Fluorescence immunoassay quenching

Using the same PAbs an optical biosensor system has been developed for 2,4,6-TCP [224]. The principle is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d=58.4 mm) produced by a piezoelectric generator system. A continuous Ar ion laser (488 nm) excites the fluorescent tracer and its fluorescence is detected by a spectrometer attached to a cooled, charge-coupled device (CCD) camera... 

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... 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

The practical and effective synthesis of photoactive lanthanide cryptates (1), capable of effective light conversion, has found application in the design of novel fluorescent immunoassay systems. The macrobicyclic bipyridyl cryptands prevent the normal solvation quenching of these cations. Remarkably effective DNA cleavage was accomplished using intercalators (2) based on 2,7-diazapyrenium cations. 

Homogeneous TR-FIAs have been reported in which proprietary lanthanide chelates are used. In a homogeneous immunoassay for T4, a fluorescent europium chelate coupled to thyroxine is quenched by antibody binding. 90 A similar approach is used for estrone-3-glucuronide. 91 TRFIAs based on homogeneous methods have not yet become widely used. 

Selected entries from Methods in Enzymology [vol, page(s)] Design, 178, 551 immunoassay, 178, 542 production, 178, 531 purification, 178, 543 substrates and enzymatic assay, 178, 544 derivatization with spectroscopic probe, 178, 567 ester cleavage assays, 178, 565 fluorescence quenching binding assay, 178,... 

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