production and characterization of chimeric monoclonal antibodies against burkholderia pseudomallei and b. mallei using the dhfr expression system生产和表征嵌合单克隆抗体对洋葱举办和b mallei使用dhfr表达系统.pdf

Production and Characterization of Chimeric Monoclonal Antibodies against Burkholderia pseudomallei and B. mallei Using the DHFR Expression System 1 1 2 1 1 Hyung-Yong Kim , Shien Tsai , Shyh-Ching Lo , Douglas J. Wear , Mina J. Izadjoo * 1 Department of Environmental and Infectious Disease Sciences, Armed Forces Institute of Pathology and American Registry of Pathology, Washington, D. C., United States of America, 2 Division of Cellular and Gene Therapies and Division of Human Tissues, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration (FDA), Bethesda, Maryland, United States of America Abstract Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65uC/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in