Terbium chelates

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

When used with europium or terbium ions, a carbostyril-based lanthanide chelate can be excited at 340 nm and provide sharp characteristic emission bands for transfer of energy to the appropriate acceptor fluor. Similar to the TMT chelator described previously, luminescence from terbium FRET signals well with Cy3 dyes and luminescence from europium can be used with APC or Cy5 dyes. Other fluorescent dyes that have similar excitation and emission ranges to these also can be used as acceptors in TR-FRET assays. For instance, terbium chelates can... 

T. K. Christopoulos and E. P. Diamandis, Enzymatically amplified time-resolved fluorescence immunoassay with terbium chelates, Anal. Chem. 64, 342-346 (1992). 

The authors point out that the fluorescent lifetime of the terbium chelate at 25°C is more than twice that of the chloride and that measurements at 0°C and 77°K showed that the lifetime for TbAn3 increases slightly as the temperature decreases. They also measured the lifetimes of each of the individual 5Z)4->7/ transitions and found the same values, as expected. 

These assays are based upon the use of a europium or terbium chelate (a transition metal-ligand complex displaying long-lived fluorescent properties) and labeled anti-phosphopeptide or anti-phos-photyrosine antibodies that can bind to phosphorylated peptides. The antibodies are usually labeled... 

Sueda, S., Yuan, J.L., and Matsumoto, K. (2002) A homogeneous DNA hybridization system by using a new luminescence terbium chelate. Bioconjugate Chemistry, 13, 200-205. 

Phimphivong, S. and Scott Saavedra, S. (1998) Terbium chelate membrane label for time-resolved, total internal reflection fluorescence microscopy of substrate-adherent cells. Bioconjugate Chemistry, 9, 350-357. 

THe excited-state kinetics of Tb3+ in vapor-phase terbium chelates 62) and terbium aluminum chloride complexes (71, 72 ) have been investigated but no laser action reported. 

To achieve the rapid diffusion limit requires a very long-lived donor. Chelates of terbium, which typically have lifetimes of 1.5-2.2 msec, are frequently used. Fluorescein and rhodamine make excellent acceptors for terbium. Chelates of europium also have long lifetimes, 0.5-2.3 msec, although these appear not to have been used. (Cronce and Horrocks have used europium in a calcium-binding site as a donor in a rapid diffusion... 

Fig. 10. Structure or terbium chelate, namely, terbium diethylenetriaminepentaacetic acid coupled to carbostyril 124. The carbostyril absorbs light (maximally at 327 nm) and transfers energy to the terbium. The net result is an increase in the effective absorbance of the terbium by several thousandfold. The DTPA chelate shields the terbium from the quenching effects of water and allows for easy attachment to macromolecules. Here the macromolecule is an 8-mer DNA oligomer modified with a primary amine on the 5 end. The acceptor, fluorescein, is attached to the 5 end of a complementary DNA oligomer. 

A wide variety of fluorescent molecular probes have been demonstrated to be suitable for excitation by the He-Cd laser 4-bromomethyl-7-methoxycoumarln has been employed for the detection of carboxylic and phosphoric acids (44,55), 7-chlorocarbonyl-methoxy-4-methylcoumarln for hydroxyl compounds (42), 7-lsothlocya-nato-4-methylcoumarln for amines and amino acids (55), 7-dlazo-4-methyl-coumarln for a variety of aromatic compounds (55), and terbium chelate molecules with long fluorescence lifetimes ( 1 ms) for protein analysis (56). In this study, we examined the utility of l-dlmethylamlnonaphthalene-5-sulfonyl chloride (dansyl chloride) as a sensitive and selective reagent for the determination of biogenic amines and amino acids. 

Diamandis EP (1999) Europium and terbium chelators for time-resolved fluorometric assay. EP0635006B1... 

Due to energy backflow problem, the decays of even the stable fluorescent Tb chelates vary rather widely from below 100 ps to as long as 2.6 ms [21, 23, 25]. Fluorescent terbium chelates have also found commercial applications in HTS in Lanthascreen product line of Invitrogen and later on by CisBio with the Lumi4 chelates of Lumiphore company. 

Surface-modified nanobeads, beads where the chelates have shorter distance to the respective acceptor-labeled reactant, are also used to create proximity in TR-FRET assays such as cAMP immtmoassay and assays of kinase activities by the Molecular Devices IMAP system. The kinase assays apply fluorescent peptides as substrates, which after phosphorylation are collected to beads by complex formation between phosphor and the trivalent metalhc ions on the beads. Fluorescent terbium chelates, substituted with a phosphor group, provide thereafter the energy-donating groups to create sensitized signal on bead surfaces [37]. 

Dual-label TR-FRET substrates have also been used for simultaneous monitoring of the enzymes related to apoptosis caspases 1, 3, and 6 [44]. Kokko et al. utilized fluorescent terbium chelates as the donors for two different acceptors, Alexa Fluor 488 and 680, in a dual-label immtmoassay of free and total PSA [45]. Free, uncomplexed PSA were measured with TR-FRET from terbium labeled (universal) antibody to free-specific Alexa 488 labeled antibody, and the total PSA was quantified with TR-FRET from terbium to another generic antibody labeled with Alexa 680. Kupcho et al. followed nuclear receptor coactivator recruitment... 

Jeyakumar and Katzenellenbogen developed a dual-acceptor assay to monitor a nuclear receptor (thyroid hormone - retinoid X receptor heterodimer) regulation by measuring both coactivator and corepressor binding by using fluorescein and Cy-5 as acceptors for a terbium chelate-labeled response element-bound receptor complex [47]. 

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