title: Derivation and characterization of monoclonal antibodies against human folypolyglutamate synthetase.
abstract: Folypolyglutamate synthetase (FPGS) plays a critical role in the cellular retention of both folates and antifolates. Resistance to antifolates is in part related to changes in FPGS enzyme activity and levels of messenger RNA, or in some instances, protein as evaluated by Western blots using polyclonal antisera. The present study was designed to derive a series of monoclonal antibodies (MAb) against the native protein, to characterize them in terms of specificity and epitope mapping, and to determine kinetic constants by Biacore. We report on 3 IgG(1) kappa MAbs-namely, 4-2, 4-3, and 4-18-with epitopes localized to the carboxyl domain of the protein. These antibodies recognize a single band on Western blots of HeLa cell lysates, which is significantly reduced following RNAi knockdown. The recognition of both the native and denatured conformations of FPGS by these MAbs should provide useful reagents for FPGS quantitation in either tumor cell lysates or in tumor biopsies.
authors.affiliation: Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46258, USA.
authors.forename: Joe
authors.initials: J
authors.lastname: Dotzlaf
authors.forename: John
authors.initials: J
authors.lastname: Carpenter
authors.forename: Shuang
authors.initials: S
authors.lastname: Luo
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Miles
authors.forename: Deborah
authors.initials: D
authors.lastname: Fisher
authors.forename: Yue-Wei
authors.initials: YW
authors.lastname: Qian
authors.forename: Mariam
authors.initials: M
authors.lastname: Ehsani
authors.forename: Xiliang
authors.initials: X
authors.lastname: Wang
authors.forename: Aimin
authors.initials: A
authors.lastname: Lin
authors.forename: Don B
authors.initials: DB
authors.lastname: McClure
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Chen
authors.forename: Steven H
authors.initials: SH
authors.lastname: Zuckerman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D018604:Epitope Mapping; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011487:Protein Conformation; D011489:Protein Denaturation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase