Time-Resolved Immunoassays

Dechaud H, Bador R, Claustrat F, Desuzinges C, MaUein R. New approach to competitive lanthanide immunoassay Time-resolved fluoroimmunoassay of progesterone with labeled analyte. Clin Chem 1988 34 501-4. 

Time-resolved fluorometry is versatile in its application. Applied in immunoassay, time-resolved fluorometry presents an unmatched combination of advantages. While sensitive and specific, radioimmunoassays present safety and stability problems. While safe, enzyme immunoassays lack sensitivity and dynamic range. 

Keywords Clinical applications Hormone assays Immunoassays Time-resolved fluorescence... 

Hildebrandt N, Charbonniere LJ, Lohmannsroben HG (2007) Time-resolved analysis of a highly sensitive forster resonance energy transfer immunoassay using terbium complexes as donors and quantum dots as acceptors. J Biomed Biotechnol 2007 79169... 

Charbonniere LJ, Hildebrandt N, Ziessel RF, Lohmannsroben HG (2006) Lanthanides to quantum dots resonance energy transfer in time-resolved fluoro-immunoassays and luminescence microscopy. J Am Chem Soc 128 12800-12809... 

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. 

Hemmila, I. (1988) Lanthanides as probes for time-resolved fluorometric immunoassays. Scand. J. Clin. Lab. Invest. 48, 389-400. 

Soini, E., and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates—A new generation of monisotopic immunoassays. Clin. Chem. 29, 65-68. 

Morrison LE (1988) Time-resolved detection of energy transfer theory and application to immunoassays. Anal Biochem 174 101-20... 

Data are expressed as percentages of users for each assay. Abbreviations ELISA, enzyme-linked immunosorbent assay EIA, electroimmunoassay RIA, radioimmunoassay TIA, turbidimetric immunoassay. Delfia, time resolved immunofluorescence assay. 

E. P. Diamindis, Immunoassays with time-resolved fluorescence spectroscopy Principles and applications, Clin. Biochem. 21, 139-150(1988). 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Figure 14.6. Time-resolved fluorescence immunoassay measurement cycle.

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

T. Lovgren, I. Hemmila, K. Pettersson and P. Halonen, Time-resolved fluorometry in immunoassay, in Alternative Immunoassays (W. P. Collins, ed.) pp. 203-217, John Wiley Sons, New York (1985). 

E. Soini, Pulsed light, time-resolved fluorometric immunoassay, in Monocolonal Antibodies and New Trends in Immunoassays (Ch. A. Bixollon, ed.) pp. 197-208, Elsevier Science Publishers, Amsterdam (1984). 

P. Helsingius, 1. Hemmila, and T. Lovgren, Solid-phase immunoassay of digoxin by measuring time-resolved fluorescence, Clin. Chem. 32, 1767-1769 (1986). 

P. Nuutila, P. Koskinen, K. Irjala, L. Linko, H.-L. Kaihola, J. U. Eskola, R. Erkkola, P. Seppcala, and J. Viikari, Two new two-step immunoassays for free thyroxine evaluated Solid-phase radioimmunoassay and time-resolved fluoroimmunoassay, Clin. Chem. 36, 1355-1360 (1990). 

E. P. Diamandis and R. C. Morton, Time-resolved fluorescence using a europium chelate of 4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labelling procedures and applications in immunoassays, J. Immunol. Methods 112, 43-52 (1988). 

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

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