Sayama Yusuke, Kaneko Mika K, Takei Junko, Hosono Hideki, Sano Masato, Asano Teizo, Kato Yukinari
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
Biochem Biophys Rep. 2021 Jan 12;25:100902. doi: 10.1016/j.bbrep.2020.100902. eCollection 2021 Mar.
TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.
TROP2是一种I型跨膜糖蛋白，最初在人滋养层细胞中被鉴定出来，在多种癌症中过表达。为了更好地理解TROP2在癌症中的作用，我们在此旨在开发一种灵敏且特异的抗TROP2单克隆抗体（mAb），用于流式细胞术、蛋白质免疫印迹和免疫组织化学，采用基于细胞的免疫和筛选（CBIS）方法。用N端带有PA标签和C端带有RAP/MAP标签的过表达TROP2的中国仓鼠卵巢（CHO）-K1细胞（CHO/PA-TROP2-RAP-MAP）免疫两只小鼠，并使用流式细胞术选择对过表达PA标签的TROP2的CHO-K1细胞（CHO/TROP2-PA）显示强信号且对CHO-K1细胞显示弱信号至无信号的杂交瘤。我们使用流式细胞术证明，所建立的抗TROP2单克隆抗体TrMab-29（小鼠IgG κ）在MCF7乳腺癌细胞系以及CHO/TROP2-PA细胞中检测到了TROP2。蛋白质免疫印迹分析显示，从CHO/TROP2-PA和MCF7细胞制备的裂解物中出现一条40 kDa的条带。此外，使用TrMab-29的免疫组织化学分析强烈检测到了TROP2，表明TrMab-29可能是检测癌症中TROP2的一种有价值的工具。
