CN101290318B - ELISA reagent kit for diagnosing liver cancer - Google Patents

ELISA reagent kit for diagnosing liver cancer Download PDF

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CN101290318B
CN101290318B CN2007100395627A CN200710039562A CN101290318B CN 101290318 B CN101290318 B CN 101290318B CN 2007100395627 A CN2007100395627 A CN 2007100395627A CN 200710039562 A CN200710039562 A CN 200710039562A CN 101290318 B CN101290318 B CN 101290318B
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antibody
gpc3
sulfate proteoglycan
epitope
kit
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CN101290318A (en
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屠红
周伟
赵新泰
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Shanghai Cancer Institute
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Abstract

本发明公开了一种肝癌的检测试剂盒,所述检测试剂盒中含有结合硫酸肝素蛋白多糖3第379-393位中的抗原表位的多克隆抗体,以及结合硫酸肝素蛋白多糖3第379-393位以外抗原表位的单克隆抗体或多克隆抗体。本发明的试剂盒检测灵敏度高,检测耗时短,有助于尽早地诊断肝癌患者,及时治疗,提高生存率。并且,与仅检测AFP标志物相比,在检测AFP标志物的同时采用本发明的试剂盒检测GPC3标记物可显著提高肝癌的诊断敏感性。The invention discloses a detection kit for liver cancer. The detection kit contains a polyclonal antibody that binds to the antigenic epitopes in positions 379-393 of heparan sulfate proteoglycan 3, and a polyclonal antibody that binds to positions 379-393 of heparan sulfate proteoglycan 3. Monoclonal or polyclonal antibodies to epitopes other than 393. The kit of the invention has high detection sensitivity and short detection time, which is helpful for early diagnosis of liver cancer patients, timely treatment and improvement of survival rate. Moreover, compared with only detecting AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer.

Description

一种用于诊断肝癌的ELISA试剂盒A kind of ELISA kit for diagnosing liver cancer

技术领域 technical field

本发明涉及生物技术和免疫学领域,具体涉及检测肝癌标记物硫酸肝素蛋白多糖3(GPC3)的抗体、试剂盒以及它们的用途。The invention relates to the fields of biotechnology and immunology, in particular to antibodies, kits and applications for detecting liver cancer marker heparan sulfate proteoglycan 3 (GPC3).

背景技术 Background technique

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。Primary liver cancer is the sixth most common cancer in the world and the third most common cancer in China. According to the estimates of the International Cancer Research Center (IARC), in 2000, the global incidence of liver cancer 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 number of cancer deaths among Chinese residents. bit. Liver cancer is mostly in the middle and late stage when symptoms appear, and the recurrence and metastasis rate after resection is high. Therefore, early diagnosis of liver cancer is of great significance to prolong the survival time of patients and reduce the mortality of liver cancer.

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,虽然影像学技术在今年来取得了巨大的进步,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。At present, imaging diagnosis, cell and histological diagnosis and chemical diagnosis are the three main methods of tumor diagnosis. 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 distinction between benign and malignant nodules. The positive results of ultrasound examination or CT, combined with the detection results of serum α-fetoprotein (AFP) level higher than 400ng/ml, can make a diagnosis of liver cancer. However, usually when these conditions are met, the best time for treatment has been missed. Whether it is ultrasonography, CT scan or MRI, the identification of small lesions has certain limitations, especially in liver cirrhosis nodules, which have many similarities with small hepatocellular carcinoma nodules in imaging. Therefore, although imaging technology has made great progress this year, it is still necessary to combine molecular markers of liver cancer clinically to distinguish benign from malignant liver diseases in more complicated cases. In addition, the clinical stage and tumor size of the tumor are the determinants of the treatment effect of liver cancer. Therefore, tumor markers can also be used for general screening of risk groups of liver cancer (such as chronic carriers of HBV and patients with liver cirrhosis).

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。At present, the most commonly used marker for the diagnosis of liver cancer is alpha-fetoprotein (AFP), which can appear abnormal several months before symptoms and imaging changes occur. The AFP content in normal human serum is generally less than 10ng/ml. When the diagnostic value of AFP is set at 20ng/ml, its sensitivity is 50-60%, but in cases with small tumors, the sensitivity is significantly reduced, and it has been reported that it is only 40%. Another problem with using AFP alone as a diagnostic marker for liver cancer is the lack of specificity. In a considerable number of patients with chronic hepatitis, especially those with liver cirrhosis, the AFP content can reach 20-200ng/ml. At present, it is believed that the markers that can be complementary to AFP diagnosis include AFP heterogeneity, γ-glutamyl transferase isoenzyme II and abnormal prothrombin, etc., but due to their insufficient sensitivity and specificity, and detection methods The cumbersome and complicated procedures make it remain at the level of laboratory research and fail to be transformed into clinical routine inspection items. Therefore, exploring new tumor molecular markers and establishing corresponding detection methods that are easy to promote are still one of the important topics in the field of liver cancer research today.

硫酸肝素蛋白多糖3(Glypican-3,GPC3)是近年来发现的一种与肝癌相关的分子。应用差异显示技术,Hsu等首次发现了cDNA100%同源于GPC3的MXR7基因在肝癌组织中有异常表达。在191例肝细胞癌(HCC)组织中,143例(74.8%)能检测到了MXR7的mRNA,而在156例癌旁“非瘤”肝组织中仅有5例(3.2%)为阳性,该5例均为有门脉或远处转移的病例。随后,多家实验室相继证实了这一结果。在HCC组织中,GPC3mRNA和蛋白阳性检出率均在73%以上,mRNA平均水平可较正常肝组织中高出21.7倍,而在正常肝脏、肝脏良性肿瘤、慢性肝炎和肝硬化等中则均呈阴性,表明该蛋白表达具有较高的肿瘤细胞特异性,是一种新型的肝癌标志物。Heparan sulfate proteoglycan 3 (Glypican-3, GPC3) is a molecule related to liver cancer discovered in recent years. Using differential display technology, Hsu et al first discovered that the MXR7 gene whose cDNA was 100% homologous to GPC3 was abnormally expressed in liver cancer tissues. In 191 cases of hepatocellular carcinoma (HCC) tissues, 143 cases (74.8%) could detect MXR7 mRNA, while only 5 cases (3.2%) were positive in 156 cases of adjacent "non-tumor" liver tissues. All 5 cases had portal vein or distant metastasis. Subsequently, many laboratories have confirmed this result. In HCC tissue, the positive detection rate of GPC3 mRNA and protein is above 73%, and the average mRNA level can be 21.7 times higher than that in normal liver tissue, while in normal liver, benign liver tumor, chronic hepatitis and liver cirrhosis, etc. Negative, indicating that the protein expression has high tumor cell specificity, and is a new type of liver cancer marker.

然而,由于正常人外周血中GPC3含量微弱,肿瘤患者血清中GPC3含量尽管明显增加但仍然处于较低水平,建立灵敏度高的检测系统非常不易,导致这样肿瘤标志物迄今未能进入临床使用。However, due to the weak GPC3 content in the peripheral blood of normal people, the GPC3 content in the serum of tumor patients is still at a low level despite a significant increase.

因此,本领域迫切需要开发可灵敏检测GPC3水平的检测系统以及检测方法,为临床肿瘤的诊断提供良好的途径。Therefore, there is an urgent need in this field to develop a detection system and a detection method that can sensitively detect the level of GPC3, so as to provide a good way for the diagnosis of clinical tumors.

发明内容 Contents of the invention

本发明的目的在于提供可灵敏、准确地检测样品中硫酸肝素蛋白多糖3含量的检测试剂。The purpose of the present invention is to provide a detection reagent capable of sensitively and accurately detecting the content of heparan sulfate proteoglycan 3 in a sample.

本发明的目的还在于提供检测样品中硫酸肝素蛋白多糖3含量的检测试剂盒。The object of the present invention is also to provide a detection kit for detecting the content of heparan sulfate proteoglycan 3 in a sample.

在本发明的第一方面,提供一种用于检测硫酸肝素蛋白多糖3的测试片,所述的测试片包括:In a first aspect of the present invention, there is provided a test piece for detecting heparan sulfate proteoglycan 3, said test piece comprising:

固相载体;和a solid support; and

包被于所述固相载体的抗硫酸肝素蛋白多糖3的多克隆抗体,所述的多克隆抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位。The anti-heparan sulfate proteoglycan 3 polyclonal antibody coated on the solid phase carrier, the polyclonal antibody specifically binds to the epitope in the 379-393 position of the heparan sulfate proteoglycan 3, and does not bind to sulfuric acid Epitopes in positions 1-378 or 394-580 of heparin proteoglycan 3.

在另一优选例中,所述固相载体是酶标反应板。In another preferred example, the solid phase carrier is an enzyme-labeled reaction plate.

在本发明的第二方面,提供一种检测试剂盒,所述的试剂盒含有所述的测试片,或者In the second aspect of the present invention, a detection kit is provided, the kit contains the test piece, or

所述的试剂盒含有:(a)固相载体;The kit contains: (a) a solid phase carrier;

(b)容器a以及位于所述容器a中的作为第一抗体的抗硫酸肝素蛋白多糖3的多克隆抗体,所述的多克隆抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位;和(b) container a and the polyclonal antibody against heparan sulfate proteoglycan 3 as the first antibody located in the container a, the polyclonal antibody specifically binds to positions 379-393 of heparan sulfate proteoglycan 3 The epitope that does not bind to the epitope in the 1-378 or 394-580 of heparan sulfate proteoglycan 3; and

(c)用于将所述的第一抗体包被于所述固相载体的试剂。(c) a reagent for coating the first antibody on the solid phase carrier.

在另一优选例中,所述的试剂盒中还装载有:In another preferred example, the kit is also loaded with:

容器b,所述的容器b中装有第二抗体,所述第二抗体是多克隆抗体,所述多克隆抗体特异性结合于硫酸肝素蛋白多糖3中除第379-393位以外的表位。Container b, said container b is equipped with a second antibody, said second antibody is a polyclonal antibody, and said polyclonal antibody specifically binds to epitopes other than 379-393 in heparan sulfate proteoglycan 3 .

在另一优选例中,所述的试剂盒中还装载有:In another preferred example, the kit is also loaded with:

容器b’,所述的容器b’中装有第二抗体,所述第二抗体是单克隆抗体,所述单克隆抗体特异性结合于硫酸肝素蛋白多糖3中除第379-393位以外的表位。Container b', the container b' is equipped with a second antibody, the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the heparan sulfate proteoglycan 3 except for positions 379-393 gauge.

在另一优选例中,所述单克隆抗体结合于硫酸肝素蛋白多糖3的以下表位:In another preferred example, the monoclonal antibody binds to the following epitope of heparan sulfate proteoglycan 3:

硫酸肝素蛋白多糖3第25-358位中的表位;Epitopes in positions 25-358 of heparan sulfate proteoglycan 3;

硫酸肝素蛋白多糖3第350-364位中的表位;或an epitope in position 350-364 of heparan sulfate proteoglycan 3; or

硫酸肝素蛋白多糖3第444-516位中的表位。Epitope in positions 444-516 of heparan sulfate proteoglycan 3.

更优选的,所述单克隆抗体结合于硫酸肝素蛋白多糖3第25-358位中的表位。More preferably, the monoclonal antibody binds to the epitope at positions 25-358 of heparan sulfate proteoglycan 3.

在另一优选例中,所述的第二抗体带有可检测的标记物。In another preferred example, the second antibody has a detectable label.

在另一优选例中,所述的可检测的标记物选自:辣根过氧化物酶(HRP)、碱性磷酸酶。In another preferred example, the detectable marker is selected from the group consisting of horseradish peroxidase (HRP) and alkaline phosphatase.

更优选的,所述的标记物是辣根过氧化物酶。More preferably, the marker is horseradish peroxidase.

在另一优选例中,所述的试剂盒中还装载有:In another preferred example, the kit is also loaded with:

容器c,所述的容器c中装有与标记物相对应的底物;container c, the substrate corresponding to the marker is housed in the container c;

容器d,所述的容器d中装有显色剂;container d, the color developer is housed in the container d;

容器e,所述的容器e中装有洗涤液;和/或a container e containing washing liquid; and/or

容器f,所述的容器f中装有终止液。Container f, said container f is filled with stop solution.

在本发明的第三方面,提供一种体外检测硫酸肝素蛋白多糖3的方法,包括以下步骤:In a third aspect of the present invention, a method for in vitro detection of heparan sulfate proteoglycan 3 is provided, comprising the following steps:

(a)将待测样品加样于包被有第一抗体的固相载体,从而使待测样品中的硫酸肝素蛋白多糖3与固相载体上的第一抗体结合,形成带有“硫酸肝素蛋白多糖3-第一抗体”二元复合物的固相载体;所述的第一抗体是特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位的多克隆抗体;(a) Load the sample to be tested on the solid phase carrier coated with the first antibody, so that the heparan sulfate proteoglycan 3 in the sample to be tested is combined with the first antibody on the solid phase carrier to form a The solid phase carrier of the binary complex of proteoglycan 3-first antibody; the first antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to heparan sulfate protein Polyclonal antibodies to epitopes in positions 1-378 or positions 394-580 of polysaccharide 3;

(b)将第二抗体加样于(a)获得的固相载体,从而形成带有“第二抗体-硫酸肝素蛋白多糖3-第一抗体”三元复合物的固相载体;所述的第二抗体是特异性结合于硫酸肝素蛋白多糖3第379-393位以外的表位的单克隆抗体或多克隆抗体,且所述的第二抗体携带一可检测标记物;和(b) adding the second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparan sulfate proteoglycan 3-first antibody"; The second antibody is a monoclonal antibody or a polyclonal antibody that specifically binds to an epitope other than the 379-393 position of heparan sulfate proteoglycan 3, and the second antibody carries a detectable label; and

(c)检测三元复合物中的可检测标记物,从而确定待检测样品中硫酸肝素蛋白多糖3的存在与否以及存在的量。(c) detecting the detectable marker in the ternary complex, thereby determining the presence or absence and the amount of heparan sulfate proteoglycan 3 in the sample to be detected.

在本发明的第四方面,提供一种多克隆抗体,所述的多克隆抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位,并且所述多克隆抗体通过将硫酸肝素蛋白多糖3第379-393位蛋白片段免疫动物而获得。In the fourth aspect of the present invention, a polyclonal antibody is provided. The polyclonal antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to the epitope of heparan sulfate proteoglycan 3. 1-378 or 394-580, and the polyclonal antibody is obtained by immunizing animals with the 379-393 protein fragment of heparan sulfate proteoglycan 3.

在另一优选例中,所述的动物是:兔。In another preferred example, the animal is: rabbit.

在另一方面,本发明还提供了上述的多克隆抗体的用途,它被用于制备检测硫酸肝素蛋白多糖3的试剂盒。In another aspect, the present invention also provides the use of the above-mentioned polyclonal antibody, which is used to prepare a kit for detecting heparan sulfate proteoglycan 3.

本发明的其它方面由于本文的公开内容,对本领域的技术人员而言是显而易见的。Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

附图说明 Description of drawings

图1显示了GPC3蛋白25-358aa氨基酸片段的编码基因。Figure 1 shows the gene encoding the 25-358aa amino acid fragment of GPC3 protein.

图2显示了GPC3蛋白25-358氨基酸序列。Figure 2 shows the 25-358 amino acid sequence of the GPC3 protein.

图3显示了pProEX Hta-GPC3(73-1074)质粒的小规模表达鉴定。Figure 3 shows the small-scale expression identification of the pProEX Hta-GPC3(73-1074) plasmid.

图4显示了Ni柱纯化后的GPC3(25-358aa)蛋白的SDS-PAGE凝胶电泳鉴定。Fig. 4 shows the SDS-PAGE gel electrophoresis identification of GPC3 (25-358aa) protein purified by Ni column.

图5显示了Western Blot检测纯化后的GPC3(25-358aa)蛋白。其中,M表示分子量marker;W-18表示采用多抗W-18(针对蛋白N端蛋白表位)检测的结果;anti-His表示采用anti-His抗体检测的结果。Figure 5 shows the Western Blot detection of purified GPC3 (25-358aa) protein. Among them, M represents the molecular weight marker; W-18 represents the result of detection using polyclonal antibody W-18 (for protein N-terminal protein epitope); anti-His represents the result of detection using anti-His antibody.

图6显示了采用鼠抗GPC3(25-358aa)单抗gp2#检测胎盘及肿瘤细胞的细胞裂解液的Western Blot结果。其中,1.胎盘组织,2.L02细胞,3.MCF-7细胞,4.Hela细胞。Figure 6 shows the Western Blot results of cell lysates of placenta and tumor cells detected by mouse anti-GPC3 (25-358aa) monoclonal antibody gp2#. Among them, 1. Placental tissue, 2. L02 cells, 3. MCF-7 cells, 4. Hela cells.

图7显示了采用兔抗GPC3-Ag3多抗检测肿瘤细胞的细胞裂解液的WesternBlot结果。其中,1.Huh-7细胞;2.HepG2细胞;3.MCF-7细胞;4.Hela细胞。Figure 7 shows the results of Western Blot of the cell lysate of tumor cells detected by rabbit anti-GPC3-Ag3 polyantibody. Among them, 1. Huh-7 cells; 2. HepG2 cells; 3. MCF-7 cells; 4. Hela cells.

图8显示了GPC3定量标准曲线。Figure 8 shows the GPC3 quantification standard curve.

图9显示了正常人、肝炎肝硬化及肝癌患者血清GPC3含量。Figure 9 shows the serum GPC3 content of normal people, hepatitis cirrhosis and liver cancer patients.

图10显示了GPC3受试者工作特征曲线(ROC曲线)。Figure 10 shows the receiver operating characteristic curve (ROC curve) of GPC3.

具体实施方式 Detailed ways

本发明人经过广泛的研究和试验,意外地发现,对应于硫酸肝素蛋白多糖3(GPC3)氨基酸序列第379-393位抗原表位的多克隆抗体可极好地识别GPC3抗原;当采用该多克隆抗体作为第一抗体捕获GPC3后,再用对应于GPC3氨基酸第379-393位以外其他区段的表位的第二抗体(包括单克隆抗体或多克隆抗体),可以极其有效地结合于GPC3,从而通过双抗夹心法高灵敏地检测GPC3抗原。与之相反,当使用结合于GPC3其他区段(如第444-516位)的多克隆抗体作为第一抗体时,则检测灵敏度较低。After extensive research and experiments, the inventors unexpectedly found that the polyclonal antibody corresponding to the 379-393 epitope of the amino acid sequence of heparan sulfate proteoglycan 3 (GPC3) can recognize the GPC3 antigen excellently; After the cloned antibody is used as the first antibody to capture GPC3, the second antibody (including monoclonal antibody or polyclonal antibody) corresponding to the epitope of the segment other than amino acid 379-393 of GPC3 can be used to bind to GPC3 extremely effectively , so as to detect GPC3 antigen with high sensitivity by double antibody sandwich method. In contrast, when using polyclonal antibodies that bind to other segments of GPC3 (such as positions 444-516) as the primary antibody, the detection sensitivity is low.

此外,本发明人还意外地发现,当使用特异性结合GPC3第379-393位抗原表位的多克隆抗体作为第一抗体时,GPC3肝癌标志物与AFP肝癌标志物的结合检测具有更高的互补性诊断价值。与单单检测AFP标志物相比,在检测AFP标志物的同时采用本发明的试剂盒检测GPC3标记物可显著提高肝癌的诊断敏感性。此基础上完成了本发明。In addition, the present inventors unexpectedly found that when a polyclonal antibody specifically binding to the epitope 379-393 of GPC3 was used as the primary antibody, the binding detection of the GPC3 liver cancer marker and the AFP liver cancer marker had a higher Complementary diagnostic value. Compared with single detection of AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer. The present invention has been accomplished on this basis.

本领域技术人员均了解,一种抗原可能含有多个抗原表位(抗原决定簇),因此,针对同一个抗原可以获得不止一个抗体(包括单克隆抗体或多克隆抗体),这些抗体对抗原的结合特性(如特异性等)均可能是不同的。因此,针对同一个抗原,本领域人员需要进行反复比较、筛选和鉴定,才能找到适合于特异性和敏感性结合的抗体。由于GPC3蛋白是一条较长的多肽,其大部分抗原决定簇被包含在空间结构的内部,因此找到适合于与之结合的抗体难度较高。Those skilled in the art understand that an antigen may contain multiple antigenic epitopes (antigenic determinants), therefore, more than one antibody (including monoclonal antibody or polyclonal antibody) can be obtained against the same antigen. Binding properties (eg, specificity, etc.) may vary. Therefore, for the same antigen, those skilled in the art need to make repeated comparisons, screenings and identifications in order to find antibodies suitable for specific and sensitive binding. Since the GPC3 protein is a long polypeptide, most of its antigenic determinants are contained in the interior of the spatial structure, so it is difficult to find antibodies suitable for binding to it.

针对上述技术难题,本发明人制备了多种对应于GPC3的单克隆抗体和多克隆抗体,通过大量的试验,检测不同种单克隆抗体或多克隆抗体对于GPC3的结合特异性以及敏感性。结果发现,对应于GPC3氨基酸序列第379-393位抗原表位的多克隆抗体可特别良好地识别GPC3抗原。In view of the above technical problems, the inventors prepared a variety of monoclonal antibodies and polyclonal antibodies corresponding to GPC3, and tested the binding specificity and sensitivity of different monoclonal antibodies or polyclonal antibodies to GPC3 through a large number of experiments. As a result, it was found that the polyclonal antibody corresponding to the 379th-393rd epitope of the GPC3 amino acid sequence can recognize the GPC3 antigen particularly well.

并且,本发明人将不同种单克隆抗体和/或不同种多克隆抗体进行两两配伍(基于双抗夹心法原理)来检测这些单克隆抗体和多克隆抗体的组合对于GPC3的结合特异性以及敏感性。最终发现:采用结合于GPC3氨基酸序列第379-393位中抗原表位的多克隆抗体(作为第一抗体),以及结合于GPC3氨基酸第379-393位以外抗原表位的单克隆抗体或多克隆抗体(作为第二抗体)作为基于双抗夹心法的检测试剂,可特别灵敏地检测GPC3抗原。而当采用结合于第379-393位以外的抗原表位的多克隆克隆抗体与其它单克隆抗体或多克隆抗体配伍时,敏感性和特异性均不高;当采用单克隆抗体与单克隆抗体配伍时,敏感性高,但是特异性很差。Moreover, the inventors combined different monoclonal antibodies and/or different polyclonal antibodies pairwise (based on the principle of double-antibody sandwich method) to detect the binding specificity of the combination of these monoclonal antibodies and polyclonal antibodies to GPC3 and sensitivity. Final discovery: use polyclonal antibodies (as primary antibodies) that bind to epitopes in amino acid positions 379-393 of GPC3, and monoclonal antibodies or polyclonal antibodies that bind to epitopes other than amino acid positions 379-393 in GPC3 The antibody (as a secondary antibody) as a detection reagent based on the double-antibody sandwich method can detect the GPC3 antigen particularly sensitively. However, when a polyclonal antibody that binds to an epitope other than 379-393 is used in combination with other monoclonal antibodies or polyclonal antibodies, the sensitivity and specificity are not high; When combined, the sensitivity is high, but the specificity is poor.

如本文所用,所述的“样品”是指分离自人体的物质,包括但不限于:血清、血浆、尿液、或其它组织提取液。优选的,所述的样品是血清或血浆。As used herein, the "sample" refers to a substance isolated from a human body, including but not limited to: serum, plasma, urine, or other tissue extracts. Preferably, the sample is serum or plasma.

如本文所用,所述的“捕获抗体”、“包被抗体”、“第一抗体”与“一抗”可互换使用,都是指所述的结合于GPC3氨基酸序列第379-393位中的抗原表位的多克隆抗体。As used herein, the "capture antibody", "coating antibody", "first antibody" and "primary antibody" are used interchangeably, and they all refer to the GPC3 amino acid sequence binding at position 379-393 polyclonal antibodies to antigenic epitopes.

所述的第一抗体可被包被在固相载体上。本发明对所采用的固相载体没有特别的限制,只要其能够与第一单克隆抗体相偶联(连接)即可。例如,所述的固相载体为酶标反应板。The first antibody can be coated on a solid carrier. The present invention has no particular limitation on the solid phase carrier used, as long as it can be coupled (linked) with the first monoclonal antibody. For example, the solid phase carrier is an enzyme-labeled reaction plate.

如本文所用,所述的“检测抗体”、“第二抗体”与“二抗”可互换使用,都是指特异性结合于GPC3第379-393位以外的抗原表位的抗体,其可以是单克隆抗体或者是多克隆抗体。As used herein, the "detection antibody", "secondary antibody" and "secondary antibody" are used interchangeably, and all refer to antibodies that specifically bind to antigenic epitopes other than positions 379-393 of GPC3, which can are monoclonal or polyclonal antibodies.

硫酸肝素蛋白多糖3Heparan Sulfate Proteoglycan 3

硫酸肝素蛋白多糖3(Glypican-3,GPC3)位于细胞膜表面,由核心蛋白和糖胺聚糖(肝素及硫酸乙酰肝素)侧链构成,是细胞表面多功能的共同受体,通过与细胞外基质、生长因子和蛋白酶等的相互作用传递信号,在细胞的生长、发育、分化和迁移等中有重要功能。GPC3在体内的存在有明显的组织特异性和时相特异性:在人胚胎期,胃肠道和中胚层来源的组织中有大量GPC3表达;但在成人,除胎盘组织外,仅肺、肾、心和卵巢等少数组织中有GPC3的低度表达,肝脏中为阴性。本发明人在研究中发现,肝细胞癌(HCC)患者血清中,GPC3平均浓度显著高于正常对照和肝硬化患者。在分化较好的HCC病例中,GPC3较AFP检测更为敏感,两者联合检测可大大提高肝癌的诊断阳性率。Heparan sulfate proteoglycan 3 (Glypican-3, GPC3) is located on the surface of the cell membrane and is composed of core protein and glycosaminoglycan (heparin and heparan sulfate) side chains. , growth factors and proteases, etc. to transmit signals, and have important functions in cell growth, development, differentiation and migration. The existence of GPC3 in vivo has obvious tissue specificity and phase specificity: in the human embryonic period, there is a large amount of GPC3 expression in tissues derived from the gastrointestinal tract and mesoderm; but in adults, except for placental tissues, only lung, kidney GPC3 was lowly expressed in a few tissues such as breast, heart and ovary, and it was negative in the liver. The inventors found in the study that the average concentration of GPC3 in the serum of patients with hepatocellular carcinoma (HCC) was significantly higher than that of normal controls and patients with liver cirrhosis. In well-differentiated HCC cases, GPC3 is more sensitive than AFP detection, and the combined detection of the two can greatly increase the positive rate of diagnosis of liver cancer.

GPC3蛋白的氨基酸序列和核苷酸序列是本领域已知的,其氨基酸序列如SEQ ID NO:7所示,其核苷酸序列如SEQ ID NO:6所示。或者,本领域人员也可参见GenBank登录号NP_004475和NM_004484.2所提供氨基酸序列和核苷酸序列。The amino acid sequence and nucleotide sequence of the GPC3 protein are known in the art, and its amino acid sequence is shown in SEQ ID NO: 7, and its nucleotide sequence is shown in SEQ ID NO: 6. Alternatively, those skilled in the art can also refer to the amino acid sequence and nucleotide sequence provided by GenBank accession numbers NP_004475 and NM_004484.2.

抗体Antibody

本发明人提供了一种可极好地识别GPC3抗原的多克隆抗体,所述抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的抗原表位。The inventors of the present invention provide a polyclonal antibody that can recognize the GPC3 antigen very well, and the antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3.

所述多克隆抗体通过采用GPC3第379-393位蛋白片段免疫动物而获得。多克隆抗体的制备是本领域技术人员所熟知的。佐剂可用于增强免疫反应,如弗氏佐剂。例如,所述多克隆抗体的制备方法如下:将GPC3第379-393位蛋白片段与弗氏佐剂按照适当比例(如1:1)混合,充分乳化后免疫动物。免疫方法可使用皮下注射。动物免疫1-4个月后,可从动物静脉血中收获抗血清并纯化,获得抗GPC3第379-393位蛋白片段的多克隆抗体。The polyclonal antibody is obtained by immunizing animals with protein fragments at positions 379-393 of GPC3. The preparation of polyclonal antibodies is well known to those skilled in the art. Adjuvants can be used to enhance the immune response, such as Freund's adjuvant. For example, the preparation method of the polyclonal antibody is as follows: mix the 379-393 protein fragment of GPC3 with Freund's adjuvant in an appropriate ratio (such as 1:1), fully emulsify and immunize animals. The method of immunization may use subcutaneous injection. After 1-4 months of animal immunization, the antiserum can be harvested and purified from the venous blood of the animal to obtain the polyclonal antibody against the 379-393 protein fragment of GPC3.

作为本发明的优选方式,所述的动物是:兔。As a preferred mode of the present invention, the animal is: rabbit.

在本发明的实施例中,本发明人制备了多种多克隆抗体(包括兔抗人GPC3-Ag3多克隆抗体(结合于GPC3第379-393位中的抗原表位)、兔抗人GPC3-Ag1(结合于GPC3第73-86位中的抗原表位)、兔抗人GPC3-Ag4多克隆抗体(结合于GPC3第444-516位中的抗原表位)),在后续的ELISA检测中发现,兔抗人GPC3-Ag3多克隆抗体与其它各种非结合于GPC3第379-393位中的表位的单克隆抗体或多克隆抗体配伍,可特别灵敏地检测GPC3抗原含量。In the embodiment of the present invention, the inventors prepared a variety of polyclonal antibodies (including rabbit anti-human GPC3-Ag3 polyclonal antibody (binding to the epitope in GPC3 379-393), rabbit anti-human GPC3-Ag3 Ag1 (binding to the epitope in GPC3 73-86), rabbit anti-human GPC3-Ag4 polyclonal antibody (binding to the epitope in GPC3 444-516)), found in the subsequent ELISA detection , Compatibility of rabbit anti-human GPC3-Ag3 polyclonal antibody with various other monoclonal antibodies or polyclonal antibodies that do not bind to the epitope in positions 379-393 of GPC3 can detect the content of GPC3 antigen particularly sensitively.

制备特异性抗GPC3蛋白的单克隆抗体的技术是本领域中众所周知的。这里,“特异性”是指抗体能结合于GPC3蛋白或其片段;更特别地,指那些能与GPC3蛋白或其片段结合但不识别和结合于其它非相关抗原分子的抗体。Techniques for preparing monoclonal antibodies specific for GPC3 proteins are well known in the art. Here, "specificity" refers to antibodies that can bind to GPC3 protein or its fragments; more specifically, refers to those antibodies that can bind to GPC3 protein or its fragments but do not recognize and bind to other irrelevant antigenic molecules.

所述的单克隆抗体是特异性结合于GPC3第379-393位以外的抗原表位的抗体。应理解,当用于作为第二抗体与所述的结合于GPC3第379-393位中的抗原表位的多克隆抗体(作为第一抗体)联合用于检测GPC3抗原时(基于双抗夹心法原理),所述的单克隆抗体可以是多种多样的,只要其能够特异性结合于GPC3第379-393位以外的抗原表位。The monoclonal antibody is an antibody that specifically binds to antigenic epitopes other than positions 379-393 of GPC3. It should be understood that when used as the second antibody in conjunction with the polyclonal antibody (as the first antibody) that binds to the epitope in GPC3 379-393 for the detection of the GPC3 antigen (based on the double-antibody sandwich method principle), the monoclonal antibody can be of various types, as long as it can specifically bind to antigenic epitopes other than positions 379-393 of GPC3.

优选的,所述的GPC3第379-393位以外的抗原表位选自:Preferably, the antigenic epitopes other than positions 379-393 of GPC3 are selected from:

GPC3第25-358位对应的抗原表位;Antigen epitopes corresponding to positions 25-358 of GPC3;

GPC3第350-364位对应的抗原表位;或The antigenic epitope corresponding to position 350-364 of GPC3; or

GPC3第444-516位对应的抗原表位。Antigen epitopes corresponding to positions 444-516 of GPC3.

本发明的单克隆抗体可以利用杂交瘤技术来制备。利用杂交瘤技术制备单克隆抗体是本领域技术人员所熟知的,例如可参见Kohler等人,Nature256;495,1975;Kohler等人,Eur.J.Immunol.6:511,1976;Kohler等人,Eur.J.Immunol.6:292,1976;Hammerling等人,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981中所描述的。The monoclonal antibodies of the present invention can be produced using hybridoma technology. Production of monoclonal antibodies using hybridoma technology is well known to those skilled in the art, for example, see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6: 511, 1976; Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981 described.

在得知了所述单克隆抗体所对应的GPC3抗原的表位位置,本领域人员可方便地通过已知的方法获得所述的单克隆抗体。例如,当需要制备抗GPC3第25-358位对应的抗原表位的单克隆抗体时,采用GPC3第25-358位蛋白片段免疫小鼠,制备杂交瘤,从而获得所需的抗体。After knowing the epitope position of the GPC3 antigen corresponding to the monoclonal antibody, those skilled in the art can conveniently obtain the monoclonal antibody through known methods. For example, when it is necessary to prepare a monoclonal antibody against the epitope corresponding to the 25th-358th position of GPC3, the mouse is immunized with the protein fragment of the 25th-358th position of GPC3 to prepare a hybridoma, so as to obtain the required antibody.

检测试剂盒Detection kit

本发明人根据双抗夹心法的原理,制备了一种可用于检测样品中GPC3蛋白水平的试剂盒。双抗夹心法常规的做法是将一抗固定于载体,然后一抗与抗原反应,洗涤后再与二抗反应(所述的二抗携带可检测标记物,或可与携带可检测标记物的物质结合),最后进行化学发光或酶联显色反应检测信号。双抗夹心法特别适用于具有两个或两个以上表位的抗原的检测。According to the principle of the double-antibody sandwich method, the inventors prepared a kit that can be used to detect the level of GPC3 protein in a sample. The routine method of the double antibody sandwich method is to immobilize the primary antibody on the carrier, then react the primary antibody with the antigen, and then react with the secondary antibody after washing (the secondary antibody carries a detectable label, or can be combined with an antibody carrying a detectable label). Substance binding), and finally perform chemiluminescence or enzyme-linked color reaction to detect the signal. The double-antibody sandwich method is especially suitable for the detection of antigens with two or more epitopes.

本发明人发现,采用结合于GPC3第379-393位中的抗原表位的多克隆抗体与特异性地结合于GPC3第379-393位以外的抗原表位的抗体来将目标抗原GPC3吸附及定位,其定位和放大效果特别好,从而具有很高的特异性和精密度。并且,相对于单抗体的竞争法来说,双抗体夹心法的测定效果更为优良,因而测定时只需很少的样品量。所以采用双抗体夹心法无论在灵敏度、精确度、准确度、特异性及稳定性上更具有优势。The present inventors found that the target antigen GPC3 can be adsorbed and localized by using a polyclonal antibody that binds to an epitope in positions 379-393 of GPC3 and an antibody that specifically binds to an epitope other than positions 379-393 in GPC3 , its positioning and amplification effects are particularly good, so it has high specificity and precision. Moreover, compared with the single-antibody competition method, the double-antibody sandwich method has a better measurement effect, so only a small amount of sample is required for the measurement. Therefore, the double-antibody sandwich method has more advantages in terms of sensitivity, precision, accuracy, specificity and stability.

本发明的检测试剂盒含有测试片,所述的测试片包括:固相载体;和包被于所述固相载体的抗硫酸肝素蛋白多糖3的多克隆抗体,所述的多克隆抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位。The detection kit of the present invention contains a test piece, and the test piece includes: a solid-phase carrier; and a polyclonal antibody against heparan sulfate proteoglycan 3 coated on the solid-phase carrier, and the polyclonal antibody has a specific It binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to the epitope in positions 1-378 or 394-580 of heparan sulfate proteoglycan 3.

或者,本发明的检测试剂盒含有(a)固相载体;(b)容器a以及位于所述容器a中的作为第一抗体的抗硫酸肝素蛋白多糖3的多克隆抗体,所述的多克隆抗体特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位;和(c)用于将所述的第一抗体包被于所述固相载体的试剂。Alternatively, the detection kit of the present invention contains (a) a solid phase carrier; (b) a container a and a polyclonal antibody against heparan sulfate proteoglycan 3 as the first antibody located in the container a, the polyclonal The antibody specifically binds to the epitope in position 379-393 of heparan sulfate proteoglycan 3, but not to the epitope in position 1-378 or position 394-580 of heparan sulfate proteoglycan 3; and (c) for A reagent for coating the first antibody on the solid phase carrier.

作为本发明的优选方式,所述的试剂盒中还装载有:As a preferred mode of the present invention, the kit is also loaded with:

容器b,所述的容器b中装有第二抗体,所述第二抗体是多克隆抗体,所述多克隆抗体特异性结合于硫酸肝素蛋白多糖3中除第379-393位以外的表位;或者,Container b, said container b is equipped with a second antibody, said second antibody is a polyclonal antibody, and said polyclonal antibody specifically binds to epitopes other than 379-393 in heparan sulfate proteoglycan 3 ;or,

容器b’,所述的容器b’中装有第二抗体,所述第二抗体是单克隆抗体,所述单克隆抗体特异性结合于硫酸肝素蛋白多糖3中除第379-393位以外的表位。Container b', the container b' is equipped with a second antibody, the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the heparan sulfate proteoglycan 3 except for positions 379-393 gauge.

作为本发明的优选方式,所述的第二抗体带有可检测的标记物。As a preferred mode of the present invention, the second antibody has a detectable label.

如本文所用,所述的“可检测的标记物”是指用于确定待检测样品中GPC3的存在与否以及存在的量的标志物。在确定了本发明的试剂盒所采用的包被抗体和/或检测抗体后,可以采用本领域常规用于与检测抗体结合来进行检测的各种标记物。本发明对所采用的标记物没有特别的限制,只要是能够与所述的检测抗体结合,且在适当处理后能够准确地指示待检测样品中GPC3蛋白的存在与否以及存在量的标记物均是可用的。所述的标记物可直接被设置于第二抗体上;或者,所述的标记物也可被设置于特异性抗第二抗体的抗抗体上,本领域人员可根据所采用的抗体的种类和特性,选择适宜的标记物。例如,所述的标记物可以选自:辣根过氧化物酶(HRP)、碱性磷酸酯酶(AP)、葡萄糖氧化酶、β-D-半乳糖苷酶、脲酶、过氧化氢酶、或葡萄糖淀粉酶。As used herein, the "detectable marker" refers to a marker used to determine the presence or absence and the amount of GPC3 in the sample to be detected. After the coating antibody and/or detection antibody used in the kit of the present invention is determined, various markers routinely used in the art for detection in combination with the detection antibody can be used. The present invention has no particular limitation on the marker used, as long as it can bind to the detection antibody and can accurately indicate the presence or absence and the amount of GPC3 protein in the sample to be detected after appropriate treatment. It is available. The label can be directly set on the second antibody; or, the label can also be set on the specific anti-second antibody anti-antibody, those skilled in the art can according to the type of antibody used and characteristics, choose the appropriate markers. For example, the marker can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

当采用如上所示的一些酶标记物时,还需要采用一些与相应的酶结合的底物,从而可通过显色等方式来报导标记物的存在情况或者存在量。如本文所用,所述的“与标记物相对应的底物”是指可被标记物所催化显色,用于显示第二抗体与GPC3发生结合的识别信号。所述的底物例如:用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS;用于碱性磷酸酯酶的对硝基苯磷酸酯(p-nitrophenyl phosphate,p-NPP);等等。本领域人员可根据所采用的标记物的种类和特性,选择适宜的底物。When using some of the above-mentioned enzyme markers, it is also necessary to use some substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be reported by means of color development and the like. As used herein, the "substrate corresponding to the label" refers to a color that can be catalyzed by the label to display the recognition signal of the binding of the second antibody to GPC3. Described substrate is for example: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP); and so on. Those skilled in the art can select an appropriate substrate according to the type and characteristics of the marker used.

作为本发明的优选方式,所述的第二抗体与标记物直接相连接。更优选的,所述的标记物是HRP。与以生物素标记检测抗体、反应后再与链亲和素-HRP反应相比较,直接在检测抗体上标记HRP、反应结束后直接加底物显色更为简单便捷。As a preferred mode of the present invention, the second antibody is directly linked to the label. More preferably, the marker is HRP. Compared with labeling the detection antibody with biotin and then reacting with streptavidin-HRP, it is simpler and more convenient to directly label HRP on the detection antibody and directly add the substrate to develop the color after the reaction is completed.

为了获得定量结果,还可以在检测过程中设置含已知浓度的多个GPC3蛋白的标准品。对于标准品的设置方法可采用常规的方法。In order to obtain quantitative results, standards containing multiple GPC3 proteins at known concentrations can also be set during the detection process. A conventional method can be used for the setting method of the standard.

为了消除假阳性和假阴性,也可在检测过程中设置质控(对照)。In order to eliminate false positives and false negatives, a quality control (control) can also be set during the detection process.

此外,为了使本发明的试剂盒在检测时更方便,所述的试剂盒中优选的还包含其它一些辅助试剂,所述的辅助试剂是ELISA试剂盒中常规使用的一些试剂,这些试剂的特性以及它们的配制方法均是本领域技术人员所熟知的。所述的试剂例如(但不限于):显色剂、洗涤液、终止液、增敏液、稀释液。In addition, in order to make the kit of the present invention more convenient during detection, the kit preferably also contains other auxiliary reagents, and the auxiliary reagents are some reagents routinely used in ELISA kits, and the characteristics of these reagents and their preparation methods are well known to those skilled in the art. The reagents are, for example (but not limited to): chromogenic reagent, washing solution, stop solution, sensitization solution, diluent.

此外,在所述试剂盒中还可包含使用说明书,用于说明其中装载的试剂的使用方法。In addition, the kit may also contain an instruction manual for explaining the method of using the reagents contained therein.

本发明的试剂盒可应用于:Test kit of the present invention can be applied to:

(A)作为独立的检测系统,进行肝癌的诊断;(A) as an independent detection system for the diagnosis of liver cancer;

(B)与影像学检查和/或AFP标志物检测等结合,进行肝癌的诊断;(B) Combined with imaging examination and/or AFP marker detection, etc., to diagnose liver cancer;

(C)进行AFP增高肝病的良恶性鉴别诊断;(C) differential diagnosis of benign and malignant liver diseases with elevated AFP;

(D)进行治疗前GPC3阳性肝癌的预后监测;(D) Prognostic monitoring of GPC3-positive liver cancer before treatment;

(E)进行肝癌高危人群的筛查;和/或(E) Screening of high-risk populations for liver cancer; and/or

(F)进行肝癌相关的基础研究。(F) Carry out basic research related to liver cancer.

检测方法Detection method

本发明提供了一种利用本发明的试剂盒体外检测GPC3蛋白的方法,包括以下步骤:The invention provides a method for using the kit of the invention to detect GPC3 protein in vitro, comprising the following steps:

(a)将待测样品加样于包被有第一抗体的固相载体,从而使待测样品中的硫酸肝素蛋白多糖3与固相载体上的第一抗体结合,形成带有“硫酸肝素蛋白多糖3-第一抗体”二元复合物的固相载体;所述的第一抗体是特异性结合于硫酸肝素蛋白多糖3第379-393位中的表位,而不结合于硫酸肝素蛋白多糖3的1-378位或394-580位中的表位的多克隆抗体;(a) Load the sample to be tested on the solid phase carrier coated with the first antibody, so that the heparan sulfate proteoglycan 3 in the sample to be tested is combined with the first antibody on the solid phase carrier to form a The solid phase carrier of the binary complex of proteoglycan 3-first antibody; the first antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to heparan sulfate protein Polyclonal antibodies to epitopes in positions 1-378 or positions 394-580 of polysaccharide 3;

(b)将第二抗体加样于(a)获得的固相载体,从而形成带有“第二抗体-硫酸肝素蛋白多糖3-第一抗体”三元复合物的固相载体;所述的第二抗体是结合于硫酸肝素蛋白多糖3第379-393位以外的抗原表位的单克隆抗体或多克隆抗体,且所述的第二抗体携带一可检测标记物;和(b) adding the second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparan sulfate proteoglycan 3-first antibody"; The second antibody is a monoclonal antibody or a polyclonal antibody that binds to an epitope other than the 379-393 position of heparan sulfate proteoglycan 3, and the second antibody carries a detectable label; and

(c)检测三元复合物中的可检测标记物,从而确定待检测样品中硫酸肝素蛋白多糖3的存在与否以及存在的量。(c) detecting the detectable marker in the ternary complex, thereby determining the presence or absence and the amount of heparan sulfate proteoglycan 3 in the sample to be detected.

作为本发明的一种优选方式,定量检测GPC3蛋白的方法具体如下:As a preferred mode of the present invention, the method for quantitatively detecting the GPC3 protein is as follows:

(i)抗原抗体反应:将本发明的第一抗体包被在多孔板上,之后在多孔板的微孔内分别加入不同浓度的标准品、质控品(可选),或待测血清样品;(i) Antigen-antibody reaction: Coat the first antibody of the present invention on a multiwell plate, and then add different concentrations of standard, quality control (optional), or serum samples to be tested in the microwells of the multiwell plate ;

(ii)酶联反应:将第二抗体(其上设置有标记物)溶液加入各孔,振荡、孵育;洗涤;(ii) Enzyme-linked reaction: adding the solution of the second antibody (with a marker on it) to each well, shaking and incubating; washing;

(iii)显色反应:每孔加入对应于标记物的底物、显色剂,孵育,每孔加入反应终止液,结束反应;(iii) Color reaction: Add substrate and chromogenic agent corresponding to the marker to each well, incubate, add reaction termination solution to each well, and end the reaction;

(iv)酶标仪测定OD值;(iv) Microplate reader measures OD value;

(v)结果计算:(v) Result calculation:

A)制作标准曲线:以GPC3蛋白标准品浓度为横坐标,标准品测定OD值为纵坐标,作出标准曲线;A) Make a standard curve: take the concentration of the GPC3 protein standard substance as the abscissa, and measure the OD value of the standard substance as the ordinate, and make a standard curve;

B)评判质控品浓度(可选):根据质控品的OD值,从标准曲线上读出相应的浓度值;质控品测定浓度值处于给定范围时,该次测定有效;B) Judging the concentration of the quality control substance (optional): read the corresponding concentration value from the standard curve according to the OD value of the quality control substance; when the measured concentration value of the quality control substance is within a given range, the determination is valid;

C)计算待测血清样品浓度:当标准曲线和质控品均被判定有效时,根据待测样本的OD值从标准曲线计算出待测血清样品的GPC3蛋白浓度。C) Calculating the concentration of the serum sample to be tested: when both the standard curve and the quality control substance are determined to be valid, the GPC3 protein concentration of the serum sample to be tested is calculated from the standard curve according to the OD value of the sample to be tested.

由于本发明的检测试剂或试剂盒敏感性高,大大缩短了样品孵育时间(约2±0.5小时),因此利用所述试剂盒只需很短的时间即可获得GPC3的定量数据,该时间一般在2-4小时。而采用常规的试剂或试剂盒检测时,样品通常需要孵育过夜以增加敏感性。Due to the high sensitivity of the detection reagent or kit of the present invention, the incubation time of the sample is greatly shortened (about 2 ± 0.5 hours), so the quantitative data of GPC3 can be obtained in a very short time using the kit, which is generally in 2-4 hours. When using conventional reagents or kits for detection, samples usually need to be incubated overnight to increase sensitivity.

本发明的主要优点在于:The main advantages of the present invention are:

(1)首次发现采用对应于GPC3氨基酸序列第379-393位抗原表位的多克隆抗体可良好地识别GPC3抗原,将其与对应于GPC3氨基酸第379-393位以外抗原表位的抗体联合应用作为基于双抗夹心法的检测试剂,可特别灵敏地检测GPC3抗原。(1) For the first time, it was found that the polyclonal antibody corresponding to the 379-393 amino acid epitope of GPC3 can well recognize the GPC3 antigen, and it was used in combination with the antibody corresponding to the antigen epitope other than the 379-393 amino acid of GPC3 As a detection reagent based on the double-antibody sandwich method, it can detect GPC3 antigen particularly sensitively.

(2)提供了一种使用方便且灵敏度高的肝癌检测试剂盒,在样品中GPC3含量较低(1ng水平)时即可灵敏地检测,并且完成检测所需的时间很短(约2-4小时)。所述试剂盒有助于尽早地诊断肝癌患者,及时治疗,提高生存率,具有积极的社会效益。在肝癌疗效随访、亚临床转移复发监控以及预后判断等方面也有广泛的应用价值。(2) An easy-to-use and highly sensitive liver cancer detection kit is provided, which can detect sensitively when the GPC3 content in the sample is low (1ng level), and the time required to complete the detection is very short (about 2-4 Hour). The kit is helpful for early diagnosis and timely treatment of liver cancer patients, improving survival rate and having positive social benefits. It also has wide application value in follow-up of liver cancer efficacy, monitoring of subclinical metastasis and recurrence, and prognosis judgment.

(3)本发明人还发现,GPC3肝癌标志物与AFP肝癌标志物的结合检测具有互补性诊断价值。GPC3与AFP联合应用,不仅可提高肝癌早期诊断的阳性率,而且可对临床上为数众多的AFP低度或中度增高患者的良恶性病变进行鉴别诊断。与单单检测AFP标志物相比,在检测AFP标志物的同时采用本发明的试剂盒检测GPC3标记物可显著提高肝癌的诊断敏感性。(3) The inventors also found that the combined detection of GPC3 liver cancer marker and AFP liver cancer marker has complementary diagnostic value. The combined application of GPC3 and AFP can not only increase the positive rate of early diagnosis of liver cancer, but also make differential diagnosis of benign and malignant lesions in a large number of patients with low or moderately increased AFP. Compared with single detection of AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer.

下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室指南(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

实施例1.多肽抗原GPC3(25-358)、GPC3(444-516)的原核制备Example 1. Prokaryotic preparation of polypeptide antigens GPC3 (25-358), GPC3 (444-516)

采用如下方法制备具有GPC3全长氨基酸序列第25-358位序列的多肽抗原GPC3(25-358):The polypeptide antigen GPC3 (25-358) having the 25th-358th sequence of the full-length amino acid sequence of GPC3 is prepared by the following method:

(1)人胎盘总RNA的提取(1) Extraction of human placenta total RNA

取人胎盘组织约1g,至1.5ml的离心管中,加1ml TRAZOL试剂,用玻璃匀浆器打碎组织,抽取RNA。Take about 1g of human placental tissue, put it into a 1.5ml centrifuge tube, add 1ml TRAZOL reagent, smash the tissue with a glass homogenizer, and extract RNA.

(2)反转录(2) reverse transcription

取5μg总RNA,用ThermoScript RT-PCR System(GIBCO BRL公司)试剂盒,参照反转录试剂盒说明以oligo(dT)20为引物,AMV反转录酶反转录合成cDNA。Take 5 μg of total RNA, use the ThermoScript RT-PCR System (GIBCO BRL company) kit, refer to the instructions of the reverse transcription kit, use oligo(dT)20 as a primer, and AMV reverse transcriptase to synthesize cDNA by reverse transcription.

(3)表达载体的构建(3) Construction of expression vector

设计如下引物:Design the following primers:

GPC3-73(SEQ ID NO:3):GPC3-73 (SEQ ID NO: 3):

5’cgcgaattcCAGCCCCCGCCGCCGCCGCC3’和5'cgcgaattcCAGCCCCCGCCGCCGCCGCC3' and

GPC3-1074(SEQ ID NO:4):GPC3-1074 (SEQ ID NO: 4):

5’gcgctcgagTTATCTATATTGGCGTTGTTGAGAATGGGCACATAAC3’5' gcgctcgagTTATCTATATTGGCGTTGTTGAGAATGGGCACATAAC3'

以新合成的人胎盘cDNA为模板,以前述设计的引物为引物,对GPC-3蛋白N端编码序列(去除24aa信号肽部分)nt.73-1074进行扩增。扩增体系为10μL;扩增条件为:94℃预变性3min;94℃变性30s,58℃退火30s,72℃延伸30s,共30个循环;72℃再延伸10min。PCR产物用10g/L的琼脂糖凝胶电泳进行鉴定,并用PCR片段回收试剂盒回收PCR产物。Using the newly synthesized human placenta cDNA as a template and the previously designed primers as primers, the N-terminal coding sequence of GPC-3 protein (with the 24aa signal peptide removed) nt.73-1074 was amplified. The amplification system was 10 μL; the amplification conditions were: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 30 cycles; extension at 72°C for 10 min. The PCR product was identified by 10g/L agarose gel electrophoresis, and the PCR product was recovered with a PCR fragment recovery kit.

PCR产物以EcoRI和Xho I位点克隆至pProEX HTa载体(购自美国Invitrogen公司),获得表达GPC-3蛋白25-358aa的质粒pProEX Hta-GPC3(73-1074),测序鉴定基因序列中无影响氨基酸改变的点突变(图1,SEQ ID NO:1),推导的氨基酸序列见图2(SEQ ID NO:2)。The PCR product was cloned into the pProEX HTa vector (purchased from Invitrogen, USA) with EcoRI and Xho I sites to obtain the plasmid pProEX Hta-GPC3 (73-1074) expressing GPC-3 protein 25-358aa, and the sequencing identified no influence on the gene sequence The point mutation of amino acid change (Fig. 1, SEQ ID NO: 1), the deduced amino acid sequence is shown in Fig. 2 (SEQ ID NO: 2).

(4)pProEX Hta-GPC3(73-1074)的表达与纯化(4) Expression and purification of pProEX Hta-GPC3(73-1074)

将重组质粒pProEX Hta-GPC3(73-1074)转化感受态大肠杆菌菌株BL21-codonplus(DE3)-RP(购自Stratagene),涂氨苄青霉素抗性琼脂平板,37℃倒置生长过夜,在含氨苄青霉素琼脂平板上用牙签挑取3个克隆于3ml含氨苄的LB中,37℃摇床过夜。过夜培养的菌按1:100比例转接种至3ml含氨苄的LB中,37℃振荡培养3h,至对数中期(A550=0.5)。加入异丙基硫代-β-d-半乳糖苷(IPTG)至终浓度为1mmol/L,继续振荡培养3h,诱导GPC3蛋白表达。取各个诱导菌及未诱导的对照菌,量为相当于A550=1时100μl菌液,沉淀后悬于10μl1×SDS上样缓冲液,沸水浴中煮5min。-20℃保存或立即用于SDS-PAGE凝胶电泳。SDS-PAGE电泳鉴定蛋白的表达。结果诱导菌在40KD处有特异性蛋白表达条带(GPC3),见图3。The recombinant plasmid pProEX Hta-GPC3 (73-1074) was transformed into competent Escherichia coli strain BL21-codonplus (DE3)-RP (purchased from Stratagene), coated with ampicillin-resistant agar plate, and grown upside down at 37°C overnight, in the ampicillin-containing On the agar plate, pick 3 clones with a toothpick and put them in 3ml LB containing ampicillin, and shake them overnight at 37°C. The bacteria cultured overnight were inoculated into 3 ml of LB containing ampicillin at a ratio of 1:100, and cultured with shaking at 37°C for 3 hours until mid-logarithmic phase (A 550 =0.5). Isopropylthio-β-d-galactoside (IPTG) was added to a final concentration of 1 mmol/L, and shaking culture was continued for 3 hours to induce GPC3 protein expression. Take each induced bacterium and uninduced control bacterium, the amount is equivalent to 100 μl bacterial liquid when A 550 =1, suspend in 10 μl 1×SDS loading buffer after sedimentation, boil in boiling water bath for 5 minutes. Store at -20°C or use immediately for SDS-PAGE gel electrophoresis. The expression of protein was identified by SDS-PAGE electrophoresis. Results The induced bacteria had a specific protein expression band (GPC3) at 40KD, as shown in Figure 3.

按照小样诱导条件,诱导200ml pProEXHta-GPC3(73-1074)-codonplus表达蛋白,并用Ni柱纯化,然后进行SDS-PAGE凝胶电泳鉴定。结果发现,40KD左右蛋白主带占总蛋白90%以上,见图4。According to the small sample induction conditions, 200ml pProEXHta-GPC3(73-1074)-codonplus expression protein was induced, purified with Ni column, and then identified by SDS-PAGE gel electrophoresis. It was found that the main band of about 40KD protein accounted for more than 90% of the total protein, as shown in Figure 4.

pProEXHta-GPC3(73-1074)表达蛋白定位于包涵体中,按不可溶蛋白的纯化步骤纯化:The protein expressed by pProEXHta-GPC3(73-1074) is located in the inclusion body and purified according to the purification steps of insoluble protein:

(a)层析柱用10倍TNA体积的GuNTA-0Buffer(20mmol/L Tris-HCl pH7.9,0.5mol/L NaCl,8mol/L Urea)洗。(a) The chromatographic column was washed with GuNTA-0Buffer (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea) with 10 times the volume of TNA.

将样品加到TNA层析柱中,流速控制在15ml/h左右,收集穿透部分,用于SDS-PAGE分析蛋白质的结合情况。The sample was added to the TNA chromatography column, the flow rate was controlled at about 15ml/h, and the breakthrough fraction was collected for SDS-PAGE analysis of protein binding.

层析用5倍TNA体积的GuNTA-0Buffer洗,流速控制在30ml/h左右。The chromatography was washed with GuNTA-0Buffer 5 times the volume of TNA, and the flow rate was controlled at about 30ml/h.

(b)分别用5倍TNA体积的GuTNA-209(20mmol/L Tris-HCl pH7.9,0.5mol/L NaCl,8mol/L Urea,20mmol/L),GuTNA-40(20mmol/L Tris-HCl pH7.9,0.5mol/L NaCl,8mol/L Urea,40mmol/L Imidazole),GuTNA-60(20mmol/LTris-HCl pH7.9,0.5mol/L NaCl,8mol/L Urea,60mmol/L Imidazole),GuTNA-100(20mmol/L Tris-HCl pH7.9,0.5mol/L NaCl,8mol/L Urea,100mmol/L Imidazole),GuTNA-500(20mmol/L Tris-HCl pH7.9,0.5mol/LNaCl,8mol/L Urea,500mmol/LImidazole)洗脱,流速控制在15ml/h,收集洗脱液,每管收集一个TNA体积。(b) GuTNA-209 (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 20mmol/L), GuTNA-40 (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 40mmol/L Imidazole), GuTNA-60 (20mmol/LTris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 60mmol/L Imidazole) , GuTNA-100 (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 100mmol/L Imidazole), GuTNA-500 (20mmol/L Tris-HCl pH7.9, 0.5mol/LNaCl , 8mol/L Urea, 500mmol/L midazole) for elution, the flow rate was controlled at 15ml/h, and the eluate was collected, and one volume of TNA was collected in each tube.

以anti-His抗体以及Santa Cruz公司抗-GPC3多抗W-18(针对蛋白N端蛋白表位)对纯化产物进行常规的Western Blot检测,结果证实表达产物为GPC3蛋白,见图5。Routine Western Blot detection was performed on the purified product with anti-His antibody and anti-GPC3 polyantibody W-18 from Santa Cruz Company (for the protein N-terminal protein epitope), and the results confirmed that the expressed product was GPC3 protein, as shown in Figure 5.

采用与前述制备多肽抗原GPC3(25-358)相似的原核表达方法,本发明人还制备了选自GPC3全长氨基酸序列的第444-516位的多肽抗原GPC3(444-516),命名为GPC3-Ag4。Using the prokaryotic expression method similar to the aforementioned preparation of polypeptide antigen GPC3 (25-358), the inventors also prepared a polypeptide antigen GPC3 (444-516) selected from the 444-516th position of the full-length amino acid sequence of GPC3, named GPC3 -Ag4.

实施例2Example 2

多肽抗原GPC3(379-393)、GPC3(73-86)、GPC3(350-364)的合成及偶联Synthesis and coupling of polypeptide antigens GPC3(379-393), GPC3(73-86), GPC3(350-364)

利用软件DNAStar对全长GPC3蛋白抗原性进行分析,主要根据蛋白的抗原性、可及性两个参数选择位于GPC3全长氨基酸序列的第379-393位的多肽抗原GPC3(379-393)位氨基酸ETLSSRRRELIQKLK(SEQ ID NO:5),命名为GPC3-Ag3。该多肽采用人工合成方法制备,GPC3-Ag3合成后采用戊二醛法与KLH(钥孔血蓝蛋白)进行偶联,得到GPC3-Ag3-KLH。Use the software DNAStar to analyze the antigenicity of the full-length GPC3 protein, and select the polypeptide antigen GPC3 (379-393) amino acids located at the 379-393th position of the full-length amino acid sequence of GPC3 mainly according to the two parameters of protein antigenicity and accessibility ETLSSRRRELIQKLK (SEQ ID NO: 5), named GPC3-Ag3. The polypeptide is prepared by artificial synthesis. After GPC3-Ag3 is synthesized, it is coupled with KLH (keyhole limpet hemocyanin) by glutaraldehyde method to obtain GPC3-Ag3-KLH.

此外,本发明人还合成了选自GPC3全长氨基酸序列的第73-86位的多肽抗原GPC3(73-86),命名为GPC3-Ag1;选自GPC3全长氨基酸序列的第350-364位的多肽抗原GPC3(350-364),命名为GPC3-Ag2。并且,将GPC3(73-86)与KLH相偶联,得到GPC3-Ag1-KLH。In addition, the inventors also synthesized the polypeptide antigen GPC3 (73-86) selected from the 73-86th position of the full-length amino acid sequence of GPC3, named GPC3-Ag1; selected from the 350-364th position of the full-length amino acid sequence of GPC3 The polypeptide antigen GPC3 (350-364), named GPC3-Ag2. Also, GPC3(73-86) was coupled to KLH to obtain GPC3-Ag1-KLH.

实施例3多克隆抗体的制备The preparation of embodiment 3 polyclonal antibody

a.兔抗人GPC3-Ag3多克隆抗体的制备a. Preparation of rabbit anti-human GPC3-Ag3 polyclonal antibody

抗原:GPC3-Ag3-KLH。Antigen: GPC3-Ag3-KLH.

动物:新西兰大白兔,一只雄性,一只雌性,4-6周,2.5-3.0kg,均购自中国科学院上海实验动物中心。Animals: New Zealand white rabbits, one male and one female, 4-6 weeks old, 2.5-3.0 kg, were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences.

(1)取一只雄性和雌性健康新西兰大白兔同时免疫。免疫前经兔耳缘静脉取血1-2ml,分离血清,分装,-80℃保存待用。初次免疫时,每只兔注射1mg抗原。将弗氏完全佐剂与1ml抗原(1mg)等体积混合,充分乳化后经背部皮内组织进行多点注射。(1) A male and a female healthy New Zealand white rabbit were immunized at the same time. Before immunization, 1-2ml of blood was collected from the rabbit's ear vein, the serum was separated, aliquoted, and stored at -80°C until use. For the first immunization, each rabbit was injected with 1 mg of antigen. Mix Freund's complete adjuvant with 1ml of antigen (1mg) in equal volume, fully emulsify, and inject at multiple points through the intradermal tissue of the back.

(2)以后每隔2周加强免疫一次,总共两次,抗原量不变,改用弗氏不完全佐剂,注射方法不变。第二次加强后第7天,用ELISA方法测定兔血清中抗体的效价。血清效价达到要求后,乙醚麻醉兔,进行颈动脉插管,取尽新西兰大白兔血液,室温放置30min,再置于4℃冰箱过夜,吸取析出的兔血清,余血于4℃,4000rpm离心10min,吸取上层血清,即得到兔抗人GPC3-Ag3多克隆抗体。(2) Afterwards, a booster immunization was given every 2 weeks, for a total of two times. The amount of antigen remained unchanged, and Freund's incomplete adjuvant was used instead, and the injection method remained unchanged. On the 7th day after the second boost, the antibody titer in the rabbit serum was measured by ELISA method. After the serum titer reached the requirement, the rabbit was anesthetized with ether, and the carotid artery was intubated. The blood of the New Zealand white rabbit was collected and kept at room temperature for 30 minutes, and then placed in a refrigerator at 4°C overnight. The separated rabbit serum was absorbed, and the remaining blood was centrifuged at 4°C and 4000rpm. After 10 minutes, the supernatant serum was drawn, and the rabbit anti-human GPC3-Ag3 polyclonal antibody was obtained.

GPC3-Ag3-KLH免疫5周后的兔血清效价见表1。See Table 1 for the serum titers of rabbits 5 weeks after GPC3-Ag3-KLH immunization.

表1Table 1

Figure S07139562720070514D000161
Figure S07139562720070514D000161

以纯化后的兔血清对细胞裂解液进行Western Blot检测。结果发现,采用肝癌细胞Huh-7(ATCC)及HepG2(ATCC)的细胞裂解液作为抗原,以所述兔血清为抗体,在分子量60KD(全长GPC3)处有特异性条带;而以MCF-7(人乳腺癌细胞,ATCC)及Hela(人宫颈癌细胞,ATCC)的裂解液作为抗原,以所述兔血清为抗体,结果为阴性,见图7。可见所述的兔抗人GPC3-Ag3多克隆抗体具有较高的特异性。并且,进一步的试验还显示,所述的多克隆抗体不结合于位于GPC3的1-378位或394-580位中的表位。The purified rabbit serum was used for Western Blot detection of the cell lysate. As a result, it was found that the cell lysates of liver cancer cells Huh-7 (ATCC) and HepG2 (ATCC) were used as antigens, and the rabbit serum was used as antibodies, and there were specific bands at molecular weight 60KD (full-length GPC3); The lysates of -7 (human breast cancer cells, ATCC) and Hela (human cervical cancer cells, ATCC) were used as antigens, and the rabbit serum was used as antibodies, and the results were negative, as shown in FIG. 7 . It can be seen that the rabbit anti-human GPC3-Ag3 polyclonal antibody has high specificity. Moreover, further tests also showed that the polyclonal antibody does not bind to the epitopes located in positions 1-378 or positions 394-580 of GPC3.

b.兔抗人GPC3-Ag1、兔抗人GPC3-Ag4多克隆抗体的制备b. Preparation of rabbit anti-human GPC3-Ag1 and rabbit anti-human GPC3-Ag4 polyclonal antibodies

采用的抗原为GPC3-Ag1-KLH以及GPC3-Ag4,采用的动物同前述a。The antigens used are GPC3-Ag1-KLH and GPC3-Ag4, and the animals used are the same as the above-mentioned a.

根据前述a同样的方法,制备获得兔抗人GPC3-Ag1多克隆抗体、兔抗人GPC3-Ag4多克隆抗体。GPC3-Ag1-KLH免疫7周后的兔血清效价见表2;GPC3-Ag4免疫5周后的兔血清效价见表3。Prepare rabbit anti-human GPC3-Ag1 polyclonal antibody and rabbit anti-human GPC3-Ag4 polyclonal antibody according to the same method as above a. See Table 2 for the titer of rabbit serum 7 weeks after GPC3-Ag1-KLH immunization; see Table 3 for the titer of rabbit serum 5 weeks after GPC3-Ag4 immunization.

表2Table 2

Figure S07139562720070514D000162
Figure S07139562720070514D000162

表3table 3

Figure S07139562720070514D000163
Figure S07139562720070514D000163

实施例4抗GPC3单克隆抗体的制备及纯化Example 4 Preparation and Purification of Anti-GPC3 Monoclonal Antibody

a.鼠抗GPC3(25-358)单克隆抗体的制备及纯化a. Preparation and purification of mouse anti-GPC3(25-358) monoclonal antibody

取6-8周龄BALB/c小鼠,初次免疫用纯化的GPC3(25-358)蛋白与等体积的福氏完全佐剂混合后,于背部及腹股沟多点皮下免疫,以后每隔2周免疫一次,用同剂量的抗原与福氏不完全佐剂混匀后于背部多点皮下免疫。第3次免疫7天后,鼠尾静脉采血,用间接ELISA法测定特异性抗体的产生。融合前3天,再用同样剂量的不加佐剂的抗原腹腔加强免疫1次。Take 6-8 week-old BALB/c mice, mix purified GPC3(25-358) protein with an equal volume of Freund's complete adjuvant for the first immunization, and subcutaneously immunize multiple points on the back and groin, and then every 2 weeks For one immunization, the same dose of antigen was mixed with Freund's incomplete adjuvant and subcutaneously immunized at multiple points on the back. Seven days after the third immunization, blood was collected from the tail vein of the rats, and the production of specific antibodies was measured by indirect ELISA. Three days before the fusion, the same dose of antigen without adjuvant was used to boost the intraperitoneal immunization once.

加强免疫三天后,断颈处死小鼠,在无菌条件下取出脾脏,用注射器注入约0.2-0.5ml无血清培养液使其胀大,再用弯曲的注射针头多点刺破脾膜,用挤压的方法使淋巴细胞从中逸出,制备免疫脾细胞悬液后计数,用不完全培养液洗涤2次。取对数生长的骨髓瘤细胞SP2/0,1000rpm/min离心5min,弃上清,用不完全培养液混悬细胞后计数,取所需的细胞数,用不完全培养液洗涤2次。将骨髓瘤细胞与脾细胞按1:10的比例混合在一起,在50ml塑料离心管内用不完全培养液洗1次,1200rpm/min离心8min。弃上清,用滴管吸净残留液体,以免影响PEG的浓度。轻轻弹击离心管底,使细胞沉淀略加松动。在室温下融合:①30s内加入预热的1ml45%PEG1000含5%DMSO,边加边搅拌;②作用90s;③加预热的不完全培养液,终止PEG作用,每隔2min分别加入1ml,2ml,3ml,4ml,5ml和10ml。离心,800rpm/min,6min。弃上清,先用6ml左右20%小牛血清RPMI1640轻轻混悬,不能用力吹打,以免使融合在一起的细胞散开。根据所用96孔培养板的数量,补加完全培养液,10ml一块96孔板。将融合后细胞悬液加入含有饲养细胞的96孔板,100μl/孔,37℃、5%CO2温箱培养。一般一块96孔板含有1×107脾细胞。在融合24h后加HAT选择培养液。HAT选择培养液维持培养两周后,改用HT培养液,再维持培养两周,改用一般培养液。当杂交瘤细胞布满孔底1/10面积时,即可开始检测特异性抗体。用包被液将通过基因工程获得的GPC3蛋白稀释为5μg/ml,以每孔100μl包被酶标板,置于4℃过夜后弃上清,用洗涤液将包被好的酶标板洗涤3次,每次5min,然后每孔加入200μl封闭液(3%BSA PBST),37℃放置2h,倾去封闭液,同上洗涤,每孔加入100μl培液上清,37℃孵育1.5h,弃上清,同上洗涤后每孔加入1:1000稀释的羊抗鼠IgG-HRP100μl,37℃孵育1h,将孔内上清弃去,同上洗涤,每孔加入100μl的显色液(OPD-H2O2),室温避光显色10-20min后,每孔加入50μl的终止液终止反应,酶标仪读出A490nm值。阳性克隆按有限稀释法进行克隆化,经三次克隆化后筛选出所需要的杂交瘤细胞系。结果获得11株阳性克隆,分别命名为gp1#-gp11#。Three days after the booster immunization, the mice were killed by neck dislocation, the spleen was taken out under aseptic conditions, and about 0.2-0.5ml of serum-free culture solution was injected with a syringe to make it swell, and then the splenic membrane was punctured at multiple points with a curved injection needle. Squeeze the lymphocytes out of it, prepare the immune spleen cell suspension, count it, and wash it twice with incomplete culture medium. Take logarithmic growth myeloma cells SP2/0, centrifuge at 1000rpm/min for 5min, discard the supernatant, suspend the cells with incomplete culture medium and count them, get the required number of cells, wash twice with incomplete culture medium. Mix myeloma cells and splenocytes at a ratio of 1:10, wash once with incomplete culture medium in a 50ml plastic centrifuge tube, and centrifuge at 1200rpm/min for 8min. Discard the supernatant, and suck up the residual liquid with a dropper, so as not to affect the concentration of PEG. Gently flick the bottom of the centrifuge tube to loosen the cell pellet slightly. Fusion at room temperature: ① Add 1ml of preheated 45% PEG1000 containing 5% DMSO within 30s, and stir while adding; ② React for 90s; ③ Add preheated incomplete culture medium to terminate the PEG effect, add 1ml and 2ml every 2min , 3ml, 4ml, 5ml and 10ml. Centrifuge, 800rpm/min, 6min. Discard the supernatant, and gently suspend it with about 6ml of 20% calf serum RPMI1640, and do not blow hard to avoid dispersing the fused cells. According to the number of 96-well culture plates used, add complete culture medium, 10ml per 96-well plate. The fused cell suspension was added to a 96-well plate containing feeder cells, 100 μl/well, and cultured in a 37°C, 5% CO 2 incubator. Generally, a 96-well plate contains 1×10 7 splenocytes. Add HAT selection medium 24h after fusion. After the HAT selection medium was maintained for two weeks, the HT medium was changed to the HT medium, and after another two weeks of maintenance, the normal medium was used. When the hybridoma cells cover 1/10 of the area of the bottom of the well, the specific antibody can be detected. Dilute the GPC3 protein obtained by genetic engineering to 5 μg/ml with the coating solution, coat the microplate with 100 μl per well, put it at 4°C overnight, discard the supernatant, and wash the coated microplate with the washing solution 3 times, 5 min each time, then add 200 μl blocking solution (3% BSA PBST) to each well, place at 37°C for 2 hours, pour off the blocking solution, wash as above, add 100 μl culture supernatant to each well, incubate at 37°C for 1.5h, discard For the supernatant, wash as above, add 100 μl of goat anti-mouse IgG-HRP diluted 1:1000 to each well, incubate at 37°C for 1 h, discard the supernatant in the well, wash as above, add 100 μl of chromogenic solution (OPD-H 2 O 2 ), after 10-20 min of color development at room temperature in the dark, 50 μl of stop solution was added to each well to terminate the reaction, and the A 490 nm value was read with a microplate reader. Positive clones were cloned by the limiting dilution method, and the required hybridoma cell lines were screened out after three clones. As a result, 11 positive clones were obtained, named gp1#-gp11# respectively.

腹腔注射液体石蜡于F1代小鼠,2周后腹腔注射1×105个杂交瘤细胞,接种细胞7-10天后产生腹水,密切观察动物的健康状况与腹水征象,待腹水尽可能多,而小鼠濒于死亡之前,处死小鼠,用滴管将腹水吸入试管中。Inject liquid paraffin intraperitoneally into the F1 generation mice, and inject 1×10 5 hybridoma cells intraperitoneally after 2 weeks, produce ascites 7-10 days after inoculating the cells, closely observe the health status of the animals and signs of ascites, wait until ascites is as much as possible, Before the mice were dying, the mice were sacrificed, and the ascites was sucked into the test tube with a dropper.

取腹水或免疫后兔血清与等体积的PBS(0.01mol/L,pH7.4,含0.15mol/L的NaCl)稀释后,再加与上面等体积的饱和硫酸铵(90g硫酸铵加到100ml蒸馏水中,80℃溶解,趁热过滤,降至室温后即有结晶析出,用硫酸调至pH7.0),置4℃,4h,然后10000rpm/min离心15min,弃上清,沉淀用1-5ml PBS溶解,移入透析袋,在PBS(0.01mol/L,pH7.4,含0.15mol/L的NaCl)中4℃透析过夜,将透析袋中的蛋白液移入Protein A Sepharose CL-4B柱循环数次,用Tris-HCl(0.05mol/L,pH8.0,含0.15mol/L的NaCl)清洗已经上样的Protein A柱,至在紫外色谱仪记录纸上显示没有蛋白再洗脱下来,然后用解离液(0.01mol/L,甘氨酸-HCl,pH3.0,含0.15mol/L的NaCl)洗脱抗体,洗脱液立即用1mol/L Tris-HCl(pH8.0)调pH至7.2,在PBS(0.01mol/L,pH7.4,含0.15mol/L的NaCl)中4℃透析过夜后进行蛋白定量和特异性验证,获得纯化的抗GPC3(25-358)单克隆抗体。所获得的单克隆抗体效价及分型见表4。Take ascites or rabbit serum after immunization and dilute it with an equal volume of PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl), then add an equal volume of saturated ammonium sulfate (90g ammonium sulfate to 100ml Dissolve in distilled water at 80°C, filter while hot, and crystals will precipitate after cooling down to room temperature, adjust to pH 7.0 with sulfuric acid), place at 4°C for 4h, then centrifuge at 10,000rpm/min for 15min, discard the supernatant, and use 1- Dissolve 5ml of PBS, transfer it into a dialysis bag, dialyze in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl) overnight at 4°C, transfer the protein solution in the dialysis bag to the Protein A Sepharose CL-4B column for circulation Wash the loaded Protein A column with Tris-HCl (0.05mol/L, pH8.0, containing 0.15mol/L NaCl) several times until no protein is eluted on the UV chromatographic recording paper. Then the antibody was eluted with a dissociation solution (0.01mol/L, glycine-HCl, pH3.0, containing 0.15mol/L NaCl), and the eluent was immediately adjusted to pH with 1mol/L Tris-HCl (pH8.0). 7.2. After dialysis overnight at 4°C in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl), perform protein quantification and specificity verification to obtain purified anti-GPC3(25-358) monoclonal antibody. The obtained monoclonal antibody titers and types are shown in Table 4.

表4Table 4

Figure S07139562720070514D000181
Figure S07139562720070514D000181

选择gp2#单克隆抗体用于后续试验,腹水纯化后的其能识别富含GPC3的胎盘裂解物中40KD左右的可溶性GPC3,但在L02(胚肝细胞,ATCC)、MCF-7(人乳腺癌细胞,ATCC)及Hela(人宫颈癌细胞,ATCC)中未见该条带(图6),可见该抗体特异性强。The gp2# monoclonal antibody was selected for subsequent experiments. After purification of ascitic fluid, it can recognize soluble GPC3 of about 40KD in GPC3-rich placental lysate, but it is not effective in L02 (embryonic liver cells, ATCC), MCF-7 (human breast cancer Cells, ATCC) and Hela (human cervical cancer cells, ATCC) did not see this band (Figure 6), showing that the antibody has strong specificity.

b.鼠抗GPC3(350-364)、GPC3(444-516)单克隆抗体的制备b. Preparation of mouse anti-GPC3(350-364), GPC3(444-516) monoclonal antibodies

以多肽抗原GPC3(350-364)为抗原,采用与前述a相同的方法,制备获得10株杂交瘤细胞株,分别命名为2G7,3D4,3F5,3G1,3G6,4B9,5H10,6H10,7C8和9D9。这些杂交瘤制备的单克隆抗体阳性腹水1:10000稀释后直接ELISA结果见表5。Using the polypeptide antigen GPC3 (350-364) as the antigen, 10 hybridoma cell lines were prepared using the same method as in the above a, and were named 2G7, 3D4, 3F5, 3G1, 3G6, 4B9, 5H10, 6H10, 7C8 and 9D9. Table 5 shows the direct ELISA results of the monoclonal antibody-positive ascites prepared by these hybridomas after dilution of 1:10000.

表5table 5

  2G7 3D4 3F5 3G1 3G6 4B9 5H10 6H10 7C8 9D9 1.459 2.456 1.824 2.142 2.56 1.500 1.124 1.443 2.39 1.112 2G7 3D4 3F5 3G1 3G6 4B9 5H10 6H10 7C8 9D9 1.459 2.456 1.824 2.142 2.56 1.500 1.124 1.443 2.39 1.112

以多肽抗原GPC3(444-516)为抗原,采用与前述a相同的方法,制备获得7株杂交瘤细胞株,分别命名为1C6,1F4,2D2,2F2,2G9,3C6和4F3。这些杂交瘤制备的单克隆抗体阳性腹水1:10000稀释后直接ELISA结果见表6。Using the polypeptide antigen GPC3 (444-516) as the antigen, 7 hybridoma cell lines were prepared using the same method as in the above a, which were named 1C6, 1F4, 2D2, 2F2, 2G9, 3C6 and 4F3. Table 6 shows the direct ELISA results of the monoclonal antibody-positive ascitic fluid prepared by these hybridomas after 1:10000 dilution.

表6Table 6

  腹水 1C6 1F4 2D2 2F2 2G9 3C6 4F3 PBS A490nm     0.491 1.211 0.888 1.249 0.633 1.099 0.588 0.103 ascites 1C6 1F4 2D2 2F2 2G9 3C6 4F3 PBS A490nm 0.491 1.211 0.888 1.249 0.633 1.099 0.588 0.103

实施例5抗体的辣根过氧化物酶(HRP)标记The horseradish peroxidase (HRP) labeling of embodiment 5 antibody

称取HRP0.6mg溶于120μl水中;加入NaIO4(12.8mg/ml)120μl,4℃30min;加入乙二醇(9μl in1ml)120μl室温30min;1mg/ml的抗体240μl混匀后装入透析袋对碳酸盐缓冲液(0.05M pH9.6)缓慢搅拌透析过夜,使之结合;第二天加NaBH4(5mg/ml)24μl,4℃2h;装透析袋,4℃对0.05M/L PBS(pH7.2)透析过夜4℃离心,取上清检测效价。Weigh 0.6 mg of HRP and dissolve in 120 μl of water; add 120 μl of NaIO 4 (12.8mg/ml), 4°C for 30 minutes; add 120 μl of ethylene glycol (9 μl in 1ml) for 30 minutes at room temperature; mix 240 μl of 1 mg/ml antibody and put it into a dialysis bag Slowly stir and dialyze carbonate buffer (0.05M pH9.6) overnight to allow it to bind; add NaBH 4 (5mg/ml) 24μl the next day, 4°C for 2h; PBS (pH7.2) was dialyzed overnight at 4°C and centrifuged, and the supernatant was taken to detect the titer.

实施例6ELISA检测Embodiment 6ELISA detects

1.以GPC3-Ag3多克隆抗体为第一抗体,以HRP标记的鼠抗GPC3单克隆抗体(gp2#)为第二抗体的ELISA1. ELISA with GPC3-Ag3 polyclonal antibody as primary antibody and HRP-labeled mouse anti-GPC3 monoclonal antibody (gp2#) as secondary antibody

将纯化的兔抗GPC3多抗GPC3-Ag3多克隆抗体用包被缓冲液((0.01mol/L磷酸酸盐缓冲液pH7.6))稀释至5μg/ml,包被酶标反应板,每孔100μl,4℃孵育过夜后洗涤1次(洗涤缓冲液PBST:100ml PBS(pH7.2)中加入0.05mlTween-20);加入200μl封闭液(封闭液:100mlPBST中加入3g的BSA)封闭酶标反应板,37℃孵育2h后洗涤1次;加入待测样本,每孔100μl(血清:PBS=1:3)(0.02mol/L PBS缓冲液pH7.2),22℃孵育2h后洗涤5次;加入1:150稀释的辣根过氧化物酶标记的鼠抗GPC3单克隆抗体(gp2#-HRP),每孔100μl,22℃孵育30min,后洗涤6次;加入底物液每孔100μl(A液、B液各50μl)(A液:0.2mol/L(19.2g)柠檬酸24.3ml;0.2mol/L Na2HPO(28.4g)425.7ml;H2O20.1%;加ddH2O至1200ml;B液:TMB3.9g;柠檬酸10.52g;EDTA1.86g;甘油300ml;加热溶解后加蒸馏水至10000ml)。37℃避光显色15min,每孔加入终止液终止反应,用Bio-Tek公司的酶标仪ELX800酶标仪读取各孔OD值(波长450nm)。Dilute the purified rabbit anti-GPC3 polyclonal anti-GPC3-Ag3 polyclonal antibody to 5 μg/ml with coating buffer ((0.01mol/L phosphate buffer pH7.6)), and coat the enzyme-labeled reaction plate, each well 100μl, incubate overnight at 4°C and wash once (washing buffer PBST: add 0.05ml Tween-20 to 100ml PBS (pH7.2)); add 200μl blocking solution (blocking solution: add 3g BSA to 100ml PBST) to block the enzyme labeling reaction Wash the plate once after incubating at 37°C for 2 hours; add the sample to be tested, 100 μl per well (serum:PBS=1:3) (0.02mol/L PBS buffer pH7.2), incubate at 22°C for 2 hours and wash 5 times; Add 1:150 dilution of horseradish peroxidase-labeled mouse anti-GPC3 monoclonal antibody (gp2#-HRP), 100 μl per well, incubate at 22°C for 30 min, and then wash 6 times; add substrate solution 100 μl per well (A 50 μl each of solution and solution B) (solution A: 0.2mol/L (19.2g) citric acid 24.3ml; 0.2mol/L Na 2 HPO (28.4g) 4 25.7ml; H 2 O 2 0.1%; add ddH 2 O to 1200ml; liquid B: TMB3.9g; citric acid 10.52g; EDTA1.86g; glycerin 300ml; add distilled water to 10000ml after heating to dissolve). Color was developed at 37°C in the dark for 15 minutes, and stop solution was added to each well to terminate the reaction, and the OD value of each well was read with a microplate reader ELX800 from Bio-Tek Company (wavelength 450 nm).

结果,以上述鼠抗GPC3单克隆抗体(gp2#)及GPC3-Ag3多克隆抗体建立夹心ELISA法,对稀释于25%正常人血清中的GPC3重组蛋白进行检测,结果最低检测浓度为2ng/ml,在5ng-125ng的范围内GPC3含量和O.D.度数呈良好的线性关系,见图8。As a result, the above mouse anti-GPC3 monoclonal antibody (gp2#) and GPC3-Ag3 polyclonal antibody were used to establish a sandwich ELISA method to detect GPC3 recombinant protein diluted in 25% normal human serum, and the minimum detection concentration was 2ng/ml , in the range of 5ng-125ng, the GPC3 content and the O.D. degree show a good linear relationship, as shown in Figure 8.

进一步地,本发明人利用上述ELISA方法,对106例正常人、96份肝炎肝硬化及364例肝癌病人的血清进行了GPC3检测,结果正常人平均值为6.04±9.21ng/ml,肝炎肝硬化为11.7±12.23ng/ml。肝癌患者血清GPC3浓度呈偏态分布,平均均值为87.22ng/ml,其中有大于2000ng/ml的有3人,最高者为5432ng/ml。正常人、肝炎肝硬化于肝癌间GPC3差异具有极显著性(P<0.000),正常人和肝炎肝硬化之间的差异无显著性(图9)。Furthermore, the present inventors used the above-mentioned ELISA method to detect GPC3 in the serum of 106 normal persons, 96 cases of hepatitis cirrhosis and 364 cases of liver cancer patients. It was 11.7±12.23ng/ml. Serum GPC3 concentration of patients with liver cancer showed a skewed distribution, with an average value of 87.22ng/ml, 3 of whom were greater than 2000ng/ml, and the highest was 5432ng/ml. The difference of GPC3 between normal people, hepatitis cirrhosis and liver cancer was extremely significant (P<0.000), but there was no significant difference between normal people and hepatitis cirrhosis (Figure 9).

根据对肝癌和肝炎肝硬化两组病人所作的受试者工作曲线(图10),当阳性值取36.8ng/ml时,诊断的敏感性和特异性分别为32%和2%;当阳性值取27.1ng/ml时,诊断的敏感性和特异性分别为45.1%和8.4%(表7)。According to the receiver operating curve (Fig. 10) done to two groups of patients with liver cancer and hepatitis cirrhosis, when the positive value was 36.8ng/ml, the sensitivity and specificity of diagnosis were 32% and 2% respectively; When taking 27.1ng/ml, the sensitivity and specificity of diagnosis are 45.1% and 8.4% respectively (Table 7).

表7Table 7

  GPC3值(ng/ml) 敏感性 1-特异性 1.5 0.864 0.663 5.2 0.814 0.589 10.3 0.716 0.463 15.5 0.655 0.379 20.3 0.572 0.295 25 0.473 0.137 26.5 0.458 0.116 27.1 0.451 0.084 30.3 0.398 0.063 33.3 0.371 0.042 36.8 0.322 0.021 37.8 0.318 0.011 40.8 0.295 0.011 45.1 0.25 0.011 48.6 0.216 0.011 50.1 0.212 0 104.1 0.114 0 GPC3 value (ng/ml) sensitivity 1- specificity 1.5 0.864 0.663 5.2 0.814 0.589 10.3 0.716 0.463 15.5 0.655 0.379 20.3 0.572 0.295 25 0.473 0.137 26.5 0.458 0.116 27.1 0.451 0.084 30.3 0.398 0.063 33.3 0.371 0.042 36.8 0.322 0.021 37.8 0.318 0.011 40.8 0.295 0.011 45.1 0.25 0.011 48.6 0.216 0.011 50.1 0.212 0 104.1 0.114 0

2.单抗与单抗,单抗与多抗,多抗与多抗之间配伍试验2. Compatibility test between monoclonal antibody and monoclonal antibody, monoclonal antibody and polyclonal antibody, polyclonal antibody and polyclonal antibody

本发明人利用前述制备的各种单克隆抗体和多克隆抗体,建立了以不同单抗或多抗作为第一抗体或第二抗体进行配伍的夹心ELISA检测方法,用于检测抗原GPC3。采用的ELISA检测步骤与1所述方法类似。The present inventors used the various monoclonal antibodies and polyclonal antibodies prepared above to establish a sandwich ELISA detection method using different monoclonal or polyclonal antibodies as the first antibody or the second antibody for the detection of antigen GPC3. The ELISA detection steps adopted are similar to the method described in 1.

结果发现,用于建立夹心ELISA方法时,各种配伍间的检测敏感性相差很远。当以兔抗人GPC3-Ag3多抗以外的其它两种多抗分别作为第一抗体和第二抗体配伍时,敏感性和特异性均差。如以兔抗人GPC3-Ag4包板,以抗人GPC3-Ag1检测,浓度为100ng/ml的标本OD450读数为0.234,而阴性标本OD450读数也有0.204,达不到诊断试剂盒的要求。It was found that when used to establish a sandwich ELISA method, the detection sensitivities of various combinations vary greatly. When two polyclonal antibodies other than the rabbit anti-human GPC3-Ag3 polyclonal antibody were used as the primary antibody and the secondary antibody respectively, the sensitivity and specificity were poor. For example, if the plate is coated with rabbit anti-human GPC3-Ag4 and detected with anti-human GPC3-Ag1, the OD450 reading of the sample with a concentration of 100ng/ml is 0.234, and the OD450 reading of the negative sample is also 0.204, which does not meet the requirements of the diagnostic kit.

当单抗与单抗(如1F4+gp2#)配伍时,敏感性高,最低检测浓度达0.5ng/ml。但是,特异性很差,正常人血清易出现假阳性结果,假阳性率甚至高达18%。When the monoclonal antibody is compatible with the monoclonal antibody (such as 1F4+gp2#), the sensitivity is high, and the minimum detection concentration reaches 0.5ng/ml. However, the specificity is very poor, and normal human serum is prone to false positive results, and the false positive rate is even as high as 18%.

当单抗与多抗配伍时,以兔抗人GPC3-Ag3多抗(第一抗体)+鼠抗GPC3单克隆抗体(gp2#)(第二抗体)配伍检测结果准确,采用多抗兔抗人GPC3-Ag3多抗(第一抗体)+3C6单抗(第二抗体)检测同一样本时的OD读数与前组接近。当采用多抗兔抗人GPC3-Ag4(第一抗体)+单抗7C8(第二抗体)检测时,检测同一样本时OD读数较兔抗人GPC3-Ag3多抗和鼠抗GPC3单克隆抗体(gp2#)配伍低10~15%,准确性差。而采用多抗兔抗人GPC3-Ag1与多种前述制备的单抗或多抗检测时,准确性更差。When the monoclonal antibody is compatible with the polyclonal antibody, the compatibility detection result of the rabbit anti-human GPC3-Ag3 polyclonal antibody (primary antibody) + mouse anti-GPC3 monoclonal antibody (gp2#) (secondary antibody) is accurate, and the polyantibody rabbit anti-human The OD reading of the same sample detected by GPC3-Ag3 polyclonal antibody (primary antibody) + 3C6 monoclonal antibody (secondary antibody) was close to that of the previous group. When polyantibody rabbit anti-human GPC3-Ag4 (primary antibody) + monoclonal antibody 7C8 (secondary antibody) was used for detection, the OD reading of the same sample was higher than that of rabbit anti-human GPC3-Ag3 polyclonal antibody and mouse anti-GPC3 monoclonal antibody ( gp2#) compatibility is low by 10-15%, and the accuracy is poor. The accuracy is even worse when polyantibody rabbit anti-human GPC3-Ag1 is used for detection with various monoclonal antibodies or polyantibodies prepared above.

实施例7GPC3与AFP的互补性诊断Embodiment 7 Complementary Diagnosis of GPC3 and AFP

采用本发明人获得的最佳配伍兔抗人GPC3-Ag3多抗和鼠抗GPC3单克隆抗体(gp2#),建立ELISA双夹心试剂盒;并且采用检测AFP指标的AFP检测放免试剂盒试剂盒(购自上海生物制品所),对151份病理诊断明确的肝细胞肝癌同时进行了AFP与GPC3的检测。The best compatibility rabbit anti-human GPC3-Ag3 polyclonal antibody and mouse anti-GPC3 monoclonal antibody (gp2#) obtained by the inventors were used to establish an ELISA double sandwich kit; and the AFP detection radioimmunoassay kit ( Purchased from Shanghai Institute of Biological Products), AFP and GPC3 were detected simultaneously on 151 cases of hepatocellular carcinoma with clear pathological diagnosis.

结果,当AFP和GPC3的阳性判定值分别定为20ng/ml和30ng/ml时,前者阳性诊断率为49%(74/151),后者为40%(61/151)(表8)。具有意义的是,在77例AFP阴性的肝癌中,有34(44%)例GPC3为阳性,表明GPC3是一和AFP互补的肝癌标志物。联合AFP和GPC3的检测,可将前者的诊断敏感性由49%提高至72%。As a result, when the positive judgment values of AFP and GPC3 were set at 20 ng/ml and 30 ng/ml respectively, the positive diagnostic rate of the former was 49% (74/151), and the latter was 40% (61/151) (Table 8). Significantly, 34 (44%) of the 77 cases of AFP-negative liver cancer were positive for GPC3, indicating that GPC3 is a liver cancer marker complementary to AFP. Combined detection of AFP and GPC3 can increase the diagnostic sensitivity of the former from 49% to 72%.

表8Table 8

Figure S07139562720070514D000221
Figure S07139562720070514D000221

在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

序列表sequence listing

<110>上海市肿瘤研究所<110>Shanghai Cancer Institute

<120>一种用于诊断肝癌的ELISA试剂盒<120>An ELISA kit for diagnosing liver cancer

<130>072225<130>072225

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<170>PatentIn version3.3<170>PatentIn version3.3

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<213>智人(Homo Sapiens)<213> Homo Sapiens

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<212>PRT<212>PRT

<213>智人(Homo Sapiens)<213> Homo Sapiens

<400>2<400>2

Figure S07139562720070514D000232
Figure S07139562720070514D000232

Figure S07139562720070514D000241
Figure S07139562720070514D000241

<210>3<210>3

<211>29<211>29

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<221>misc_feature<221>misc_feature

<223>引物<223> Primer

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<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<220><220>

<221>misc_feature<221>misc_feature

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Figure S07139562720070514D000251
Figure S07139562720070514D000251

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<211>15<211>15

<212>PRT<212>PRT

<213>人工序列<213> Artificial sequence

<220><220>

<221>MISC_FEATURE<221>MISC_FEATURE

<223>GPC3蛋白第379-393位氨基酸序列<223>GPC3 protein amino acid sequence 379-393

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Figure S07139562720070514D000252
Figure S07139562720070514D000252

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<213>智人(Homo Sapiens)<213> Homo Sapiens

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Figure S07139562720070514D000253

Figure S07139562720070514D000261
Figure S07139562720070514D000261

<210>7<210>7

<211>580<211>580

<212>PRT<212>PRT

<213>智人(Homo Sapiens)<213> Homo Sapiens

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Figure S07139562720070514D000262
Figure S07139562720070514D000262

Figure S07139562720070514D000271
Figure S07139562720070514D000271

Claims (9)

1. A test strip for detecting heparin sulfate proteoglycan 3, comprising:
a solid support; and
a polyclonal antibody against heparin sulfate proteoglycan 3 coated on the solid phase carrier, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3, and is obtained by immunizing an animal with the protein fragment in 379-393 of the heparin sulfate proteoglycan 3.
2. The test strip of claim 1, wherein the solid support is an enzyme-labeled reaction plate.
3. A test kit comprising the test strip according to claim 1, or
The kit comprises: (a) a solid support;
(b) a container a and a polyclonal antibody against heparin sulfate proteoglycan 3 as a first antibody in the container a, wherein the polyclonal antibody specifically binds to the epitope in 379-position 393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-position 580 of the heparin sulfate proteoglycan 3, and is obtained by immunizing an animal with the protein fragment in 379-position 393 of the heparin sulfate proteoglycan 3; and
(c) a reagent for coating said first antibody on said solid support.
4. The kit of claim 3, wherein the kit is further loaded with:
a container b, wherein the container b contains a second antibody, and the second antibody is a polyclonal antibody, and the polyclonal antibody specifically binds to the epitope except for the 379-position 393 in the heparin sulfate proteoglycan 3.
5. The kit of claim 3, wherein the kit is further loaded with:
and a container b ', wherein the container b' is filled with a second antibody, and the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the epitope in the heparin sulfate proteoglycan 3 except the 379-393 position.
6. The kit of claim 5, wherein the monoclonal antibodies bind to the following epitopes of heparin sulfate proteoglycan 3:
an epitope in positions 25-358 of heparin sulfate proteoglycan 3;
an epitope in position 350-364 of heparin sulfate proteoglycan 3; or
An epitope in position 444-516 of heparin sulfate proteoglycan 3.
7. The kit of claim 4 or 5, wherein the second antibody is detectably labeled.
8. The kit of claim 7, wherein the detectable label is selected from the group consisting of: horseradish peroxidase, alkaline phosphatase.
9. A polyclonal antibody, characterized in that the polyclonal antibody specifically binds to the epitope in 379-393 position of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 position or 394-580 position of the heparin sulfate proteoglycan 3, and the polyclonal antibody is obtained by immunizing an animal with the protein fragment in 379-393 position of the heparin sulfate proteoglycan 3.
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