Homogeneous fluorescence immunoassay

Using the same PAbs an optical biosensor system has been developed for 2,4,6-TCP [224]. The principle is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d=58.4 mm) produced by a piezoelectric generator system. A continuous Ar ion laser (488 nm) excites the fluorescent tracer and its fluorescence is detected by a spectrometer attached to a cooled, charge-coupled device (CCD) camera... 

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. 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

Alternatively, Briggs et al. (B4) reported a fluorescent immunoassay based on the correlation of fluctuations in particle number measures of the amount of tagged species bound to micrometer-sized beads. A homogeneous competitive assay based on this principle can detect 1 ng of gentamicin per ml from a total sample volume of 10 p.1. 

B6. Burd, J. F., Wong, R. C., Feeney, J. E., Carrico, R. J., and Boguslaski, R., Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum. Clin. Chem. 23, 1402-1408 (1977). 

Fan and Zhang determined acetylcholine and choline in rat brain tissue by a fluorescence immunoassay method, making use of immobilized enzymes and chemiluminescence detection [50]. Tissue was homogenized with a 10 fold volume of 0.6 M HC104, the homogenates were kept on ice for 30 min, and then centrifuged at 2000 G for 20 min. The pellets were... 

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. 

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

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

T. A. Kelly and G. D. Christian, Homogeneous Enzymatic Fluorescence Immunoassay of Serum IgG by Continuous Flow Injection Analysis. Ta-lanta, 29 (1982) 1109. 

Kuningas K, Pakkila H, Ukonaho T, Rantanen T, Lovgren T, Soukka T. Upconversion fluorescence enables homogeneous immunoassay in whole blood. Clin Chem 2007 53(1) 145. 

Ullmann EF, Ballet NF, Brinkley JM, et al. (1980) Homogeneous fluorescence immunoassays. In Nakamura RM, Dito WR, and Tucker ES (eds.) Immunoassays Clinical Laboratory Techniques for the 1980s, pp. 13-43. New York Alan R. Liss. 

---