Searching for just a few words should be enough to get started. If you need to make more complex queries, use the tips below to guide you.
Article type: Research Article
Authors: Sui, Jianhuaa | He, Yixina | Jiang, Xueyinga | Dübel, Stefanb | Han, Zhongchaoa | Song, Zengxuana; *
Affiliations: [a] State key laboratory of Experimental Hematology, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, 300020, China | [b] Department of Biotechnology, Technical University of Braunschweig, 38106 Braunschweig, Germany
Correspondence: [*] Corresponding author: Prof. Zhengxuan Song, State key laboratory of Experimental Hematology, Chinese Academy of Medical Sciences, Peking Union Medical College, Tianjin, 300020, China
Abstract: The aim of this study was to establish a method to obtain antibodies against cell-surface molecules of hematopoietic stem and progenitor cells in situ. A phage-displayed scFv antibody library with a size of 2 × 106 clones was constructed from spleens of mice immunized with living KG1a human leukemia cells. Living cell panning was used to screen anti-KG1a cell antibody fragments. After four rounds of panning, 27 out of 126 scFv fragments showed detectable binding to KG1a cells without cross-reaction to non-hematopoietic cell lines. Individual clones were analyzed by cell-based ELISA and flow cytometry for their binding specificity. Their sequence analysis showed highest homology to mouse gamma heavy chain subgroup II and mouse Kappa subgroup III genes, with four amino acids difference in VH and identical VL. Further, a fusion protein of scFv 5C1 to core-streptavidin was cloned and produced. It was used to search for cell-surface antigen on immunoblots and to test its effect on KG1a cell growth in cell culture. The scFv5C1::core-streptavidin fusion protein recognized a molecule with MW 85/125 KDa in immunoblots of KG1a cell membrane proteins and inhibited KG1a cell homoaggregation in cell cultures. The results validate an efficient approach of making antibodies against living cells and searching for functional inhibitors of cell-surface molecules.
Keywords: phage display, single chain antibody, cancer, leukemia, cell panning
DOI: 10.3233/HAB-2004-13403
Journal: Human Antibodies, vol. 13, no. 4, pp. 111-118, 2004
IOS Press, Inc.
6751 Tepper Drive
Clifton, VA 20124
USA
Tel: +1 703 830 6300
Fax: +1 703 830 2300
sales@iospress.com
For editorial issues, like the status of your submitted paper or proposals, write to editorial@iospress.nl
IOS Press
Nieuwe Hemweg 6B
1013 BG Amsterdam
The Netherlands
Tel: +31 20 688 3355
Fax: +31 20 687 0091
info@iospress.nl
For editorial issues, permissions, book requests, submissions and proceedings, contact the Amsterdam office info@iospress.nl
Inspirees International (China Office)
Ciyunsi Beili 207(CapitaLand), Bld 1, 7-901
100025, Beijing
China
Free service line: 400 661 8717
Fax: +86 10 8446 7947
china@iospress.cn
For editorial issues, like the status of your submitted paper or proposals, write to editorial@iospress.nl
如果您在出版方面需要帮助或有任何建, 件至: editorial@iospress.nl