Chemiluminescence assays

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). 

Ma Y, Zhou M, Jin X et al (2004) Flow-injection chemiluminescence assay for ultra-trace determination of DNA using rhodamine B-Ce(IV)-DNA ternary system in sulfuric acid media. Anal Chim Acta 501 25-30... 

Human embryo lung fibroblasts infected with a reference laboratory strain of herpes simplex virus (HS V) type 2 were used to detect antibody to HSV type 2 in serum samples. After treatment of cells with serial dilutions of sera, HRP-labeled immunoglobulins to human IgG (class G immunoglobulins) were added and detected with CL substrate [36], In both cases a sharp detection of the specific antibodies was achieved with chemiluminescent assays, which proved more sensitive than the colorimetric immunoperoxidase assays. 

Kawasaki, S., Yamashoji, S., Asakawa, A., Isshiki, K., and Kawamoto, S. (2004). Menadione-catalyzed luminol chemiluminescence assay for the rapid detecHon of viable bacteria in foods under aerobic conditions. /. Food Prot. 67, 2767-2771. 

Table 3.11 Chemiluminescent assays using luminol and its derivatives...

Kooy and Royall (1994) have shown that cultured endothelial cells produce peroxynitrite when stimulated by bradykinen. They used an ultrasensitive chemiluminescent assay based on luminol developed by Radi et al. (1993). Peroxynitrite is a potent toxin to trypanosomes, attacking both sulfhydryl dependent enzymes and respiratory enzymes (Rubbo et al., 1994). Radi et al. (1994) have also shown that it is far more damaging to mitochondria than nitric oxide. 

Bronstein, I., Voyta, J. C., Thorpe, G. H. G., Kricka, L. J., and Armstrong, G (1989) Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin. Clin Chem 35, 1441—1446. 

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. 

Fang and co-workers [115] have reported a flow injection chemiluminescence assay for the detection of maleic hydrazide (MH). The imprinted poly(MAA-co-EDMA) polymer selectively retained the herbicide that was further treated with alkaline luminol-potassium periodate to produce a strong CL signal. Upon reaction, the absorbed MH was destroyed and removed by the flowing solution. The polymer was reused up to ten times and the linear response was between 3.5 x 10 and 5.0 x 10 2 mg mL-1 with a detection limit of 6.0 x 10 5 mg mL-1. The CILA flow injection system was used for the analysis of potato and onion samples spiked with 1.0, 2.0, and 4.0 x 10-3 mg mL-1 of MH. Recoveries ranged from 98% to 103% (RSD 2.3%), demonstrating the successful application of the method. 

Enhanced chemiluminescent assay Luminol Amersham ECL Antioxidant Detection Pack Luminometric (induction time) W10... 

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

Ternaux and Chamoin described an enhanced chemiluminescence assay method for the determination of acetylcholine [48]. Reaction medium was prepared by mixing 250iu/mL of choline oxidase (100 pL), 2mg/mL of horse-radish peroxidase (50 pL) and 10-120 pM luminol in 100 pL of 0.1 M Tris buffer (pH 8.6), or 100 pL of 10-100 pM 7-dimethylamino-naphthalene-l,2-dicarbonic acid hydrazide, for 10min in 5mL of 0.1 M sodium phosphate buffer (pH 8.6). Aqueous 0.325-80 pmol of acetylcho-lineesterase (50 pL) purified on a Sephadex G50 coarse column was mixed with 450 pL of reaction mixture, and the chemiluminescence was measured at 21° C. 

Israel and Lesbats reported a chemiluminescence method for the determination of acetylcholine, and continuous detection of its release from torpedo electric organ synapses and synaptosomes [55], Birman described a new chemiluminescence assay method for the determination of acetylcholineesterase activity with the natural substrate [56], The method involved monitoring the increase in light emission produced by accumulation of choline, or measuring the amount of choline generated. 

The immediate appearance of lipid hydroperoxides after irradiation in this study contrasts with several other studies, which have either reported no increase in lipid-peroxidation products in skin after UV irradiation [21] or an increase only several hours after irradiation [27-29], In these studies the background level of lipid-peroxidation products has been high, even in control, non-irradiated skin. In contrast, in our experiments lipid hydroperoxides were undetectable before irradiation, but appeared at very high concentrations in skin immediately after irradiation. The discrepancies between various studies may be due to differences in technique. The HPLC-chemiluminescence assay used in the present study is specific for lipid hydroperoxides, and has less potential for artifact than the TBARS assay, which is known to suffer from many possibilities for artifact [30] -... 

Massey [1] functionalized graphitic nanotubes including Ceo-fiiHerenes, (I), which were then used in electrogenerated chemiluminescence assays. 

Luminescent endpoints are available for all of the commonly used enzyme labels, including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, glucose oxidase, and P-galactosidase. Detection limits in the subattomolar range have been attained (34). Chemiluminescent assays for novel enzyme labels such as xanthine oxidase have also been developed (50). 

Chemiluminescent assays are conventionally monitored using photomultiplier-based instruments. However, portable instruments are becoming available that use as photodetectors silicon photodiodes, charge-coupled devices, and instant photographic or x-ray film (58, 63-68). Spacially resolved, quantitative light measurements are pcirticularly advantageous for assays based on membranes or microtiter plates. 

Lackey, D. B. (1998). A homogeneous chemiluminescent assay for telomerase. y4na/. Biochem. 263 57-61. 

Figure 15.5 HRP-acridan chemiluminescence assay on both glass and silvered slides. Reproduced from Ana Chem 78 8020-8027, (2006).

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