Immunoassays 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).]...

Walter, B. Greenquist, A.C. Howard, W.E., III. Solid-phase reagent strips for detection of therapeutic drugs in serum by substrate-labelled flourescent immunoassay. Anal. Chem. 1983, 55, 873-878. 

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. 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

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. 

Homogeneous competitive enzyme immunoassays using substrate-labeled antigen The method of Ngo et al. (1981) to... 

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 ... 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

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. 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

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