Bioluminescent immunoassay

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Hamilton Umicon Lumicon chemi- and biolumium assay luminometer This equipment is used in test-tube scale luminescent immunoassays. With its sample compartment (thermostatted by means of Peltier elements, which allow the temperature to be set from 15°C to 40°C with a precision of 0.1°K) this instrument is suitable for the measurement of temperature-sensitive bioluminescence resulting from enzymic reactions and also in phagocyte-mediated luminescence measurements. 

Yamakawa Y, Ueda H, Kitayama A et al (2002) Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. J Biosci Bioeng 93 537-542... 

Several other examples of 1,2-dioxetane derivatives containing easily oxidizable groups have been reported and the high singlet quantum yield observed in their decomposition was attributed to the occurrence of the intramolecular CIEEL sequence Based on this concept, Schaap and coworkers have introduced the concept of induced chemiluminescence, which is very relevant for investigations into firefly luciferin bioluminescence and has led to the development of chemiluminescent probes widely used in immunoassays (Section N. 

Ito, K. Nakagawa, K. Murakami, S. Arakawa, H. Maeda, M. Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay. Anal. Sci., 19, 105-109 (2003)... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

J. C. Lewis and S. Daunert, Bioluminescence Immunoassay for Thyroxine Employing Genetically Engineered Mutant Aequorins Containing Unique Cysteine Residues, Anal. Chem. 2001, 732, 3227. 

The LIA is an immunoassay in which the antigen or antibody are labeled with either a chemiluminescent or bioluminescent tags (41, 58). Luminescent molecules are produced by oxidation reactions. Bis-phenyl oxalates in presence of hydrogen peroxides are used for chemiluminescent assays and luciferin in presence of luciferase enzyme is used for bioluminescent assays. The sensitivity of the LIA s are in the pg/ml or lower range. 

Quantum Yield Efficiency of fluorescence percentage of incident energy emitted after absorption. The higher the quantum yield, the greater the intensity of the fluorescence, luminescence, or phosphorescence. See Papp, S. and Vanderkooi, J.M., Tryptophan phosphorescence at room temperature as a tool to study protein structure and dynamics, Photochem. Photobiol. 49, 775-784, 1989 Plasek, J. and Sigler, K Slow fluorescent indicators of membrane potential a survey of different approaches to probe response analysis, J. Photochem. Photobiol. 33, 101-124, 1996 Vladimirov, Y.A., Free radicals in primary photobiological processes, Membr. Cell Biol. 12, 645-663, 1998 Maeda, M., New label enzymes for bioluminescent enzyme immunoassay, J. Pharm. Biomed. Anal. 30, 1725-1734, 2003 Imahori, H., Porphyrin-fullerene linked systems as artificial photosynthetic mimics, Org. Biomol. Chem. 2, 1425-1433, 2004 Katerinopoulos, H.E., The coumarin moiety as chromophore of fluorescent ion indicators in biological systems, Curr. Pharm. Des. 10, 3835-3852, 2004. 

Chemiluminescence immunoassay,a technique that has rapidly gained popularity because its sensitivity is comparable to that of radioimmunoassay, is in a sense a variation of FIA. In the 1930s, the first work on chemiluminophores was published, but it was not until the 1980s that chemiluminescence was first tried in immunoassays. Due to the increased use of automated immunoassay analyzers, chemiluminescence has become one of the most common immunoassay detection methods used in the clinical laboratory setting. Chemiluminescence (also called bioluminescence when it occurs in fireflies and some dinoflagellates, coelente-rates, and fungi) is fluorescence. However, what is... 

Geiger, R. Miska, W. Bioluminescence enhanced enzyme-immunoassay new ultrasensitive detection systems for enzyme immunoassays. J. Clin. Chem. Biochem. 1987, 25, 31-38. 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

Hoyle NR. The application of electrochemilumines-cence to immunoassay-based analyte detection. In Campbell AK, Kricka LJ, Stanley PE, eds. Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects. Chichester Wiley, 1994 28-31. 

Krodel E, Boland J, Carey G, et al. Technical challenges in the development of the CIBA Corning ACS 180 benchtop immunoassay system. In Stanley PE, Kricka L], eds. Bioluminescence and chemiluminescence Current status. Chichester John Wiley Sons, 1991 107-10. 

Rigi CT, Rivera HN, Patel MT, et al. Bioluminescence immunoassays for human endocrine hormones based on Aqualite , a calcium-activated protoprotein. Clin Chem 1995 41 1363-4. 

Sekiya K, Saito Y, Ikegami T, et al. Fully automated analyzer for chemiluminescent enzyme immunoassay. In Stanley PE, Kricka LJ, eds. Bioluminescence and... 

Wannlund J, DeLuca M. 1982. Sensitive bioluminescent immunoassay for dinitrophenol and trinitrotoluene. Anal Biochem 122 385-393. 

Bioluminescence has been used in immunoassays (Terouanne et al., 1986) but its complexity and high background, arising from ATP contamination, thus far make this system less attractive. Biolumines-cent assays have also been proposed with alkaline phosphatase using luciferin phosphate as the substrate (Hauber and Geiger, 1987) and... 

Fukoda S, Tatsumi H, Maeda M. Bioluminescent enzyme immunoassay with biotinylated firefly luciferase. J Clin Ligand Assay 1998 21 358-362. 

So there were obvious implications to apply obelin as a label in immunoassay the protein accessibility, high sensitivity, an unlimited linear range of bioluminescence, the lack of background, the simplicity with which the reaction is triggered, the absence of hazard and the availability of modem registration devices. 

Obelin-biotin complex was successfully obtained using succinimide derivatives of biotin, with less than 30% of its bioluminescent activity lost under the synthesis conditions. The biotinylated obelin is a universal label, suitable for any immunoassay through the avidin bridge. As an example, Fig.l gives the scheme of a solid-phase microanalysis of alphafetoproteins in standard human sera and displays the results of this analysis. 

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