抗人羧酸酯酶-Ⅱ单克隆抗体的制备与鉴定

目的制备鼠抗人羧酸酯酶-Ⅱ(humancarboxyles-terases-Ⅱ,hCE-Ⅱ)单克隆抗体(mAb)并对其特性进行鉴定。方法以含hCE-Ⅱ的人肝脏微粒体蛋白混合抗原免疫BALB/c小鼠,采用杂交瘤技术制备鼠抗hCE-Ⅱ的mAb,并用Protein-G亲和层析法纯化mAb。用间接ELISA法测定mAb的效价,以Westernblot鉴定mAb的特异性、免疫组化染色法对mAb相应的抗原进行组织定位。用免疫沉淀和基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtime-of-flightmassspectrometry,MALDI-TOF-MS)得到相应的肽质量指纹谱(peptidemassfingerprint,PMF),再通过Mascot在人类蛋白质数据库中分析比对鉴定mAb对应的抗原。结果获得1株可持续、稳定分泌抗hCE-Ⅱ的mAb的杂交瘤细胞系。杂交瘤细胞分泌的mAbIg的亚类(型)为IgG1(κ),腹水mAb的效价达1×10-7。Westernblot分析显示,在Mr为62000处有1条清晰的带。免疫组化染色显示,该mAb可与肝细胞的浆蛋白结合(hCE-Ⅱ存在于肝细胞浆内微粒体),而不与血管平滑肌细胞内的蛋白结合。该mAb免疫沉淀物的Mr为62000,与hCE-Ⅱ的Mr相符。对免疫沉淀的蛋白质进行质谱鉴定证实,该mAb对应的抗原为hCE-Ⅱ。结论制备出的鼠抗hCE-Ⅱ的mAb效价高、特异性良好,为进一步研究hCE-Ⅱ的功能及其在肝癌等癌症的诊断和治疗中的作用提供了工具。

Preparation and characterization of monoclonal antibody against human carboxylesterases-Ⅱ

AIM: To prepare monoclonal antibody(mAb) against human carboxylesterases-II (hCE-II) and characterize its properties. METHODS: BALB/c mice were immunized with human liver microsome protein which contained hCE-II. The mAb was prepared by hybridoma technique and purified by protein-G affinity chromatography. The titer and specificity of mAb was detected by ELISA and Western blot respectively. Tissue localization of antigen was detected by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) matched with Mascot human protein database. PMF was obtained by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: One clone of hybridoma secreting specific mAb against hCE-II was obtained. The Ig subclass of the mAb was IgG1(kappa). The titer of the mAb was 1 x 10(-7). Western blot analysis showed one clear belt in the Mr of 62,000. Immunohistochemistry demonstrated that the mAb had special combination with the liver cytoplasm protein, but not with the vascular smooth muscle cell protein. Immunoprecipitation showed one clear band in the Mr of 62,000, which was in conformity with the Mr of hCE-II and the antigen was confirmed to be hCE-II after being analyzed with mass spectrometry. CONCLUSION: The mAb against hCE-II with high titer and specificity has been obtained, which lays the foundation for investigation of hCE-II function and diagnosis and therapy of liver cancer.