Enzyme-labelled fluorescence

Note ELF = enzyme labeled fluorescence. ECL = enhanced chemiluninescence. CL = chemiluminescence. RES = biotin amplification, sAV-Cy3. RCA = rolling circle amplification. 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

Figure 6.24 Enzyme-labeled fluorescence. ELF-97 is a soluble phosphorylated substrate cleaved by alkaline phosphatase into a highly fluorescent, insoluble product. (Molecular Probes, Inc., Eugene, OR.)...

Examples of its use include protein and nucleic acid detection, enzyme-labelled fluorescence, in the intrinsic fluorescence of normal and cancer cells, as external... 

Enzyme-labelled fluorescence substrate (ELF 97 phosphate, also known as ELFP) used to detect phosphatase activity by staining the site of cleavage. 

Enzyme-labeled fluorescence (ELF) Various phosphate-labeled fluorescent dyes, ELF 97 Uses phosphatase-based signal amplification. Applicable to immunohisto-chemical and cytological staining, mRNA in situ hybridization, detection of endogenous phosphatase activity, and blot analyses. 34... 

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. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

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. 

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

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. 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The enzyme label must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). The biotin label can be detected using streptavidin conjugated with an enzyme or fluorophore (see Sect. 6.2.1). 

The hydrolysis of a nonfluorescent enzyme substrate to a fluorescent product is widely utilized to measure the activity of a large number of enzymes. Binding of enzyme substrates by antibodies often protects the enzymatically labile bond from hydrolysis. By the combination of these two formats, the substrate-labeled fluorescent immunoassay (SLFIA) was developed. ... 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Immunohistochemical stains use antibodies to identify specific constituents in tissue sections. In order to detect the site of reaction, the antibody is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. The alternative is to use a fluorescent label. The advantage of an enzyme label is that the nuclei can be counter stained thereby revealing the tissue architecture, and that the stain fades slowly, if at all, allowing the slides to be stored. 

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. 

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