Enzyme linked fluorescence

The most critical and demanding requirement is that the fluorescent product generated by an enzyme is retained quantitatively within the cell. A number of ingenious strategies for product retention have been developed including intracellular precipitation (the Enzyme-Linked Fluorescence or ELF family of fluorescent substrates), attachment of lipophilic anchors, substrates that yield insoluble chromophores, and retention via formation of complexes with intracellular proteins (for examples, see footnote 3 and Zlokamik et al., 1998). 

Sohm et al. (in press) have extended their studies of APA and P uptake kinetics by Trichodesmium colonies in the N. Atlantic to a comparative study of these two indices in the tropical N. Pacific and coastal northern Australian waters as well. They detected sharp contrasts among the sites with much higher Riax and APA values in their tropical N. Atlantic stations compared to the N. Pacific and northern AustraHa indicating more severe P hmitation in the tropical N. Atlantic. Dyhrman et al. (2002) also reported evidence of severe P stress in Trichodesmium in the N. Atlantic based on a enzyme linked fluorescence (ELF) assay. A follow up study also saw less evidence of P stress in samples from the N. Pacific (Hynes et al., Pers. Comm.). 

A fully automated system derivated from ELISA method has been evaluated by Vemozy-Rozand et al. (1998), tested by the Association of Agricultural Chemists (AOAC) and validated by the Erench national organization for standardization (AENOR). This system, the VIDAS E. coli 0157 (BioMerieux, France) is an Enzyme Linked Fluorescent Assay (ELEA) using two ready-to-use components ... 

Diagnostic procedures include dark-field microscopy12, non-treponemal exams10 (i.e., the Venereal Disease Laboratory and the rapid plasma reagin test), and treponemal exams (i.e., enzyme immunoassay, the T. pallidum hemagglutination test, the fluorescent treponemal antibody test, and the enzyme-linked immunosorbent assay). 

A very versatile piece of equipment that is affordable for individual laboratories is the microplate reader. This allows multiple samples to be analyzed at once, commonly in a 96-well format, although 384- and 1536-well formats are available. Typical measurements that can be performed include UV-Vis absorbance, fluorescence, or luminescence, allowing a range of assays to be performed, such as cell growth, enzyme kinetics, enzyme stability, or enzyme-linked immunosorbent assay [60-62]. Functionality can be increased by the use of liquid dispensing systems or automatic plate handling. 

Another method which uses capillary electrophoresis with laser-induced fluorescence detection can also be employed to detect zearalenone (Maragos and Appell 2007). In order to analyse trace amounts of zearalenone in plants, a sensitive, quick and accurate method, the enzyme-linked immunosorbent assay (ELISA) was developed by Chen et al. 1989. 

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). 

In addition to these research applications of fluorescence, there is a continuing use of fluorescence detection to replace analytical methods based on radioactivity, as can be judged from the recent books and conferences on fluorescence sensing methods. (7 n) These emerging applications of fluorescence can be seen by the growth and introduction of improved methods for immunoassays, enzyme-linked immunoassays... 

Fluoroimmunoassays comprise a subclass of extrinsic labehng methods where various selective antigen (Ag)- antibody (Ab) immunoassay fluorescent labeling schemes yield a emission signal. One common scheme involves an enzyme-linked immunosorbent assay (ELISA) depicted in Figure 11.2 where the free Ab is tagged with a fluorophore. Numerous analytes can be detected via these types of selective lock-and-key methods. ... 

Cell components or metabolites capable of recognizing individual and specific molecules can be used as the sensory elements in molecular sensors [11]. The sensors may be enzymes, sequences of nucleic acids (RNA or DNA), antibodies, polysaccharides, or other reporter molecules. Antibodies, specific for a microorganism used in the biotreatment, can be coupled to fluorochromes to increase sensitivity of detection. Such antibodies are useful in monitoring the fate of bacteria released into the environment for the treatment of a polluted site. Fluorescent or enzyme-linked immunoassays have been derived and can be used for a variety of contaminants, including pesticides and chlorinated polycyclic hydrocarbons. Enzymes specific for pollutants and attached to matrices detecting interactions between enzyme and pollutant are used in online biosensors of water and gas biotreatment [20,21]. 

Fluorescent Antibodies Enzyme-linked immunosorbant assays... 

Wolbers R, Landrey G (1987) The use of direct reactive fluorescent dyes for the characterization of binding media in cross sectional examinations. Preprints of 15th Annual Meeting of the American Institute for Conservation and Artistic Works, Vancouver, 168-202. Heginbotham A, Millay V, Quick M (2006) The use of immunofluorescence microscopy and enzyme-linked immunosorbent assay as complementary techniques for protein identification in artist s materials. J Am Inst Conserv 45 89-105. 

Use of a surrogate end point that is quick and easy to obtain Permeation experiments using a radiolabeled, fluorescent, HPLC-detectable, or radio immuno assay/enzyme linked immuno sorbent assay-detectable marker necessitate the need of extensive sample handling and sample analysis. This accentuates the cost of sample analysis and overall time spent in characterizing the efficacy of formulations. Furthermore, current state of the art fluidics systems put a fundamental limit on the number of samples handled in a given time. 

Benzodiazepines are an important group of drugs with tranquilizing properties. Available immunochemical methods include radioimmunoassays (164, 165), a radioreceptor assay (166), and nonseparation immunoassays such as the widely used enzyme-monitored immunotest (EMIT) and fluorescent polarization immunoassays (167, 168). Such assays generally require sophisticated apparatus and dedicated laboratories. However, a relatively simple enzyme-linked immunosorbent assay was recently described for screening benzodiazepines in urine (169). 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

Because of concerns about the safety of radioisotope use, researchers are developing fluorescent and chemiluminescent methods for detection of small amounts of biomolecules on gels. One attractive approach is to label biomolecules before analysis with the coenzyme biotin. Biotin forms a strong complex with enzyme-linked streptavidin. Some dynamic property of the enzyme is then measured to locate the biotin-labeled biomolecule on the gel. These new methods approach the sensitivity of methods involving radiolabeled molecules, and rapid advances are being made. 

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