Markers, fluorescent radioactive

The most critical step in the interaction studies is a sensitive and reproducible method for quantification of the marker substrates and the corresponding metabolite. Analytical methods are usually applied to time- and resources-consuming HPLC (UV/fluorescence/radioactivity detection) or LC/MS/MS detection. 

In this method, specific functional groups reagents containing a marker - halogens, radioactive isotopes, fluorescent moieties... 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

The origin of the microarray or biochip can be traced to a seminal publication by Edwin Southern over 30 years ago. Southern described a method by which DNA could be attached to a solid support following electrophoresis and interrogated for sequences of interest by hybridization with a complementary DNA sequence (16). The complementary DNA sequence, termed a probe, was labeled with either a radioactive or a fluorescent marker and hybridized to the DNA target sample, which was immobilized on a sohd support, such as a nitrocellulose filter membrane. 

With a limited ability to sample the brain s chemicals directly, researchers have turned to other methods. As described in the sidebar on pages 82-83, proteins such as enzymes and receptors play a vital role in neurotransmitter function. Since these proteins are specific to a specific neurotransmitter, they serve as markers for its use. By attaching various molecules such as dyes, radioactive substances—which emit energetic particles or radiation—or fluorescent molecules—which emit light—to... 

Figure 6. Elution profile obtained on examination of the gel permeation behavior of 14C acetylated S. aureus Factor III. Cytochrome-c and fluorescein labeled E. coli HPr were included in the applied sample as internal standards. Absorbance at 410 nm (cytochrome-c), fluorescein fluorescence (HPr), and radioactivity (Factor III) are plotted vs. fraction number. Inset shows the elution profiles for the external (blue dextran) and internal (fiuoroglycine) column markers. Data in this figure are from unpublished work (20).

Access to nucleic acid dendrimers is initiated by a zip-fastener like dissociation of the DNA double strand by heating. The double strand separates into the two individual strands by thermal motion (denaturation). Subsequent association, hybridisation of complementary sequences, is followed by stepwise cross-linking to form DNA dendrimers, which can contain up to two million oligonucleotide-end group strands (Fig. 8.19). The latter can be labelled with fluorescence or radioactive markers. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

Sensitivity of immunoassays is largely conditioned by markers applied for conjugation with the antibody. Traditional immunodetection methods ELISA with radioactive markers (radioimmunoassay—RIA), enzymatic markers (enzyme immunoassays—El A), or fluorescent markers (fluoroenzyme immunoassays FEIA) are currently the most widely used techniques in laboratory analysis of allergens as well as in clinical studies for determination of general and specific IgE and other subclasses of immunoglobulines, e.g., IgG4 in the Immuno-CAP system (Samson, 2001 Duran-Tauleria et al., 2004 Lidholm et al., 2006). 

Encapsulation efficiency is commonly measured by encapsulating a hydrophilic marker (i.e. radioactive sugar, ion, fluorescent dye), sometimes using single-molecule detection. The techniques used for... 

Examples of fluorescence labels for hgands are carboxyfluorescein, Cy3, a commercially available fluorescent marker based on a cyanine dye or tetramethyl-rhodamine. They are chemically introduced into a ligand. As with the radioactive labels, a possible influence of the labels on the binding behavior of the labeled hgands has to be considered, especially as the fluorescent dyes are complex molecules. Furthermore, the receptors themselves can be fluorescent labeled, which is done recombinantly. The respective receptors are expressed as fusion proteins with fluorescent proteins, e.g., green fluorescent protein (GFP) from Aequorea victoria, one of its mutant variants, or DSRed from Discosoma striata [26, 35]. 

One can place in some positions on the protein radioactive, fluorescent, or spin-labeled markers. For the same purpose one may also use antibodies and proteolytic enzymes. With these procedures one can determine those parts of the protein that are outside the membrane. Next, efforts are made to determine the position of the membrane part in the sequence. A complementary method is the hydrophobic marking of the protein by building into it hydrophobic photolabeled parts from the class of phospholipid compounds in order to determine those parts that are situated in the membrane. Another method uses proteolytic enzymes that split the protein in a specific position in the segment that is not located in the membrane. ... 

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