Derivation and characterization of monoclonal antibodies against human folypolyglutamate synthetase.
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.
Dotzlaf
Joe
J
Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46258, USA.
Carpenter
John
J
Luo
Shuang
S
Miles
Rebecca R
RR
Fisher
Deborah
D
Qian
Yue-Wei
YW
Ehsani
Mariam
M
Wang
Xiliang
X
Lin
Aimin
A
McClure
Don B
DB
Chen
Victor J
VJ
Zuckerman
Steven H
SH
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
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase