Time resolved fluorescence fluoroimmunoassay

Fluoroimmunoassay makes use of the above behaviour. One of the common commercial methods is dissociation-enhanced fluoroimmunoassay (DELFIA). In this, a nonfluorescent Eu(III) EDTA-like complex is attached by a simple chemical reaction to an antibody or antigen, in a process called labelling. An immunoreaction is next initiated to bind the target, and then a (3-diketone and trioctylphosphine oxide (TOPO) mixture are added to the immunocomplex formed, at pH 3, to promote release of the Eu(III) from the antibody and its complexation as the strongly fluorescent complex [Eu((3-diketonate)3(TOPO)2], which is then measured by time-resolved fluorescence methods. The signal size relates to the amount of europium complexed, which in turns relates directly to the amount of the specifically formed target immunocomplex. This process is represented schematically in Figure 9.5. 

Keywords Fluoroimmunoassays High throughput screening Homogeneous time-resolved fluorescence Lanthanide chelates Lanthanide cryptates... 

Yuan, J. Wang, G. Majima, K. Matsumoto, K. Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay. Anal. Chem. 2001, 73(8), 1869-1876. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Wang, G., J. Yuan, K. Matsumoto, et al. 2001. Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer. Talanta 55 1119-1125. 

Luminescent RE + chelates have been successfully developed as labels and probes for highly sensitive and selective bioassays in the past two decades. Time-resolved Inmines-cence detection has been widely applied in fluoroimmunoassay, DNA hybridization assay, enzyme assay, cell activity assay and fluorescence imaging microscopy. ... 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

Time-resolved techniques (time-resolved fluoroimmunoassay (TRFIA)) make use of the fact that some compounds have long decay times resulting in phosphorescence or delayed fluorescence (e.g., lanthanide chelate complexes) (Figure 1). Apparatus for time-resolved measurements use normally a pulsed excitation source and electronically gated detectors. By this way background fluorescence can be eliminated. 

Ye ZQ, Tan MQ, Wang GL et al (2005) Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application. Talanta 65 206-210... 

Diamandis E. P., R. C. Morton, E. Reichstein, M. J. Khosravi. Multiple fluorescence labeUng with europium chelators. Application to time-resolved fluoroimmunoassays, Anal. Chem., 61, 48-53 (1989). 

Yuan J. L., G. Wang, H. Kimura, K. Matsumoto. Sensitive time-resolved fluoroimmunoassay of human thyroid- stimulating hormone hy using a new europium fluorescent chelate as a label. 

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