Fluorescence Immunoassay Reagents

Fluorescence immunoassays require labeling of antibodies, antigens, or both. The structure and some of the properties of antibodies that are important to the construction of immunoassays are briefly discussed first in this section, followed by a general discussion of probes and some of their characteristics. 

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. 

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Fluorescence Immunoassay. Basic FIA follows the same formats and approaches as EIA. The difference Hes in the indicator a fluotophote is used instead of an enzyme. This allows direct quantification of the indicatot—antibody—antigen complex, or free indicator-reagent, without the need for a substrate. 

The aim of this chapter is to discuss fluorescence concepts that are used in selected immunoassay applications. The primary focus is on fluorescence topics of recent interest that provide insight into the characteristic properties of antibodies and antigens in immunoassays, or that describe enhancements in immunoassay technologies. The basic reagents and instrumentation required for immunoassay purposes are discussed first, followed by a brief description of immunoassay formats. The principles that are utilized in various fluorescence immunoassay technologies are outlined with specific examples and their significance. Since it is beyond the scope of this chapter to review all of the applications of fluorescence immunoassays, apologies are extended to authors that this chapter fails to cite. A number of comprehensive treatments of fluorescence immunoassay (FIA) applications and related topics are available. 18 ... 

The principle approach to immunoassay is illustrated in Figure 1, which shows a basic sandwich immunoassay. In this type of assay, an antibody to the analyte to be measured is immobilized onto a solid surface, such as a bead or a plastic (microtiter) plate. The test sample suspected of containing the analyte is mixed with the antibody beads or placed in the plastic plate, resulting in the formation of the antibody—analyte complex. A second antibody which carries an indicator reagent is then added to the mixture. This indicator may be a radioisotope, for RIA an enzyme, for EIA or a fluorophore, for fluorescence immunoassay (FIA). The antibody-indicator binds to the first antibody—analyte complex, free second antibody-indicator is washed away, and the two-antibody—analyte complex is quantified using a method compatible with the indicator reagent, such as quantifying radioactivity or enzyme-mediated color formation (see Automated instrumentation, clinical chemistry). 

Fluorescent immunoassay was also conducted. For instance, physisorption of goat anti-mouse IgG (biotin-conjugated) was first made to the PDMS surface for the immunoassay of IgG. Then neutravidin was introduced to bind to the biotin on the surface. Immobilization of biotinylated goat anti-human IgG on the neu-travidin-coated channel was first achieved by confining the reagent flow at a T-intersection on the PDMS chip. The antigen (Cy5-human IgG) was applied via a perpendicular channel. Fluorescence measurement allowed the... 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

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. 

Walter, B., Greenquist, A.C. and Howard, W.E. (1983). Solid-phase reagent strips for detection of therapeutic drugs in serum by substrate-labeled fluorescent immunoassay. Anal. Chem. 55, 873-878. 

In an immunoflorescence assay, fluorescent dyes such as fluorescein and rhodamine can be visualized on a microscope slide through the use of ultraviolet light or detected with instruments which measure their emissions. Fluorescent immunoassays are widely used in clinical laboratories. They are quite sensitive but may be time-consuming and reagents may be unstable and require special handling. 

On this basis, the dissociation-enhanced lanthanide fluorescence immunoassay was developed. Mostly a two-site assay format is used whereby antibodies are immobilized at microtiter plates and after applying the sample antigen, europium-chelate-labeled antibodies bind to the sample. A commonly used chelating reagent is Nl-(p-isothiocyanato-benzyl)-diethylene triamine. After the addition of a S-diketone as a dissociation enhancer, at acidic pH, europium is released and fluorescence of Eu is measured. Other TRFIAs use stabilized lanthanide chelates or stable chelates. 

Qin QP, Lovgren T, Pettersson K (2001) Development of highly fluorescent detection reagents for the constmction of ultrasensitive immunoassays. Anal Chem 73 1521-1529... 

Five anticonvulsants including valproic acid were determined by the Abbott TD x fluorescence polarization immunoassay automatic analyzer. Recoveries were 94.8-106% and the coefficients of variations were 1.0-9.7% [23], Fluorescence polarization immunoassay and enzyme immunoassay were compared for the determination of free valproic acid in serum [24], Good correlation (R = 0.9992) was obtained between the two assays. Higgins [25] reported on the determination of valproic acid in serum by enzyme immunoassay with use of EMIT reagents and the Abbot ABA-200 analyzer. Responses were rectilinear up to 150 mg/L. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

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