Fluorescence immunoassay substrate-labeled

The hydrolysis of a nonfluorescent enzyme substrate to a fluorescent product is widely utilized to measure the activity of a large number of enzymes. Binding of enzyme substrates by antibodies often protects the enzymatically labile bond from hydrolysis. By the combination of these two formats, the substrate-labeled fluorescent immunoassay (SLFIA) was developed. ... 

Figure 6.2. Chemical structure of 8-[3-(7-/)-galactosylcoumarin-3-carboxyamido )propyl ] theophylline (B) used in the homogeneous substrate-labeled fluorescent immunoassay for theophylline (A). (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

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. 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Fig. 4. Schematic representation of the principle of substrate-labeled fluorescent immunoassay (tda, Ames). [Cited and modified from Fig. B-5, Miyai, K., in Miyai etal., eds. (M10).]...

HPLC = high-performance liquid chromatography. SLFIA = substrate-labelled fluorescent immunoassay. 

S28 Walter, B. (1981). A unitized solid phase analytical element for detection of therapeutic drugs in serum by homogeneous substrate-labeled fluorescent immunoassay. Clin. Chem. 27, 1086, Abstr. 311. 

Burd, J.F., Ellis, P.B., Greenquist, A.C., Li, T.M., Morris, D.L., Rupchock, P.A., Sommer, R.G., Tyhach, R.J., Walter, B. and Zipp, A.P. (1983). Measurement of theophylline with the substrate-labeled fluorescent immunoassay and apoenzyme reactivation immunoassay system in solution and in solid phase reagent strips. In Avrameas, S. et al. (Eds), Immunoenzymatic Techniques Proc. 2nd Int. Symp. Immunoenzymatic Techniques, Cannes, 16-18 March. Elsevier Science Publishers, Amsterdam, pp. 239-246. 

Enzyme immunoassays that require physical separation of free and bound T4 are generally based on principles analogous to those of conventional RIAs except that enzyme activity rather than radioactivity is measured. Most enzyme immunoassays use labeled T4 as antigen. An assortment of photometric, fluorescent, and luminescent substrates have been used to monitor the enzyme activity of the antibody-bound fraction. 

TM Li, JL Benovic, RT Buckler, JF Burd. Homogeneous substrate labeled fluorescent immunoassay for theophylline in serum. Clin Chem 27 22, 1981. 

SG Thompson, JF Burd. Substrate labeled fluorescent immunoassay for amikacin in human serum. Antimicrob. Agents Chemother 18 264, 1980. 

B Rollman, et al. Dibekacin assay in serum by automated fluorescence polarization immunoassay (Abbot TDX)—Comparison with high performance liquid chromatography, substrate labeled fluorescent immunoassay and radioimmunoassay. J Pharm Biomed Anal 4 53, 1986. 

Among homogeneous fluoroimmunoassays based on conventional fluorimetry, substrate-labeled fluoro-immunoassay is another alternative for both hapten and protein determination using a fluorescent label. As in other homogeneous competitive immunoassays, the sensitivity is limited by the serum background fluorescence and the limited amount of tracer that can be used. 

A Comparison of Serum Phenytoin Determination by the Substrate-Labeled Fluorescence Immunoassay with Gas Chromatography, Liquid Chromatography, Radioimmunoassay and "EMIT ... 

Several types of labels have been used in immunoassays, including radioactivity, enzymes, fluorescence, luminescence and phosphorescence. Each of these labels has advantages, but the most common label for clinical and environmental analysis is the use of enzymes and colorimetric substrates. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

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