Chen Zhi-cheng, Yang Jin-ju, Liu Rong, Wang Wan, Liu Xiao-lan, Liu Ying, Gao Jian-en, Sun Qi-hong
Department of Immunology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Sep;23(9):844-6.
To generate monoclonal antibodies (mAb) against glyoxylate reductase/hydroxypyruvate reductase (GRHPR).
Normal human liver tissues were homogenized, and human liver cytosolic proteins were isolated by centrifugation. The total human liver cytosolic proteins were used to immunize BALB/c mice to prepare mAb by hybridoma technique. The mAbs were detected by ELISA, Western blot, and immunohistochemistry. The antibody specificities were identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening.
One hybridoma cell line, ADB291, secreting specific mAb against GRHPR was established. The Ig subclass of the mAb was IgG1(kappa). Data from immunohistochemistry showed that ADB291 can recognize hepatocyte cytoplasm. ADB291 mAb was used to isolate its protein antigen by IP. Proteins captured by the mAb were loaded to SDS-PAGE and subjected to Western blot and MALDI-TOF MS analysis. lambda expression Uni-ZAP XR pre-made liver cDNA library was screened with ADB291 hybridoma supernatants. All of our data demonstrated that ADB291 mAb specially reacted with GRHPR.
A hybridoma cell line stably secreting specific mAb against GRHPR is established. The specific mAb against GRHPR would be very useful for the studies of GRHPR functions and distribution.
制备针对乙醛酸还原酶/羟基丙酮酸还原酶（GRHPR）的单克隆抗体（mAb）。
将正常人肝脏组织匀浆，通过离心分离人肝脏胞质蛋白。用人肝脏胞质总蛋白免疫BALB/c小鼠，采用杂交瘤技术制备单克隆抗体。通过酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western blot）和免疫组织化学法检测单克隆抗体。在免疫沉淀（IP）后通过质谱（MS）鉴定抗体特异性，并通过Uni-ZAP表达文库筛选进行确认。
建立了一株分泌针对GRHPR的特异性单克隆抗体的杂交瘤细胞系ADB291。该单克隆抗体的免疫球蛋白亚类为IgG1（κ链）。免疫组织化学数据显示ADB291可识别肝细胞胞质。利用ADB291单克隆抗体通过免疫沉淀法分离其蛋白抗原。将该单克隆抗体捕获的蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），并进行蛋白质免疫印迹法和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析。用ADB291杂交瘤细胞培养上清液筛选λ表达型Uni-ZAP XR预构建肝脏cDNA文库。我们所有的数据均表明ADB291单克隆抗体与GRHPR发生特异性反应。
建立了一株稳定分泌针对GRHPR的特异性单克隆抗体的杂交瘤细胞系。该针对GRHPR的特异性单克隆抗体对于GRHPR功能和分布的研究将非常有用。
