WO2022083582A1 - 半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物作为结肠直肠癌诊断标志物的应用 - Google Patents
半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物作为结肠直肠癌诊断标志物的应用 Download PDFInfo
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Definitions
- the present application relates to the field of medical diagnostics, in particular, to the application of a cysteine protease inhibitor SN and Cathepsin B complex as a diagnostic marker for colorectal cancer.
- Colorectal cancer is one of the common malignant tumors in the gastrointestinal tract.
- Tumorgenesis is a multifactorial and multistage process, and tumor invasion and metastasis include adhesion, degradation and penetration. Tumor invasion and metastasis are important characteristics of malignant tumors.
- proteolytic enzymes play a key role in tumor invasion.
- Proteolytic enzymes are a class of endopeptidases or exopeptidases capable of hydrolyzing or modifying the peptide bonds of proteins or polypeptides.
- CTSB Cathepsin B
- mammalian and human lysosomes are cysteine protease present in mammalian and human lysosomes. Recent studies have shown that abnormal expression of CTSB is often found in tumor tissues, such as gastric cancer, colon cancer, cervical cancer, lung cancer, breast cancer and prostate cancer.
- Cystatin SN (cystatins SN, CST1) is a kind of cathepsin inhibitor. It is a protein of 141 amino acids encoded by CST1 gene with a molecular weight of 16.4kDa. The CST1 molecule contains two disulfide bonds and is a typical secreted protein. It can inhibit intracellular and extracellular cathepsin activity and play an important role in tumor growth, angiogenesis, infiltration and metastasis. High expression is associated with a variety of cancers.
- CTSB is not only highly expressed in colorectal cancer, but also expressed in gastric cancer, glioma, melanoma and other patients, CTSB has poor tissue specificity when used as a tumor diagnostic marker, making it difficult to identify tumors. specific type.
- CST1 also has a certain degree of discrimination in colorectal cancer, due to its high sequence homology with the same family of cysteine protease inhibitors, CST1 also has certain limitations as a colorectal cancer target . Studies have shown that CTSB is one of the main inhibitory substrates of CST1, and CTSB can be inhibited by CST1 to form a complex.
- a quantitative detection agent of cystatin SN and Cathepsin B complex in the preparation of a kit for diagnosis, auxiliary diagnosis or prognostic analysis of colorectal cancer.
- a quantitative detection agent for the complex of cystatin SN and Cathepsin B for the diagnosis, auxiliary diagnosis or prognostic analysis of colorectal cancer.
- the quantitative detection agent is an antibody specific for the Cystatin SN and the Cathepsin B, and the antibody can be used to perform co-immunoprecipitation or enzyme-linked immunosorbent assay to detect all the The cysteine protease inhibitor SN and Cathepsin B complex were detected.
- the quantitative detection agent is an antibody specific for the complex of Cystatin SN and Cathepsin B.
- the specific antibody is a monoclonal antibody or a polyclonal antibody.
- the specific antibody is obtained by immunization with the amino acid sequence shown in SEQ ID NO:1.
- the specific antibody has a label for indicating signal strength.
- the label for indicating signal strength is selected from any one or more of chromophores, digoxigenin-labeled probes, electron-dense substances, colloidal gold, or enzymes.
- a colorectal cancer diagnostic, auxiliary diagnostic or prognostic analysis kit comprising a specific antibody as defined above.
- the kit further comprises at least one of a solid phase carrier, a blocking solution, a chromogenic reagent, a calibrator for the fusion antigen of cystatin SN and Cathepsin B, and a washing buffer.
- the solid support is a chemiluminescent plate.
- a method for diagnosis, auxiliary diagnosis or prognostic analysis of colorectal cancer comprising: measuring cysteine protease using the quantitative detection agent or kit as described above The content of the inhibitor SN in complex with Cathepsin B.
- FIG. 1 is an SDS-PAGE electrophoresis image after purification of CST1-CTSB recombinant protein in an embodiment of the application;
- Fig. 2 is the calibration curve of CST1-CTSB detection kit in an embodiment of the application
- FIG. 4 is the ROC curve of CST1-CTSB for colorectal cancer diagnosis in one embodiment of the present application.
- the present application relates to the application of a quantitative detection agent of cystatin SN and Cathepsin B complex (CST1-CTSB complex) in the preparation of a kit for diagnosis, auxiliary diagnosis or prognostic analysis of colorectal cancer.
- CST1-CTSB complex Cathepsin B complex
- colorectal cancer Colon and rectal cancer are collectively known as colorectal cancer. Both share many similar characteristics and are therefore discussed in this application as one cancer type.
- CST1-CTSB complex provides a new marker for diagnosis: CST1-CTSB complex.
- CTSB can be inhibited by CST1 to form a complex, while CST4 does not have this ability. Therefore, the detection of CST1-CTSB complex can effectively improve the tissue specificity of the marker, and can also effectively improve the detection rate of early colorectal cancer.
- the term "marker” as used herein refers to a molecule to be used as a target for analyzing a patient's experimental sample.
- molecular targets are proteins or polypeptides.
- Proteins or polypeptides used as markers in the present application are intended to include naturally occurring variants of said proteins as well as fragments of said proteins or said variants, particularly immunologically detectable fragments.
- the immunologically detectable fragment comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 contiguous amino acids of the marker polypeptide.
- proteins released by cells or present in the extracellular matrix may be damaged (eg, during inflammation) and may be degraded or cleaved into such fragments.
- markers are synthesized in an inactive form, which can then be activated by proteolysis.
- proteins or fragments thereof can also be present as part of a complex.
- Such complexes can also be used as markers in the sense of the present application.
- marker polypeptides or variants thereof may carry post-translational modifications.
- Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation.
- the marker should be located at the binding site of CST1 and CTSB in the CST1-CTSB complex. This "binding site" refers to the site where the amino acid sequences contact each other when CST1 and CTSB interact, which can be a linear epitope or a spatial epitope. bit.
- the quantitative detection reagent is a specific antibody of the Cystatin SN and the Cathepsin B, and the antibody can be used to perform co-immunoprecipitation or enzyme-linked immunosorbent assay to detect all the The CST1-CTSB complex was detected.
- Quantitative detection reagents can detect CST1-CTSB complexes by methods known in the art; possible methods such as biological mass spectrometry, native polyacrylamide gel electrophoresis, chromatography, enzyme-linked immunosorbent assay, immunofluorescence, Immunochemiluminescence, immunoturbidimetry, immunoblotting, and dot blot. Common methods are co-immunoprecipitation and enzyme-linked immunosorbent assay.
- CST1 can be captured by A antibody, unbound components can be washed away, and CTSB can be detected by B antibody with signal substance; of course, CTSB can also be captured and then CST1 can be detected. , which is easy for those skilled in the art.
- Quantitative detection reagents are usually reagents that specifically detect the CST1-CTSB complex, for example, a lectin that specifically binds to the CST1-CTSB complex, an aptamer that specifically binds to the CST1-CTSB complex, or that specifically binds to the CST1-CTSB complex Complex antibodies and antibody fragments.
- a specific binding agent has an affinity of at least 10 7 l/mol for its corresponding target molecule. In some embodiments, a specific binding agent may have an affinity of 10 8 1/mol for its target molecule. In some embodiments, a specific binding agent may have an affinity of 10 9 1/mol for its target molecule.
- specific means that other biomolecules present in the sample do not bind significantly to the quantitative detection agent of the CST1-CTSB complex, such biomolecules in particular being free CST1 and CTSB alone.
- the quantitative detection agent is an antibody specific for the CST1-CTSB complex.
- the specific antibody is a monoclonal antibody or a polyclonal antibody.
- the specific antibody is obtained by immunization with the amino acid sequence shown in SEQ ID NO: 1.
- the CST1-CTSB recombinant protein can be used to screen the antibody that specifically binds to the CST1-CTSB complex, and optionally an antibody with high antibody titer can be selected.
- the specific antibody has a label for indicating signal strength.
- the label for indicating signal strength is selected from any one or more of chromophores, digoxigenin-labeled probes, electron-dense substances, colloidal gold, or enzymes.
- Enzymes that produce a detectable signal such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
- Chromophores such as fluorophores, quantum dots, fluorescent microspheres, luminescent compounds (eg acridinium esters or derivatives thereof) and dyes.
- a detectable group such as its molecular size is sufficient to induce a modification detectable in its physical and/or chemical properties; such detection can be achieved by optical methods (eg diffraction, surface plasmon resonance, surface variation and contact variation angle) or Physical methods (such as atomic force spectroscopy and tunneling) are implemented.
- optical methods eg diffraction, surface plasmon resonance, surface variation and contact variation angle
- Physical methods such as atomic force spectroscopy and tunneling
- Electron dense substances such as radioactive molecules (eg 32 P, 35 S or 125 I).
- the present application also relates to a colorectal cancer diagnostic kit comprising a specific antibody as defined above.
- the kit further comprises at least one of a solid phase carrier, a blocking solution, a color developing agent, a calibrator for the CST1-CTSB fusion antigen, and a washing buffer.
- the calibrator of the CST1-CTSB fusion antigen preferably has the amino acid sequence shown in SEQ ID NO:1.
- the blocking solution can be one or more of BSA, bovine serum, skim milk, TBST and other components.
- the color developing solution can be determined according to the substance labeled on the antibody, for example, when the labeled substance is horseradish peroxidase, the color developing agent can be luminol.
- the washing buffer can be PBS, TBS and other components.
- the blocking solution, color developing solution, washing buffer can be packaged in the kit in the form of working concentration, or can be packaged in the form of their concentrated mother solutions (for example, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 20, 30, 40, 50 times concentrated mother liquor).
- the solid phase carrier is usually used to coat the antibody, and the solid phase carrier material used for coating the antibody can be polystyrene, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, agar Glycogel and other materials, the carrier can be in the form of test tubes, Eppendorf tubes, multi-well plates (especially chemiluminescence plates), wells of micro-reaction plates, beads (especially magnetic beads), small discs, and the like.
- the solid support is a chemiluminescent plate. It may contain 16, 32, 48, 64, 96 or more wells.
- the present application also provides a method for diagnosis, auxiliary diagnosis or prognostic analysis of colorectal cancer, the method comprising: measuring CST1- Content of CTSB complex.
- the detected sample can be at least one of blood, serum, cerebrospinal fluid, tissue or tissue lysate, semen, and saliva samples of the subject.
- the subject is usually a mammal.
- the subject can be a primate.
- the subject can be a human.
- Protein expression The gene was synthesized according to the amino acid sequence of SEQ ID NO: 1 in the sequence listing and optimized for mammalian expression codons. The gene was inserted into pcDNA3.1 vector containing 6 ⁇ His tag to obtain pcDNA3.1-CST1-CTSB. Then pcDNA3.1-CST1-CTSB was transformed into DH5 ⁇ , positive clones were picked and cultured in large quantities, and the recombinant plasmid pcDNA3.1-CST1-CTSB was extracted with a high-purity plasmid extraction kit. The recombinant plasmid was transferred into 293t cells, and at the same time, the pcDNA3.1 empty vector was transfected as a negative control. The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO 2 for 72 h, and the supernatant was collected. The supernatant was filtered using a 0.22 ⁇ m filter.
- Protein purification The obtained 500 mL filtrate was subjected to Ni-NTA affinity chromatography under native conditions. Equilibration was performed with equilibration buffer (50 mM PBS, 10 mM imidazole, 150 mM NaCl, pH 7.6). After loading, wash with 10 mL washing buffer; elute with elution buffer (50 mM PBS, 250 mM imidazole, 150 mM NaCl, pH 7.6), and collect the eluate. The protein solution was concentrated using a 3kD ultrafiltration tube, and the protein was stored in 50mM PBS buffer (pH 7.4) at -80°C. The purity of the purified protein was identified by SDS-PAGE, the molecular weight was about 52kD, and the grayscale analysis showed that the protein purity reached more than 95% (Fig. 1).
- Antibody pairing chemiluminescent plates were coated with 1 ⁇ g/ml capture antibody. Add 100 ⁇ l of CST1-CTSB calibrator at different concentrations (5-1000 pg/mL) to each well, incubate at 37 °C for 60 min, add 100 ⁇ L of HRP-labeled detection antibody at a concentration of 100 ng/ml to each well after washing, and incubate at 37 °C for 60 min. After washing, a chemiluminescent substrate was added and the luminescence intensity of each well was measured. From the results, the capture antibody and the detection antibody are well paired and can be used for the construction of a double-antibody sandwich system.
- Calibration curve drawing First, the capture antibody was coated on a chemiluminescent plate overnight at 4°C at a concentration of 1 ⁇ g/mL.
- the recombinant human CST1-CTSB calibrator protein was diluted with protein stabilizer to 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 500pg/mL, 1500pg/mL, and 100 ⁇ L was added to each well for incubation. , after washing, add HRP-labeled detection antibody at a concentration of 5ng/ml, 100 ⁇ L per well, and incubate at 37°C for 1 hour.
- FIG. 1 shows the calibration curve of the CST1-CTSB detection kit.
- the linear range of the calibration curve is 10-1500 pg/mL, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic concentration value of the CST1-CTSB calibrator.
- CST1-CTSB detection kit for colorectal cancer diagnosis. Fifty preoperative sera from colorectal cancer patients were collected from the hospital; at the same time, the serum of 50 healthy blood donors was collected from the blood bank. The CST1-CTSB detection kit was used to detect the CST1-CTSB concentration in colorectal cancer and normal human serum, and the sample concentration scatter diagram was drawn. It can be seen that CST1-CTSB can statistically distinguish the detection results of serum from colorectal cancer patients and normal people ( Figure 3).
- the kit provided in this application uses a monoclonal antibody specific for CST1-CTSB as the capture and detection antibody, so that the kit also has the characteristics of high sensitivity, good specificity, low detection limit and good stability. .
- the linear range of detection can reach 10 ⁇ 1500pg/mL, and the lowest detection limit can reach 5pg/mL.
- the CST1-CTSB complex itself has the tissue specificity of both CST1 and CTSB proteins, with a specificity of 94.1% and a sensitivity of 87.8% when detecting colorectal cancer samples.
- the CST1-CTSB detection kit can be used for early diagnosis of colorectal cancer, evaluation of efficacy during treatment, and monitoring of metastasis and recurrence after treatment.
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Description
Claims (24)
- 半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物(CST1-CTSB复合物)的定量检测剂在制备用于结肠直肠癌的诊断、辅助诊断或预后分析的试剂盒中的应用。
- 根据权利要求1所述的应用,其中,所述定量检测剂为所述半胱氨酸蛋白酶抑制剂SN以及所述Cathepsin B的特异性抗体,所述抗体可用于执行免疫共沉淀或酶联免疫吸附试验以对所述半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物复合物进行检测。
- 根据权利要求1所述的应用,其中,所述定量检测剂为所述CST1-CTSB复合物的特异性抗体。
- 根据权利要求3所述的应用,其中,所述特异性抗体为单克隆抗体或多克隆抗体。
- 根据权利要求4所述的应用,其中,所述特异性抗体由SEQ ID NO:1所示的氨基酸序列免疫得到。
- 根据权利要求3~5任一项所述的应用,其中,所述特异性抗体具有用于指示信号强度的标记。
- 根据权利要求6所述的应用,其中,所述用于指示信号强度的标记选自发色团、地高辛标记探针、电子致密物质、胶体金或酶中的任一种或多种。
- 一种用于结肠直肠癌的诊断、辅助诊断或预后分析的半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物的定量检测剂。
- 根据权利要求8所述的定量检测剂,其中,所述定量检测剂为所述半胱氨酸蛋白酶抑制剂SN以及所述Cathepsin B的特异性抗体,所述抗体可用于执行免 疫共沉淀或酶联免疫吸附试验以对所述半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物进行检测。
- 根据权利要求8所述的定量检测剂,其中,所述定量检测剂为所述半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物的特异性抗体。
- 根据权利要求10所述的定量检测剂,其中,所述特异性抗体为单克隆抗体或多克隆抗体。
- 根据权利要求11所述的定量检测剂,其中,所述特异性抗体由SEQ ID NO:1所示的氨基酸序列免疫得到。
- 根据权利要求10~12中任一项所述的定量检测剂,其中,所述特异性抗体具有用于指示信号强度的标记。
- 根据权利要求13所述的定量检测剂,其中,所述用于指示信号强度的标记选自发色团、地高辛标记探针、电子致密物质、胶体金或酶中的任一种或多种。
- 结肠直肠癌诊断、辅助诊断或预后分析试剂盒,其中其中,包含权利要求3~7任一项中所定义的特异性抗体。
- 根据权利要求15所述的试剂盒,其中,还包含固相载体、封闭液、显色剂、半胱氨酸蛋白酶抑制剂SN与Cathepsin B融合抗原的校准品以及洗涤缓冲液中的至少一种。
- 根据权利要求16所述的试剂盒,其中,所述固相载体为化学发光板。
- 一种结肠直肠癌的诊断、辅助诊断或预后分析的方法,所述方法包括:使用根据权利要求8所述的定量检测剂或根据权利要求15所述的试剂盒测量半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物的含量。
- 根据权利要求18所述的方法,其中,所述定量检测剂为所述半胱氨酸蛋 白酶抑制剂SN以及所述Cathepsin B的特异性抗体,所述抗体可用于执行免疫共沉淀或酶联免疫吸附试验以对所述半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物进行检测。
- 根据权利要求19所述的方法,其中,所述定量检测剂为所述半胱氨酸蛋白酶抑制剂SN与Cathepsin B复合物的特异性抗体。
- 根据权利要求20所述的方法,其中,所述特异性抗体为单克隆抗体或多克隆抗体。
- 根据权利要求21所述的方法,其中,所述特异性抗体由SEQ ID NO:1所示的氨基酸序列免疫得到。
- 根据权利要求20~22中任一项所述的方法,其中,所述特异性抗体具有用于指示信号强度的标记。
- 根据权利要求23所述的方法,其中,所述用于指示信号强度的标记选自发色团、地高辛标记探针、电子致密物质、胶体金或酶中的任一种或多种。
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