Fluorescent ELISA

The absorbance measurement conducted in these assays can be replaced by fluorescence detection (fluorescence ELISA). In this case, the enzymatic conjugate chosen is an alkaline phosphatase that converts the specific substrate into a fluorescent compound. 

Vascular endothelial growth factor 2000 C Factor 5 (IPCR 0.2 pg/ml fluorescence-ELISA 1 pg/ml) Sims et al. [49], real-time IPCR (see Section 2.2.3) detection of IPCR-amplificate was carried out online during PCR according to the TaqMan-principle, AbiPrism7700 sequence detector (Applied Biosystems)... 

In a fluorescent ELISA technique (such as the one employed in the Pharmacia Delphia test for B2M), the labeled enzyme is bound to a strong fluorescent dye. In the Pharmacia Delphia test, an antigen bound to a fluorescent dye competes with unlabeled antigen in the sample for a predetermined amount of specific, immobile... 

ELISA Enzyme-linked immunosorbent assay. A sensitive biotechnology analytical technique in which an enzyme is complexed to an antigen or antibody. The analyte is bound and complexed, and then removed for quantification via color development or other instrumental analysis techniques (i.e., fluorescence). ELISA is used as a rapid and accurate technique to quantify most mycotoxins. 

The rapid turnover rate of some enzymes allows ELISAs to be designed that surpass the sensitivity of radiolabeling techniques. In addition, substrates can be chosen to produce soluble products that can be accurately quantified by their absorbance or fluorescence. Alternatively, substrates are available which form insoluble, highly colored precipitates, excellent for localizing antigens in blots, cells, or tissue sections. The flexibility of enzyme-based assay systems makes the chemistry of enzyme conjugation one of the most important application areas in bioconjugate techniques. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

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). 

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. ... 

An international intercomparison exercise in the determination of microcystin, carried out by using the most common methods (LC/DAD, ELISA and LC/MS) indicated that LC/DAD is affected by lower precision [234], while the coupling of the LC technique with ELISA permit the achievement of high sensitivity and specificity in the determination of microcystins and nodularin [235] without the need of pre-concentration the method meets the World Health Organization guidelines (1 pg L ). The combination of ELISA characterization and LC analysis with fluorescence, UV, and tandem MS detections, allowed the first identification of cylindrospermopsin, an algal toxin that caused the poisoning of up to 148 persons in Australia [236],... 

Molecular weight of heavy and light chains Peptide mapping Amino acid analysis Intrinsic fluorescence spectroscopy Thermal denaturation monitored by fluorescence Fourier transfrome infrared spectroscopy Binding (e.g., ELISA, BiaCore, etc) Potency (e.g., cell based, ELISA)... 

Because of the requirement to use radioactive substances, RIAs are frequently being replaced by other immunologic assays, such as ELISA and fluorescence polarization immunoassays (FPIA) (Niemann et al. 1985). These have similar degrees of sensitivity. FPIAs are highly quantitative, as are RIAs, and ELISAs can be designed to be quantitative. 

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