Li Yue-Hui, Jiang Yan, Xiong Jin, Zhou Xin-Fu, Wang Yan-Jiang, Li Guan-Cheng
Cancer Research Institute, Central South University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education and Health, Changsha, China.
Monoclon Antib Immunodiagn Immunother. 2013 Feb;32(1):55-9. doi: 10.1089/mab.2012.0091.
The aim of this study was to prepare and identify a monoclonal antibody against the extracellular domain of p75 neurotrophin receptor (p75NTR-ECD), which will be used in diagnostics, therapeutics, and as a tool in understanding the role of P75NTR in pathogenesis of neuronal degenerative diseases and cancers. In this study, hybridoma technique was used for production of anti-p75NTR-ECD monoclonal antibody. BALB/c mice were immunized with p75NTR-ECD recombinant protein. Hybridoma clones were screened using indirect enzyme-linked immunosorbent assay (ELISA). Anti-p75NTR-ECD monoclonal antibody was produced by ascites revulsion. Protein A affinity chromatography was used for the purification of anti-p75NTR-ECD monoclonal antibody. Titer of anti-p75NTR-ECD was assessed by ELISA. Specificity of anti-p75NTR-ECD was detected by immunofluorescence and immunohistochemistry. As a result, one stable hybridoma cell clone (3B5F9) producing anti-p75NTR-ECD monoclonal antibody was established. The titer of anti-p75NTR-ECD monoclonal antibody is 1:51200. A 2.91 mg monoclonal antibody against p75NTR-ECD with high specificity was prepared. Immunofluorescence and immunohistochemistry showed that p75NTR-ECD positive staining occurs in the plasma membrane of glioma cell and tissue, which results in an advantage in diagnostic and therapeutic targeting of P75NTR expressing neuronal degenerative diseases and cancers.
本研究的目的是制备并鉴定一种针对p75神经营养因子受体细胞外结构域（p75NTR-ECD）的单克隆抗体，该抗体将用于诊断、治疗，并作为一种工具来理解P75NTR在神经元退行性疾病和癌症发病机制中的作用。在本研究中，采用杂交瘤技术制备抗p75NTR-ECD单克隆抗体。用p75NTR-ECD重组蛋白免疫BALB/c小鼠。使用间接酶联免疫吸附测定（ELISA）筛选杂交瘤克隆。通过腹水抽取制备抗p75NTR-ECD单克隆抗体。采用蛋白A亲和层析法纯化抗p75NTR-ECD单克隆抗体。通过ELISA评估抗p75NTR-ECD的效价。通过免疫荧光和免疫组织化学检测抗p75NTR-ECD的特异性。结果，建立了一个产生抗p75NTR-ECD单克隆抗体的稳定杂交瘤细胞克隆（3B5F9）。抗p75NTR-ECD单克隆抗体的效价为1:51200。制备了2.91 mg具有高特异性的抗p75NTR-ECD单克隆抗体。免疫荧光和免疫组织化学显示，p75NTR-ECD在胶质瘤细胞和组织的质膜中呈阳性染色，这为诊断和治疗靶向表达P75NTR的神经元退行性疾病和癌症提供了优势。
