title: Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.
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 (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.
authors.affiliation: Department of Environmental and Infectious Disease Sciences, Armed Forces Institute of Pathology and American Registry of Pathology, Washington, DC, United States of America.
authors.forename: Hyung-Yong
authors.initials: HY
authors.lastname: Kim
authors.forename: Shien
authors.initials: S
authors.lastname: Tsai
authors.forename: Shyh-Ching
authors.initials: SC
authors.lastname: Lo
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Wear
authors.forename: Mina J
authors.initials: MJ
authors.lastname: Izadjoo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D000942:Antigens, Bacterial / Q000276:immunology; D042726:Burkholderia mallei / Q000276:immunology*; D016957:Burkholderia pseudomallei / Q000276:immunology*; D016466:CHO Cells; D002846:Chromatography, Affinity; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D008322:Mammals / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D013762:Tetrahydrofolate Dehydrogenase / Q000276:immunology*; D014162:Transfection
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase