 括特异性单抗， 生物素标记的所述捕获剂为特异性多抗；
或 3) 竞争 ELISA 试剂盒 包括固相载体、 固定在固相载体上的抗原； 生物素标记 的捕获剂及显色底物； 特异性单抗； 生物素标记的所述捕获剂为特异性多抗。
所述诊断试剂盒还包括阳性对照、 阴性对照或 /和空白对照。
所述双抗夹心 Elisa试剂盒中， 所述特异性单抗为鼠单抗， 抗- CYSTATIN S 蛋白， 所 述固相载体为酶标板， 所述生物素标记的特异性多克隆抗体为加入生物素标记的兔抗- CYSTATIN S 蛋白的多抗。
所述试剂盒为基于双抗夹心法的 ELISA诊断试剂盒， 所述固相载体为酶标板， 所述固 定在固相载体上的捕获剂为鼠抗- CYSTATIN S 蛋白单抗 （R&D, MAB1296 ) (5ug/ml), 生物 素标记的捕获剂为兔抗 - CYSTATIN S 蛋白的多抗 （1 : 1000)， 显色底物为碱性磷酸酶； 或所述试剂盒为基于竞争 Elisa 法的诊断试剂盒， 所述固相载体为酶标板， 所述 CYSTATIN S 蛋白的浓度为 5ug/ml,所述特异性单抗为鼠抗- CYSTATIN S 蛋白单抗 ( R&D, MAB 1296, 1 : 2000)， 所述酶标二抗为碱性磷酸酶标记的山羊抗 -小鼠 IgG， 所述酶 标二抗效价为 1 : 2000 (Jackson, 1： 2000, 溶于 TBS中的 0. 3%BSA中）， 所述显色底物为 碱性磷酸酶底物， 所述 CYSTATIN S蛋白、 酶标二抗及显色底物的体积比为 1 : 2;
或所述试剂盒为基于免疫印记的诊断试剂盒， 所述固相载体为硝酸纤维素膜， 捕获剂 为 CYSTATIN S 鼠单抗 （1 : 1000)， 酶标二抗为缀合至过氧化物酶 （Jackson) 的山羊抗-兔 IgG , 显色底物为商品化的 TMB 溶液 ( Kirkegaard and Perry Laboratories Inc. (Gaithersburg; MD)) " TMB Peroxidase Substrate" solution Cat. No。 50-76-01)。
所述的试剂盒的使用方法， 其特征在于， 具体步骤包括：
将 Corning ELISA 平板孔用 CYSTATIN S ( Abnova, cat. No H00001472-P01 ) ( 5ug/ml ) 覆盖且用 3%BSA封闭。 将包含抗- CYSTATIN S 鼠单抗 (R&D, cat. No MAB 1296) ( 1： 2000) 与 8个倍比稀释的血清样品于 4度温育过夜。 随后加至覆盖的 ELISA板中且 37度温育 1小时。 每孔用 TBS (溶于 PH=7. 5的 10 mM Tris-HCl中的 154mM的 NaCl ) 洗 涤且缀合至碱性磷酸酶中的山羊抗 -小鼠 IgG (Jackson, 1： 2000， 溶于 TBS中的 0. 3%BSA 中） 在 37度反应 1小时。 加入碱性磷酸酶底物 （KPL， lOOul每孔， Blue Phos solution cat. No 508805 Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) )， 利 用 ELISA读数器在 405nm波长处定量 0D。
本发明的目的之四在于提供一种乳腺癌的体外诊断方法和体外诊断试剂盒， 该方法操 作简单， 特异性和灵敏性高。
为实现上述目的， 本发明的技术方案为：
运用所述的诊断试剂盒检测或预示乳腺癌的方法， 用所述诊断试剂盒检测待测样品中 所述乳腺癌标志物的含量或表达水平， 将得到的含量或表达水平与正常人进行 比较， 判定所述待测样品是否为阳性； 或直接判定所述含量或表达水平是否超过阈 值， 当超过阈值时， 判定为阳性； 所述阈值是通过对比正常人与乳腺癌患者 体液或组织中标志物的含量或表达水平统计得到； 所述待测样品为血液、 尿 液、 骨髓、 乳腺癌细胞株或乳腺癌及癌旁手术组织、 淋巴结中的任一种或多种。 如 所述 CYSTATIN S蛋白的阈值为在 3. 434ng/ml。
用于诊断和预示乳腺癌的试剂盒， 所述试剂盒用于检测 CYSTATIN S 蛋白水平， 包括 固相载体、 固定在固相载体上的所述捕获剂、 生物素标记的所述捕获剂及显色底物； 固定 在固相载体上的所述捕获剂为特异性单抗， 生物素标记的所述捕获剂为特异性多抗；
或所述试剂盒用于检测 CYSTATIN S 蛋白的水平， 所述试剂盒包括固相载体、 包被于 固相载体的 CYSTATIN S蛋白、 CYSTATIN S特异性鼠单抗、 酶标二抗及显色底物；
或所述试剂盒用于检测 CYSTATIN S 蛋白水平， 包括固相载体、 捕获剂、 酶标二抗及 显色底物， 捕获剂包括特异性单抗， 生物素标记的所述捕获剂为特异性多抗。
所述试剂盒为基于双抗夹心法的 ELISA诊断试剂盒， 其特征在于， 所述固相载体为酶 标板， 所述固定在固相载体上的捕获剂为鼠抗- CYSTATIN S 蛋白单抗， 生物素标记的捕 获剂为效价为 1 : 1000的兔抗 - CYSTATIN S 蛋白的多抗， 显色底物为碱性磷酸酶；
或所述试剂盒为基于竞争 Elisa法的诊断试剂盒， 其特征在于， 所述固相载体为酶标 板， 所述 CYSTATIN S蛋白的浓度为 5ug/ml,所述特异性单抗为鼠抗- CYSTATIN S 蛋白单 抗， 其效价为 1 : 2000， 所述酶标二抗为碱性磷酸酶标记的山羊抗 -小鼠 IgG， 所述酶标二 抗效价为 1 : 2000， 所述显色底物为碱性磷酸酶底物， 所述 CYSTATIN S蛋白、 酶标二抗及 显色底物的体积比为 1 : 2;
或所述试剂盒为基于免疫印记的诊断试剂盒， 其特征在于， 所述固相载体为硝酸纤维 素膜， 捕获剂为效价为 1： 1000的 CYSTATIN S鼠单抗， 酶标二抗为缀合至过氧化物酶的山 羊抗-兔 IgG , 显色底物为 TMB溶液。
本发明的主要优点在于： 1 ) 首次发现并证实 CST4 mRNA和蛋白表达水平与乳腺癌 诊断、 动态监测以及预后判断具有密切相关性， 验证的样本量多， 结果准确。 该相关性的 提出为乳腺癌诊断、 动态监测以及预后判断提供了新的途径。 2) 开发了适合于乳腺癌诊 断、 动态监测以及预后判断的试剂或试剂盒， 检测灵敏度良好。
附图说明 下面结合附图及实施例对本发明作进一步描述：
图 1 为 CST4与 Pmdl8-T重组质粒图谱；
图 2 为 CST4在人体正常组织中的表达情况 （芯片）， 所述组织包括扁桃体、 垂体后 叶、 甲状腺、 唾液腺、 骨骼肌、 骨髓、 除去红细胞和血小板的外周血、 肺、 胃、 肝脏、 心 脏、 肾、 肾上腺、 肠、 结肠、 胰脏脾脏、 膀胱、 前列腺、 卵巢、 子宫、 胎盘、 睾丸以及乳 腺癌细胞株 HCC1937, SK-BR-3, MCF-7, 人正常乳腺细胞株 Hs578Bst ;
图 3为染料法实时定量 PCR给出的 CST124; CST1 ; CST2; CST4在 20对乳腺癌和 癌旁组织中的表达量差异；
图 4为 Real-time PCR绝对定量法给出的 CST4在 100例乳腺癌和癌旁组织中的表达 量分布；
图 5为 Real-time PCR绝对定量法给出的 CST4在 40例乳腺癌以及 40例穿刺活检标 本的表达量分布；
图 6为 Real-time PCR绝对定量法给出的 CST4在 30枚乳腺癌病理阳性淋巴结， 30枚 病理阴性淋巴结中表达量分布；
图 7为通过 Real-time PCR绝对定量法检测外周血游离乳腺癌细胞的准确度与细胞学 检测的对比；
图 8为通过 Real-time PCR绝对定量法检测乳腺癌骨髓转移的准确度与细胞学检测的 对比；
图 9为乳腺癌病人血浆 Cell-free RNA中 CST4量与炎 /正常人的差异分析图， A为 通过 Real-time PCR绝对定量法， 乳腺癌病人 （50例） 血浆 Cell-free RNA中 CST4量与炎 ( 30例） /正常人 (30例)的差异; B为通过 Real-time PCR绝对定量法获得的 ROC曲线， 区 分乳腺癌 /炎 /正常的敏感性和特异性； 图 10 为通过连接酶链反应 （Ligase chain reaction, LCR ) 方法， 乳腺癌病人 （50 例） 血浆 Cell-free RNA中 CST4量与炎 （30例） /正常人 (30例)的差异；
图 11 为通过适温链置换反应 (reverse transcription strand displacement amplification, rtSDA) 方法， 乳腺癌病人 （50例） 血浆 Cell-free RNA中 CST4量与炎 （30例） /正常人 (30例)的差异；
图 12 为通过核酸序列扩增法 (Nucleic Acid based Amplificatin, NASBA), 30例乳腺 癌， 20例乳腺炎， 20例正常人尿样中 CST4的表达差异。
图 13 为通过转录介导的扩增 （Transcription-mediated amplification, TMA), 检测 80 例乳腺癌 （pTNM分期， Ι+Π 30例， III+IV50例） 不同分期 CST4表达量的差异。
图 14显示了 CYSTATIN S 在乳腺癌细胞株培养上清和正常人血清中的表达情况。
图 15显示了 CYSTATIN S 在乳腺癌细胞株及正常人血清中的表达情况。
图 16 显示了应用单克隆抗体竞争法 ELISA检测 20例正常人、 30例乳腺癌患者血清中 CYSTATIN S 的表达情况。
图 17 显示了 ELISA方法检测乳腺癌 CYSTATIN S 蛋白和 CEA的在灵敏性和特异性方面 的比较。
图 18 显示了乳腺癌患者经治疗后高于 CYSTATIN S 蛋白表达水平中位数组及低于该 中位数组的无病生存曲线。 具体实施方式
第一部分分子检测
就分子生物学领域中， 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施 例仅用于说明本发明而不用于限制本发明的范围。 下列实施例中为注明具体条件的实验方 法， 通常按照常规条件如 Sambrook等人， 分子克隆： 实验室指南 （New York:Cold Spring Harbor Laboratory Press, 1989) 中所述的条件， 或按照制造商所建议的条件。 除非另外说 明， 否则百分比和份数按重量计算。
材料和方法：
实施例中所用标本均与病人签署知情同意书后按医院规定途径获取， 来源于北京友谊 医院。
穿刺活检获取的病理部位标本和非病理部位样本作为对比， 也可是手术中获取的淋巴 结等样本应立即提取 RNA或置于液氮或 RNAlater(Ambion公司产品)中保存。 外周血、 骨髓或尿液样本首先通过离心机 4000rpm， 4°C， 离心 20 分钟， 取上清， 13000rpm, 4°C， 离心 10分钟， 分离上清和沉淀， 立即提取 RNA或置于 -20°C至 -80°C保 存。
TaqMan水解探针的 Real-time PCR法： 上述样本用商业化的核酸抽提方法进行， 一个 常用的非限制性例子是酚 /氯仿抽提方法， 如用 Invitrogen 公司生产的 Trizol 方法抽提总 RNA , 并进行 RNA 质量鉴定， 相关方法可参照 《分子生物学实验》 等参考书描述。 mRNA 的逆转录过程采用商业化逆转录试剂盒并按操作说明书进行， 所得 cDNA 按比例 稀释成需要的工作浓度。 所用特异性引物经优化设计， CST4 的上下游引物设计在外显子 1 上。 制备包含 CST4扩增子的重组质粒， 所需的质粒为商业化的 Pegm-TXpromega) (图 1 )， 上下游引物设计在外显子 1， 3上。 采用基于 TaqMan水解探针的 Real-time PCR法进 行 CST4扩增。
针对 CST4的引物和探针， 最优的如下：
上游引物： gctctcaccctcctctcctg ( SEQ ID No: 1 )
下游引物： tatcctattctcctccttgg ( SEQ ID No:2)
探针： 5'-fam-ctccagctttgtgctctgcctctg-tamra-3' ( SEQ ID No:3 ) 扩增长度为 142bp。
针对制备包含 CST4扩增子的重组质粒的引物， 最优的如下：
上游引物： tgcctcgggctctcaccctcctct ( SEQ ID No :22 )
下游引物： tgggtggtggtcggtgtgactggc ( SEQ ID No:23 )
实验组， 阳性对照组， 阴性对照组与重组质粒标准品一起扩增。 依据重组质粒浓度梯度 和扩增后对应的 CP (cross point)作标准曲线。 依据该标准曲线给出实验组、 阳性对照组和 阴性对照组的拷贝数。
染料法实时定量 PCR : 样本处理同 TaqMan水解探针的 Real-time PCR法， CST1 , 2, 4 (可以同时扩增 CST1， CST2, CST4的引物） 引物， 上游引物： agtcccagcccaacttgga ( SEQ ID No: 24) ， 下游弓 I物： gggaacttcgtagatctggaaaga ( SEQ ID No: 25 ) ； CST4上游弓 | 物： agtacaacaa ggccaccgaa gat ( SEQ ID No: 4) ， 下游弓 |物： agaagcaaga aggaaggagg gag ( SEQ ID No: 5 ) 或 CST4上游弓 |物： tacaacaagg ccaccgaaga tga ( SEQ ID No: 6 ) ， 下游弓 I物： agaagcaaga aggaaggagg gag ( SEQ ID No: 7 ) 或 CST4上游弓 |物： tgctactcct gatggctacc ctg ( SEQ ID No: 8 ) ， 下游弓 |物： gtggccttgt tgtactcgct gat ( SEQ ID No: 9) 或 CST4上游弓 I物： agtacaacaa ggccaccgaa gat ( SEQ ID No: 10) ， 下游弓 |物： taccaggtct attagaagca agaagga ( SEQ ID No: 11 ) 或 CST4上游弓 |物： tgctactcct gatggctacc ctg (SEQ ID No: 12) ， 下游弓 |物： catcttcggt ggccttgttg tac (SEQ ID No: 13) 或 CST4上游引物： tgctactcct gatggctacc ctg (SEQ ID No: 14) ， 下游引物: tactcatctt cggtggcctt gtt (SEQ ID No: 15) 或 CST4上游弓 |物： tgggattatc ctattctcct ccttg (SEQ ID No: 16) ， 下游弓 |物： ctccagcttt gtgctctgcc tct (SEQ ID No: 17) 或 CST4上游引物： tgctactcct gatggctacc ctg (SEQ ID No: 18) ， 下游引物: ctcatcttcg gtggccttgt tgt (SEQ ID No: 19) 或 CST4上游弓 |物： tacagtgggt gggagtgggt ggt (SEQ ID No: 20) ， 下游弓 |物： gagtgggtac agcgtgccct tea (SEQ ID No: 21) ； CST2引物， 上游引物： cagaagaaacagttgtgctc (SEQ ID No: 26) 下游引物： ggagtaggaggtggtcag (SEQ ID No: 27) CSTl弓 |物， 上游弓 |物： tctcaccctcctctcctg (SEQ ID No: 28) 下游弓 I物： ttatcctatcctcctccttgg (SEQ ID No: 29) ， 内参基因 β - actin, 上游弓 I物： aagatcattgctcctcctg (SEQ ID No: 30) ， 下游弓 |物： cgtcatactcctgcttgc
(SEQ ID No: 31) 。 癌和癌旁组织目的基因和内参基因一起扩增， 通过 2 (癌组织 細 癌 翻 /2 (癌 织目隨目" -癌旁 細《。" ， ct为荧光定量 PCR反应孔信号刚刚突破背景时的循 环数。 公式计算目的基因在癌和癌旁组织中的相对表达量。 荧光染料如 SYBR Green, Eve Green, LC Green等。
核酸序列扩增法 (Nucleic Acid based Amplificatin, NASBA)的体外 RNA扩增技术: T7 RNA聚合酶， RNase H， 禽骨髓白血病病毒 （AMV) 逆转录酶， 核糖核苷酸 （NTP), 脱 氧核糖核苷酸 （dNTP)， CST4上下游引物同 TaqMan水解探针的 Real-time PCR法， RNA 荧光染料 （Ribo-Green fluorescent dye)。 42度， 2小时， 可将 RNA模板扩增 2 (912)， 反应 产物放入荧光检测仪内检测荧光强度 （U)， 从而反应初始模板量。
实施例 1. CST4与 CST家族其他成员的表达差异
1. 人体不同组织中 CST4的表达差异
所用人体组织标本除正常乳腺组织为发明人从北京友谊医院收集外， 其它各组织均从 商业化机构购买。 采用 Affymetrix的核苷酸芯片 HG_U95Av进行各正常组织 CST4 mRNA表 达比较， 操作过程参照产品说明书。 本实验的相对表达量的值是通过管家基因 β -actin 荧光值进行归一化处理后的标准化信号值。
结果如图 2所示， 除唾液腺中 CST4表达量较高外， 其它组织中 CST4不表达， 说明 CST4 mRNA表达在正常组织中的背景值很低， 这对 CST4 用于病理条件下检测极为有利。 在结乳腺癌细胞株 HCC1937, SK-BR-3, MCF-7 中过表达， 在人正常乳腺细胞 Hs578Bst 不 表达， 说明 CST4可作为乳腺癌分子诊断的 Marker。 2.乳腺癌及癌旁手术组织样本 CST4与 CST124, CST1, CST2 mRNA表达量比较 通过比较 20 对编号 C1-C20 的乳腺癌及癌旁组织中 CST124, CST1, CST2, CST4 mRNA 表达量， 发现 CST4 mRNA 在乳腺癌和癌旁组织中表达量差异较大， 仅次于 CST1。 结果见图 3。 上述标本均通过病理证明， 采用染料法实时定量 PCR， 阳性样本正 常扩增， 阴性样本未扩增。
基于染料法的 CST4 mRNA实时定量检测试剂盒
试剂盒中含有：
(1) CST4弓 I物， 包括：
上游引物： agtacaacaa ggccaccgaa gat (SEQ ID No:4)
下游弓 I物： agaagcaaga aggaaggagg gag (SEQ ID No:5)
或
上游弓 I物： tacaacaagg ccaccgaaga tga (SEQ ID No:6)
下游弓 I物： agaagcaaga aggaaggagg gag (SEQ ID No:7)
或
上游引物： tgctactcct gatggctacc ctg (SEQ ID No:8)
下游弓 I物： gtggccttgt tgtactcgct gat (SEQ ID No:9)
或
上游弓 I物： agtacaacaa ggccaccgaa gat ( SEQ ID No:10)
下游引物： taccaggtct attagaagca agaagga ( SEQ ID No: 11 )
或
上游引物： tgctactcct gatggctacc ctg ( SEQ ID No: 12 )
下游引物： catcttcggt ggccttgttg tac (SEQ ID No:13)
或
上游引物： tgctactcct gatggctacc ctg ( SEQ ID No: 14 )
下游引物： tactcatctt cggtggcctt gtt (SEQ ID No: 15)
或
上游引物： tgggattatc ctattctcct ccttg (SEQ ID No:16)
下游引物： ctccagcttt gtgctctgcc tct ( SEQ ID No: 17)
或
上游引物： tgctactcct gatggctacc ctg ( SEQ ID No: 18 ) 下游引物： ctcatcttcg gtggccttgt tgt ( SEQ ID No:19)
或
上游引物： tacagtgggt gggagtgggt ggt ( SEQ ID No:20)
下游引物： gagtgggtac agcgtgccct tea ( SEQ ID No:21 )
β -actin:
上游引物： aagatcattgctcctcctg ( SEQ ID No: 30)
下游引物： cgtcatactcctgcttgc ( SEQ ID No: 31 )
( 2) 常规的核酸抽提液及逆转录试剂， SYBR Green 染料， dNTP， Taq 酶， 无 RNA 酶的水 （RNase-free H20 )、 标准品、 阳性对照品、 阴性对照品 10*Buffer， Mgcl2。
实施例 2 CST4在不同部位与组织的表达差异
1 乳腺癌及癌旁手术组织样本 CST4表达差异
使用 Taqman水解探针法的 CST4 mRNA实时定量检测试剂盒
制备试剂盒， 所述的试剂盒中含有：
( 1 ) 引物， 探针， 包括：
上游引物： gctctcaccctcctctcctg ( SEQ ID No: 1 )
下游引物： tatcctattctcctccttgg ( SEQ ID No:2)
探针： 5'-fam-ctccagctttgtgctctgcctctg-tamra-3' ( SEQ ID No:3 )
(2) 常规的核酸抽提液及逆转录试剂， dNTP、 Taq 酶， 无 RNA 酶的水 （Rnase- free H20) CST4重组质粒标准品、 阳性对照品、 阴性对照品， 10*Buffer， Mgcl2。
通过手术取样的样本均经病理验证后才进行 RNA抽提和逆转录 cDNA， Real-time PCR绝对定量法鉴定 CST4在乳腺癌和癌旁组织中的表达差异， 测试样本数为 100对， 标准曲线线性、 扩增效率符合要求， 阳性样本检测阳性， 阴性样本检测阴性， 无模板对 照未扩增。
图 4的结果表明， 乳腺恶性病理条件下特异性高表达 CST4 mRNA, 而癌旁组织低 表达。 癌拷贝的中位数是癌旁拷贝中位数的约 7.08倍， 提示 CST4 mRNA可作为良好的 乳腺癌分子标志物。 在 264.92拷贝处划线， 可将癌与正常区分开来， 因此 264.92拷贝 可作为乳腺癌临床诊断的一个参考值。
2穿刺活检取样的乳腺癌、 乳腺炎中 CST4的表达差异。
穿刺活检取样与手术取样的样本具有较大差别， 主要表现在取样组织中乳腺癌细 胞的比例变动很大， 有时可能仅占到整块组织的很小一部分， 有的甚至没有。 因此本发 明人统计比较了 40例乳腺癌以及 40例乳腺炎病人穿刺活检取样标本中 CST4的表达差 异， 癌标本拷贝数的中位数是炎标本拷贝数中位数约 9.15倍， 如果在 113.795拷贝处划 线， 可以将癌和炎区分开来， 可为穿刺活检取样乳腺癌诊断提供参考。
结果见图 5， 方法同样采用 Real-time PCR 绝对定量的方法， 标准曲线线性、 扩增 效率符合要求， 阳性样本检测阳性， 阴性样本检测阴性， 无模板对照未扩增。
3 CST4在乳腺癌转移阳性淋巴结和阴性淋巴结中的表达差异
手术中获得的经病理证明为乳腺癌转移阳性的淋巴结 30枚， 且转移灶大小不等。 病理阴性淋巴结 30 枚主要取自早期乳腺癌病人， 原因是尽可能减少病理诊断阴性但实 际有微转移存在的淋巴结， 以避免实验误差。 实验采用 Real-time PCR检测方法， 具体 材料和过程同实施例 2， 标准曲线线性、 扩增效率符合要求， 阳性样本检测阳性， 阴性 样本检测阴性， 无模板对照未扩增。
图 6的结果表明， CST4在转移阳性淋巴结中高表达， 而在阴性淋巴结中只有较低 表达。 转移阳性淋巴结 CST4 mRNA表达量是阴性淋巴结中的 8.458倍。 如果从 120.66 拷贝处划线， 基本可区分乳腺癌转移阳性和阴性淋巴结。 病理阴性淋巴结中有两例检出 CST4 弱阳性的淋巴结， 经病理医师连续切片细致观察， 鉴定有微转移的存在。 因此， 从 CST4 mRNA表达程度划分不仅 100%区分了细胞学检测结果， 而且能检测细胞学不 能检出的有微转移存在的淋巴结， 相比细胞学检测该检测方法具有更高的灵敏度。
4定量 PCR检测外周血中游离乳腺癌细胞的准确度与细胞学检测对比
除去红细胞和血小板的外周血有核细胞提取 RNA， 经 Real-time PCR方法定量检 测 CST4 mRNA表达水平， 通过和乳腺炎病人和正常人表达量的对比， 判断乳腺癌患者 的外周血中是否有游离乳腺癌细胞的存在， 并和细胞学检测比对。
图 7的结果表明， 本发明的检测方法 100%确认细胞学鉴定的阳性结果， 同时可检 测出细胞学鉴定阴性的病例中有部分转移病例， 说明该方法比细胞学检测具有更高的灵 敏度， 能检测到细胞学无法检测的微转移的存在。
5定量 PCR方法检测乳腺癌骨髓转移与细胞学检查结果对比
穿刺等方法获得的乳腺癌患者骨髓经 Real-time PCR方法定量检测 CST4 mRNA表 达水平， 通过和正常骨髓的比较判断 CST4 mRNA是否高表达， 确认骨髓是否存在转移 和微转移， 并与细胞学结果进行了比较。
图 8 的结果表明， 本发明的检测方法可 95%确认细胞学阳性结果， 同时阳性率高 于细胞学结果， 表明敏感性比细胞学检测方法要高。 实施例 3 CST4量与炎 /正常人的差异
1 乳腺癌病人血浆 Cell-free RNA中 CST4量与炎 /正常人的差异
应用商品化的试剂盒， 抽提血浆中 Cell-free RNA， Real-time PCR检测乳腺癌病人 ( 50例） 血浆 Cell-free RNA中 CST4量与炎 （30例）， 正常人 (30例)的差异。
结果如图 9所示， 图 9-A显示 CST4在癌中拷贝数的中位数是炎的约 8.87倍， 正 常的 25.62， 在拷贝数 71.218处划线， 可将癌和炎 /正常区分开来。 图 9-B的 ROC曲线 显示基于 CST4 表达量诊断乳腺癌的方法具有较高的灵敏度和特异性 （曲线下面积 0.987)， 因此 CST4可作为非侵入性的血浆样本乳腺癌诊断的特异性分子标记。
2连接酶链反应 （Ligase chain reation, LCR) 方法检测乳腺癌病人血样 Cell-free RNA中 CST4量与炎 /正常人的差异
基于连接酶链反应 （Ligase chain reation, LCR) 的 CST4 mRNA定量检测试剂盒 所述的试剂盒中含有：
( 1 ) 探针 4条 （半抗原标记）
gggctctggcctcgagctccaagga ( SEQ ID No:33 );
ataggataatcccaggtggcatctatgatg ( SEQ ID No :34 )；
tctcctccttggagctcgaggccagagccc ( SEQ ID No:35 )；
catcatagatgccacctgggattatcctat ( SEQ ID No:36 )；
(2) 商业化的核酸提取试剂、 逆转录试剂， 其它试剂同 Abbott LabOTatorieS(LCX) 应用商品化的试剂盒， 抽提血浆中 Cell-free RNA， LCR检测乳腺癌病人 （50例） 血浆 Cell-free RNA中 CST4表达量与炎 （30例）， 正常人 (30例）的差异。
结果如图 10所示， CST4在癌中相对荧光强度 （relative light units, RLU) 的中位 数是炎的 10.881倍、 正常的 35.286倍， 在相对荧光强度 17.458RLU处划线，可将癌和炎 /正常区分开来。
3适温链置换扩增 (reverse transcription strand displacement amplification, rtSDA) 方法检测乳腺癌病人血样 Cell-free RNA中 CST4量与炎 /正常人的差异
适温链置换扩增 （thermophilic strand displacement amplification, tSDA) 的 CST4 mRNA定量检测试剂盒
(1)试剂盒中含有： CST4 B 1弓 I物： cccggcctctgtgtaccctgcta ( SEQ ID No:37 )
CST4 S1 引物： gaa-ctcgagctaccctggctggggctctgg ( SEQ ID No:38 )
CST4 B2弓 I物： ggtggccttgttgtactcgctgat ( SEQ ID No:39)
CST4 S2弓 I物： get -ctcgag agtgaagggcacgctgtac ( SEQ ID No:40)
检测探针：
5， -32P-ttactcgag ctccaaggaggagaatagga-3， ( SEQ ID No:41 )
(2)常规的核酸抽提液及逆转录试剂， dCTP a S , dATP , dGTP , dTTP , Bsob I 酶， exo-Bca E
应用商品化的试剂盒， 抽提血浆中 Cell-free RNA, rtSDA 检测乳腺癌病人 （50 例） 血浆 Cell-free RNA中 CST4量与炎 （30例）， 正常人 (30例)的差异。
结果见图 11， CST4在癌中相对荧光强度 （relative light units, RLU) 的中位数是 炎的 34.58倍， 正常的 35.89倍， 在相对荧光强度 24.095RLU处划线， 其作为阈值可将 癌和炎 /正常区分开来。
4乳腺癌病人尿样 Cell-free RNA中 CST4量与炎 /正常人的差异
核酸序列扩增法 (Nucleic Acid based Amplificatin, NASBA)法的 CST4 mRNA定量 检测试剂盒
试剂盒中含有：
( 1 ) CST4弓 I物， 探针， 包括：
上游引物： aattctaatacgactcactataggg-gctctcaccctcctctcctg ( SEQ ID No:32 )
下游引物： tatcctattctcctccttgg ( SEQ ID No:2)
分子信标探针：
5'-FAM-gcggcctccagctttgtgctctgcctctggccgc-dabsyl-3 ' ( SEQ ID NO: 3 )
常规的核酸抽提液及逆转录试剂， T7 RNA聚合酶， RNase H,
禽骨髓白血病病毒 （AMV ) 逆转录酶， 核糖核苷酸 （NTP ) , 脱氧核糖核苷酸 (dNTP), RNA荧光染料 （Ribo-Green fluorescent dye )。
应用商品化的试剂盒， 抽提尿液中 Cell-free RNA, 核酸序列扩增法 (Nucleic Acid based Amplificatin, NASBA)检测乳腺癌病人 （30例） 尿样 Cell-free RNA中 CST4量与 炎 (20例）， 正常人 (20例)的差异。
结果如图 12所示， CST4在癌中荧光强度的中位数是炎的约 15.86倍， 正常的约 38.35 倍， 在拷贝数 30.92 处划线， 可将癌和炎 /正常区分开来， 即可作为非侵入性的尿 液样本乳腺癌诊断的参考值。 实施例 4 CST4作为 pTNM分期、 治疗过程中动态监测及预后判断中的应用 转录介导的扩增 （Transcription-mediated amplification, TMA ) 的 CST4 mRNA 定量检测试剂盒。
制备试剂盒， 所述的试剂盒中含有：
( 1 ) 引物， 探针， 包括：
上游引物： aattctaatacgactcactataggg-gctctcaccctcctctcctg ( SEQ ID No:32 ) 下游引物： tatcctattctcctccttgg ( SEQ ID No:2)
分子信标探针：
5'-FAM-gcggcctccagctttgtgctctgcctctggccgc-dabsyl-3 ' ( SEQ ID NO: 3 )
(2) 其它反应试剂为 Gen-probe TMA assay中除引物和探针之外的试剂。
1. CST4表达量与乳腺癌 pTNM分期的关系
应用商品化的试剂盒， 抽提血浆中 Cell-free RNA, 应用 Gen-probe转录介导的扩增 ( Transcription-Mediated amplification, TMA) 方法， 检测 80 例乳腺癌 （pTNM 分期， Ι+Π 30例， III+IV50例） 不同分期 CST4表达量的差异。
结果见图 13， CST4在乳腺癌 III+IV中相对荧光强度 （relative light units, RLU) 的中位数是 Ι+Π的 7.2倍， 因此 CST4表达量可用于乳腺癌 pTNM分期。
2 CST4表达量用于乳腺癌治疗过程中动态监测
应用实时定量 PCR方法， 通过检测病人血液中 CST4表达量， 实时监测乳腺癌化 疗病人 6人， 放疗病人 4人， 治疗效果。
结果见表 1， 治疗有效的病人 CST4表达量随着疗程的增加逐渐降低， 影像学结果 也显示， 肿块逐渐减小； 而治疗无效的病人 CST4表达量随着疗程的增加逐渐增加， 影 像学结果显示， 肿块有变大的趋势。 因此， CST4 可作为乳腺癌患者治疗过程中一个动 态监测的指标。
表 1为通过实时定量 PCR检测放化疗过程中乳腺癌患者血液中 CST4的量 病人 CST4表达 ί l(copy) 影像学 （ cm)
疗程 1 疗程 2 疗程 3 疗程 1 疗程 2 疗程 3 治疗 化疗 1 781.32 521.78 80.64 2.5 1.5 <1 有效 化疗 3 1533.6 1314.6 125.25 3.5 2.5 <1
化疗 5 1213.5 439.89 66.64 3 2 <1 放疗 2 1434.58 1160.33 116.83 3 1.5 1 放疗 3 2062.66 1689.44 189.65 3 2.5 1.5 治疗 化疗 2 1466.14 1984.62 2433.57 3 3 3.5 无效 化疗 4 956.92 1156.34 1846.21 2 2 2.5
化疗 6 646.2 826.7 1032.55 1 1 1.5
放疗 1 1032.4 1246.8 1989.61 2 2.5 3 放疗 4 936.4 1048 1678 1 1.5 2
3 CST4表达量与乳腺癌患者预后判断的关系
基于实时定量 PCR技术， 分别在术后或放化疗后的 1个月、 3个月、 1年， 跟踪 随访了 5个乳腺癌患者血液中 CST4表达量的情况。
结果如表 2所示， 一年后复发的 2个病人， CST4表达量随着时间的延长逐渐增 高， 当达到 1000个拷贝左右时才被影像学发现确诊； 而 1年内没有复发的病人， CST4 表达量并无明显变化， 影像学也无异常。 因此， CST4 表达量变化可作为乳腺癌患者预 后判断的一个指标。 表 2为通过实时定量 PCR检测手术后或放化疗后 1个月、 3个月、 1年， 乳腺癌患者血液中 CST4的量。 表 2为通过实时定量 PCR检测随访过程中乳腺癌患者血液中 CST4的量
病人 CST4 (copy) 影像学 （ cm)
1个月 3个月 1年 1个月 3个月 1年
复发 患者 1 56.84 198.87 1135.24 无 无 1
患者 5 15.25 64.34 786.59 无 无 <1
未 复 患者 2 23.6 40.5 36.3 无 无 无
发 患者 3 52.43 39.8 48.65 无 无 无
患者 4 67.9 79.6 86.79 无 无 无 第二部分蛋白质部分
就蛋白质领域中， 本发明涉及用于乳腺疾病诊断和监控以及治疗效果评价的非侵入性标 记的抗体、 试剂盒、 测定法及其使用方法。
本发明 CYSTATIN S 重组蛋白购自 Abnova (浓度 0. 06ug/ul,货号 H00001472- P01)； CYSTATIN S 鼠单克隆抗体购自 R&D (效价为 1 : 2000,货号 MAB 1296)； CYSTATIN S兔多克隆抗 体购自 Abeam (效价为 1 : 800， 货号为 ab58515)。
本发明提供了诊断受试者样品中乳腺状况的测定法及是否转移或复发； 此外， 还提供了 根据受试者样品来评价治疗是否有效的测定法。 该测定法优选检测患者样品中至少一种所选 蛋白， 优选 CYSTATIN S 蛋白 ， 更优选该测定法是定量或至少半定量地检测患者样品中 CYSTATIN S 水平。 虽然任意类型的报道分子都可用于检测蛋白， 但优选该报道分子为 CYSTATIN S 特异性抗体或其片段。 任选地， 本发明的方法、 测定法和试剂盒可用于发现早 期处于无症状前或无症状的人。
测定法任选和优选利用至少一种， 且优选多种特异性结合至少一种 CYSTATIN S 表位肽 的抗体或其片段， 用于检测受试者样品中对 CYSTATIN S 的免疫反应的存在。 抗体可以是多 克隆抗体或单克隆抗体。 优选该单克隆抗体可结合至 SEQ ID NO : 50, 和 /或可由任意一个该 序列得到。 更优选 CYSTATIN S 的量是确定的。 因此， 本发明还提供了通过利用至少一种上 述抗体定量样品中对 CYSTATIN S 的免疫反应的水平而定量样品中 CYSTATIN S 的水平的测 定法。
任选和优选受试者为人， 且检测的免疫反应以抗体和人多肽为特征。 该免疫反应可任 选地通过利用任何合适的测定法而检测， 所述测定法包括但不限于 ELISA (酶联免疫吸附测 定）或免疫印迹如蛋白印迹或其组合。 优选利用竞争性 ELISA和双抗夹心 ELISA。 根据优选的实施方案， 乳腺状况的诊断或监控、 乳腺疾病治疗效果的监控或监控受到检 测的免疫反应定量的影响， 其代表样品中标记的多肽 （ CYSTATIN S ) 的量。 在其它任选实施方案中， 本发明还涉及检测样品中标记蛋白的检测试剂盒。 由此能诊断 或监控乳腺状况、 监控乳腺疾病的治疗效果或评价是否转移复发， 其中检测试剂盒包含本发 明的至少一种抗体或其片段， 其能与存在与所检查流体中的标记多肽 （ CYSTATIN S ) 反 应， 以及包含至少一种报告组分， 其能检测由抗体或其片段和标记蛋白组成的复合物。 如上 所述， 抗体可以任选为多克隆抗体或单克隆抗体的。 优选检测试剂盒， 报告组分可以是抗体， 其试剂盒中所用的抗体或其片段， 且显示标 记。 在这里， 报告组分优选为合适的 IgG抗体或合适的 IgM抗体。 标记任选和优选为能催化 显色反应的酶， 例如过氧化物酶， 以及更优选能共价结合至第二抗体。 或者， 标记可以是荧 光部分， 或比色抗体。 本发明优选的实施方案中， 检测试剂盒为 ELISA检测试剂盒。 在本发明特别优选的实施方案中， 该 ELISA检测试剂盒为如下所列实施例部分所述的竞 争性 ELISA试剂盒和双抗夹心 ELISA试剂盒。 优选至少一种抗体或其片段与所检查的样品预 温育。 优选该抗体为单克隆抗体。 用于生产抗- CYSTATIN S 抗体的蛋白与用作示例性固相 物质的微量滴定板 （此处也称为基质） 偶联， 且预温育的样品随后灌注平板且之前未与血清 中存在的表位结合的抗体可与平板上的蛋白结合。 报告组分由合适的免疫球蛋白组成， 尤其 是抗 -IgG抗体和 /或抗 -IgM抗体， 其可检测结合至平板的抗体， 且与催化显示的酶和 /或荧 光标记偶联。 在本发明又一优选实施方案中， 该检测试剂盒为免疫印迹， 也称为蛋白印迹 （Western blot 在该特点的检测试剂盒中， 样品中的蛋白利用电泳凝胶， 例如聚丙烯酰胺凝胶转移 至固相基质上 (例如硝酸纤维素膜）。 转移可例如利用电转移进行。 随后存在于基质上的蛋白 和抗该蛋白的抗体间发生免疫反应。 优选利用单克隆抗体或其片段。 免疫反应可随后通过合 适的方法检测， 例如利用酶标记的和 /或荧光标记的抗-抗体的抗体。 在本发明的又一个优选实施方案中， 该检测试剂盒用于流通测定法。 在具有该特点的用 于测定法的试剂盒中， 抗体或其片段结合于柱上， 待测样品经过所述柱进行灌注。 如上所述 抗体可以是多克隆或单克隆的， 且优选所用的单克隆抗体包括本发明提及的上述特异性抗 体。 将待测样品灌注到柱中， 且流过所述柱。 如果样品包含特异性结合柱上的抗体或抗体级 分的蛋白， 则其将在柱中截留而不再流过。 优选一旦所有样品流体流过该柱。 结合至柱的蛋 白通过竞争性抗体流过或通过改变缓冲液条件而洗下。 优选如果不同的蛋白结合至柱上， 则 将其在不同的时期洗下。 随后测量从洗涤柱获得的或在柱洗涤特定时期发现的蛋白的量。 纯 化的可溶性蛋白定量的技术为本领域所熟知， 例如通过测量包含蛋白的流体的光密度。 在本发明进一步优选的实施方案中， 提供了测定取自受试者样品中 CYSTATIN S 水平的 检测试剂盒， 其中双指示剂指示 CYSTATIN S 水平处于正常水平还是高于正常水平。 优选取 自受试者的样品为流体样品。 该检测试剂盒优选包括容纳样品流体的容器、 特异性抗体或其 片段以及指示剂。 优选该抗体为单克隆的， 且更优选为本发明如上所述的单克隆抗体。 任选 和优选该试剂盒进一步包括进行检测所需的溶液和缓冲液， 以及任选包含进行检测和解释结 果的说明书的印刷品。 该检测试剂盒可由本领域技术人员使用， 且可用于任何实际场所， 包 括但不限于医院、 诊所和私人住宅。
本发明试剂盒所针对的受试者可以为乳腺部不适就医者， 且检测包括对受试者进行筛 查以检测乳腺癌。
本发明试剂盒所针对的受试者可以为乳腺炎患者， 且检测包括对受试者进行筛查以检 测乳腺癌。
本发明试剂盒所针对的受试者可以为有家族性乳腺癌者， 且检测包括对受试者进行筛 查以检测乳腺癌。
本发明所针对的样品包括但不限于血清样品、 血浆样品、 尿样或血样。 血样可包括全血 样品或血液级分样品。
如上述， 本发明知道的抗体可用于乳腺疾病诊断或乳腺癌是否转移复发的评估。 本领域 技术人员熟知的一些方法可用来应用该指导， 如利用此处所述缀合至可检测部分 （例如荧光 部分） 的抗体试剂盒可用于评估获自检测个体的血清， 血浆， 尿液样品中的 CYSTATIN S 标 记水平， 并且由此确定疾病的存在 /不存在或者甚至是状态。
在参阅下列并不意欲限制的实施例后， 本发明另外的目的， 优点， 和新的特征对本领域 技术人员将是显而易见的， 另外， 上文所述以及权利要求部分请求保护的本说明的各个实施 方案和方面的每一个都将在下列实施例中找到实验性的支持。
材料和方法
CYSTATIN S重组蛋白： 购自 Abnova (浓度 0. 06ug/ul,货号 H00001472- P01)；
抗体： CYSTATIN S 兔多克隆抗体， 购自 Abeam (效价为 1 : 800， 货号为 ab58515 ) ; CYSTATIN S鼠单克隆抗体购自 R&D (效价为 1： 2000,货号 MAB 1296)
免疫沉淀法： 直接通过添加 2mM PMSF, A蛋白 -琼脂糖凝胶和抗- CYSTATIN S 抗体进行 细胞上清液 （60mm皿的 1. 5mL) 的免疫沉淀， 随即在 4度旋转过夜。 利用通过庚二亚氨酸二 甲脂 （dimethl pimel imidate ) 偶联至 A 蛋白-琼脂糖凝胶的抗 -Cystatin 抗体， 以类似的 方法进行血清样品 （200ul ) 的免疫沉淀。 如早期研究所述进行免疫沉淀物的洗涤， 免疫沉 淀物用 N-糖苷酶 F的处理和 SDS_PAGE
简而言之， 为了用 N-糖苷酶处理， 洗涤免疫沉淀物且随后在 lOul用于 ίΉ=6. 0的 50mM 柠檬酸钠中的 0. 5%SDS 中煮沸， 随后加入 lOul 包含 200mM PH=8. 0 的磷酸钠， PH=8. 0 的 40mM EDTA, 3%N_辛基葡糖苷的溶液连同 40 毫单位 N-聚糖酶， 且在 37 度温育过夜， 加入 20ul 样品缓冲液且上样进行 SDS-PAGE 前煮沸样品。 除非另外说明， 在 15%聚丙烯酰胺 ( Lae l i gel ) 上进行 SDS_PAGE。 凝胶通过利用 20% 2, 5_二苯基噁唑的荧光自自显影进 行分析且通过之前所述的光密度测定法进行定量。
蛋白电转和免疫印迹： 将蛋白点转入硝酸纤维素膜上。 用溶于 PBS 中的 5%脱脂奶粉和 0. l%Brij-35在室温下进行 2小时的封闭。 封闭后用 CYSTATIN S兔多克隆抗体 4度温育过 夜。 在包含 0. 1% Brij-35 的 PBS 中洗涤印迹三次， 且用 0. 27ul 的缀合至过氧化物酶 ( Jackson) 的山羊抗-兔 IgG在 37度下温育 1小时， 印迹用溶于 PBS中的 0. l%Brij-35洗 涤 4 次， 且用 PBS 洗涤一次， 之后用商品化的 TMB 溶液 （Kirkegaard and Perry Laboratories Inc. ( Gaithersburg； MD ) ) " TMB Peroxidase Substrate " solution Cat. No 50-76-01 和" Peroxidase Solution B " Cat . No. 50-65-00)进行检测， 并且对 印迹照相。 或者通过 ECL 方法进行检测， 其中利用溶于 PH=8. 5 的 100 mM Tris-HCl 中的 5. 4mM双氧水混合的用于 PH=8. 5的 100 mM Tris-HCl 中的 2. 5nM 鲁米诺， 400mM对香豆酸 的新鲜制备液， 且曝光于 Agfa CP-BU膜。
竞争法 ELISA :将 Corning ELISA平板孔用 CYSTATIN S ( 5ug/ml ) 覆盖且用 3%BSA封 闭。 将包含 CYSTATIN S鼠多克隆抗体 （1 : 1000) 与 8个二重稀释的血清样品于 4度温育过 夜。 随后加至覆盖的 ELISA板中且 37度温育 1 小时。 每孔用 TBS (溶于 PH=7. 5 的 10 mM Tris-HCl 中的 154mM的 NaCl ) 洗涤且缀合至碱性磷酸酶中的山羊抗 -小鼠 IgG (Jackson, 1： 2000， 溶于 TBS 中的 0. 3%BSA 中） 在 37度反应 1 小时。 加入碱性磷酸酶底物 （KPL， lOOul 每孑 L， Blue Phos solution cat. No 508805 Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) )， 利用 ELISA读数器在 405nm波长处定量 0D。
双抗夹心 ELISA: 将 Corning ELISA 平板孔用鼠单抗抗- CYSTATIN S 蛋白 （5ug/ml ) 覆盖且用 3% BSA封闭，将 8个二重稀释的血清样品加至覆盖的 ELISA板中且 37度温育 1小 时， 每孔用 TBS (溶于 PH=7. 5的 10 mM Tris-HCl中的 154mM的 NaCl ) 洗涤， 加入生物素标 记的兔抗- CYSTATIN S 蛋白的多抗 （1 : 1000 (实验前已用生物素标记好并纯化））， 37 度 温育 1小时。 每孔用 TBS (溶于 PH=7. 5的 10 mM Tris-HCl中的 154mM的 NaCl ) 洗涤， 加入 亲合素一生物素一过氧化物酶复合物 （ABC复合物）， 37度温育 1 小时。 每孔用 TBS (溶于 PH=7. 5 的 10 mM Tris-HCl 中的 154mM 的 NaCl ) 洗涤。 加入碱性磷酸酶底物 （KPL， lOOul 每孑 L， Blue Phos solution cat. No 508805 Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) )， 利用 ELISA读数器在 405nm波长处定量 0D。
实施例 1
1 乳腺癌细胞株培养上清液中 CYSTATIN S 检测
从第一部分可以得出， CST4 mRNA 在乳腺癌中异常高表达， 而 CYSTATIN S 为分泌蛋 白， 分布于多种体液和分泌物中， 为了确定 CYSTATIN S 是否可作为乳腺癌血液检测的标 记， 取 CST4 mRNA 高表达的乳腺癌细胞株 HCC1973, 如图 14 所示中泳道 1_2 的细胞上清 液， 1个供体 （S1，泳道 3-4) 的正常人血清， 且进行 15%的 SDS_PAGE。 蛋白随后转入硝酸纤 维素膜且印迹与抗 CYSTATIN S 抗体反应， 随后与山羊抗兔过氧化物酶反应。 利用 TMB膜过 氧化物酶底物 （3， 3' , 5, 5' -四甲联苯胺） 进行条带的检测。 所有方法基本上如上述材料 和方法部分中所述的进行。
结果如图 14所示， 下方为内参 β -actin蛋白的条带。 在泳道 1，2中， 其分别代表乳腺 癌细胞株培养上清， 检测到约 16Kda 条带的存在。 泳道 3， 4， 则代表正常血清样本， 仅检 测到淡淡的 16Kda条带的存在。
2 乳腺癌病人血清中 CYSTATIN S 检测
方法同 " 1 "， 结果如图 15所示， 下方为内参 β -actin蛋白的条带。 泳道 3， 4代表正 常血清样本， 仅有很淡的条带； 泳道 1， 2代表乳腺癌病人血清样本均有较明显的条带。
3利用单克隆抗体定量检测 CYSTATIN S 血清水平的 ELISA测定法
实验方法： 将 5 μ g/ml CYSTATIN S 肽加入 ELISA平板， 4 度过夜； 正常人血清 （20 例）、 乳腺癌患者血清 （30 例） 与抗 CYSTATIN S 单克隆抗体 （1 : 2000 , 溶于 TBS (溶于 PH7. 5的 10mM Tris-HCl 中的 154mM NaCl)中的 0. 3%BSA中） 预温育， 4度过夜。 随后将与 单克隆抗体温育后的样本加入包被了 CYSTATIN S 肽的 ELISA平板， 室温孵育 1小时。 随后 与溶于 TBS中的 0. 3%BSA中的 0. 08 μ g/ml缀合至 AP的山羊抗兔抗体温育， 且与憐酸对硝基 苯酯（p-NPP) (CHEMICON International) 反应。 显影的颜色随后在 ELISA读数器分光光度计中 定量。
实验结果， 如图 16 所示， 正常人血清 CYSTATIN S 浓度中位数为 1. 35ng/ml,乳腺癌 患者血清 CYSTATIN S浓度中位数为 2. 95 ng/ml , 在 3. 105 ng/ml处划线基本可以将癌和正 常区分开来， 因此可作为乳腺癌临床诊断的一个参考值。
4 血清 /血浆中 CYSTATIN S 和 CEA (癌胚抗原） 检测灵敏度和特异性比较
CYSTATIN S 蛋白检测方法同 " 3 "， CEA 检测应用商品化的试剂盒 （德国 DRG,货号 EIA5071 ), 按照其说明书操作。
结果如图 17及表 3所示， CYSTATIN S曲线下面积为 0. 832大于 CEA， 即 ELISA方法检 测血清 /血浆中 CYSTATIN S比检测 CEA在灵敏性和特异性方面更具有优越性。 表 3 曲线下面积
Figure imgf000024_0001
检验的变量: CYSTATIN S , CEA在实际阳性组与实际阴性组之间有至少一个变叉的话，统
计可能会有偏差。
a.根据非参数假设
b.无效假设面积 = 0.5
5 测定法、 试剂盒和其使用方法
该实施例提供了本发明的许多关于测定法、 试剂盒和其使用方法的非限制性、 例证性的 实施方案。
检测乳腺疾病的方法包括： 将可溶性蛋白 CYSTATIN S 抗原固定至支持物上， 使用 R&D 抗体 （MAB1296 ) , 洗涤， 添加标记的第二抗体 （碱性磷酸酯酶标记的山羊抗兔 IgG,碧云 天， 货号 A0239), 洗涤且直接或间接检测和 /或测量标记， 其反应了结合至抗体的检测的目 的蛋白的量。
支持物的实例包括但不限于乳胶颗粒、 纤维素材料例如纤维素片、 塑料测定平板和颗粒 所用的抗原可任选例如通过共价结合或物理吸附固定在支持物上。 待测样品为人血清等 等。 任选和优选支持物的表面在样品添加之前通过与牛血清血蛋白（BSA)等等预温育而 "封 闭"， 以至少降低样品中其他抗体非特异性结合支持物的可能性。 随后支持物用合适的缓冲 液， 例如包含表面活性剂的磷酸盐缓冲液等等进行洗涤。
标记的第二抗体的非限制性实例为标记的抗 -小鼠多克隆抗体。 有效的标记包括但不限 于各种类型的酶如碱性磷酸酶、 荧光素酶、 过氧化物酶、 β _半乳糖苷酶等等以及各种荧光 化合物如荧光素等等。 化合物如生物素、 抗生物素蛋白、 链霉抗生物素蛋白、 洋地黄苷等等 可插入抗体和标记之间。
当标记为酶时， 其存在可任选通过添加底物并且检测和 /或测量由于酶的催化作用而发 生的发光或显色和 /或通过测量光吸收的变化而检测和 /或测量。 当标记为荧光化合物时， 可 任选通过用紫外光照射该反应体系并且检测和 /或测量发射的荧光而检测和 /或测量。 如果需 要可使用感光剂。 利用本发明的结合该标记的至少一个表位 （SEQ ID NO : 50) 的抗体检测和 /或测量标记 蛋白 CYSTATIN S 的试剂优选包括抗体或其片段， 必要量的第二抗体或基质 （如果需要的 话）， 以及任选一种或多种 "支持"试剂， 其为之前所述试剂作用所必需。 这些试剂任选和 优选提供于试剂盒中。 上述试剂可用作诊断乳腺癌症、 分期乳腺癌症、 评估是否转移、 评估 治疗效果的试剂。
例如， 试剂盒优选以特异性抗体或其片段为特征。 任选和更优选该试剂盒还以能检测到 受试者样品中标记蛋白存在的报告组分为特征。 该报告组分优选为合适第二抗体， 任选和优 选还包括检测第二抗体的标记 （如果需要）。 该试剂盒还可任选和优选以一种或多种缓冲 液， 如和基质预温育以封闭与基质和 /或固定其上的蛋白的非特异性反应的 "封闭" 缓冲 液； 以及一种或多种用于样品、 第二抗体、 基质和 /或用于在与之前所述的试剂温育之间洗 涤基质的缓冲液。 当然， 还可酌情任选其它试剂。
任选地， 该试剂盒提供用于竞争测定法， 其中对照蛋白结合固相物质。 在这种情况下， 抗体或其片段与血清样品预温育， 随后加于平板。 通过报告组分检测抗体与平板的结合， 据 此本领域技术人员可确定抗体是否结合来自血清的表位， 且优选确定结合血清中表位的抗体 的量， 以及由此确定样品中标记蛋白的量。
任选地， 该试剂盒提供用于双抗夹心测定法， 其中抗 CYSTATIN S 抗体结合固相物质， 随后标准品 CYSTATIN S 和处理过的待测样品血清分别加入平板， 通过含报告组分的抗 CYSTATIN S 多克隆抗体与平板的结合， 据此本领域技术人员可确定抗体是否结合来自血清 的表位， 且优选确定结合血清中表位的抗体的量， 以及由此确定样品中标记蛋白的量。
试剂和 /或试剂盒的类型， 和 /或对除该试剂盒之外的其他类型设备和 /或与该试剂盒组 合的其他类型设备的需求， 其每个都取决于使用该试剂盒进行的测定法的类型。 如前所述， 上述测定法非限制性的实例包括 ELISA、 蛋白印迹或流式细胞术。 用于进行 ELISA测定法的 实例性方法如上所述。 蛋白印迹常常比 ELISA更精确但操作需要更多的时间和 /或设备。
6、 例证性的试剂盒及其使用方法
标本的采集及保存 ： 血清： 全血标本请于室温放置 2 小时或 4°C过夜后于 lOOOxg离 心 20 分钟， 取上清即可检测， 或将标本放于 -20 °C或 -80 °C保存， 但应避免反复冻融； 血 浆： 可用 EDTA或肝素作为抗凝剂， 标本采集后 30分钟内于 2-8°C 1000xg离心 15分钟， 或 将标本放于 -20°C或 -80°C保存， 但应避免反复冻融。
样本处理： 血清或血浆标本推荐稀释 10倍， 如： 稀释 10倍， 取 lOOuL血清或血浆加 Λ 900uL PBS o 标本使用 0. 1 M 的 PBS稀释 （PH=7. 0-7· 2)。
试剂盒中包含以下内容： 1 ) 酶联板， 覆膜。 2 ) 标准品： 浓度为 lOOng/ml CYSTATIN S ， 先用 1%BSA 的 PBST稀释至 10 ng/ml CYSTATIN S ， 再做系列倍比稀释后， 分别稀释 成 10 ng/ml CYSTATIN S ， 5 ng/ml CYSTATIN S , 2. 5 ng/ml CYSTATIN S ， Ing/mL CYSTATIN S， 0. 5 ng/ml CYSTATIN S， 0. 25 ng/ml 的 CYSTATIN S ， 样品稀释液直接作为 标准浓度 0 ng/ml CYSTATIN S ， 临用前 15分钟内配制。 如配制 4ng/ml CYSTATIN S 标 准品： 取 0. 5ml (不要少于 0. 5ml ) 8 ng/ml CYSTATIN S 的上述标准品加入含有 0. 5ml样 品稀释液的 Eppendorf 管中， 混匀即可， 其余浓度以此类推。 3 ) 3%BSA 的 PBST 封闭液。
4) 包被抗体 5 g/ml 鼠抗人 CYSTATIN S 单抗， 包被抗体稀释液 0. 05M PH=9. 0 NaHC03。
5 ) 生物素标记的兔抗人 CYSTATIN S 多抗 （1 : 200 ) , 1%BSA 的 PBST 抗体稀释液。 6 ) ABC (亲合素一生物素一过氧化物酶复合物）。 7) TMB 显色液。 8 ) PBST ( 0. 05% Tween-20 in PBS)。 9) 终止液： 2NH2S04 o
具体步骤包括：
1 ) 包被： 用 0. 05M PH9, 碳酸氢钠包被缓冲液将鼠抗人 CYSTATIN S 单抗稀释至蛋白 质含量为 5 g / ml， 在每个聚苯乙烯板的反应孔中加 0. lml， 4°C过夜。 次日， 弃去孔内溶 液， 用洗涤缓冲液洗 3次， 每次 3分钟。
2) 封闭： 每孔加 200ul含 3%BSA的 PBST, 37度 lh或 4度过夜。 弃去孔内溶液， 用洗 涤缓冲液洗 3次， 每次 3分钟
3) 加样： 分别设空白孔、 标准孔、 待测样品孔。 空白孔加样品稀释液 100 μ 1， 余孔 分别加标准品或待测样品 100 μ 1， 注意不要有气泡， 加样将样品加于酶标板孔底部， 尽量 不触及孔壁， 轻轻晃动混匀， 酶标板加上盖或覆膜， 37°C反应 120分钟。 为保证实验结果有 效性， 每次实验请使用新的标准品溶液。
4) 弃去液体， 甩干， 不用洗涤。 每孔加生物素标记的兔抗人 CYSTATIN S 多抗 lOOul (在使用前用 1%BSA的 PBST 1： 200稀）， 酶标板加上覆膜， 37°C反应 60分钟。
5) 温育 60分钟后， 弃去孔内液体， 甩干， 洗板 3次， 每次浸泡 1-2分钟， 大约 300 μ ΐ/每孔， 甩干 （也可轻拍将孔内液体拍干）。
6) 每孔加 ABC 100 μ L， 酶标板加上覆膜 37°C反应 60分钟。
7) 温育 60分钟后， 弃去孔内液体， 甩干， 洗板 5次， 每次浸泡 1-2分钟， 300 μ !7每 孔， 甩干 （也可轻拍将孔内液体拍干）。 8) 依序每孔加底物溶液 50 L， 酶标板加上覆膜 37°C避光显色 （15 分钟内， 此时肉 眼可标准品的前 4-5孔有明显的梯度兰色， 后 4-5孔梯度不明显， 即可终止）。
9) 依序每孔加终止溶液 50 L， 终止反应， 此时蓝色立转黄色。 终止液的加入顺序应 尽量与底物液的加入顺序相同。 为了保证实验结果的准确性， 底物反应时间到后应尽快加入 终止液。
10) 用酶联仪在 405nm波长依序测量各孔的光密度 （0D值）。 在加终止液后立即进行 检测。 结果计算： 根据标准品浓度和对应的 0D值绘制散点曲线， 标出 R平方和曲线方程式。 如 R平方大于等于 0. 95， 则将待测样品的 0D值代入曲线方程式中， 算出待测样品浓度。
7. CYSTATIN S蛋白表达水平用于乳腺癌 pTNM分期
实验方法同上述 "7"， 病理确诊的乳腺癌 T期病人 20人， N期病人 30人， M期病人 30， 结果如表 4 所示， 随着病程的进展， CYSTATIN S 蛋白表达水平亦随之提高， 提示， CYSTATIN S蛋白表达水平可用于乳腺癌 pTNM分期
表 4 不同病理分期的乳腺癌患者 CYSTATIN S蛋白表达水平 病例信息 CYSTATIN S 蛋白表达中位数
(ng/ml )
T期（20例） 1. 16
N期 （30例） 2. 89
M期 （30例） 4. 34
8. CYSTATIN S蛋白表达水平用于评估乳腺癌是否转移
实验方法同上述 "7"， 病理确诊的乳腺癌未转移病人 20人， 转移病人 30人结果如表 5 所示， 转移患者 CYSTATIN S蛋白表达水平高于未转移者， 提示 CYSTATIN S蛋白表达水平 可用于指示乳腺癌是否转移。
表 5 转移与未转移乳腺癌患者 CYSTATIN S蛋白表达水平
病例信息 CYSTATIN S 蛋白表达中位数 (ng/ml ) 未转移患者 (20例） 1. 43
转移患者 ( 30例) 4. 19
9. CYSTATIN S蛋白表达水平用于评估乳腺癌辅助内分泌治疗联合化疗治疗效果 实验方法同上述 "7"， 研究共纳入 2885例 N0-1期乳腺癌患者，行 4个周期的 AC (多 柔比星 +环磷酰胺） 或 AT (多柔比星 +紫杉醇） 方案化疗，再根据激素受体 (HR)状况选择是否 行辅助内分泌治疗，随访时间为 76个月。 检测治疗周期结束后 CYSTATIN S蛋白表达水平， 有效数据 776例， CYSTATIN S蛋白表达中位数 3. 96ng/ml。 结果如图 19 所示， 高于该中位 数的患者无病生存 49% ( Disease-free survival , DFS )， 低于该中位数的患者无病生存 64%。
可以理解的是本发明为了清楚而在上下文各个实施例中描述的某些特征， 也可组合提 供于单个实施方案中。 反之， 本发明为了简便起见而在上下文单个实施方案中所述的不同特 征也可单独或以任何的亚组合形式提供。
虽然本发明结合其具体实施方案进行了描述， 但显然许多替换、 修饰、 和改变对于本领 域技术人员都是显而易见的。 因此， 其试图包括所有这样的替换、 修饰、 和改变， 其落入附 加权利要求的精神和较宽的范围内。 说明书提及的所有出版物、 专利和专利申请均此处整体 引入说明书作为参考， 其程度就如每个单独的出版物、 专利或专利申请此处具体和单独地引 入作为参考一样。 此外， 该申请中任何参考文献的引用或鉴定将不会被理解为承认所述参考 文献可作为发明的现有技术。

Claims

权利要求书
1.CST4 基因、 CST4 基因的 mRNA、 CST4 基因剪切子的 cDNA、 CST4 特异性引物对应的扩增子、 CST4基因编码的 CYSTATIN S蛋白和 CYSTATIN S蛋白 的表位肽在制备诊断和预示乳腺癌标志物中的应用， 所述 CST4 基因的核苷酸序列 如 SEQ ID No: 42所示。
2.根据权利要求 1 所述的应用， 其特征在于， CST4 基因、 CST4 基因的 mRNA、 CST4基因剪切子的 cDNA的探针核苷酸序列如 SEQ ID No: 3。
3.根据权利要求 1 所述的应用， 其特征在于， 所述扩增子的特异性引物， 其上 游引物如 SEQ ID No: 1， 4,6,8,10,12,14,16,18,20; 所示， 下游引物如 SEQ ID No: 2, 5,7,9,11,13,15,17,19, 21 所示， 上游引物 SEQ ID No: 1 与下游引物 如 SEQ ID No: 2配对； 上游引物 SEQ ID No: 4与下游引物如 SEQ ID No：
5配对； 上游引物 SEQ ID No: 6与下游引物如 SEQ ID No: 7配对； 上游弓 I 物 SEQ ID No: 8与下游引物如 SEQ ID No: 9配对； 上游引物 SEQ ID No: 10与下游引物如 SEQ ID No: 11配对； 上游引物 SEQ ID No: 12与下游引物 如 SEQ ID No: 13 配对； 上游引物 SEQ ID No: 14 与下游引物如 SEQ ID No: 15 配对； 上游引物 SEQ ID No: 16 与下游引物如 SEQ ID No: 17 配 对； 上游引物 SEQ ID No: 18与下游引物如 SEQ ID No: 19配对； 上游引物 SEQ ID No: 20与下游引物如 SEQ ID No: 21配对。
4.根据权利要求 1 所述的应用， 其特征在于， 所述 CYSTATIN S 蛋白的表位肽的氨基 酸序列如 SEQ ID No: 50所示。
5.根据权利要求 1 所述的应用， 其特征在于， 所述诊断和预示具体为： 乳腺癌 的转移、 微转移、 pTNM分期、 治疗过程中的动态检测及预后判断。
6.乳腺癌标志物的捕获剂， 其特征在于： 所述捕获剂为所述诊断和预示乳腺癌 标志物的捕获剂， 所述乳腺癌标志物为 CST4 基因、 CST4 基因的 mRNA、 CST4 基因剪切子的 cDNA、 CST4 特异性引物对应的扩增子、 CST4 基因编码的 CYSTATIN S蛋白禾 B CYSTATIN S蛋白的表位肽。
7.根据权利要求 6 所述的捕获剂， 其特征在于， 所述 3) 中的所述引物的核苷酸 序列如 SEQ ID No: 1-2所示。
8.根据权利要求 6所述的捕获剂， 其特征在于， 所述 2) 中的所述探针的核苷酸序 列如 SEQ ID No: 3所示。
9.根据权利要求 6 所述的捕获剂， 其特征在于， 所述 3) 中的所述扩增子的核苷 酸序列如 SEQ ID No : 43所示。
10.根据权利要求 6 所述的捕获剂， 其特征在于， 所述捕获剂为所述 CYSTATIN S 蛋白或所述识别 CYSTATIN S蛋白的表位肽的特异性抗体。
11.根据权利要求 6所述的捕获剂， 其特征在于： 所述 CYSTATIN S表位肽的氨基 酸序列如 SEQ ID No : 50所示。
12.所述捕获剂在制备乳腺癌检测试剂或试剂盒中的应用。
13.含有权利要求 6所述的捕获剂的诊断试剂盒。
14.根据权利要求 13所述的诊断试剂盒， 其特征在于， 所述诊断试剂盒具体为：
1 ) 为基于 Taqman 水解探针法的 CST4 mRNA 实时定量检测试剂盒， 其上游引物如 SEQ ID No:l， 其下游引物 SEQ ID No:2所示， 所述探针的核苷酸序列如 SEQ ID No:3所 示； 或
2 ) 为基于染料法的 CST4 mRNA 实时定量检测试剂盒， 其上游引物如 SEQ ID No:l， 其下游引物 SEQ ID No:2所示； 及内参引物， 所述内参引物的上游引物如 SEQ ID No: 30所示， 下游引物如 SEQ ID No: 31所示； 或
3) 为基于核酸序列扩增法的 CST4 mRNA定量检测试剂盒或基于转录介导的扩增的 CST4 mRNA定量检测试剂盒， 两者均含有 CST4引物和探针， 所述 CST4的上游引物如 SEQ ID No:32所示， 所述下游引物如 SEQ ID No:2所示， 所述探针如 SEQ ID NO: 3所 示； 或
4) 为基于连接酶链反应的 CST4 mRNA定量检测试剂盒， 其含有 4条探针， 所述探 针的核苷酸序列分别如 SEQ ID No:33-36的核苷酸序列所示； 或
5) 为基于适温链置换扩增的 CST4 mRNA 定量检测试剂盒， 其含有引物和探针， 所 述引物如 SEQ ID No:37-40所示， 所述探针如 SEQ ID No:41所示。
15.根据权利要求 13所述的诊断试剂盒， 其特征在于， 所述诊断试剂盒具体为：
1) 双抗夹心 Elisa试剂盒， 包括固相载体、 固定在固相载体上的所述捕获剂、 生物素 标记的所述捕获剂及显色底物； 固定在固相载体上的所述捕获剂为特异性单抗， 生物素标 记的所述捕获剂为多抗；
或 2) 蛋白印迹试剂盒包括固相载体、 捕获剂、 酶标二抗及显色底物， 捕获剂包括特 异性单抗， 生物素标记的所述捕获剂为特异性多抗；
或 3) 竞争 ELISA试剂盒包括固相载体、 固定在固相载体上的抗原； 生物素标记的捕 获剂及显色底物； 特异性单抗； 生物素标记的所述捕获剂为特异性多抗。
17.根据权利要求 14所述的诊断试剂盒， 其特征在于， 所述诊断试剂盒还包括阳性对 照、 阴性对照或 /和空白对照。
18.根据权利要求 14 所述的诊断试剂盒， 其特征在于， 所述双抗夹心 Elisa试剂盒 中， 所述特异性单抗为鼠单抗， 抗- CYSTATIN S 蛋白， 所述固相载体为酶标板， 所述生 物素标记的特异性多克隆抗体为加入生物素标记的兔抗- CYSTATIN S 蛋白的多抗。
19.权利要求 18所述的试剂盒的使用方法， 其特征在于， 具体步骤包括：
将 Corning ELISA平板孔用鼠抗- CYSTATIN S 蛋白单抗覆盖且用 3% BSA封闭，将 8 个倍比稀释的血清样品加至覆盖的 ELISA板中且 37度温育； 每孔用 TBS洗涤， 加入生物 素标记的兔抗- CYSTATIN S 蛋白的多抗， 37度温育； 每孔用 TBS洗涤， 加入亲合素一生 物素一过氧化物酶复合物， 37 度温育； 每孔用 TBS 洗涤， 加入碱性磷酸酶底物， 利用 ELISA读数器在 405nm波长处定量 0D。
20.运用权利要求 14所述的诊断试剂盒检测或预示乳腺癌的方法， 其特征在于： 用所 述诊断试剂盒检测待测样品中所述乳腺癌标志物的含量或表达水平， 将得到的含量 或表达水平与正常人进行比较， 判定所述待测样品是否为阳性； 或直接判定所述含 量或表达水平是否超过阈值， 当超过阈值时， 判定为阳性； 所述阈值是通过 对比正常人与乳腺癌患者体液或组织中标志物的含量或表达水平统计得到； 所述待测样品为血液、 尿液、 骨髓、 乳腺癌细胞株或乳腺癌及癌旁手术组织、 淋巴 结中的任一种或多种。
21.用于诊断和预示乳腺癌的试剂盒， 其特征在于， 所述试剂盒用于检测 CYSTATIN S 蛋白水平， 包括固相载体、 固定在固相载体上的所述捕获剂、 生物素标记的所述捕获剂及 显色底物； 固定在固相载体上的所述捕获剂为特异性单抗， 生物素标记的所述捕获剂为特 异性多抗；
或所述试剂盒用于检测 CYSTATIN S 蛋白的水平， 所述试剂盒包括固相载体、 包被于 固相载体的 CYSTATIN S蛋白、 CYSTATIN S特异性鼠单抗、 酶标二抗及显色底物；
或所述试剂盒用于检测 CYSTATIN S 蛋白水平， 包括固相载体、 捕获剂、 酶标二抗及 显色底物， 捕获剂包括特异性单抗， 生物素标记的所述捕获剂为特异性多抗。
23.根据权利要求 22所述的用于诊断和预示乳腺癌的试剂盒， 所述试剂盒为基于双抗 夹心法的 ELISA诊断试剂盒， 其特征在于， 所述固相载体为酶标板， 所述固定在固相载体 上的捕获剂为鼠抗- CYSTATIN S 蛋白单抗， 生物素标记的捕获剂为效价为 1 : 1000 的兔 抗 - CYSTATIN S 蛋白的多抗， 显色底物为碱性磷酸酶；
或所述试剂盒为基于竞争 Elisa法的诊断试剂盒， 其特征在于， 所述固相载体为酶标 板， 所述 CYSTATIN S蛋白的浓度为 5ug/ml,所述特异性单抗为鼠抗- CYSTATIN S 蛋白单 抗， 其效价为 1 : 2000， 所述酶标二抗为碱性磷酸酶标记的山羊抗 -小鼠 IgG， 所述酶标二 抗效价为 1 : 2000， 所述显色底物为碱性磷酸酶底物， 所述 CYSTATIN S蛋白、 酶标二抗及 显色底物的体积比为 1 : 2;
或所述试剂盒为基于免疫印记的诊断试剂盒， 其特征在于， 所述固相载体为硝酸纤维 素膜， 捕获剂为效价为 1： 1000的 CYSTATIN S鼠单抗， 酶标二抗为缀合至过氧化物酶的山 羊抗-兔 IgG , 显色底物为 TMB溶液。
PCT/CN2012/070151 2012-01-09 2012-01-09 诊断和预示乳腺癌的标志物 WO2013104104A1 (zh)

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