Time-Resolved Fluorescence Immunoassay

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. 

Homogeneous TR-FIAs have been reported in which proprietary lanthanide chelates are used. In a homogeneous immunoassay for T4, a fluorescent europium chelate coupled to thyroxine is quenched by antibody binding. 90 A similar approach is used for estrone-3-glucuronide. 91 TRFIAs based on homogeneous methods have not yet become widely used. 

---

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. 

Figure 14.6. Time-resolved fluorescence immunoassay measurement cycle.

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

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

B2. Barnard, G., and Kohen, E., Monitoring ovarian function by the simultaneous time-resolved fluorescence immunoassay of two urinary steroid metabolites. Clin. Chem. 44,1520-1528 (1998). 

E. P. Diamandis and T. K. Christopoulos, Europium Chelate Labels in Time-Resolved Fluorescence Immunoassays and DNA Hybridization Assays, Anal. Chem. 1990, 62, 1149A. 

Reimer, G.J., S.J. Gee, and B.D. Hammock (1998). Comparison of a time-resolved fluorescence immunoassay and an enzyme-linked immunosorbent assay for the analysis of atrazine in water. J. Agric. Food Chem., 46 3353-3358. 

Butcher, H., W. Kennette, O. Collins, et al. 2003. A sensitive time-resolved fluorescent immunoassay for metallothionein protein. J. Immunol. Methods 272 247-256. 

Two rather interesting variations on fluorescence immunoassay that do not require the fluorescence spectra of free and bound labeled materials to have different spectral positions have fairly recently become rather popular in clinical analysis. These are fluorescence polarization immunoassay and time-resolved fluorescence immunoassay. 

Laboratory testing for ricin is limited, especially for inhalational exposures. The two common methods that can detect ricin in blood or other body fluids are the radioimmunoassay and the enzyme-linked immunosorbent assay (ELISA). Because ricin binds quickly and the body metabolizes it efficiently before excretion, the length of time necessary for these tests limits their usefulness for inhalation exposures (35). Besides testing body fluids, the CDC and member LRN state public health laboratories have a time-resolved fluorescence immunoassay that can test preparations of suspected ricin-containing substances and environmental specimens for the presence of ricin (40). 

The original inhibition test has been replaced by an immunoradiometric assay and a time-resolved fluorescent immunoassay (the DELFIA method developed by Pharmacia, Uppsala, Sweden). The cut-off values for healthy subjects vary from 14 to 20 kU/L, depending on the method. 

Wong T, McLaurin J, Makela S, Makela SK, Elefthe-rios E. Development of a time-resolved fluorescence immunoassay for 17a-hydroxyprogesterone. Clin Chem 1990 36 1150. 

PapanastasioU-Diamandi A, Khosravi M. Total triiodothyronine (Tj) measurement in serum by time-resolved fluorescence immunoassay. Clin Chem 1991 37 1029. 

Kakabakos SE, Khosravi MJ. Direct time-resolved fluorescence immunoassay of progesterone in serum involving the biotin-streptavidin system and the immobihzed-antibody approach. Clin Chem 1992 38 725-30. 

Aggerbeck, H., Norgaard Pedersen, B., and Heron, I. (1996) Simultaneous quantitation of diphtheria and tetanus antibodies by double antigen, time resolved fluorescence immunoassay. Journal of Immunological Methods, 190, 171 183. 

Figure 18-27. Fluorescence decay profile of an europium chelate as used in a time-resolved fluorescence immunoassay. Background fluorescence disappears after a few nanoseconds, whereas the chelate decays in the millisecond time range. Reproduced by permission, courtesy of LKB Produkter, Bromma, Sweden.

Rickett, G. et al. (2003) Development of a high throughput time resolved fluorescence immunoassay to support discovery of HIV-1 cell entry Inhibitors. 43rd Annual Interscience Conference on Antimicrobial Agents and Chemotherapy. 2003 Chicago, IL. 

See also Chemiluminescence Overview. Fluorescence Time-Resolved Fluorescence. Immunoassays, Techniques Enzyme Immunoassays. Phosphorescence Room-Temperature. Sequential Injection Analysis. 

Morton RC, Diamandis EP (1990) Streptavidin-based macromolecular complex labeled with a Europium chelator suitable for time-resolved fluorescence immunoassay applications. Anal Chem 62 1841-1845... 

---