Luciferase detection

Microtiter plates HRP/H202/luminol AP/dioxetanes Firefly luciferin/luciferase Bacterial luciferin/luciferase Detection of enzymes and metabolites by direct or coupled enzyme reactions Determination of antioxidant and enzyme inhibitory activities Immunoassay... 

Immunoassay and nucleic acid probe assays Labels. recApoaequorin, firefly luciferase, marine bacterial luciferase, Vargula luciferase Detection reactions, alkaline phosphatase label (luciferin-O-phosphate/firefly luciferase), glucose 6-phosphate dehydrogenase label (marine bacterial luciferase/NADH FMN oxidoreductase reaction)... 

Add 10 pL of Cypridina luciferin substrate to each well and detect CL activity immediately using a luminometer see Notes 13-15). Note that the suggested manufacturer s protocol for the detection CL differs from a typical luciferase detection system (say for FL) in that it requires the addition of buffer to samples prior to the addition of the substrate. 

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. 

As httle as lO " g of ATP can be detected with carefiiUy purified luciferase. Commercial luciferase contains enough residual ATP to cause background emission and increase the detection limit to 10 g (294). The method has been used to determine bacterial concentrations in water. As few as lO" cells/mL of Lscherichia coli, which contains as Httle as 10 g of ATP per cell, can be detected (294). Numerous species of bacteria have been studied using this technique (293—295). 

It should be noted that Cypridina luciferin emits a fairly strong chemiluminescence in aqueous solutions in the presence of various lipids and surfactants, even in the complete absence of luciferase. The luminescence is especially conspicuous with cationic surfactants (such as hexadecyltrimethylammonium bromide) and certain emulsion materials (such as egg yolk and mayonnaise). Certain metal ions (especially Fe2+) and peroxides can also cause luminescence of the luciferin. Therefore, great care must be taken in the detection of Cypridina luciferase in biological samples with Cypridina luciferin. 

A note on the assays of coelenterazine and luciferase activity. The methods for measuring coelenterazine and the corresponding luciferases are given in Appendix C5. Special attention must be paid to the fact that coelenterazine in aqueous buffer solutions spontaneously emits a low level of chemiluminescence in the absence of any luciferase, which is greatly enhanced by the presence of various substances, including egg yolk, BSA and various surfactants (especially, hexadecyltrimethylammonium bromide). Therefore, the utmost care must be taken in the detection and measurement of a low level of... 

H2O2 was 2.9 x 10-6 M. A requirement for O2 could not be detected, suggesting that 02 is not involved in the luminescence reaction. The luciferase was found to be a peroxidase that catalyzes peroxidation of the luciferin, and it can be substituted with horseradish peroxidase. 

Coelenterazine can be detected and measured with a coelenterazine luciferase, i.e. a luciferase specific to coelenterazine. As the coelenterazine luciferase, the luciferases from the sea pansy Renilla and the copepods Gaussia and Pleuromamma are commercially available. Certain kinds of decapod shrimps, such as Oplophoms and Heterocarpus, contain a large amount of luciferase, and the luciferases purified from them are most satisfactory for the assay of coelenterazine considering their high activities and high quantum yields. Even partially purified preparations of these luciferases are satisfactory for most measurements. The author routinely uses purified Oplophoms luciferase. 

The amount of Cypridina luciferase is measured with Cypridina luciferin. However, the detection and measurement of a trace amount of the luciferase is extremely difficult, for the reason explained below. 

Coelenterazine and the corresponding luciferase can be easily tested in the field. A small piece of tissue sample is put in a test tube with methanol (for coelenterazine) or water (for luciferase), and crushed with a spatula. To measure coelenterazine, a buffer solution containing a coelenterazine luciferase is injected into a small amount of the fluid part of the crushed sample mixture. Similarly, luciferase can be measured with a buffer solution containing coelenterazine. The presence of Cypridina luciferin can be tested in the same fashion, with the methanol extract of samples and crude Cypridina luciferase. However, the detection of a very weak Cypridina luciferase activity in the field is not recommended (see Section C5.6). To test the presence of a Ca2+-sensitive photoprotein, crush a sample in a neutral buffer solution containing 20-50 mM EDTA, and then add lOmM calcium acetate to a small portion of the fluid part of the crushed sample to detect any light emission. 

Macheroux, P., Ghisla, S., and Hastings, J. W. (1993). Spectral detection of an intermediate preceding the excited state in the bacterial luciferase reaction. Biochemistry 32 14183-14186. 

Palmer, L. M., and Colwell, R. R. (1991). Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction. Appl. Environ. Microbiol. 57 1286-1293. 

Rees, J. F., Thompson, E. M., Baguet, F., and Tsuji, F. I. (1990). Detection of coelenterazine and related luciferase activity in the tissues of the luminous fish, Vinciguerria attenuata. Comp. Biochem. Physiol. 96A 425-430. 

DNA binding domain (DBD). When ligand binds to the NR, it translocates into the nucleus, the DBD binds to the GAL4 response element (GAL4RE) driving lucifer-ease transcription. The produced luciferase provides a subsequent detectable fluorescent signal. 

Cruz-Aguado, J.A., Chen, Y., Zhang, Z., Elowe, N.H., Brook, M.A. and Brennan, J.D. (2004) Ultrasensitive ATP detection using firefly luciferase entrapped in sugar-modified sol-gel-derived silica. Journal of the American Chemical Society, 126, 6878-6879. 

Although numerous luminous organisms are known, only a few of them has been studied and really exploited. Analytical applications of bioluminescence concern mainly the detection of ATP with the firefly luciferase and of NADH with some marine bacteria systems. Luciferase from the North American firefly, i.e., Photinus pyralis, has been extensively studied10-12 and afterwards, attention has been paid to the luciferase from Luciola mingrelica, i.e., the North Caucasus firefly13 15. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

ATP measured by luciferin-luciferase BL assay was used to examine the effect of toxic substances on whole microbial communities in activated sludge mixed liquid samples [114], It was used to detect whether wastewater had an effect on the biodegradation capability of the resident population of microorganisms. Actually ATP BL represents an important rapid toxicity test that utilizes waste treatment natural microorganisms to determine the toxicity of wastes discharged to the sewer [132],... 

Other applications dealt with the development of a luciferin ester substrate to measure the luciferase activity in living cells [141], the detection of toxic compounds such as sodium azide, fluoroacetic acid, and antibiotics [142], the development of a biosensor for the determination of bioavailable mercury [143], the use of eukaryotic luciferases as bacterial markers with different colors of luminescence [144], the determination of complement-mediated killing of bacteria [145], and the development of a bioassay for the determination of HIV type 1 virus and HIV-1 Tat protein activity, valuable also for analysis of HlV-inhibi-tory agents [146],... 

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