Lanthanide chelates, assays

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

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... 

Other fluorescent probes also may be used to label (strept)avidin molecules for detection of biotinylated targeting molecules. Chapter 9 reviews many additional fluorescent labels, such as quantum dots, lanthanide chelates, and cyanine dye derivatives, all of which may be used in similar protocols to create detection conjugates for (strept)avidin-biotin-based assays. 

Another example of improved sensitivity due to modulation of lanthanide photophysics by ancillary ligands can be found in the europium and terbiiun chelates used in time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays (100,101). Due to their line-type emissions and long decay times, the lanthanide chelate is used as a donor, with some visible-absorbing dye such as Alexa 647 or a rhodamine derivative as the acceptor. Without the helper ligand, the lanthanides would be unprotected from solvent and have much shorter decay times, making them unsuitable for such an assay. 

Diamandis EP, Christopoulos TK, Immunological assays based on time-resolved fluorometry with lanthanide chelates as labels. Endocrinology and Metabolism In-Service Training and Continuing Education Program. Washington, DC AACC Press 1992 10 9-19. 

This time-resolved fluorescence technique allows a measure of the time dependence of fluorescence intensity after a short excitation pulse. It consists of obtaining a spectrum measured within a narrow time window during the decay of the fluorescence of interest. The usefulness of this technique is now well proven for biochemical assays and immunoassays. Lanthanide chelates have luminescence decay times over 600 ps, which allows time-gated fluorecence detection, with a complete rejection of other fluorecence signals. For these quantitative applications, the primary source is generally a quartz lamp associated with a splitter. 

DELFIA or time-resolved fluorometty (TRF) is a well-established technology that exploits the unique fluorescence properties of lanthanide chelates and provides a powerful alternative to radioisotopic assays in many high throughput-screening applications. 

Since the mid 1970 s many different structures and classes of lanthanide chelates have been synthesised (Rgure 5). For high sensitivity assays the dissociation enhanced system is currently the best approach, however for screening assays where the sensitivity or detection limitation is not an issue the fluorescent chelates can be used to simplify the assay. Some of these chelates can be even used to develop homogeneous and non-separation assays. This assay format is essential for the measurement of a reaction where the components have only a weak binding affinity thus cannot withstand a wash or separation step. 

The structures of fluorescent lanthanide chelates used for separation and homogeneous time resolved fluorometric assays. 

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