Fluorescence immunoassay hydrolysis

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. ... 

Figure 6.3. Substrate-labeled fluorescent immunoassay for theophylline. (A) Effect of theophylline rabbit antiserum (O) and normal rabbit serum (A) on the enzymatic hydrolysis of 8-[3-(7-/l-galactosylcoumarin-3-carboxyamido)propyl]theophylline. (B) Effect of various concentrations of theophylline on the fluorescence resulting from enzymatic hydrolysis of 8-[3-(7-/)-galactosyl-coumarin-3-carboxyamido)propyI]theophylline. (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

Figure 6.4. Schematic representation of a fluorescent immunoassay for theophylline utilizing enzymatic hydrolysis of an intramolecularly quenched theophylline conjugate of flavin adenine dinucleotide. (Reprinted from Ref. 5, with permission from Academic Press.)...

Many fluorescence immunoassays have utilized umbelliferone, a 7-hydroxycoumarin (excitation and emission maxima of 360 nm and 447 nm, respectively), as the fluorescence reporter group. Normal serum, however, exhibits emission maximum at 520 nm and excitation maxima of 330 nm and 440 nm. Even with this overlap between the spectra of umbelliferone and normal serum, umbelliferone has one property that makes it suitable for fluorescence immunoassays conversion of the free hydroxyl group to an ester or a glucoside effectively quenches the fluorescence of umbelliferone, and hydrolysis of the ester or glycoside allows for recovery of fluorescence. It is... 

Coumarins also have a C6-C3 skeleton, but they possess an oxygen heterocycle as part of the C3-unit. There are numerous coumarins, many of which play a role in disease and pest resistance, as well as UV-tolerance. The coumarin umbelliferone (1.21) is popular in enzyme assays. Umbelliferone esters can be used as a substrate for non-specific esterase enzyme assays and in fluorescent immunoassays (Jacks and Kircher, 1967). In order to quantify the enzyme activity of the popular reporter gene P-glucuronidase (GUS), plant extracts can be incubated with 4-methylumbelliferyl P-D-glucuronide (4-MUG 1.22), which upon hydrolysis... 

Enzymatic Hydrolysis of Antigen-Fluorescent Dye Conjugate Immunoassay... 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

Sensitive immunoassays specific for PAH-DNA adducts allow the detection of one adduct per 10 nucleotides. Fluorimetry has also been used as an alternative detection method to immunoassays. In another method, isomeric tetrols of PAH are liberated by acid hydrolysis of the DNA-PAH adducts and analyzed by LC with fluorescence detection. Structural studies and detailed characterization of PAH metabolites and their conjugates has been performed by trapping the corresponding fractions at the exit of the LC system. As an example, the total characterization of the in t /tro-formed benzo(a)pyrenetetra-hydrodiol-epoxide-guanosine adduct has been achieved by a combination of nuclear magnetic resonance, circular dichroism, and mass spectroscopic techniques. 

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