Fluorescence Immunoassay Applications

FIAs can be based on steady-state intensity measurements without probe amplification, owing to the sensitivity of detection that is possible with fluorescence instrumentation, which exceeds that of spectrophotometers by two or three orders of magnitude. A sensitive fluorometer has been described for an estradiol assay(36) in which the limit of estradiol detection is 3 x KT11 M. Estradiol antibody labeled with rhodamine B is reacted with estradiol samples. Unreacted labeled antibody is removed with Sepharose-estradiol-casein beads, and the remaining fluorescence is directly proportional to the analyte concentration. The detection limit of rhodamine B on the same fluorometer is 5 x 1(T12 M. This instrument uses a 0.75 mW green helium-neon (HeNe) laser to irradiate the sample from above, at the air-liquid interface, to increase the light path and to decrease surface reflections. The sample compartment has a top-mounted photon trap, and a mirror mounted on the side of the sample compartment opposite the PMT to enhance detection. 

In space-resolved immunoassays, a smooth metal slide is coated with an antibody monolayer, and a parallel laser beam is used to quantitate surface bound fluorophore.1(37,38) Scattered light is low since the excitation is reflected into a different space, although scatter still remains the principal source of background. 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D- 

In a particle concentration fluoroimmunoassay system that is based on microtiter plates with 0.22-/ filters on the bottoms, microparticles are used with a flowthrough wash system. Either fluorescein-labeled antibodies or cells serve as labels.(41) Car-boxyfluorescein diacetate, which crosses the cell membrane, is hydrolyzed by cytoplasmic esterases, thereby trapping carboxyfluorescein within the cell. The method is also useful for screening and isotyping antibodies. 

Phase-separation immunoassays have been reported, in which the solid phase particles are formed after the immunoreaction is completed.(42) Phase-separation immunoassays are advantageous since the unstirred layer of solution near a solid surface alters diffusion and binding kinetics at the surface in comparison with the properties of the bulk solution. In phase-separation assays for IgG and IgM, capture antibodies are bound with monomers suitable for styrene or acrylamide polymerization.(42) Monomer-labeled capture antibodies are reacted with analyte and with fluorescein- and/or phycoerythrin-labeled antibodies in a sandwich assay, followed by polymerization of the monomers. Fluorescence of the resulting particles is quantitated in a FACS IV flow microfluorometer, and is directly proportional to analyte concentration. 

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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... 

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 ... 

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. 

Lanthanide luminescence applications have already reached industrial levels of consumption. Additionally, the strongly specific nature of the rare-earths eneigy emissions has also led to extensive work in several areas such as photostimulable phosphors, lasers (qv), dosimetry, and fluorescent immunoassay (qv) (33). 

X. Gao, M. Schawaller, and H. J. Mathieu, Covalent Neutravidin Immobilization on NH3 RF Plasma Surface Modified Polystyrene Biochip and Its Application in Fluorescence Immunoassay, to be submitted to Surf. Interface Anal. 

Aslan K, Holley P, Geddes CD (2006) Microwave-accelerated metal-enhanced fluorescence (MAMEF) with silver colloids in 96-well plates Application to ultra fast and sensitive immunoassays, high throughput screening and dmg discovery. J Immunol Methods 312 137-147 Matveeva E, Gryczynski Z, Malicka J et al (2004) Metal-enhanced fluorescence immunoassays using total internal reflection and silver-coated surfaces. Anal Biochem 334 303-311 Blue R, Kent N, Polerecky L (2005) Platform for enhanced detection efficiency in luminescent-based sensors. Electron Lett 41 682-684... 

Yang, W., Trau, D., Renneberg, R., Yu, N. T., and Caruso, F. (2001). Layer-by-Layer construction of novel biofunctional fluorescent microparticles for immunoassay applications. J. Colloid Interface Sci. 234 356-362. 

Sdva DP Jr, Landt Y, Porter SE, Ladenson JH. Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay. Clin Chem 1991 37 1356-64. 

Finally, fluorescence immunoassay is a method of major importance for biochemical and biomedical applications. 

See also Bloassays Overview. Derivatizatlon of Analytes. Extraction Solvent Extraction Principles. Fluorescence Food Applications. Food and Nutritional Analysis Contaminants. Gas Chromatography Detectors Mass Spectrometry. Immunoassays Production of Antibodies. Immunoassays, Applications Food. Immunoassays, Techniques Enzyme Immunoassays. Liquid Chromatography Instrumentation Liquid Chromatography-Mass Spectrometry Food Applications. Sampling Theory. 

See also Chemiluminescence Overview. Chiroptical Analysis. Derivatizatlon of Analytes. Enzymes Enzyme-Based Assays. Fluorescence Overview Ciinicai and Drug Appiications. Gas Chromatography Coiumn Technoiogy Mass Spectrometry. Immunoassays Overview. Immunoassays, Applications Clinical. Immunoassays, Techniques Enzyme Immunoassays Luminescence Immunoassays. Infrared Spectroscopy Overview. Liquid Chromatography Column Technology Normal... 

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