Liu Jungui, Zhou Ningxin, Zhang Xiaodong
Institute of Hepatobiliary and Gastrointestinal Surgery, General Hospital of the Second Artillery, Beijing, China.
Hybridoma (Larchmt). 2011 Oct;30(5):487-90. doi: 10.1089/hyb.2011.0039.
Previous research has shown that FXYD6 (FXYD domain-containing ion transport regulator 6) is highly increased in bile duct tumor. However, the biological function of FXYD6 is unclear. We aim to prepare and identify a monoclonal antibody against FXYD6, which will be used in diagnostics and as a tool in understanding the role of FXYD6 in pathogenesis of hepatobiliary cancer. In this study, hybridoma cell fusion technology is used for production of FXYD6 monoclonal antibody. BALB/c mice are immunized with FXYD6 synthetic peptide fragment. Hybridoma clones are screened using indirect enzyme-linked immunosorbent assay (ELISA). FXYD6 monoclonal antibody is produced by ascites revulsion. Protein A affinity chromatography is used for the purification of FXYD6 monoclonal antibody. Titer and specificity of monoclonal antibody are assessed by ELISA. Expression of FXYD6 in pancreatic cancer is detected by immunohistochemistry. As a result, one stable hybridoma cell clone producing FXYD6 monoclonal antibody has been established. 2.86 mg monoclonal antibody against FXYD6 with high specificity is prepared with titer of 1:5400. Immunohistochemistry shows that FXYD6 positive staining occurs in the cell membrane of pancreatic cancer, which results in an advantage in investigating the tissue distribution and biological function of FXYD6.
先前的研究表明，FXYD6（含FXYD结构域的离子转运调节蛋白6）在胆管肿瘤中高度上调。然而，FXYD6的生物学功能尚不清楚。我们旨在制备并鉴定一种抗FXYD6单克隆抗体，用于诊断以及作为了解FXYD6在肝胆癌发病机制中作用的工具。在本研究中，采用杂交瘤细胞融合技术制备FXYD6单克隆抗体。用FXYD6合成肽片段免疫BALB/c小鼠。使用间接酶联免疫吸附测定（ELISA）筛选杂交瘤克隆。通过腹水提取制备FXYD6单克隆抗体。采用蛋白A亲和层析法纯化FXYD6单克隆抗体。通过ELISA评估单克隆抗体的效价和特异性。采用免疫组织化学法检测胰腺癌中FXYD6的表达。结果，建立了一个稳定的产生FXYD6单克隆抗体的杂交瘤细胞克隆。制备出了2.86 mg具有高特异性的抗FXYD6单克隆抗体，效价为1:5400。免疫组织化学显示，FXYD6在胰腺癌细胞膜呈阳性染色，这有利于研究FXYD6的组织分布和生物学功能。
