WO2013120394A1

WO2013120394A1 - Kit for detection or diagnosis of prostate cancer

Info

Publication number: WO2013120394A1
Application number: PCT/CN2013/000118
Authority: WO
Inventors: 曾燕; 钱泽
Original assignee: 昂科生物医学技术(苏州)有限公司
Priority date: 2012-02-14
Filing date: 2013-02-04
Publication date: 2013-08-22
Also published as: CN102998455B; CN102998455A

Abstract

A kit for the detection or diagnosis of prostate cancer, comprising a δ-catenin adsorbed antibody, a coating buffer solution, a washing buffer solution, a blocking buffer solution, a δ-catenin detection antibody and an enzyme-labeled antibody, or comprising a δ-catenin adsorbed antibody, a coated buffer solution, a washing buffer solution, a blocking buffer solution, a δ-catenin detection antibody and nano-quantum dot marking. The kit is able to detect in urine δ-catenin protein changes for use in prostate cancer diagnosis, and has the advantages of non-invasiveness, and high sensitivity and specificity.

Description

FIELD OF THE INVENTION The present invention relates to a kit, and more particularly to an agent for detecting or diagnosing prostate cancer.

Product. Book

BACKGROUND OF THE INVENTION Prostate cancer (PCa) 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. Currently widely used screening methods are serum prostate specific antigen (PSA), digital rectal examination (DRE) and transrectal ultrasound (TRUS). However, the key statistical experiments of PSA showed that the positive predictive value of PSA for PCa was only 34%, in the gray area 4-l (25% of patients with ^g/L had hidden PCa. The male with PSA concentration < 4 g/L had 15 % has PCa. Benign prostatic hyperplasia (BPH), which can also increase PSA, can lead to unnecessary pathological biopsy and other expensive and uncomfortable invasive examination interventions. As the only prostate cancer serum currently used in clinical practice. Markers, PSA has obvious shortcomings (Hessels D and Schalken JA. The use of PCA3 in the diagnosis of prostate cancer. 2009. Nature Reviews Urology. 6: 255-261). So looking for a simple and easy Markers with a significant increase in PCa specificity, sensitivity, and detection rate will have a profound impact on the early detection, diagnosis, and treatment of PCa. 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. Other urine biomarkers are currently under investigation, including PCADM-1, sarcosine, Engrailed gene, microchromosome 5 protein, prostate specific membrane antigen, and the like. An ideal urine biomarker must meet some important criteria, in addition to being able to distinguish between normal tissue and cancer tissue, scientific support for prostate cancer pathology, and its detection method should be simple and painless, and should be easily accessible to clinicians. Explanation.

δ-Catenin/NPRAP/Neurojungin (δ-cyclonectin/NPRAP/neuronectin) is an adhesion junction protein in the β-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. However, studies have shown that δ-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. Increased expression of δ-catenin/neural Plakophilin-related armadillo protein (NPRAP) is associated with the downregulation and redistribution of E-cadherin and l20 ^ctn in human prostate cancer. 2005. Human Pathology. 36: 1037-1048; Burger, MJ Tebay MA, Keith PA, Samaratunga HM, Clements J, Lavin MF et al" Expression analysis of delta-catenin and prostate-specific membrane antigen: their potential as diagnostic markers for prostate cancer. Int J Cancer. 2002. 100(2): 228-237). Tissue microarray study It was shown that the expression of S-catenin in prostate intraepithelial neoplasia (PIN) began to increase and was consistent with the increase in Gleason score, indicating that δ-catenin is a potential indicator of prostate cancer progression (Lu Q, Dobbs LJ, Christopher WG, Lanford GW, Revelo MP, Shappell S and Chen YH. Increased expression of δ-catenin/neural plakophilin-re Lated armadillo protein (NPRAP) is associated with the downregulation and redistribution of E-cadherin and l20 ^ctn in human prostate cancer. 2005. Human Pathology. 36: 1037-1048; Lu Q and Chen YH. Method of detecting cancer using delta-catenin . United States Patent: 7,445,906. 2008).

Using 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) δ- 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 δ-catenin alone is 91.7%. Specificity is 75.4% (Lu Q, Dobbs LJ, Christopher WG, Lanford GW, Revelo MP, Shappell S and Chen YH. Increased expression of δ-catenin/neural plakophilin-related armadillo protein (NPRAP) is associated with the downregulation and redistribution ^{of E-cadherin and l20 ctn in} human prostate cancer 2005. human Pathology 36:... 1037-1048) recent studies on human urine precipitate in the cell-free urine display prostasomes al detectable Δ-catenin, PCa disease There was a significant increase in δ-Catenin in human urine (P < 0.0005) (8). These studies suggest a possibility for non-invasive detection in patients with PCa (Lu Q, Zhang J, Allison R, Gay H, Yang WX, Bhowmick N, Frelix G, Shappell S, Chen YH. Identification of extracellular -catenin accumulation for prostate cancer detection. 2009. The Prostate. 69(4):411-418) SUMMARY OF THE INVENTION It is an object of the present invention to provide a simple and easy detection or A kit for diagnosing and diagnosing prostate cancer, which mainly comprises δ-catenin adsorbing antibody, coating buffer, washing buffer, blocking buffer, δ-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 δ-catenin adsorbing antibody, a coating buffer, a washing buffer, a blocking buffer, a δ-catenin detecting antibody, and a nano quantum dot label.

Standards and negative controls are also included in the above kits provided by the present invention.

Wherein the standard is preferably a human δ-catenin standard protein. Δ-catenin adsorption resistance The body may be rabbit anti-human, mouse anti-human, chicken anti-human or other δ-catenin antibody, and the δ-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.

Wherein the δ-catenin detecting antibody may be rabbit anti-human, mouse anti-human, chicken anti-human or other δ-catenin antibody. Preferably, the δ-catenin recognition antibody recognizes a different δ-catenin protein sequence from the δ-catenin detection antibody.

The invention further relates to the use of a δ-catenin protein for the preparation of a preparation for detecting or diagnosing prostate cancer.

The kit provided by the invention can detect δ-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 δ-catenin protein in the urine for diagnosis of prostate cancer.

The above and other objects, features, and advantages of the invention will be apparent from BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1B show the δ-catenin protein in urine by ELISA. Figure 1A shows the purified standard protein δ-catenin (ng/ml), and Figure 1B shows the δ-catenin enzyme reaction in urine.

Figure 2 shows the detection of δ-catenin protein in urine by nanometer quantum dot labeling. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a simple and easy kit for detecting or diagnosing prostate cancer, which mainly comprises δ-catenin adsorption antibody, coating buffer, washing buffer, blocking buffer, δ-catenin detection antibody, enzyme Target antibody. Or mainly comprising δ-catenin adsorption antibody, coating buffer, washing buffer, blocking buffer, δ-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 δ-catenin standard protein of the present invention is a purified recombinant human δ-catenin protein, and an anti-human δ-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.

Example h Detecting δ-catenin protein in urine by ELISA

Step 1: Coat plate

Dilute the δ-catenin antibody to a protein content of 1 to 10 (^g / ml) with carbonate or dPBS coating buffer. Add 100 μl 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.

1) Coating buffer (carbonate buffer):

NaC0 ₃ 1.59 g

NaHC0 ₃ 2.93g

Add distilled water to 1000ml

Or Dulbecco's PBS (Invitrogen)

2) Wash Buffer:

KH ₂ P0 ₄ 0.2 g

Na ₂ HP0 ₄ 12H ₂ 0 2.9 g

NaCl 8.0 g

KC1 0.2 g

Tween-20 0.05 % 0.5ml

Add distilled water to 1000ml Step 2: Closed

Add 200 μl of blocking buffer to each well and incubate for 1 hour at room temperature. Wash with a washer. Blocking solution: (Blocking buffer)

Bovine serum albumin (BSA) 2 g

Add wash buffer to 100ml.

Step 3: Load the sample

Standard: Human δ-catenin standard protein.

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 μl 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 δ-catenin detection antibody diluted with Block solution, 50 μl 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.

Step 6: Color development

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.

Step 7: Stop the reaction

The reaction was terminated by adding 3⁄4 S0 ₄ per well.

Step 8: Result determination

The reader reads the board.

Experiments have shown that at least 2 ng can be detected in the urine by double antibody sandwich enzyme-linked immunosorbent assay. S-catenin. As shown in Figures 1A-1B, wherein Figure 1A shows the purified standard protein δ-catenin (ng/ml), Figure 1B shows the δ-catenin enzyme reaction in urine. As can be seen from Figure 1B, the δ-catenin in the urine of patients with prostate cancer is three times higher than that of normal people. The sensitivity of urine and normal people in 48 patients with prostate cancer was 65~85%, and the specificity was over 90%.

Example 2: Determination of δ-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 δ-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 δ-catenin antibody was added to form a covalent bond with the nano quantum dots.

Step 6: Result determination

As shown in Figure 2, nano-quantum dot labeling experiments show that nano-quantum dots (QD) combined with δ-catenin antibodies can also measure δ-catenin protein in solution.

All of the above experiments have demonstrated that δ-catenin antibody binding enzymes or binding to nano quantum dots can detect changes in δ-catenin protein which is highly correlated with prostate cancer, and thus can be used for diagnosis of prostate cancer.

Although the invention has been disclosed above in the preferred embodiments, it is not intended to limit the invention. In any human or animal tissues and various body fluids, δ-catenin antibodies bind to or bind to nano quantum dots as long as δ-catenin proteins are recognized by δ-catenin antibodies or δ-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.

Claims

Claim

A kit for detecting or diagnosing prostate cancer, which comprises a δ-catenin absorbing antibody, a coating buffer, a washing buffer, a blocking buffer, a δ-catenin detecting antibody, and an enzyme-labeled antibody.

A kit for detecting or diagnosing prostate cancer, which comprises a δ-catenin absorbing antibody, a coating buffer, a washing buffer, a blocking buffer, a δ-catenin detecting antibody, and a nano quantum dot label.

3. A kit according to claim 1 or 2, further comprising a standard and a negative control.

The kit according to claim 3, wherein the standard is a human δ-catenin standard protein.

The kit according to claim 1 or 2, wherein the δ-catenin-adsorbing antibody is a δ-catenin antibody.

The kit according to claim 1 or 2, wherein the coating buffer is carbonate or dPBS.

The kit according to claim 1 or 2, wherein the washing buffer is detergent-containing dPBS.

The kit according to claim 1 or 2, wherein the δ-catenin detecting antibody and the δ-catenin adsorbing antibody recognize different δ-catenin protein sequences.

9. Use of δ-catenin protein in the preparation of a preparation for detecting or diagnosing prostate cancer.