Chemiluminescence, enhanced

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

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). 

Sakamoto [243] determined picomolar levels of cobalt in seawater by flow injection analysis with chemiluminescence detection. In this method flow injection analysis was used to automate the determination of cobalt in seawater by the cobalt-enhanced chemiluminescence oxidation of gallic acid in alkaline hydrogen peroxide. A preconcentration/separation step in the flow injection analysis manifold with an in-line column of immobilised 8-hydroxyquinoline was included to separate the cobalt from alkaline-earth ions. One sample analysis takes 8 min, including the 4-min sample load period. The detection limit is approximately 8 pM. The average standard deviation of replicate analyses at sea of 80 samples was 5%. The method was tested and inter calibrated on samples collected off the California coast. 

The enhanced chemiluminescence associated with the autoxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) in the presence of trace amounts of iron(II) is being used extensively for selective determination of Fe(II) under natural conditions (149-152). The specificity of the reaction is that iron(II) induces chemiluminescence with 02, but not with H202, which was utilized as an oxidizing agent in the determination of other trace metals. The oxidation of luminol by 02 is often referred to as an iron(II)-catalyzed process but it is not a catalytic reaction in reality because iron(II) is not involved in a redox cycle, rather it is oxidized to iron(III). In other words, the lower oxidation state metal ion should be regarded as a co-substrate in this system. Nevertheless, the reaction deserves attention because it is one of the few cases where a metal ion significantly affects the autoxidation kinetics of a substrate without actually forming a complex with it. 

Lueking et al. (1999) arrayed recombinant proteins on NC membranes and screened them with different antibodies. Joos and coworkers (2000) printed down autoantigens onto NC membranes and compared performance relative to silylated (aldehyde) and PLL glass slides. Protein arrays could be stored at room temperature for a month without significant loss in activity. Huang (2001) hand spotted down IgC species and antibodies directed toward various cytokines onto membranes. The properties of various commercial membranes were assessed in terms of absorption, background, and sensitivity levels based upon detection by enhanced chemiluminescence (ECL). 

The separated proteins were transferred to a polyvinylidene difluoride membrane, and nonspecific IgC binding sites were blocked by incubation with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. Immune complexes were detected by enhanced chemiluminescence (Amersham Biosciences). 

Energy transfer quantum yields, chemiluminescence, 1223 Enhanced chemiluminescence, 1219-20, 1221 Enol esters, dioxirane asymmetric epoxidation, 1150... 

Constantine, N. T. Bansal, J. Zhang, X. Hyams, . C. Hayes, C. Enhanced chemiluminescence as a means of increasing the sensitivity of western blot assays for HIV antibody. J. Virol. Methods 1994, 47(1-2), 153-164. 

Heinicke, E. Kumar, U. Munoz, D. G. Quantitative dot-blot assay for proteins using enhanced chemiluminescence. J. Immunol. Methods 1992,152(2), 227-236. 

Nitrocellulose membrane enhanced chemiluminescence (ECL Amersham Biosciences). 

Horseradish peroxidase (HRP)-coupled secondary anti-IgG antibodies (species depending on primary antibody) enhanced chemiluminescence substrate... 

In common with all other sensitive detection systems, maintenance of the label enzyme in its active state is important. The precautions detailed in Notes 1—3 should be observed to maximize the sensitivity achieved. Reagents for enhanced chemiluminescence can be prepared in the laboratory or ure available commercially (see Note 4). The purity of the substrate solution is important in achieving maximum sensitivity. Therefore, the precautions detailed in Notes 5-7 should be followed if preparing substrate solutions. The free base form of lummoi undergoes rearrangement ro a mixture of luminol and a series of contaminants. Therefore, luminol should be purified by recrystaliistation as the sodium salt before use (see Note 8). 

Working enhanced chemiluminescence substrate solution (see Note 9)—either a. /Modophenol-enhanced substrate luminol 1.25 mM, p-iodophenol 4 pM,... 

Add 150 pL of enhanced chemiluminescent substrate to each well in the same order and preferably with the same timing as used by the plate reader. Allow at least 2 min for the light output to stabilize before reading the plate. 

DMSO is a colorless, odorless compound. However, it is hygroscopic and acquires an onion-like smell. DMSO in this state has been found to be inhibitory in the enhanced chemiluminescent reaction. Therefore, the highest available grade should be purchased in small amounts and carefully stored to minimize water uptake. 

Thorpe, G H. G and Kricka, L. J. (1986) Enhanced chemiluminescent reactions catalyzed by horseradish peroxidase. Methods Enzymol. 133, 331-354. 

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