Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence.
Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.
Enomoto
Koji
K
Discovery Research Laboratories, Shionogi & Company, Fukushima-ku, Osaka 553-0002, Japan. koji.enomoto@shionogi.co.jp
Nagasaki
Tohru
T
Yamauchi
Akira
A
Onoda
Junji
J
Sakai
Katsunori
K
Yoshida
Tetsuya
T
Maekawa
Kazuhiko
K
Kinoshita
Yuko
Y
Nishino
Ikuko
I
Kikuoka
Shino
S
Fukunaga
Takahiro
T
Kawamoto
Keiko
K
Numata
Yoshito
Y
Takemoto
Hiroshi
H
Nagata
Kiyoshi
K
D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004791:Enzyme Inhibitors / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009942:Organometallic Compounds; D010798:Phycocyanin; D013050:Spectrometry, Fluorescence / Q000379:methods*; D011436:Spermidine Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D014230:Triazoles / Q000138:chemical synthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D014230:Triazoles; C002497:allophycocyanin; D010798:Phycocyanin; C070734:europium(III) trisbipyridine cryptate; C067418:5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione; D011436:Spermidine Synthase