Alkaline phosphatase, chemiluminescent

Pande, R., S. Kamtekar, M.S. Ayyagari, M. Kamath, K.A. Marx, J. Kumar, S.K. Tripathy, and D.L. Kaplan. 1996. A biotinylated undecylthiophene copolymer bioconjugate for surface immobilization Creating an alkaline phosphatase chemiluminescence-based biosensor. Bioconjugate Chem 7 159-164. 

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). 

Alkaline phosphatase-labeled probes are synthesized so that 18 bases are complementary to sequences on the arms of the bDNA. Three hybridization sites are located on each branch for a total binding capacity of 45 labeled probes per bDNA molecule. The alkaline phosphatase catalyzes the dephosphorylation of chemiluminescent substrate, dioxetane (Lumi-Phos Plus, Lumigen, Detroit, MI). The intensity of the light emission is measured with a plate luminometer as relative luminescent units. 

In the Hybrid-Capture assay (Digene), a full-length RNA probe is hybridized to denatured HBV DNA in solution and the hybrids are captured on the surface of a tube coated with anti DNA RNA hybrid antibody. The bound hybrids are reacted with antihybrid antibody labeled with alkaline phosphatase. A chemiluminescent substrate is converted to a luminescent compound by the bound alkaline phosphatase. Light emission is measured in a luminometer and the concentration of HBV DNA, in pg/ml, is determined from a standard curve. The concentrations of the standards are determined spectrometrically (A260nm/A280nm). 

A.N. Diaz, F.G. Sanchez, M.C. Ramos, and M.C. Torijas, Horseradish peroxidase sol-gel immobilized for chemiluminescence measurements of alkaline-phosphatase. Sens. Actuat. B 82, 176-179 (2002). 

Recently, two major enzyme-catalyzed chemiluminescent reactions have become popular. These use either luminol as a substrate of peroxidase or 3-(2 -spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) as a substrate of alkaline phosphatase (ALP). 

Aminophthalate anion Atmospheric pressure active nitrogen Analyte pulse perturbation-chemiluminescence spectroscopy Arthromyces rasomus peroxidase Ascorbic acid Adenosine triphosphate Avalanche photodiode 5-Bromo-4-chloro-3-indolyl 2,6-Di-t< r/-bu(yl-4-mclhyl phenol Bioluminescence Polyoxyethylene (23) dodecanol Bovine serum albumin Critical micelle concentration Calf alkaline phosphatase Continuous-addition-of-reagent Continuous-addition-of-reagent chemiluminescence spectroscopy Catecholamines Catechol... 

Figure 2 illustrates the reaction mechanisms of acridinium ester label probes and alkaline phosphatase probes using dioxetane chemiluminescent detection. Table 2 summarizes approaches for labeling DNA. 

An elegant approach is to capture the target DNA or RNA with specific oligonucleotides on to a microwell plate. Synthetic branched DNA bearing multiple alkaline phosphatase-labeled probes hybridizes to the target. A chemiluminescent substrate is added to produce signal. This branched DNA assay has been used in infectious disease detection (W3). 

Schaap, A. P., Akhavan, H., and Romano, L. J., Chemiluminescent substrates for alkaline phosphatase Application to ultrasensitive enzyme-linked immunoassays and DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1863-1864 (1989). 

The rational design of effective and efficient dioxetane-based bioanalytical probes requires the in-depth mechanistic understanding of the enzymatically triggered chemiluminescence. In this context, alkaline-phosphatase-triggered CIEEL furnishes a textbook example , which will be examined below in detail. 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

Applied Biosystems Ltd. CDP-Star <6 CSPD Chemiluminescent Substrates for Alkaline Phosphatase , 2003. www.appliedbiosystems.com/products/productde-tail.cfm procLid = 114. 

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. 

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