WO2013120394A1 - Kit for detection or diagnosis of prostate cancer - Google Patents
Kit for detection or diagnosis of prostate cancer Download PDFInfo
- Publication number
- WO2013120394A1 WO2013120394A1 PCT/CN2013/000118 CN2013000118W WO2013120394A1 WO 2013120394 A1 WO2013120394 A1 WO 2013120394A1 CN 2013000118 W CN2013000118 W CN 2013000118W WO 2013120394 A1 WO2013120394 A1 WO 2013120394A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- catenin
- antibody
- prostate cancer
- kit
- buffer
- Prior art date
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Classifications
-
- 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
- G01N33/57434—Specifically defined cancers of prostate
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
Definitions
- the present invention relates to a kit, and more particularly to an agent for detecting or diagnosing prostate cancer.
- Prostate cancer is one of the common cancers and ranks second among male lethal cancers (Jemal, A., Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010. 60 ( 5): 277-300; Lu Q, Zhang J and Chen YH. Prostate cancer cell growth and death: complex roles of pro- and anti-oncogenic protein signaling. 2009. In: Handbook of Prostate Cancer Cell Research (Editor: Alan T Meridith). Nova Science Publishers, Inc. 431-447; Hessels D and Schalken JA. The use of PCA3 in the diagnosis of prostate cancer. 2009. Nature Reviews Urology. 6: 255-261). Due to the complexity of prostate cancer performance, early diagnosis of prostate cancer is difficult.
- PSA serum prostate specific antigen
- DRE digital rectal examination
- TRUS transrectal ultrasound
- Urine biomarker detection is very attractive because it provides a simple, Non-invasive Early prostate cancer screening methods allow a wide range of people to be screened. In recent years, great progress has been made in the discovery of urine markers for prostate cancer. A urine biomarker was validated using PCA3 (prostate cancer gene 3) non-coding m NA and TMPR S2:ERG fusion gene technology. However, they need to take urine after prostate massage or the positive value is lower.
- PCA3 prostate cancer gene 3
- ⁇ -Catenin/NPRAP/Neurojungin ( ⁇ -cyclonectin/NPRAP/neuronectin) is an adhesion junction protein in the ⁇ -catenin superfamily (Lu, Q, Paredes M, Medina M, Zhou J, Cavallo R, Jifer Biol, 1999. 144(3): 519-532), originally identified as a specific protein in brain tissue.
- ⁇ -catenin mRNA is overexpressed in prostate cancer compared with benign prostatic hyperplasia (Lu Q, Dobbs LJ, Christopher WG, Lanford GW, Revelo MP, Shappell S and Chen YH.
- NPRAP Plakophilin-related armadillo protein
- tissue microarray TMA to analyze 90 specimens of prostate cancer after prostatectomy and 90 specimens of normal prostate tissue, 92% of patients with Cap showed strong (46/72) or moderate (20/72) ⁇ - Catenin staining (immune score ⁇ 2, only 6 of the 72 specimens (8%) were negative for the immune score or ⁇ 2. 65 of the 65 benign specimens (75%) had an immune score ⁇ 2, and 65 of the benign specimens were Six patients (9%) had an immune score equal to 2, and 10 of 15 65 (15%) benign specimens had an immune score higher than 2. These data indicate that the clinical sensitivity of ⁇ -catenin alone is 91.7%.
- a kit for diagnosing and diagnosing prostate cancer which mainly comprises ⁇ -catenin adsorbing antibody, coating buffer, washing buffer, blocking buffer, ⁇ -catenin detecting antibody, and enzyme labeling antibody.
- the invention also provides a simple and easy method A kit for detecting or diagnosing a prostate, which mainly comprises a ⁇ -catenin adsorbing antibody, a coating buffer, a washing buffer, a blocking buffer, a ⁇ -catenin detecting antibody, and a nano quantum dot label.
- the standard is preferably a human ⁇ -catenin standard protein.
- ⁇ -catenin adsorption resistance The body may be rabbit anti-human, mouse anti-human, chicken anti-human or other ⁇ -catenin antibody, and the ⁇ -catenin antibody is diluted to a protein content of l ⁇ 10 ( ⁇ g / ml) when used.
- the coating buffer may be For carbonate or dPBS or other buffer.
- the wash buffer is preferably detergent-containing dPBS or other buffer.
- the ⁇ -catenin detecting antibody may be rabbit anti-human, mouse anti-human, chicken anti-human or other ⁇ -catenin antibody.
- the ⁇ -catenin recognition antibody recognizes a different ⁇ -catenin protein sequence from the ⁇ -catenin detection antibody.
- the invention further relates to the use of a ⁇ -catenin protein for the preparation of a preparation for detecting or diagnosing prostate cancer.
- the kit provided by the invention can detect ⁇ -catenin protein which is highly correlated with prostate cancer by ELISA in the urine of the patient, and the ELISA method is mature, sensitive and specific, and non-invasive to the patient, Can be repeated, the patient is willing to accept. Further, the nanoquantum dot (QD) labeling method has a small sample size, and the antibody used in the ELISA method is combined with QD to more sensitively detect changes in the ⁇ -catenin protein in the urine for diagnosis of prostate cancer.
- QD nanoquantum dot
- Figures 1A-1B show the ⁇ -catenin protein in urine by ELISA.
- Figure 1A shows the purified standard protein ⁇ -catenin (ng/ml)
- Figure 1B shows the ⁇ -catenin enzyme reaction in urine.
- Figure 2 shows the detection of ⁇ -catenin protein in urine by nanometer quantum dot labeling.
- the present invention provides a simple and easy kit for detecting or diagnosing prostate cancer, which mainly comprises ⁇ -catenin adsorption antibody, coating buffer, washing buffer, blocking buffer, ⁇ -catenin detection antibody, enzyme Target antibody. Or mainly comprising ⁇ -catenin adsorption antibody, coating buffer, washing buffer, blocking buffer, ⁇ -catenin detection antibody, nano quantum dot labeling.
- the kit also contains standards and a negative control.
- the following detailed description uses the kit of the present invention to detect or diagnose prostate cancer.
- the human ⁇ -catenin standard protein of the present invention is a purified recombinant human ⁇ -catenin protein, and an anti-human ⁇ -catenin antibody has been disclosed in the following literature: Paffenholz R, Franke WW. Identification and localization of a neurally expressed member of the Plakiglobin/armadillo multigene family. Differentiation. 1997 Aug;61(5):293-304 ; Lu Q, Paredes M, Medina M, Zhou J, Cavallo R, Peifer M Orecchio L, Kosik KS. delta-catenin, an adhesive junction -associated protein which promotes cell scattering. J Cell Biol. 1999 Feb 8; 144(3): 519-32.
- ⁇ -catenin antibody Dilute the ⁇ -catenin antibody to a protein content of 1 to 10 ( ⁇ g / ml) with carbonate or dPBS coating buffer. Add 100 ⁇ l to the reaction well of each polystyrene plate, overnight at 4 ° C. On the next day, the solution in the well was discarded, washed 5 times with washing buffer, and washed by a plate washer.
- Blocking buffer (Blocking buffer)
- Step 3 Load the sample
- Negative control Normal healthy adult male urine specimens.
- Prostate cancer urine specimens - 37 patients with prostate cancer aged 50 to 80 were diagnosed by urology ultrasound and pathologically diagnosed Gleason grade 7 ⁇ 10. Their urine is stored at -20 degrees after collection.
- Each specimen was given 100 ⁇ l of urine and incubated at 37 ° C for 2 hours. Wash the plate.
- Urine specimens can also be centrifuged at low temperature and high speed (14,000) for 15 minutes, and the sediment is filled with 3 wells and the urine is cleared to three wells.
- Step 4 Add ⁇ -catenin detection antibody diluted with Block solution, 50 ⁇ l per well. Wash the plate.
- Step 5 Add the enzyme-labeled antibody (goat anti- or other anti-HRP) (Sigma, USA). Add freshly diluted anti-HRP enzyme-labeled antibody (Sigma, USA) to each well. After incubating for 1 hour at room temperature, the plate washer was washed.
- TMB (Sigma T0440) was added to each well and stored at room temperature in the dark. Or Shanghai Kehua Company coloring liquid A and B liquid.
- the reaction was terminated by adding 3 ⁇ 4 S0 4 per well.
- Step 8 Result determination
- the reader reads the board.
- Example 2 Determination of ⁇ -catenin protein in urine by nanometer quantum dot labeling
- Steps 1 to 4 are the same as in the first embodiment.
- Step 5 Nano-quantum dot labeling is combined with the anti-human ⁇ -catenin antibody of step 4 using Evitas Technologies (Troy, New York, USA) Evitags nano-quantum dot water-soluble.
- EDC (1 -ethyl-3-[3-dimethylaminopropyl] carbodiimide) and sulfo-NHS were used to form an activity on the surface of nano quantum dots. After the excess EDC and sulfo-NHS were removed by a filter, an anti-human ⁇ -catenin antibody was added to form a covalent bond with the nano quantum dots.
- Step 6 Result determination
- nano-quantum dot labeling experiments show that nano-quantum dots (QD) combined with ⁇ -catenin antibodies can also measure ⁇ -catenin protein in solution.
- QD nano-quantum dots
- ⁇ -catenin antibody binding enzymes or binding to nano quantum dots can detect changes in ⁇ -catenin protein which is highly correlated with prostate cancer, and thus can be used for diagnosis of prostate cancer.
- ⁇ -catenin antibodies bind to or bind to nano quantum dots as long as ⁇ -catenin proteins are recognized by ⁇ -catenin antibodies or ⁇ -catenin antibodies and nano quantum dot conjugates. Can be used for testing.
- the scope of protection of the present invention is defined by the scope of the claims, and the scope of the invention is defined by the appended claims.
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- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
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- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
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Applications Claiming Priority (2)
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CN201210032258.0 | 2012-02-14 | ||
CN201210032258.0A CN102998455B (en) | 2012-02-14 | 2012-02-14 | The kit of a kind of detection or diagnosing prostate cancer |
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WO2013120394A1 true WO2013120394A1 (en) | 2013-08-22 |
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PCT/CN2013/000118 WO2013120394A1 (en) | 2012-02-14 | 2013-02-04 | Kit for detection or diagnosis of prostate cancer |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112574955A (en) * | 2019-09-29 | 2021-03-30 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell LCZ9H4, monoclonal antibody secreted by hybridoma cell LCZ9H4 and application of monoclonal antibody |
US11391744B2 (en) | 2015-06-08 | 2022-07-19 | Arquer Diagnostic Limited | Methods and kits |
US11519916B2 (en) | 2015-06-08 | 2022-12-06 | Arquer Diagnostics Limited | Methods for analysing a urine sample |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103983791A (en) * | 2014-05-29 | 2014-08-13 | 深圳市柏明胜医疗器械有限公司 | Human CA125 protein quantum dot labeled double-sandwiched immunoassay detection kit |
CN107522780A (en) * | 2017-10-12 | 2017-12-29 | 南京精竞生物科技有限公司 | A kind of preparation method and applications method of human prostate Immunodominant Antigenic |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11391744B2 (en) | 2015-06-08 | 2022-07-19 | Arquer Diagnostic Limited | Methods and kits |
US11519916B2 (en) | 2015-06-08 | 2022-12-06 | Arquer Diagnostics Limited | Methods for analysing a urine sample |
CN112574955A (en) * | 2019-09-29 | 2021-03-30 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell LCZ9H4, monoclonal antibody secreted by hybridoma cell LCZ9H4 and application of monoclonal antibody |
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CN102998455B (en) | 2016-01-13 |
CN102998455A (en) | 2013-03-27 |
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