Assay time-resolved

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Ferguson, R. A., Diamandis, E. P. Ultrasensitive detection of prostate-specific antigen by a time-resolved immunofluorometric assay and the immunochemiluminescent third-generation assay. Clin. Chem. 42,203-212 (1996). 

Overall, it can be envisioned that the Py-G group 47 represents an important label for the time-resolved studies of DNA dynamics and stacking interaction [123] and could be applied especially for assays in which conformational changes or base-flipping processes are essential in observation, such as in the investigation of DNA-protein complexes with DNA repair proteins. 

Product and services of interest include genomics, proteomics, custom research and services, and instruments, accessories, consumables and software. PerkinElmer proprietary technologies include time-resolved fluorescence which is employed in the sensitive Wallac DELFIA system and Wallac LANCE homogeneous assay system, and fluorescence polarization, [FP]2 , a robust fluorescence-based technique for receptor binding assays that is both fast and easy-to-use. 

Cencic, R., Yan, Y., and Pelletier, J. (2007). Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation. Comb. Chem. High Throughput Screen 10, 181-188. 

Blomberg, K., Hurskainen, P. and Hemmila, I. (1999). Terbium and rhodamine as labels in a homogeneous time-resolved fluorometric energy transfer assay of the b-subunit of human chorionic gonadotropin in serum. Clin. Chem. 45, 855-61. 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

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.

Diamandis, E.P., and Christopoulos, T.K. (1990) Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays (Review). Anal. Chem. 62, 1149-1157. 

Liang, A.M., Claret, E., Ouled-Diaf, J., Jean, A., Vogel, D., Light, D.R., Jones, S.W., Guilford, W.J., Parkinson, J.F., and Snider, R.M. (2007) Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of Leukotriene A4 hydrolase. J. Biomol. Screen. 12, 536-545. 

Soini, E., Hemmila, I., and Dahlen, P. (1990) Time-resolved fluorescence in biospecific assays [Review], Ann. Biol. Clin. 48, 567-571. 

H. Harma, T. Soukka, S.Lonnberg, J. Paukkunen, P. Tarkkinen, and T. Lovgren, Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence. Luminescence 15, 351-355 (2000). 

Soukka T, Paukkunen J, Harma H, Lonnberg S, Lindroos H, Lovgren T (2001) Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology. Clin Chem 47 1269-1278... 

Hennig A, Florea M, Roth D, Enderle T, Nau WM (2007) Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore. Anal Biochem 360 255-265... 

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