+

WO2019189990A1 - Composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et kit de diagnostic du cancer du sein l'utilisant - Google Patents

Composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et kit de diagnostic du cancer du sein l'utilisant Download PDF

Info

Publication number
WO2019189990A1
WO2019189990A1 PCT/KR2018/005304 KR2018005304W WO2019189990A1 WO 2019189990 A1 WO2019189990 A1 WO 2019189990A1 KR 2018005304 W KR2018005304 W KR 2018005304W WO 2019189990 A1 WO2019189990 A1 WO 2019189990A1
Authority
WO
WIPO (PCT)
Prior art keywords
breast cancer
protein
fragment
txnl2
cast
Prior art date
Application number
PCT/KR2018/005304
Other languages
English (en)
Korean (ko)
Inventor
송진수
김대원
김묘덕
김동일
이준경
정다윤
강주성
강호철
정용식
김지영
정지민
김소연
Original Assignee
신일제약주식회사
아주대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 신일제약주식회사, 아주대학교산학협력단 filed Critical 신일제약주식회사
Publication of WO2019189990A1 publication Critical patent/WO2019189990A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a breast cancer diagnostic composition using multiple autoantibodies and a breast cancer diagnostic kit using the same, and more particularly, to a diagnostic composition comprising an agent capable of measuring the autoantibody, and a kit for diagnosing breast cancer comprising the composition. It is about.
  • Breast cancer is an advanced disease that is the most common cancer reported in the United States and affects one in eight women. South Korea is the second most common cancer after thyroid cancer among women, with breast cancer accounting for 15.4% of all cancers in 2010, according to the Ministry of Health and Welfare.
  • stage 0 99%, stage 1 98.4%, stage 2 91.4%, stage 3 69.7%, stage 4 30.2%. The importance can be confirmed.
  • the present inventors have studied the sensitivity and specificity of the TXNL2 protein or fragment and the sensitivity and specificity of the CAST protein or fragment to improve the low specificity and sensitivity in diagnosing breast cancer. Based on the clinical evaluation of the group and the group of breast cancer patients, a diagnostic kit was developed that is more accurate in the diagnosis of breast cancer through the combination of TXNL2 protein or fragment, CAST protein or fragment.
  • the present inventors attempted to increase the effectiveness of breast cancer diagnosis by simultaneously detecting antigen-specific autoantibodies in the blood of breast cancer patients with two selected biomarkers.
  • an object of the present invention is to detect breast cancer-specific autoantibodies using plasma samples from breast cancer patients and plasma samples from normal individuals determined to be non-breast cancer, and select biomarkers with high specificity and sensitivity from dozens of antigen candidate groups. It was.
  • Another object of the present invention was to identify an effective method for diagnosing breast cancer by combining different biomarkers to improve the specificity and sensitivity of the identified biomarkers.
  • the present inventors performed the following method.
  • Biomarker combination for effective biomarker selection and efficiency through analysis of specificity and sensitivity and specificity of other carcinomas based on clinical results of breast cancer of selected biomarkers.
  • breast cancer diagnostic composition using the multiple autoantibodies and the breast cancer diagnostic kit using the same when a recombinant protein related to breast cancer-related autoantibodies is used as a diagnostic marker for breast cancer, blood, plasma, Alternatively, breast cancer can be diagnosed only by using non-invasive biological samples such as serum.
  • the diagnosis of breast cancer is higher than that of diagnosis using a single recombinant protein and may be used as a breast cancer diagnosis kit having excellent specificity for other carcinomas.
  • FIG 1 shows breast cancer specific autoantibody identification using 22K protein chip according to an embodiment of the present invention.
  • Figure 2 shows a schematic diagram of the construct for constructing TXNL2 and CAST proteins or fragments thereof in accordance with an embodiment of the present invention.
  • FIG. 3 shows the cloning results of TXNL2 and CAST protein or fragments thereof in accordance with an embodiment of the present invention.
  • FIG. 4 shows the expression state of the TXNL2 and CAST protein or fragment thereof of the present invention according to an embodiment of the present invention.
  • Figure 5 shows the results of size exclusion chromatography purification of TXNL2 protein or fragments thereof (BCM102) according to an embodiment of the present invention.
  • Figure 6 shows the results of size exclusion chromatography purification of TXNL2 protein or fragments thereof (BCM113) according to an embodiment of the present invention.
  • Figure 7 shows the results of size exclusion chromatography purification of TXNL2 protein or fragments thereof (BCM114) according to an embodiment of the present invention.
  • Figure 8 shows the results of size exclusion chromatography purification of TXNL2 protein or fragments thereof (BCM115) according to an embodiment of the present invention.
  • Figure 9 shows the results of size exclusion chromatography purification of CAST protein or fragments thereof (BCM1001) according to an embodiment of the present invention.
  • Figure 10 shows the results of size exclusion chromatography purification of CAST protein or fragments thereof (BCM1004) according to an embodiment of the present invention.
  • Figure 11 shows the antigen adsorption plate configuration according to an embodiment of the present invention.
  • Figure 12 shows the results of specificity, sensitivity to the diagnosis of breast cancer according to the final screened breast cancer biomarkers BCM113 and BCM1004 and combination according to an embodiment of the present invention.
  • the present invention relates to a breast cancer diagnostic composition
  • a breast cancer diagnostic composition comprising a TXNL2 protein or fragment thereof and specifically a CAST protein or fragment thereof that specifically binds to a breast cancer autoantibody.
  • the TXNL2 protein or fragment thereof includes any one of SEQ ID NO: 1 to SEQ ID NO: 4, and the CAST protein or fragment thereof includes a protein or fragment represented by the amino acid sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 6.
  • the TXNL2 and CAST proteins or fragments thereof are preferably fused at least one of GST-tag at the N-terminus or 6X His-tag at the C-terminus.
  • the TXNL2 and CAST proteins or fragments thereof are GST-tag or 6X His- in proteins or fragments in which GST-tag or GST-tag and 6X His-tag are fused by constructing a construct in a host cell. Can be obtained by removing the tag.
  • a breast cancer diagnostic kit comprising the breast cancer diagnostic composition.
  • the breast cancer diagnostic kit detects autoantibodies of TXNL2 (Thioredoxin-like 2) protein or fragment and CAST (Calpastatin) protein or fragment.
  • autoantibody detection may be performed simultaneously or at the same time in one breast cancer diagnosis kit.
  • the breast cancer diagnosis kit may include an Enzyme-linked immunosorbent assay (ELISA), Radioimmnoassay (RIA), Sandwich assay, Western blot on polyacrylamide gel, Immunoblot assay or Immune. At least one or more autoantibody detection methods may be included during immunohistochemical staining.
  • ELISA Enzyme-linked immunosorbent assay
  • RIA Radioimmnoassay
  • Sandwich assay Western blot on polyacrylamide gel
  • Immunoblot assay or Immune.
  • At least one or more autoantibody detection methods may be included during immunohistochemical staining.
  • the method for providing breast cancer diagnostic information of the present invention comprises the steps of 1) measuring the level of autoantibodies for TXNL2 and CAST proteins or fragments from biological samples isolated from the subject; 2) comparing the measured autoantibody expression levels with autoantibody expression levels for TXNL2 and CAST proteins or fragments measured from biological samples isolated from normal individuals; And 3) determining that the cancer is positive if the test level of the test subject is higher than the test level of the normal test subject. It includes.
  • the biological sample is preferably at least one of tissue, whole blood, plasma, serum, lymph, bone marrow fluid, saliva, milk, urine or ascites.
  • TXNL2 protein or fragment thereof may include any one of SEQ ID NO: 1 to SEQ ID NO: 4
  • the CAST protein or fragment thereof may include an amino acid sequence represented by any one of SEQ ID NO: 5 to SEQ ID NO: 6.
  • BCM Breast cancer-specific autoantibodies were detected using plasma samples from breast cancer patients and plasma samples from normal humans with 22K protein chips, and several dozen antigen candidates were identified. Among them, TXNL2 and CAST autoantigens were finally selected.
  • the biomarker candidate was named BCM (Breast Cancer Marker), and a total of six constructs were constructed, including the full domains of TXNL2 and CAST, and (1) BCM 102 (GST-TXNL2 aa 1-334), (2) BCM 113 (6XHis-TXNL2 aa 2-130), (3) BCM114 (6XHis-TXNL2 aa 132-239), (4) BCM 115 (6XHis-TXNL2aa 229-334), (5) BCM 1001 (GST -CAST aa 1-708), (6) BCM 1004 (GST-CAST aa 1-356-6X His).
  • the BCM 102, BCM 113, BCM 114, BCM 115, BCM 1001, and BCM 1004 were commissioned by a specialized synthesis company based on the amino acid sequence of TXNL2 and CAST proteins (Cosmogenetech, Korea). Synthesized DNA sequences of the completed TXNL2 and CAST were prepared by PCR primers and then cloned into pGEX4T1 vector or pET-28a vector.
  • FIG. 2A is a cloning schematic diagram of BCM 102
  • FIG. 2B is a clone schematic diagram of BCM 113
  • FIG. 2C is a clone schematic diagram of BCM 114
  • FIG. 2D is a clone schematic diagram of BCM 115
  • FIG. 2E is a cloning schematic diagram of BCM 1001
  • 2F is a schematic diagram of cloning of BCM 1004.
  • 3A is a cloning result of BCM 102
  • FIG. 3B is a cloning result of BCM 113
  • 3C is a cloning result of BCM 114
  • FIG. 3D is a cloning result of BCM 115
  • FIG. 3E is a cloning result of BCM 100
  • 3F shows the cloning results of BCM 1004.
  • BCM102 (aa 2-334), BCM113 (aa 2-130), BCM114 (aa 132-239), BCM115 (aa 229-334), BCM1001 (aa 1-708), BCM1004 (aa 1-356) Constructs
  • PCR primers for TXNL2 and CAST gene amplification were prepared and cloned into pGEX4T1 vector or pET-28a vector (FIGS. 2 and 3).
  • Example 2 In order to express the breast cancer biomarker (BCM 102, BCM 113, BCM 114, BCM 115, BCM 1001, BCM 1004) constructs prepared in Example 2, a 42 ° C heat shock was applied to C43 (DE3) cells, an E. coli-expressing cell line. After transfection, the cells were incubated at 37 ° C. in 1 mM IPTG in LB medium, and the six types of breast cancer markers (BCM 102, BCM 113, BCM 114, BCM 115, BCM 1001, BCM 1004) were Expression was confirmed. The results are shown in FIG.
  • Figure 4a shows the expression state of BCM 102
  • Figure 4b shows the expression state of BCM 113 and BCM 114
  • Figure 4c shows the expression state of BCM 115
  • Figure 4d shows the expression state of BCM 1001
  • 4e shows the expression state of BCM 1004.
  • the six types of breast cancer biomarkers (BCM 102, BCM 113, BCM 114, BCM 115, BCM 1001, and BCM 1004) constructs were all well expressed.
  • the host cell for breast cancer biomarker protein purification was E. coli C43 (DE3) strain that can lower the level of T7 RNA polymerase to reduce cell death due to toxicity of overexpressed recombinant protein, plasmid (plasmid) was expressed using pGEX-4T1 vector. Affinity chromatography was generated using the GST protein tag included in the plasmid.
  • Antibiotic ampicillin was added to LB medium commonly used for Escherichia coli expression and cultured overnight at 37 ° C. and 200 rpm using a shaking incubator. Since the cell culture medium grown on the new LB medium the day before to inoculate to about 1% and put the antibiotic ampicillin and further cultured at 37 °C, 200 rpm conditions using a shaker incubator. After inoculation, when the OD600 value was 0.6, it was induced using 1 mM IPTG and incubated for 3 hours at 37 ° C. and 200 rpm. Subsequently, the culture medium was centrifuged at 4 ° C. and 8000 rpm to obtain pellets.
  • cell lysis buffer 50 mM Tris-HCl, 20 mM NaCl, 10% glycerol
  • 2 mM ⁇ -mercaptoethanol, 0.2 mM PMSF, pH 8.0 was added and mixed with the cells.
  • the cells were lysed for 10 minutes (pulse on 3 seconds, pulse off 3 seconds) using an ultrasonic crusher.
  • the protein in the aqueous solution was centrifuged for 1 hour at a speed of 20,000 rpm.
  • Affinity chromatography was performed to purify only BCM102 from the BCM102 and protein aqueous solution separated from the cells.
  • the affinity chromatography is a method of purifying GST fusion protein by inducing binding with GST protein fused to the N-terminus of BCM102 using a closed GST column (GST Hitrap FF column, GE).
  • Equilibrium buffer 50 mM Tris-HCl, 20 mM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, pH 8.0 was flowed into equilibrium.
  • the resin and the GST fusion BCM102 were bound by flowing BCM102 and an aqueous solution of protein to the column, and then 10 CV (column volume) equilibration buffer (equilibrium buffer) was flowed to remove nonspecifically bound impurities.
  • Elution buffer 50 mM Tris-HCl, 20 mM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, 10 mM reduced glutathionine, 10 mM reduced glutathionine, to separate GST fused BCM102 from GST selective resin pH 8.0
  • size exclusion chromatography to confirm the homogeneity of the purified GST fusion BCM102 as a result of the primary purification.Connect a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) to the AKTA prime FPLC system.
  • the host cell for breast cancer biomarker protein purification used E. coli C43 (DE3), which can lower the level of T7 RNA polymerase and reduce cell death due to toxicity of overexpressed recombinant protein. It was used to express, and was generated to enable affinity chromatography using (His) x6 tag (or 6x Histag) included in the plasmid.
  • Antibiotic kanamycin was added to LB medium commonly used for E. coli expression and cultured overnight at 37 ° C. and 200 rpm using a shaking incubator. After inoculating the cell culture medium grown in the new LB medium to about 1% the day before, the antibiotic kanamycin was added and further cultured at 37 °C, 200 rpm conditions using a shake incubator.
  • Affinity chromatography was performed to purify only BCM113 from the BCM113 and protein aqueous solution separated from the cells.
  • the affinity chromatography is a method of purifying 6xHis-fusion protein by inducing binding with 6x histidine protein fused to the N-terminus of BCM113 using a closed histidine column (HisTrap FF column, GE).
  • Equilibrium buffer 50 mM Tris-HCl, 20 mM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, pH 8.0 was flowed into equilibrium.
  • BCM101 and aqueous solution of protein were flowed to the column to allow resin and 6xHis fusion BCM113 to bind, followed by 10 CV (column volume) of washing buffer (50 mM Tris-HCl, 20 mM NaCl, 10% glycerol, 2 mM).
  • Non-specifically bound impurities were removed by flowing ⁇ -mercaptoethanol, 35 mM imidazole (Imidazol, pH 8.0) Elutionbuffer (50 mM Tris-HCl, to separate 6xHis fused BCM101 from histidine selective resin) 150 mM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, 350 mM imidazole, pH 8.0 were purified and histidine fusion BCM113 purified as a result of the first purification was subjected to size exclusion chromatography to confirm homogeneity.
  • Elutionbuffer 50 mM Tris-HCl, to separate 6xHis fused BCM101 from histidine selective resin
  • 150 mM NaCl, 10% glycerol, 2 mM ⁇ -mercaptoethanol, 350 mM imidazole, pH 8.0 were purified and histidine fusion BCM113 purified as a result of the first purification was subjected to size
  • a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) was connected to the AKTA prime FPLC system and equilibrated by flowing an equilibration buffer (PBS, pH 7.2).
  • the retention volume was determined to be 99.5 ml, which was predicted to form the monomeric form by GPC data, based on the 280 nm absorbance and SDS-PAGE experiments showing histidine fusion BCM113 in the chromatogram. Homogeneity was evaluated to be very high, and electrophoresis confirmed that about 16 kDa BCM113 protein was expressed (FIG. 6).
  • BCM114 a breast cancer biomarker of the present invention, was cultured and purified in the same manner as in BCM113 of Example 4-2.
  • a histidine fused BCM114 aqueous solution eluted during the second purification was flowed into the column, and the retention volume was measured at 100.4 ml. It was predicted that the monomer form was formed by GPC data. From the results of the 280 nm absorbance and SDS-PAGE experiment showing histidine fusion BCM114 in the chromatogram, the homogeneity was evaluated to be very high. Electrophoresis confirmed that about 14 kDa BCM114 protein was expressed (FIG. 7).
  • BCM115 a breast cancer biomarker of the present invention, was cultured and purified in the same manner as in BCM113 of Example 4-2.
  • a histidine fused BCM115 aqueous solution eluted during the second purification was flowed into the column, and the retention volume was measured to be 99.3 ml. It was predicted that the monomer form was formed by GPC data. From the 280 nm absorbance and SDS-PAGE experiments showing histidine fusion BCM115 in the chromatogram, the homogeneity of the antigen was evaluated to be very high. Electrophoresis confirmed that about 13 kDa BCM114 protein was expressed (FIG. 8).
  • the host cell for the purification of breast cancer biomarker protein was a strain of E. coli C43 (DE3) that lowers the level of T7 RNA polymerase to reduce cell death due to toxicity of the overexpressed recombinant protein.
  • plasmid was expressed using pGEX-4T1 vector. It was generated to enable affinity chromatography using the GST protein tag included in the plasmid.
  • Ampicillin was added to LB medium generally used for E. coli expression and cultured overnight at 37 ° C. and 200 rpm using a shake incubator. Thereafter, the cell culture medium incubated in fresh LB medium was inoculated to about 1% and further cultured at 37 ° C. and 200 rpm using a shake incubator. When the OD600 value was 0.5-0.6 after inoculation, expression was induced for 4 hours under conditions of 1 mM IPTG, 37 ° C, and 200 rpm.
  • Cells were then recovered by centrifugation at 4 ° C. and 8000 rpm to recover the cells, and about 20 ml of cell lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 1 mM) per 1 L of the culture medium was recovered. PMSF, pH7.5) was added and mixed with the cells. Then, the cells were lysed for 10 minutes (pulse on 3 seconds, pulse off 3 seconds) using an ultrasonic crusher. In order to separate BCM1001 and proteins and cell debris in aqueous solution, the solution was centrifuged at a speed of 20,000 rpm for 1 hour using a centrifuge.
  • cell lysis buffer 50 mM Tris-HCl, 500 mM NaCl, 1 mM
  • Affinity chromatography was performed to purify only BCM1001 from BCM1001 and protein aqueous solution separated from the cell debris.
  • the affinity chromatography is a method of purifying GST fusion protein by inducing binding with GST protein fused at the N-terminus of BCM1001 using a closed GST column (GST Hitrap FF column, GE).
  • equilibration buffer 50 mM Tris-HCl, 500 mM NaCl, pH7.5
  • BCM1001 and aqueous protein solution were flowed to the column to allow the resin and GST fusion BCM1001 to bind, and then 10 CV (column volume) of washing buffer (50 mM Tris-HCl, pH 7.5) was flowed to remove nonspecifically bound impurities. .
  • Elution buffer 50 mM Tris-HCl, 10 mM reduced glutathione, pH 7.5 was flowed to separate the GST fusion BCM1001 from the resin of the GST column. Electrophoresis resulted in the expression of about 120 kDa BCM1001 protein (Fig. 9).
  • the host cell for the purification of breast cancer biomarker protein was a strain of E. coli C43 (DE3) that lowers the level of T7 RNA polymerase to reduce cell death due to toxicity of the overexpressed recombinant protein.
  • plasmid was expressed using pGEX-4T1 vector. Affinity chromatography was enabled by using the GST protein tag and the 6X His tag included in the plasmid.
  • Ampicillin was added to LB medium generally used for E. coli expression and cultured overnight at 37 ° C. and 200 rpm using a shaker incubator. Then, the cell culture medium incubated in fresh LB medium was inoculated to about 1% and further cultured at 37 ° C. and 200 rpm using a shaker incubator. When the OD600 value was 0.5-0.6 after inoculation, expression was induced for 4 hours under conditions of 1 mM IPTG, 37 ° C, and 200 rpm.
  • Cells were then recovered by centrifugation at 4 ° C. and 8000 rpm to recover the cells, and about 20 ml of cell lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 1 mM) per 1 L of the culture medium was recovered. PMSF, pH7.5) was added and mixed with the cells. Then, the cells were lysed for 10 minutes (pulse on 3 seconds, pulse off 3 seconds) using an ultrasonic crusher. In order to separate BCM1004 and proteins and cell debris in aqueous solution, the solution was centrifuged at a speed of 20,000 rpm for 1 hour using a centrifuge.
  • cell lysis buffer 50 mM Tris-HCl, 500 mM NaCl, 1 mM
  • Affinity chromatography was performed to purify only BCM1004 from the aqueous solution of BCM1004 and protein isolated from the cell debris.
  • the affinity chromatography uses a closed GST column (GST Hitrap FF column, GE) to induce binding with GST protein fused at the N-terminus of BCM1004 to purify the GST fusion protein and closed histidine column (HisTrap FF). column, GE) was used to induce binding with 6X His tag fused to the C-terminus of BCM1004 to purify the 6X His fusion protein.
  • equilibration buffer 50 mM Tris-HCl, 500 mM NaCl, pH7.5
  • BCM1004 and aqueous solution of protein were flowed to the column to bind the resin and GST fusion BCM1001, and then 10 CV (column volume) of washing buffer (50 mM Tris-HCl, pH 7.5) was flowed to remove non-specifically bound impurities.
  • Elution buffer 50 mM Tris-HCl, 10 mM reduced glutathione, pH 7.5 was flowed to separate GST fusion BCM1004 from the resin of the GST column.
  • GST fusion BCM1004 purified by primary purification was once more purified using a closed histidine column.
  • equilibrate buffer 50 mM Tris-HCl, pH7.5
  • CV column volume
  • wash buffer 50 mM Tris-HCl, 30 mM imidazole, pH7.5
  • Elution buffer 50 mM Tris-HCl, 500 mM imidazole, pH7.5
  • GST-BCM1004-6X His aqueous solution eluted during the second purification was subjected to size exclusion chromatography to confirm homogeneity.
  • a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) was connected to the AKTA prime HPLC system and equilibrated by flowing equilibration buffer (PBS, pH7.4).
  • the GST-BCM1004-6X His aqueous solution purified as a result of the secondary purification was flowed to the column, and the GST-BCM1004-6X His was measured at an absorbance of 280 nm in the chromatogram.
  • electrophoresis of the eluted aqueous solution confirmed that the BCM1004 protein of about 93 kDa was expressed (FIG. 10).
  • Purified antigen BCM102 (100 ng / well) was diluted with PBS and added to 100 ⁇ l in each well of a 96 well ELISA plate. After overnight reaction at 4 ° C. to immobilize the antigen on the plate surface, the supernatant was removed and 200 ⁇ l of 2 ⁇ casein buffer (Sigma, B6429-500ML) was dispensed into each well and blocked for 1 hour at room temperature. It was. Serums of breast cancer patients obtained from Ajou University Medical Center IRB were diluted 1/500 using 2X casein buffer, and 100 ⁇ l of each was dispensed and reacted for 1 hour at room temperature to bind antigen.
  • 2 ⁇ casein buffer Sigma, B6429-500ML
  • Purified antigen BCM113 (100 ng / well) was diluted with PBS and added to 100 ⁇ l in each well of a 96 well ELISA plate. After reacting overnight at 4 ° C. to immobilize antigen on the plate surface, the supernatant was removed and 200 ⁇ l of 2 ⁇ casein buffer (Sigma, B6429-500ML) was dispensed into each well and blocked for 1 hour at room temperature. Clinical samples were diluted 1/500 using 2X casein buffer, and 100 ⁇ l of each was dispensed and reacted for 1 hour at room temperature to bind antigen. After the reaction was washed 7 times with PBST (PBS, 0.2% tween 20, pH 7.4).
  • PBST PBS, 0.2% tween 20, pH 7.4
  • Purified antigen BCM114 (100 ng / well) was diluted with PBS and added to 100 ⁇ l in each well of a 96 well ELISA plate. After reacting overnight at 4 ° C. to fix the antigen on the plate surface, the supernatant was removed and 200 ⁇ l of 2 ⁇ casein buffer (Sigma, B6429-500ML) was dispensed into each well and blocked for 1 hour at room temperature. Clinical samples were diluted 1/500 using 2 ⁇ casein buffer, 100 ⁇ l each of which was allowed to react for 1 hour at room temperature to bind antigen. After the reaction was washed 7 times with PBST (PBS, 0.2% tween 20, pH 7.4).
  • PBST PBS, 0.2% tween 20, pH 7.4
  • Purified antigen BCM115 (100 ng / well) was diluted with PBS and added to 100 ⁇ l in each well of a 96 well ELISA plate. After overnight reaction at 4 ° C. to immobilize antigen on the plate surface, the supernatant was removed and 200 ⁇ l of 2 ⁇ casein buffer (Sigma, B6429-500ML) was dispensed into each well and blocked for 1 hour at room temperature. Clinical samples were diluted 1/500 using 2X casein buffer, and 100 ⁇ l of each was dispensed and reacted for 1 hour at room temperature to bind antigen. After the reaction was washed 7 times with PBST (PBS, 0.2% tween 20, pH 7.4).
  • PBST PBS, 0.2% tween 20, pH 7.4
  • Sensitivity and specificity of the four purified TXNL2 proteins or fragments BCM 102, BCM113, BCM114, and BCM115 showed that BCM 102 was positive in 1 of 14 healthy patients and 72 of 180 breast cancer patients. Positive predicted 98.63%, negative predicted 10.74%, specificity 92.86%, and sensitivity 40.00% for the normal group, and BCM 113 was evaluated as positive in 1 out of 14 healthy patients, as a result of analyzing the sensitivity and specificity of BCM 113. 73 out of 180 patients were selected as BCM113, which was evaluated as positive, 98.65% positive, 10.83% negative, 92.86% specific, and 40.56% sensitive. Sensitivity analysis for carcinoma was performed.
  • the purified BCM113 was diluted in PBS at a concentration of 10 ug / ml, dispensed at 100 ul / well on the surface of a 96well plate, and then fixed overnight at 4 ° C. Each clinical sample was then diluted 250-fold and reacted at room temperature for 1 hour. After washing three times with 0.2% PBS-t buffer, the secondary antibody Peroxidase-conjugated Affinity Pure Mouse Anti-Human IgG (H + L), (Jackson, 209-035-088) diluted 5000-fold, 100 ul was dispensed into the wells and allowed to react at room temperature for 1 hour.
  • the washing step was carried out in the same manner as before, and the TMB coloring reagent was dispensed by 100 ul for each well for 5 minutes. After 5 minutes, 100 ul of 1M HCl reaction stop reagent was dispensed into each well to complete the reaction. Absorbance was measured at 450 nm using a Versamax microplate reader.
  • the sensitivity of other carcinomas of BCM 113 was 10% for liver cancer, 10% for thyroid cancer, 10% for colon cancer, and 10% for stomach cancer.
  • the specificity was 70% for liver cancer, 70% for thyroid cancer, 63% for stomach cancer, and large intestine. Cancer was identified as 63%.
  • BCM 1001 was evaluated as positive in 4 of 14 healthy patients and 6 out of 30 breast cancer patients.
  • the positive predictive value was 60.00%, negative predictive value 29.41%, specificity 71.43%, and sensitivity 20.00%.
  • the sensitivity and specificity of BCM 1004 were evaluated. Among them, 67 patients were evaluated as positive, with 97.10% positive predictive value, 37.12% negative predictive value, 96.08% specificity, 44.67% specificity, and selected the final one (BCM1004) with high specificity and sensitivity to breast cancer. Sensitivity analysis was performed for other carcinomas.
  • the purified BCM1004 was diluted in PBS at a concentration of 1 ug / ml, dispensed at 100 ul / well onto the surface of a 96well plate, and then fixed overnight at 4 ° C. Each clinical sample was then diluted 250-fold and reacted at room temperature for 1 hour. After washing three times with 0.2% PBS-t buffer, the secondary antibody Peroxidase-conjugated Affinity Pure Mouse Anti-Human IgG (H + L), (Jackson, 209-035-088) diluted 5000-fold, 100 ul was dispensed into the wells and allowed to react at room temperature for 1 hour.
  • the washing step was carried out in the same manner as before, and the TMB coloring reagent was dispensed by 100 ul for each well for 5 minutes. After 5 minutes, 100 ⁇ l of 1M HCl reaction stop reagent was dispensed into each well to complete the reaction. Absorbance was measured at 450 nm using a Versamax microplate reader.
  • Purified standards BCM113 and BCM1004, respectively, were prepared by diluting in PBS at a concentration of 5 ug / ml using the antigen adsorption plates shown in FIG. 11.
  • dispense 100 ul / well into two rows of plates 1 and 2 and dispense 100 cm / well into the row marked B with BCM113 and 100 ul / well into the row labeled B and fix overnight at 4 ° C.
  • each clinical sample is prepared by diluting 250 times, and the standard solution (stock: 1 ug / ml) is diluted to be 250 concentrations of 31250 pg / ml, and then diluted 1/2 sequentially.
  • the diluted clinical sample and the standard solution were incubated in the rows marked with plates A and B, and the standard solutions were dispensed with 100 ul into each well of 1 and 2 rows and reacted at room temperature for 1 hour. After washing three times with 0.2% PBS-t buffer, the secondary antibody Peroxidase-conjugated Affinity Pure Mouse Anti-Human IgG (H + L), diluted at 5000-fold, (Jackson, 209-035-088) was plated. Dispense 100 ul into each well of the lines marked A and B, and dilute the secondary antibody of Goat Poly Anti-Mouse IgG (H + L), ( Koma, K0211589) to 100 ul on two rows of plates 1 and 2.
  • the solution was dispensed at / well and reacted at room temperature for 1 hour. After the reaction, the washing step was performed in the same manner as before, and the reaction was performed for 5 minutes by dispensing 100 ⁇ l of each TMB coloring reagent. After 5 minutes, 100 ⁇ l of 1M HCl reaction stop reagent was dispensed into each well to complete the reaction. Absorbance was measured at 450 nm using a Versamax microplate reader. As a result, after classifying negative / positive with cutoff value of 0.09 for BCM113, BCM1004, and two combined (logistic model), respectively, sensitivity and specificity for breast cancer were calculated and logistic regression analysis (logistic) regression analysis). In addition, AUC (Area Under the Curve) of BCM113, BCM1004, and Two combined for breast cancer were calculated. The results are shown in FIG.
  • the probability of breast cancer was 11.907 times higher in positive than in negative, P ⁇ 0.001.
  • the sensitivity of BCM113 was 94% and specificity 40%.
  • the probability of breast cancer was 19.777 times higher in positive than in negative, P ⁇ 0.001.
  • Calpastatin had a sensitivity of 97% and specificity of 45%.
  • the probability of breast cancer was 16.356 times higher in positive than in negative, P ⁇ 0.001.
  • the sensitivity of the two combined was 94% and specificity 65%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Rheumatology (AREA)
  • Rehabilitation Therapy (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et un kit de diagnostic du cancer du sein l'utilisant. Quand elle est utilisée comme marqueur diagnostique du cancer du sein, une protéine recombinée pertinente pour un auto-anticorps lié au cancer du sein permet de diagnostiquer le cancer du sein à l'aide d'un échantillon biologique obtenu de manière non invasive, tel que le sang, le plasma ou le sérum, seul, sans biopsie. L'utilisation combinée de protéines recombinées capables de détecter des auto-anticorps liés au cancer du sein permet d'obtenir une plus grande précision dans le diagnostic du cancer du sein que l'utilisation individuelle desdites protéines recombinées et peut être utilisée dans un kit de diagnostic du cancer du sein ayant une excellente spécificité pour d'autres carcinomes.
PCT/KR2018/005304 2018-03-27 2018-05-09 Composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et kit de diagnostic du cancer du sein l'utilisant WO2019189990A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180035016 2018-03-27
KR10-2018-0034991 2018-03-27
KR20180034991 2018-03-27
KR10-2018-0035016 2018-03-27

Publications (1)

Publication Number Publication Date
WO2019189990A1 true WO2019189990A1 (fr) 2019-10-03

Family

ID=68059263

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/005304 WO2019189990A1 (fr) 2018-03-27 2018-05-09 Composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et kit de diagnostic du cancer du sein l'utilisant

Country Status (1)

Country Link
WO (1) WO2019189990A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHUNG, JEE MIN ET AL.: "Identification of the Thioredoxin-Like 2 autoantibody as a specific biomarker for triple-negative breast cancer", JOURNAL OF BREAST CANCER, vol. 21, no. 1, 2018, pages 87 - 90, XP055640174 *
DATABASE GenBank 23 March 2015 (2015-03-23), "thioredoxin-like 2, isoform CRA_a [Homo sapiens]", XP055640180, retrieved from NCBI Database accession no. EAW49152.1 *
DATABASE Protein 15 June 2010 (2010-06-15), "calpastatin [Homo sapiens]", XP055640185, Database accession no. BAA03747.1 *
STORR, SARAH J ET AL.: "Use of autoantibodies in breast cancer screening and diagnosis", EXPERT REVIEW OF ANTICANCER THERAPY, vol. 6, no. 8, 2006, pages 1215 - 1223, XP008175876, doi:10.1586/14737140.6.8.1215 *
STORR, SARAH J. ET AL.: "Calpastatin is associated with lymphovascular invasion in breast cancer", THE BREAST, vol. 20, 2011, pages 413 - 418, XP028285220, doi:10.1016/j.breast.2011.04.002 *

Similar Documents

Publication Publication Date Title
Jungblut et al. Proteomics in human disease: cancer, heart and infectious diseases
CN109142755B (zh) 一种诊断早期食管鳞状细胞癌的四种自身抗体联合检测试剂盒及应用
WO2009113814A2 (fr) Marqueur protéique utilisé pour le diagnostic précoce du cancer du foie
CN107643405B (zh) 一种基于自身抗体检测的肺癌诊断试剂盒及其应用
CN101297046B (zh) 用于检测尿路上皮癌的试剂盒及方法
CN107561288B (zh) 一种检测血液自身抗体的肺癌诊断试剂盒及其应用
CN103540657A (zh) 食道癌确定用组合物及方法
JP2003517483A (ja) 血中ヘリコバクターピロリ菌抗原
CA2708209A1 (fr) Dosage pour le diagnostic de streptococcus pneumoniae
WO2019013394A1 (fr) Anticorps monoclonal reconnaissant spécifiquement une protéine en2 ou composition pour le diagnostic du cancer de la prostate le contenant
WO2011046309A2 (fr) Marqueur de diagnostic pour carcinome hépatocellulaire comprenant des auto-anticorps anti-fasn et composition de diagnostic pour carcinome hépatocellulaire comprenant des antigènes de celui-ci
WO2015174585A1 (fr) Kit comprenant un anticorps se liant spécifiquement à la protéine du facteur b du complément et anticorps se liant spécifiquement à la protéine d'antigène carbohydrate 19-9 pour le diagnostic du cancer du pancréas
WO2019189990A1 (fr) Composition de diagnostic du cancer du sein utilisant de multiples auto-anticorps et kit de diagnostic du cancer du sein l'utilisant
WO2019013392A1 (fr) Anticorps monoclonal obtenu à partir d'un antigène spécifique reconnaissant spécifiquement une protéine en2 ou composition pour le diagnostic du cancer de la prostate le contenant
JP2012017334A (ja) クラミジア・ニューモニア検出用抗体
WO2017155160A1 (fr) Kit de diagnostics multiples
KR20240126079A (ko) 신규한 펩타이드 및 진단에 있어서의 이의 용도
US20100330554A1 (en) Diagnostic kit for solid cancer and medicament for solid cancer therapy
WO2012060636A2 (fr) Procédé pour le diagnostic du cancer de l'estomac
WO2013039166A1 (fr) Procédé de mesure d'anticorps anti-wt1
KR20220074286A (ko) 흰반점증후군 바이러스에 특이적인 재조합 항체 및 이의 용도
JP5211315B2 (ja) 腫瘍マーカ、腫瘍診断キットおよび腫瘍マーカの測定方法
CN107163131B (zh) 肿瘤抑制因子p16的抗原多肽和其应用
KR101974205B1 (ko) 자가항체를 이용한 유방암 진단키트
CN118530365B (zh) 特异性检测mmp12蛋白的单克隆抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18912813

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18912813

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载