WO2017113565A1 - 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 - Google Patents
基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 Download PDFInfo
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- WO2017113565A1 WO2017113565A1 PCT/CN2016/081858 CN2016081858W WO2017113565A1 WO 2017113565 A1 WO2017113565 A1 WO 2017113565A1 CN 2016081858 W CN2016081858 W CN 2016081858W WO 2017113565 A1 WO2017113565 A1 WO 2017113565A1
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- 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
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- 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
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- the invention relates to the application of a protein marker PSG3 in assisting diagnosis of liver cancer or digestive tract patients, and the application in preparing corresponding detection kits.
- liver cancer is the sixth most common cancer in the world and the third most common cancer in China. According to estimates by the International Agency for Research on Cancer (IARC), the number of cases of liver cancer in the world in 2000 was 564,000, of which about 55% occurred in China, that is, the incidence of liver cancer in China was 306,000, and the death of liver cancer was 300,000, accounting for the second largest cancer death in our country. Bit. Most of the symptoms of liver cancer are in the middle and late stage, and the recurrence and metastasis rate after resection are high. Therefore, the early diagnosis of liver cancer is of great significance for prolonging the survival time of patients and reducing the mortality of liver cancer.
- IARC International Agency for Research on Cancer
- Imaging diagnosis plays an important role in the diagnosis of liver cancer, but it has certain limitations in the diagnosis of small liver cancer and the differentiation of benign and malignant nodules.
- Ultrasound or CT-positive results combined with serum alpha-fetoprotein (AFP) levels above 400 ng / ml, can confirm the diagnosis of liver cancer. However, usually when these conditions are met, the best time to treat has been missed.
- AFP serum alpha-fetoprotein
- tumor markers can also be used for the screening of patients at risk for liver cancer (such as chronic carriers of HBV and patients with cirrhosis).
- AFP alpha-fetoprotein
- the AFP content in normal human serum is generally less than 10 ng/ml.
- the diagnostic value of AFP was set at 20 ng/ml, the sensitivity was 50-60%, but in cases with small tumors, the sensitivity was significantly reduced, as reported by only 40%.
- Another problem with AFP alone as a diagnostic marker for liver cancer is the lack of specificity, at considerable In many patients with chronic hepatitis, especially those with cirrhosis, the AFP content is also 20-200 ng/ml.
- markers that can be diagnosed in complementary with AFP include AFP heterogeneity, ⁇ -glutamyltransferase isoenzyme II and abnormal prothrombin, but due to their lack of sensitivity and specificity, and detection methods.
- the cumbersome complexity makes it always stay at the level of laboratory research and fails to translate into clinical routine examination items. Therefore, exploring new tumor molecular markers and establishing corresponding easy-to-promote detection methods is still one of the important topics in the field of liver cancer research.
- colon cancer is insidious, and there are no obvious clinical symptoms in the early stage, and the disease develops slowly. Most of the patients have obvious symptoms and have reached the middle and late stage.
- the treatment of colon cancer is mainly based on surgery, but the chemotherapy effect after colon cancer metastasis is poor. About 40% of patients have recurrence after operation, and the postoperative survival rate is lower in advanced patients.
- many scholars have done a lot of research on the molecular level of colon cancer, there is still no effective molecular marker for early diagnosis of colon cancer. Tumor markers can be effectively used as a clinical diagnosis basis.
- Human blood contains a variety of cellular components and molecular substances, which can well reflect the physiological and pathological state of different tissues and organs of the body, and its specimens are easy to obtain. It is an ideal non-invasive diagnostic tool for tumors. Therefore, markers with good sensitivity and specificity have yet to be developed.
- PSG3 Human pregnancy specific protein 3
- PSG family contains 11 proteins, including PSG1, PSG2, and PSG3.
- PSG proteins generally include a structure similar to the N-terminal immunoglobulin variable region, followed by 2-3 immunoglobulin constant region C2-like domains, and a short hydrophobic tail at the C-terminus.
- PSG can be classified into four types according to its structural composition, type I (N-A1-A2-B2-C), type IIa (N-A1-B2-C) and type IIb (N-A2-B2-C), Type III (N-B2-C), Type IV (A1-B2-C).
- type I N-A1-A2-B2-C
- type IIa N-A1-B2-C
- type IIb N-A2-B2-C
- Type III N-B2-C
- Type IV Type 1-B2-C
- the PSG3 protein is 428 amino acids in length and has a molecular weight of 47,945 Da.
- the amino acid sequence of the protein is as follows:
- a first aspect of the invention relates to the use of a molecule for detecting a PSG3 protein for the preparation of a kit or detection reagent for use in the diagnosis of a cancer, in particular a liver cancer or a gastrointestinal cancer patient.
- a molecule refers to a molecule capable of specifically detecting the expression of a PSG3 protein, and may be, for example, a nucleic acid, a protein or a compound.
- the protein is an antibody which is capable of specifically binding to a PSG3 protein, preferably a monoclonal antibody.
- the molecule that detects the PSG3 protein is a monoclonal antibody that is capable of specifically binding to the PSG3 protein.
- the PSG3 protein is detected by immunochemical methods.
- liver cancer is hepatocellular carcinoma.
- kit or detection reagent for the detection of a plasma sample.
- auxiliary diagnosis is judged by the fact that the content of PSG3 in the plasma is higher than 551.65 ⁇ g/ml, and the subject is a candidate hepatocellular carcinoma or colorectal cancer patient.
- the molecule for detecting the PSG3 protein further carries a detectable label.
- kit or the detection reagent further comprises a buffer and a color developer.
- a second aspect of the invention relates to a kit or detection reagent for assisting in the diagnosis of cancer, in particular liver cancer or digestive tract cancer, comprising a product for detecting the marker PSG3 protein.
- the product of the detection marker PSG3 protein refers to a molecule capable of specifically detecting the expression of PSG3 protein.
- it can be a nucleic acid, a protein or a compound.
- kits or detection reagent according to any one of the second aspects of the invention, wherein the protein is an antibody which is capable of specifically binding to a PSG3 protein, preferably a monoclonal antibody.
- the molecule that detects the PSG3 protein is a monoclonal antibody that is capable of specifically binding to the PSG3 protein.
- the PSG3 protein is detected by immunochemical methods.
- liver cancer is hepatocellular carcinoma
- the digestive tract cancer is colorectal cancer
- kits or detection reagent according to any one of the second aspects of the invention, the kit or detection reagent for use in the detection of a plasma sample, preferably the detection method is an immunochemical method.
- kit or the detection reagent according to any one of the second aspect of the present invention, wherein the auxiliary diagnosis is judged by the method that the plasma PSG3 protein expression is higher than 551.65 ⁇ g/ml, and the candidate is a candidate hepatocellular carcinoma or colorectal Cancer patients.
- kits or detection reagent according to any one of the second aspects of the invention, wherein the molecule for detecting the PSG3 protein further carries a detectable label.
- kit or detection reagent according to any one of the second aspects of the invention, wherein the kit or the detection reagent further comprises a buffer and a color developer.
- the molecule for detecting the PSG3 protein refers to a molecule capable of detecting whether the PSG3 protein is overexpressed, and may be, for example, a nucleic acid, a protein or a compound.
- the presence of a protein marker can be quantitatively or qualitatively detected using an immunoassay method.
- the immunoassay typically involves incubating a biological sample with an antibody and detecting the bound antibody by a variety of well known techniques.
- the compound has a meaning well known in the art, which can specifically bind to the PSG3 protein and thereby be used to detect the expression of the PSG3 protein.
- the molecule for detecting the PSG3 protein may be linked to a detectable label molecule.
- the kit or the detection reagent may further comprise a molecule capable of binding to a molecule detecting the PSG3 protein, such as an antibody, such as a second antibody, which may also be linked to a detectable label molecule.
- a molecule capable of binding to a molecule detecting the PSG3 protein such as an antibody, such as a second antibody, which may also be linked to a detectable label molecule.
- the detectable label molecule may be, for example, an enzyme, a fluorescein, a metal ion or an isotope or the like.
- the present invention also relates to two monoclonal antibodies against PSG3, which are secreted by hybridoma cell line P201 or P209, respectively, which can specifically bind to PSG3 protein, wherein the hybridoma cell strain corresponding to the preservation number is CGMCC NO. .11597 or CGMCC NO.11598.
- the use of these two monoclonal antibodies to construct a kit for detecting the expression level of PSG3 in a sample provides an auxiliary diagnosis for liver cancer and colon cancer, and has important commercial value.
- the invention also relates to the use of the above monoclonal antibody or hybridoma cell in the preparation of an immunodiagnostic test kit.
- the present invention also relates to an immunoassay diagnostic kit comprising the above monoclonal antibody or a monoclonal antibody prepared from hybridoma cells.
- the present invention also relates to a kit for assisting in the diagnosis of cancer, in particular a liver cancer or a digestive tract cancer patient, comprising the monoclonal antibody or the hybridoma cell preparation antibody according to any of the preceding claims.
- liver cancer is hepatocellular carcinoma and the digestive tract cancer is colorectal cancer.
- kit as described above, wherein the kit is for the detection of a plasma sample.
- kits described above are a chemiluminescence immunoassay kit, an enzyme-linked immunosorbent assay kit, a colloidal gold immunoassay kit, and a fluorescent immunoassay kit.
- Figure 1 shows a linear relationship between the antibody 201 and the standard protein of the enzyme-labeled capture antibody 209.
- Figure 2 shows the ROC curve of PSG3 distinguishing between normal and hepatocellular carcinoma patients (HCC).
- Figure 3 shows the ROC curve of PSG3 distinguishing between normal and colorectal cancer patients (CC).
- the PSG3 protein was expressed in E. coli prokaryotic expression, and the expression vector was pET30a.
- BALB/c mice were taken at 5-6 weeks old, and PSG3 antibody was prepared by immunization with Freund's adjuvant using PSG3 full-length protein as antigen. Each mouse was immunized 4 times, each time 2 weeks apart, using 15-30 ⁇ g of antigen protein each time, using the complete adjuvant for the first immunization and the incomplete adjuvant for the last three times.
- Three days before cell fusion one of the three spleen cell donor mice was selected for intravenous immunization with PSG3 antigen for further immunization. Cell fusion and hybridoma screening were performed according to the method of J.M.
- the mixture was mixed upside down, transferred to a 35 mm plate, and each plate was about 2 ml, and cultured at 37 ° C in a 5% CO 2 atmosphere. After 7 days, the clones were transferred into 96-well plates and cultured again by adding IMDM medium containing 15% fetal bovine serum and 2% HT (containing hypoxanthine and thymidine). When the cell fusion degree reached 50% to 70%, the culture supernatant was collected, and the presence or absence of the antibody was detected by ELISA and Western blot.
- the specific steps are as follows: PSG3 standard protein was freeze-dried to dry powder at 4 ° C, and diluted with 50 mM Tris-HCl pH 8.5-9.0 buffer.
- PSG3 standard protein coated with ELISA plate add 100 ⁇ l of medium supernatant for 1 hour; remove reaction liquid, wash plate with 0.5% PBST 5 times, 300 ⁇ l/well; add TMB coloring solution, 200 ⁇ l/well, avoid light reaction for 15 minutes
- the reaction was terminated by adding 2 M sulfuric acid, 100 ⁇ l/well, and the absorbance was measured with a microplate reader at OD 450 nm, and the well having an OD value of more than 0.5 was a positive reaction antibody.
- the antibody positive for ELISA was further determined by western blot.
- the specific steps were as follows: 40 ⁇ g total placental protein, separated by 10% SDS polyacrylamide gel electrophoresis, electroporated onto PVDF membrane, 5% milk blocked, PSG3 monoclonal
- the cell culture supernatant was diluted 1:5 and reacted with PVDF membrane at 4 °C overnight.
- the mouse secondary antibody was reacted for 1 hour, and the luminescence signal was detected by ELC luminescence method.
- the signal identifying the molecular weight of 48 KDa is a positive antibody.
- the monoclonal cell strain expressing the positive antibody was subjected to the next antibody preparation.
- the establishment of the enzyme-labeled double antibody sandwich method for detecting PSG3 antigen Firstly, a monoclonal antibody was produced by using the mouse abdominal cavity, and the obtained positive hybridoma cells were separately injected into the abdominal cavity of the mouse to produce ascites, and the planting of the phytoane, each mouse 0.5 ml; after 1 week of injection of pristane, hybridoma cells were injected, and 10 6 cells were injected per mouse. After 7-10 days, ascites was collected separately, and the antibody was purified by n-octanoic acid-ammonium sulfate precipitation method, and the antibody was subjected to horseradish peroxidase labeling (enzyme-labeled antibody) by sodium periodate method.
- the ELISA plate was separately coated with the purified monoclonal antibody, and the PSG3 standard protein and plasma (200-fold dilution) were detected by pairing with the enzyme-labeled antibody, respectively.
- the specific loading sequence and ELISA reaction results are shown in Table 1.
- the OD value is greater than 3.500.
- the capsid antibody P201 and the enzyme-labeled capture antibody P209 were produced by hybridoma cell lines P201 and P209, respectively.
- the hybridoma cell lines P201 and P209 were deposited on November 27, 2015 at the General Microbiology Center of the China Microbial Culture Collection Management Committee.
- the deposit numbers are CGMCC NO.11597 and CGMCCNO.11598, respectively.
- the deposit address is Beichenxi, Chaoyang District, Beijing. No. 3, No. 1 Road.
- PSG3 coated antibody P201 is coated with 96-well microtiter plate, 100 ⁇ l/well, overnight at 4 ° C; Plate the antibody P201, 0.5% PBST for 3 times, 300 ⁇ l/well; add 2% BSA blocking solution, 300 ⁇ l/well, room temperature for 4 hours; remove the blocking solution, wash the plate with 0.5% PBST 3 times, 300 ⁇ l/well ; each well in the ELISA plate Do not add PSG3 standard protein and sample to be tested, 100 ⁇ l/well; then add PSG3 enzyme detection antibody P209, 100 ⁇ l/well, mix and mix at 4°C overnight; remove the reaction liquid, wash the plate with 0.5% PBST 5 times, 300 ⁇ l/well Add TMB coloring solution, 200 ⁇ l/well, avoid light reaction for 15 minutes; add 2M sulfuric acid to stop the reaction, 100 ⁇ l/well, measure
- the concentration of PSG3 protein was measured in the plasma of pregnant women using the prepared ELISA kit. Samples tested included 11 early pregnant women (11-12 weeks) and 17 late pregnant women (28-38 weeks). The results of the ELISA are shown in Table 3. The protein concentration of PSG3 in the plasma of pregnant women was significantly higher than that in the late pregnancy (P ⁇ 0.001).
- the protein concentration of PSG3 was measured in large-scale tumor patients and normal population plasma samples using the prepared ELISA kit.
- the samples tested included 178 cases of hepatocarcinoma (HCC), 121 cases of lung cancer (Lung cancer, LC), 159 cases of ovarian cancer (Ovarian Cancer, OC), and 88 cases of colorectal cancer (CC), and 115 healthy controls matched by age and sex ratio (Health Control).
- HCC hepatocarcinoma
- 121 cases of lung cancer Lung cancer, LC
- 159 cases of ovarian cancer Ovarian Cancer, OC
- OC colorectal cancer
- 115 healthy controls matched by age and sex ratio (Health Control).
- the results of ELISA showed that plasma protein levels of PSG3 in patients with hepatocellular carcinoma and colorectal cancer were significantly higher than those in the normal control group (P ⁇ 0.001, Table 4).
- Example 3 The role of PSG3 plasma protein concentration in the diagnosis of hepatocellular carcinoma or colorectal cancer
- hepatocellular carcinoma marker alpha-fetal protein (AFP) was also detected in the plasma of the same group of liver cancer patients.
- the clinical routine detection limit was 25 ng/ml, there were 107 patients with hepatocellular carcinoma AFP negative, accounting for 60.1% (107/178) of the total number of specimens.
- the number of PSG3-positive (PSG3 protein concentration in plasma > 551.65 ⁇ g/ml) was 66, which improved the detection rate of liver cancer patients and clinically improved hepatocellular carcinoma patients. Diagnostic rate.
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Abstract
本发明提供了一种基于蛋白质标记物PSG3辅助诊断癌症,尤其是肝癌与结肠癌的试剂盒及其应用。本发明还提供了一种抗PSG3蛋白的单克隆抗体,其由杂交瘤细胞株CGMCC NO.11597或CGMCC NO.11598产生。这两种单克隆抗体可用于PSG3蛋白的体外检测及肝癌和结直肠癌的辅助诊断。
Description
本发明涉及一种蛋白标志物PSG3在辅助诊断肝癌或消化道患者中的应用,以及制备相应的检测试剂盒中的应用。
原发性肝癌是全球第6位、中国第3位最常见的癌症。据国际癌症研究中心(IARC)估计,2000年全球肝癌发病数为56.4万人,其中约55%发生在中国,即中国肝癌发病30.6万人,肝癌死亡30.0万,占我国居民肿瘤死亡的第二位。肝癌出现症状时多属中晚期,切除后复发、转移率高。因此,肝癌的早期诊断对延长患者的生存时间和降低肝癌死亡率具有重要意义。
目前,影像学诊断、细胞与组织学诊断及化学诊断是肿瘤诊断的三大主要方法。影像学诊断在肝癌诊断中起重要的作用,但是在诊断小肝癌及区分良恶性结节中均具有一定的局限。超声检查或CT阳性结果,结合血清甲胎蛋白(α-fetoprotein,AFP)水平高于400ng/ml的检测结果,可对肝癌做出确诊。但是,通常当这些条件都符合时,已经错过了治疗的最佳时机。无论是超声波检查、CT扫描还是核磁共振,对小病灶的鉴定都有一定的局限性,尤其是肝硬化结节在影象学上与小肝癌结节有许多的相似之处。因此,虽然影像学技术取得了巨大的进步,但临床上仍需结合肝癌的分子标志物,在较为复杂的病例中将良恶性肝病进行区分。此外,肿瘤的临床分期及肿瘤大小是肝癌治疗效果的决定因素,因此,肿瘤标志物还可用于肝癌危险人群(如HBV慢性携带者和肝硬化患者)的普查。
目前肝癌诊断最常用的标志物是甲胎蛋白(AFP),在症状和影像学改变发生前数月即可出现异常。正常人血清中AFP含量一般小于10ng/ml。当AFP的诊断值定为20ng/ml时,其敏感性为50~60%,但在肿瘤较小的病例中,敏感性显著降低,有报道仅为40%。单独使用AFP作为肝癌诊断标志物的另一个问题是缺乏特异性,在相当
多的慢性肝炎患者尤其是肝硬化患者中,AFP含量也达20-200ng/ml。目前认为可与AFP互补诊断的标志物有AFP异质体、γ-谷氨酰转移酶同工酶II和异常凝血酶原等,但由于它们在敏感性和特异性上的不足,以及检测方法的繁琐复杂,使其始终停留在实验室研究水平,未能转化成临床常规检查项目。因此,探索新的肿瘤分子标志物,以及建立相应的易于推广的检测方法,仍是当今肝癌研究领域的重要课题之一。
同样,消化道肿瘤在我国也是导致死亡的主要病因之一。其中的结直肠癌起病隐匿,早期常无明显的临床症状,而且病情发展较慢,病人出现明显症状时大多已经到了中晚期。目前,结肠癌的治疗主要是以手术为主,但结肠癌转移后的化疗效果差,术后约有40%的患者出现复发,且晚期患者术后生存率较低。尽管有许多学者已经做了大量的关于结肠癌分子水平的研究,但是目前仍然缺乏有效地早期诊断结肠癌的分子标志物。肿瘤标志物可以有效地作为临床诊断依据,可以监测高危人群、早期诊断、指导治疗、判断治疗疗效、检测复发及转移,是肿瘤患者的重要检查指标、具有重要的临床意义。结肠癌的发生前期会有一段时间的癌前病变,如结肠息肉,因此在早期发现并阻断或延迟癌症的发展,有极其重要的意义。因此,寻找发现有价值的肿瘤标志物对于提高消化道肿瘤、尤其是结直肠癌的诊断和预测发生、发展及改善预后具有重要的临床指导意义。
人类血液中包含丰富的各种细胞成分和分子物质,能很好地反映机体不同组织和器官的生理、病理状态,且其标本容易获得,是一种理想的肿瘤无创诊断手段。因此,仍有待开发敏感性和特异性良好的标志物。
妊娠特异蛋白3(Human pregnancy specific beta-1-glycoprotein 3,PSG3),是一种分泌到细胞外的糖蛋白。PSG家族中共包含11个蛋白,包括PSG1、PSG2、PSG3等。PSG蛋白一般都包括N端同免疫球蛋白可变区类似的结构,随后是2-3个免疫球蛋白恒定区C2样结构域,以及一个短的疏水尾,位于C端。PSG根据其结构组成可分为4类,I型(N-A1-A2-B2-C)、IIa型(N-A1-B2-C)和IIb型(N-A2-B2-C)、
III型(N-B2-C)、IV型(A1-B2-C)。目前PSG家族的作用机制尚不清楚,在正常情况下,胎盘的合胞体滋养层细胞中大量表达,在妊娠期女性外周血中PSG蛋白含量升高,可达200-400μg/ml。关于PSG家族在癌症中的研究鲜有报道。
PSG3蛋白长度428个氨基酸,分子量47,945Da,蛋白的氨基酸序列如下:
有关PSGs在肿瘤中的研究报道较少。Salahshor等应用差异基因表达谱分析发现结直肠癌、肝转移癌、子宫肿瘤组织中均可检测到异常升高的PSG9转录本,与正常结直肠粘膜相比,PSG9在腺瘤中上调2倍,且结直肠癌PSG9表达异常上调呈APC突变依赖性(Salahshor et al,2005)。Kamarli ZP等的研究表明,PSG1在骨恶性肿瘤患者血清中蛋白浓度升高(Kamarli et al,2004)。而关于PSG3在肿瘤中的研究还未见报道。
发明内容
本发明第一方面涉及检测PSG3蛋白的分子在制备用于辅助诊断癌症,尤其是肝癌或消化道癌症患者的试剂盒或检测试剂中的用途。
根据本发明第一方面任一项的用途,其中所述检测PSG3蛋白的
分子是指能够特异性检测PSG3蛋白表达的分子,例如可以为核酸、蛋白质或化合物。
根据本发明第一方面任一项的用途,其中所述蛋白质为抗体,其能够与PSG3蛋白特异性结合,优选为单克隆抗体。
在本发明的实施方案中,所述检测PSG3蛋白的分子为单克隆抗体,其能够与PSG3蛋白特异性结合。
在本发明的具体实施方案中,采用免疫化学的方法检测PSG3蛋白。
根据本发明第一方面任一项的用途,其中所述的肝癌为肝细胞肝癌。
根据本发明第一方面任一项的用途,其中所述的消化道癌症为结直肠癌。
根据本发明第一方面任一项的用途,所述试剂盒或检测试剂用于血浆样品的检测。
根据本发明第一方面任一项的用途,其中所述辅助诊断的判断方法为,血浆中PSG3的含量高于551.65μg/ml,则待检者为候选的肝细胞肝癌或结直肠癌患者。
根据本发明第一方面任一项的用途,其中所述检测PSG3蛋白的分子还带有可检测的标记。
根据本发明第一方面任一项的用途,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
根据本发明第二方面涉及一种用于辅助诊断癌症,尤其是肝癌或消化道癌症的试剂盒或检测试剂,其含有检测标记物PSG3蛋白的产品。
根据本发明第二方面涉及一种用于辅助诊断癌症,尤其是肝癌或消化道癌症的试剂盒或检测试剂,所述检测标记物PSG3蛋白的产品是指能够特异性检测PSG3蛋白表达的分子,例如可以为核酸、蛋白质或化合物。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述蛋白质为抗体,其能够与PSG3蛋白特异性结合,优选为单克隆抗体。
在本发明的实施方案中,所述检测PSG3蛋白的分子为单克隆抗体,其能够与PSG3蛋白特异性结合。
在本发明的具体实施方案中,采用免疫化学的方法检测PSG3蛋白。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述的肝癌为肝细胞癌,所述的消化道癌症为结直肠癌。
根据本发明第二方面任一项的试剂盒或检测试剂,所述试剂盒或检测试剂用于血浆样品的检测,优选地,所述检测方法为免疫化学方法。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述辅助诊断的判断方法为,血浆PSG3蛋白表达高于551.65μg/ml,则待检者为候选的肝细胞肝癌或结直肠癌患者。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述检测PSG3蛋白的分子还带有可检测的标记。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
在本发明中,其中所述检测PSG3蛋白的分子是指能够检测PSG3蛋白是否过表达的分子,例如可以为核酸、蛋白质或化合物。
在本发明中,可以使用免疫测定的方法定量或定性检测蛋白标志物的存在。所述免疫测定通常包括将生物样品与抗体一起孵育,并通过多种熟知技术检测结合抗体。
在本发明中,所述化合物具有本领域公知的含义,其可以和PSG3蛋白特异性结合,进而用于检测PSG3蛋白的表达。
在本发明中,所述检测PSG3蛋白的分子上可以连接有可检测的标记分子。
在本发明中,所述试剂盒或检测试剂中还可以包含能够与检测PSG3蛋白的分子结合的分子,例如抗体,例如第二抗体,该分子也可以连接有可检测的标记分子。
在本发明中,所述可检测的标记分子例如可以为酶、荧光素、金属离子或同位素等。
本发明还涉及2株抗PSG3的单克隆抗体,分别由杂交瘤细胞株P201或P209分泌制备,其能与PSG3蛋白特异结合,其中所述的杂交瘤细胞株所对应的保藏编号分别为CGMCC NO.11597或CGMCC NO.11598。利用这两株单抗构建用于检测样本中PSG3表达量的试剂盒,为肝癌和结肠癌提供辅助诊断,具有重要的商业价值。
本发明还涉及上述单克隆抗体或杂交瘤细胞在制备免疫诊断检测试剂盒中的应用。
本发明还涉及一种免疫检测诊断试剂盒,其中含有上述的单克隆抗体或由杂交瘤细胞制备的单克隆抗体。
前面任一项所述的单克隆抗体或杂交瘤细胞在制备用于辅助诊断癌症,尤其是肝癌或消化道癌症患者的试剂盒中的应用。
本发明还涉及一种用于辅助诊断癌症,尤其是肝癌或消化道癌症患者的试剂盒,其中含有前面任一项所述的单克隆抗体或杂交瘤细胞制备的抗体。
前面任一项所述的应用或试剂盒,其中所述肝癌为肝细胞癌,所述的消化道癌症为结直肠癌。
前面所述的应用或试剂盒,其中所述试剂盒用于血浆样品的检测。
进一步而言地,前面所述的试剂盒为化学发光免疫试剂盒、酶联免疫检测试剂盒、胶体金免疫试剂盒、荧光免疫试剂盒。
图1包被抗体201与酶标的捕获抗体209的标准蛋白线性关系。
图2为PSG3区分正常人和肝细胞肝癌患者(HCC)的ROC曲线。
图3为PSG3区分正常人和结直肠癌患者(CC)的ROC曲线。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限
定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1制备PSG3检测的ELISA试剂盒
采用大肠杆菌原核表达PSG3蛋白,表达载体为pET30a。取5-6周龄BALB/c小鼠,利用PSG3全长蛋白作为抗原添加弗氏佐剂免疫制备PSG3抗体。每只小鼠免疫4次,每次间隔2周,每次利用15-30μg抗原蛋白,第一次免疫注射时利用完全佐剂,后三次利用不完全佐剂。细胞融合前三天,从三只脾细胞供体小鼠挑选一只进行静脉注射PSG3抗原做进一步免疫。细胞融合、杂交瘤筛选均依据J.M.Davis的方法(Davis et al 1982)。简略过程如下:加强免疫后的小鼠脾细胞和SP2/0骨髓瘤细胞混合,慢慢加入促融合剂50%聚乙二醇0.7ml。37℃培养1min,10ml IMDM培养基稀释,低速离心。然后用40ml含1.25%甲基纤维素、25%胎牛血清、2%HAT(含次黄嘌呤、甲氨蝶呤和胸腺嘧啶核苷)的IMDM培养基重悬。上下颠倒混合,转移到35mm平板中,每板约2ml,于37℃、5%CO2环境中培养。7天后克隆转移入96孔板,加入含15%胎牛血清和2%HT(含次黄嘌呤和胸腺嘧啶核苷)的IMDM培养基再次培养。当细胞融合度达到50%~70%,收集培养基上清,利用ELISA和Western blot检测是否存在抗体。具体步骤如下:将PSG3标准蛋白冷冻干燥为干粉4℃保存,临用时用50mM Tris-Hcl PH8.5-9.0的缓冲液稀释。PSG3标准蛋白包被ELISA板,加入100μl培养基上清反应1小时;甩去反应液体,0.5%PBST洗板5次,300μl/孔;加入TMB显色液,200μl/孔,避光反应15分钟;加入2M硫酸终止反应,100μl/孔,用酶标仪OD450nm处检测吸光度,OD值大于0.5的孔为阳性反应抗体。ELISA检测为阳性的抗体再进一步用western blot方法确定,具体步骤如下:胎盘总蛋白40μg,用10%SDS的聚丙烯酰胺凝胶电泳分离,电转至PVDF膜上,5%牛奶封闭,PSG3单克隆细胞株培养上清1:5稀释后与PVDF膜4℃反应过夜,加鼠二抗反应1小时,ELC发光法检测发光信号,
识别48KDa分子量的信号为阳性抗体。将表达阳性抗体的单克隆细胞株进行下一步的抗体制备。
检测PSG3抗原的酶标双抗体夹心法的建立:首先利用小鼠腹腔生产单克隆抗体,将得到的阳性杂交瘤细胞分别注入小鼠腹腔,使其产生腹水,注射降植烷,每只小鼠0.5ml;注射降植烷1周后,注射杂交瘤细胞,每只小鼠注射106个细胞。7-10天后分别收集腹水,用正辛酸-硫酸铵沉淀法分别提纯抗体,再用过碘酸钠法将抗体分别进行辣根过氧化物酶标记(酶标抗体)。用纯化的单克隆抗体分别包被酶标板,并分别与酶标抗体配对检测PSG3标准蛋白和血浆(200倍稀释)。具体加样顺序和ELISA反应结果(OD450nm吸光度)请见表1。
表1.筛选配对抗体ELISA分布表及ELISA反应结果(OD450nm吸光度)
***为跳出酶标仪检测限,OD值大于3.500。
进一步优化ELISA反应,包被抗体201包被ELISA板,酶标的捕获抗体209(1:1000),由此获得的标准蛋白线性关系很好,R2=0.9976,具体参见表2,图1。
表2.包被抗体201与酶标的捕获抗体209的标准蛋白线性关系
由此我们获得最佳配对的包被抗体P201以及酶标的捕获抗体P209。其中包被抗体P201和酶标的捕获抗体P209分别由杂交瘤细胞株P201和P209制备产生。杂交瘤细胞系P201和P209已于2015年11月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号分别为CGMCC NO.11597和CGMCCNO.11598,保藏地址为北京市朝阳区北辰西路一号院3号。
进一步地,利用获得的最佳配对抗体建立检测PSG3抗原的酶标双抗体夹心法试剂盒,具体步骤如下:将PSG3包被抗体P201包被96孔酶标板,100μl/孔,4℃过夜;甩去包被抗体P201,0.5%PBST洗板3次,300μl/孔;加入2%BSA封闭液,300μl/孔,室温4小时;甩去封闭液,0.5%PBST洗板3次,300μl/孔;酶标板中每孔分
别加入PSG3标准蛋白和待测样本,100μl/孔;再加入PSG3酶标检测抗体P209,100μl/孔,混匀后4℃过夜;甩去反应液体,0.5%PBST洗板5次,300μl/孔;加入TMB显色液,200μl/孔,避光反应15分钟;加入2M硫酸终止反应,100μl/孔,用酶标仪OD450nm处检测吸光度;利用标准蛋白吸光度的值绘制标准曲线,并计算待测样本中PSG3的蛋白浓度。
实施例2在大规模的肿瘤患者和正常人群血浆样本中检测PSG3的蛋白浓度
利用制备的ELISA试剂盒在怀孕妇女血浆中检测PSG3蛋白的浓度。检测的样本包括:11例怀孕早期妇女(11-12周),17例怀孕晚期妇女(28-38周)。ELISA的结果如表3显示,PSG3在怀孕早期妇女血浆中的蛋白浓度显著高于怀孕晚期(P<0.001)。
表3.PSG3在怀孕妇女血浆中的蛋白浓度(μg/ml)
利用制备的ELISA试剂盒在大规模的肿瘤患者和正常人群血浆样本中检测PSG3的蛋白浓度。检测的样本包括:178例肝细胞肝癌(Hepatocarcinoma,HCC),121例肺癌(Lung cancer,LC),159例卵巢癌(Ovarian Cancer,OC),88例结直肠癌(Colorectal Cancer,CC),以及115例年龄与性别比例匹配的健康正常人(Health Control)。ELISA的结果显示,PSG3在肝细胞肝癌、和结直肠癌患者的血浆蛋白水平显著高于正常对照组(P<0.001,表4)。
表4.PSG3在各组肿瘤患者和正常人群血浆中的蛋白浓度(μg/ml)
实施例3PSG3血浆蛋白浓度在辅助诊断肝细胞癌或结直肠癌症中的作用
当PSG3血浆蛋白浓度值为551.65μg/ml时,检测肝细胞肝癌的敏感性为64.2%,特异性为74.8%,ROC曲线下面积为0.774,95%CI=0.719-0.828,P<0.001(图2)。同一组肝癌病人血浆中也检测了肝细胞肝癌标志物甲胎蛋白(alpha-fetal protein,AFP)的水平。当使用临床常规检测限值为25ng/ml时,肝细胞肝癌AFP阴性的病人为107例,占标本总数的60.1%(107/178)。而在此107例AFP阴性的肝癌病人中,PSG3阳性(血浆中PSG3蛋白浓度>551.65μg/ml)的病例数为66例,提高了肝癌病人的检出率,临床上可以提高肝细胞肝癌病人的诊断率。
当PSG3血浆蛋白浓度值为551.65μg/ml时,检测结直肠癌(n=88)的敏感性为92.0%,特异性为74.8%,ROC曲线下面积为0.873,95%CI=0.819-0.828,P<0.001(图3)。这88例病人血浆也检测了结直肠癌标志物癌胚抗原(carcinoembryonic antigen,CEA)的蛋白水平。当使用临床常规检测限值为5ng/ml时,结直肠癌患者中CEA阴性的病人为67例,占标本总数的76.1%(67/88)。而在此67例CEA阴性的结直肠癌病人中,PSG3阳性(血浆中PSG3蛋白浓度>551.65μg/ml)的病例数为60例,显著的提高了结直肠癌患者的检出率,临床上有较好的应用前景。
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Claims (11)
- 蛋白标志物PSG3在制备用于辅助诊断癌症患者的试剂盒中的应用。
- 权利要求1的应用,其中所述癌症为肝癌或消化道癌症。
- 用于辅助诊断癌症患者的试剂盒,其中含有检测蛋白标记物PSG3的产品。
- 权利要求3的试剂盒,其中所述癌症为肝癌或消化道癌症。
- 权利要求2的应用或权利要求4的试剂盒,其中所述肝癌为肝细胞癌,所述的消化道癌症为结直肠癌。
- 前述任一项权利要求所述的应用或试剂盒,所述试剂盒用于血浆样品的检测,优选地,所述检测方法为免疫化学方法。
- 前述任一项权利要求所述的应用或试剂盒,其中检测蛋白标志物PSG3的产品是指能够特异性检测PSG3蛋白表达的分子,例如可以为核酸、蛋白质或化合物。
- 权利要求7所述的应用或试剂盒,其中所述特异性检测PSG3蛋白表达的分子为抗体,优选为单克隆抗体。
- 权利要求8所述的用途或试剂盒,其特征在于所述的单克隆抗体由杂交瘤细胞株P201或P209分泌制备,其中所述杂交瘤细胞株对应的保藏编号分别为CGMCC NO.11597和CGMCC NO.11598。
- 权利要求7或8任一项所述的用途或试剂盒,其中所述检测PSG3蛋白表达的分子还带有可检测的标记。
- 前述任一项权利要求所述的用途或试剂盒,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
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CN201511017748.3A CN106928352B (zh) | 2015-12-29 | 2015-12-29 | 一种抗psg3蛋白的单克隆抗体及其杂交瘤细胞株与应用 |
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