Fluorescence immunoassay FIA

Binding assays including the following immunoassays such as radio immunoassay (RIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIO), enzyme-linked immunoassay (ELISA and EMIT)... 

Beckman Robotic Biomek 1000 automated laboratory The Biomek 1000 integrates the work formerly done by four instruments sample preparation system, diluter/dispenser, plate washer and a spectrometer finish. In can handle assays such as radio-immunoassays (RIA), fluorescence immunoassays (FIA), enzyme immunoassays EIA and enzyme-linked immunoassays (ELISA). 

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). 

Most immunoassays currently employed in the biomedical field are either radioimmunoassays, enzyme immunoassays, or luminescence immunoassays (including fluorescence immunoassays [FIA] and chemiluminescence immunoassays). Although radioimmunoassay is currently the most sensitive of these (10 -10 M concentrations are often detectable), due to the problems inherent to dealing with radioactive materials, such as licensing, radiation hazard, short shelf-life of expensive radioisotopes, the expense of the counting equipment, and the tedium associated with heterogeneous immunoassay, it has fallen, in popularity, behind the non-isotopic methods of analysis. 

Enzyme activities may also be measured in urine, cerebrospinal fluid, bone marrow cells or fluid, amniotic cells or fluid, red blood cells, leukocytes, and tissue cells. Cytochemical localization is possible in leukocytes and biopsy specimens (e.g., from liver and muscle). Under ideal conditions, both the concentration of the enzyme and its activity would be measured. Radioimmunoassay (RIA) and its alternative modes such as fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), and chemiluminescence immunoassay (CLIA) (discussed later), can be used to measure enzyme concentration as well as other clinically important parameters. 

In fluorescent immunoassay (FIA), a fluorescent molecule is covalently bound to a molecule of analytical interest. The labeled analyte interacts reversibly with an antibody (usually derived from a rabbit or goat) which is specific for the analyte and will bind with about equal affinity to the labeled or unlabeled analyte. The labeled analyte usually shows different fluorescent properties according to whether it is bound by the antibody or free to diffuse in solution. The serial additional of different concentrations of the analyte to the labeled analyte-protein... 

Alternative labels that have been investigated are reviewed in Table 6.1. As can be seen from the table, certain of these methods along with RIA are unsuitable for application to sensor development and will not be mentioned further. Other methods, particularly fluorescence immunoassay (FIA), will be discussed in more detail later with particular reference to their application in sensors. 

Fluorescence immunoassay (FIA) Fluorophores Fluorimetry Simple labelling homogeneous methods possible background effects possible (23), (24), (25), (26), (27)... 

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. 

FIA Fluorescence immunoassay uses a fluorescent tag on the antibody or antigen. Fluorescent labels absorb light of one wavelength and reemit it at another wavelength. The label is excited by UV and emits visible light. Common fluorescent labels are fluorescein, Texas red, and GFP (green fluorescent protein). 

Homogeneous TR-FIAs have been reported in which proprietary lanthanide chelates are used. In a homogeneous immunoassay for T4, a fluorescent europium chelate coupled to thyroxine is quenched by antibody binding. 90 A similar approach is used for estrone-3-glucuronide. 91 TRFIAs based on homogeneous methods have not yet become widely used. 

A variety of immunoassay techniques employ changes in the fluorescent properties of molecules to measure drug concentrations. Labelling of drugs with such molecules avoids the hazards of working with radioisotopes. Fluoroimmunoassays (FIA) also offer enhanced sensitivity in comparison to enzyme immunoassays they may be homogeneous or heterogeneous. 

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