Joichi Usui1,2, Mitsujiro Osawa1, Satoshi Yamazaki1, Yohei Morita1, Akio Koyama2, Hiromitsu Nakauchi3. 1. Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. 2. Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, 305-3525, Japan. 3. Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan. nakauchi@ims.u-tokyo.ac.jp.
Abstract
BACKGROUND: For identifying an antigen recognizable by a monoclonal antibody (mAb), the cDNA cloning method using expression cDNA library has become increasingly popular. For analysis of the mAb clone 2E3, which recognizes the cell membrane surface of immature podocytes, we developed an expression cloning strategy that identified the targeted antigen using retrovirus-mediated gene transfer and enrichment by cell sorting. METHODS: In this experiment, the NIH3T3 cell line was infected by a retrovirally amplified cDNA library derived from the same fetal murine kidneys that provided the immunized antigen. Infected NIH3T3 staining for mAb clone 2E3 was concentrated by cell sorting, followed by identification of the integrated gene. RESULTS: The infected cells reacted with mAb were highly enriched by two rounds of cell sorting. As a result of sequencing the inserted gene from the enriched cells, we isolated the low-affinity nerve growth factor receptor (NGFR) gene. Furthermore, the NIH3T3 cell line that was enforced by recovery of NGFR cDNA reacted with mAb clone 2E3. CONCLUSIONS: Monoclonal antibodies recognizing cell membrane surface molecules are essential tools for immunohistochemistry and flow cytometry analysis, and the application of a combination of retrovirus-mediated gene transfer and cell sorting is beneficial for identifying the targeted antigen.
BACKGROUND: For identifying an antigen recognizable by a monoclonal antibody (mAb), the cDNA cloning method using expression cDNA library has become increasingly popular. For analysis of the mAb clone 2E3, which recognizes the cell membrane surface of immature podocytes, we developed an expression cloning strategy that identified the targeted antigen using retrovirus-mediated gene transfer and enrichment by cell sorting. METHODS: In this experiment, the NIH3T3 cell line was infected by a retrovirally amplified cDNA library derived from the same fetal murine kidneys that provided the immunized antigen. Infected NIH3T3 staining for mAb clone 2E3 was concentrated by cell sorting, followed by identification of the integrated gene. RESULTS: The infected cells reacted with mAb were highly enriched by two rounds of cell sorting. As a result of sequencing the inserted gene from the enriched cells, we isolated the low-affinity nerve growth factor receptor (NGFR) gene. Furthermore, the NIH3T3 cell line that was enforced by recovery of NGFR cDNA reacted with mAb clone 2E3. CONCLUSIONS: Monoclonal antibodies recognizing cell membrane surface molecules are essential tools for immunohistochemistry and flow cytometry analysis, and the application of a combination of retrovirus-mediated gene transfer and cell sorting is beneficial for identifying the targeted antigen.
Authors: A Shibuya; N Sakamoto; Y Shimizu; K Shibuya; M Osawa; T Hiroyama; H J Eyre; G R Sutherland; Y Endo; T Fujita; T Miyabayashi; S Sakano; T Tsuji; E Nakayama; J H Phillips; L L Lanier; H Nakauchi Journal: Nat Immunol Date: 2000-11 Impact factor: 25.606
Authors: K Yamasaki; T Taga; Y Hirata; H Yawata; Y Kawanishi; B Seed; T Taniguchi; T Hirano; T Kishimoto Journal: Science Date: 1988-08-12 Impact factor: 47.728