Enzyme-labeled fluorescence assay

Enzyme-Labeled Fluorescence (ELF) Phosphatase-based signal amplification assay also applicable to nucleic acid labeling (see description under proteins) 43... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

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. 

Urdea, M.S., Warner, B.D., Running, J.A., Stempien, M., Clyne, J., and Horn, T. (1988) A comparison of non-radioactive hybridization assay methods using fluorescent, chemiluminescent and enzyme-labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 4937-4956. 

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. ... 

Here, the antibody-antigen reactions are observed indirectly by use of labels which are attached to either the antibody or antigen. The labels can be conjugated covalently and include radioisotopes, enzymes, labeled second antibodies, fluorescent tags, luminescent molecules and phages (12, 41, 45, 48). The use of labels helps in increasing the sensitivity of the assays considerably compared to the precipitation or agglutination assays. 

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

The method may also be used the other way round with GOx as the enzyme label, glucose as substrate, and [Eu(Tc)] as the fluorescent indicator. In that type of assay, the presence of enzyme-labeled antibodies is indicated by an increase in the observed fluorescence intensity. It should be noted that commercially available fluorescence microplate readers, preferable equipped with time resolution, are also suited for the screening of all the microwell plate-based assays presented in this chapter. Nevertheless, the imaging process is much faster, accomplished in the order of one second, and enables ratiometric measurements. 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

All of the homogeneous fluoroimmunoassay systems suffer from the possibility of background interference from other drugs and metabolites. Enzyme labels may be inhibited by endogenous molecules, and there may be fluorescence enhancement or quenching from the same sources. This may be overcome by careful choice of assay conditions and the use of high dilutions of the biological sample. 

The determination of the quantity of protein bound to the insoluble carrier sometimes causes difficulties. The methods usually applied are laborious or somewhat inaccurate. Labeling of assayed protein, for instance with C-acet-anhydride, makes it possible to carry out a very fast and exact determination of immobilized protein The determination of bound enzyme C-labeled aldolase after its immobilization on polyacrylamide can serve as an example The concentration measurements of certain proteins are based on their ability to bind certain ligands. Radiolabels such as or H-biotin have been used for the determination of avidin by direct binding or for biotin assay by isotopic dilution Cofactor and fluorescent labeled ligands have been also used for the monitoring of specific protein binding reactions. 

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