Preparation of NSE monoclonal antibody and establishment of a double-antibody sandwich ELISA assay

神经元特异性烯醇化酶(Neuron-specificenolase，NSE)是烯醇化酶的一种同工酶。目前已发现5种烯醇化酶的同工酶αα、ββ、γγ、αβ、αγ、γγ、αγ组成的同工酶为神经元和神经内分泌细胞所特有，故命名为神经元特异性烯醇化酶。当缺血、缺氧、中毒或损伤时，细胞膜的完整性受到破坏，NSE便从细胞内释放出来。从而进入血液循环。目前NSE已成为小细胞肺癌及神经母细胞瘤诊断中最敏感的肿瘤标志物，它对神经系统及神经内分泌系统肿瘤的诊断、治疗及预防都有很高的临床价值。 我们通过基因工程技术克隆人的神经元特异性烯醇化酶，通过工程菌株的表达来获得足够量的NSE融合蛋白，对其纯化后，免疫Bal...Neuron-specific enolase (NSE) family is consisted of αα, ββ, γγ, αβ and αγ subunits. The γγ and αγ isoenzymes are found in neurons and neuroendocrine cells, they were accordingly designated neuron-specific enolases. NSE would be released from cells when neuronal damage occurred at the cell membrane after ischemia, hypoxia, poisoning or injury. It became the most sensitive tumor marker in diagnosis...学位：理学硕士院系专业：生命科学学院生物化学与生物技术系_生物化学与分子生物学学号：2172009115205

Characterization of NSE monoclonal antibodies and establishment of a double-antibody sandwich ELISA assay

通讯作者，E-mail: xtli@ xmu.edu.cn 作者简介: 丁焕弟( 1985 年－ ) ，女，在读硕士，主要从事抗肿瘤单克 隆抗体及诊断试剂盒的研究，E-mail: dinghuandi1125 @163.com。[中文文摘]目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。[英文文摘]Objective: Preparation and characterization of monoclonal antibodies against NSE protein，and establishment of a double-antibody sandwich ELISA assay． Methods: BALB/c mice were immunized by using purified recombinant NSE，and monoclonal antibodies were generated by hydridoma technique． These antibodies were characterized with ELISA，Western blot，Immunofluorescent and Immunohistochemical staining． The isotypes of these antibodies were determined with an antibody isotyping kit． With Horseradish Peroxidase labelled NSE monoclonal antibody，we were able to establish a double-antibody sandwich ELISA to detect NSE protein． Results: Two positive hybridoma cell lines were selected for test，the titers of these two monoclonal antibodies could reach 4． 2 × 107 －6. 5 × 107，and their isotypes were IgG2b． Our NSE antibodies could detect not only endogernous NSE protein from cells，but also secreted NSE protein from cells in culture medium by Western blot，in addition，they could be used for immunofluorescent and immunohistochemical staining． The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8． 85 ng /ml． Conclusion: Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers，and we established a doubleantibody sandwich ELISA assay which could be used for clinical test in future．福建省科技重点项目(编号项目No.2011Y0050); 厦门市科技计划项目(No.3502Z20123009

Preparation,identification and application of monoclonal antibody against urease subuint B of Helicobacter pylori

目的制备抗幽门螺杆菌(HP)的单克隆抗体(MAb),并对该HP MAb进行分析鉴定,建立一种检测患者由于HP感染而在血清中产生HP抗体的竞争ElISA检测方法。方法用灭活的HP免疫bAlb/C小鼠,通过杂交瘤技术制备HP MAb。我们采用HP混合蛋白包括毒素相关蛋白A(CAgA)、空泡毒素A(VACA)和尿素酶以及灭活的HP菌体筛选阳性杂交瘤细胞株,用ElISA和WESTErn blOT等技术对所获得的HP MAb进行鉴定。利用辣根过氧化物酶(HrP)标记所筛选的HP MAb来建立一个可检测患者血清中HP抗体的竞争ElISA。结果通过大规模的杂交瘤筛选,我们选择了1株将其命名为C3 HP MAb,其抗体亚型为Igg2A,腹水效价可达1x107。WESTErn blOT法、ElISA和质谱检测结果显示,该C3 HP MAb能特异地识别HP的尿素酶b亚单位。用这个C3 HP MAb,我们建立了一种可检测患者血清中HP抗体的竞争ElISA。结论成功获得一种可以特异识别HP尿素酶b亚单位的MAb,建立了一种可检测患者血清中HP抗体的竞争ElISA。Objective To prepare and characterize the monoclonal antibody(mAb) against Helicobacter pylori(Hp)and establish a competitive ELISA used for detection of Hp antibodies in the sera of Hp-infected patients.Methods BALB /c mice were immunized with inactivated Hp to generate Hp mAb using the hybridoma technology.Hp mixed proteins including cytotoxin-associated gene A(CagA),vacuolating cytotoxin A(VacA) and urease,as well as inactivated Hp were applied to screen positive hybridoma.Selected Hp mAb was analyzed and characterized with ELISA and Western blotting,and then labeled with horseradish peroxidase( HRP) for establishing a competitive ELISA to detect Hp antibodies in the sera of Hp-infected patients.Results One Hp mAb named as C3 was selected after screening large amount of hybridoma,and the C3 Hp mAb was special for IgG2 a subtype with the affinity titer of 1 × 107.Western blotting,ELISA and mass spectrum analysis indicated that the C3 Hp mAb could recognize Hp urease subunit B specifically.Using the C3 Hp mAb,we developed a competitive ELISA which could be used to detect Hp antibodies in the sera of Hp-infected patients.Conclusion We successfully obtained one mAb that could specifically recognize Hp urease subunit B and developed a competitive ELISA using the Hp mAb.福建省科技重点项目(2011Y0050); 厦门市科技计划项目(3502Z20123009); 国家基础科学人才培养基金(J1310027