Chemiluminescence and Bioluminescence, Analysis by

Abstract

Bioluminescent (BL) and chemiluminescent (CL) reactions are used as analytical detection tools in immunoassay, protein and nucleic acid blotting, nucleic acid sequencing, and hybridization assays, and in reporter gene studies. In a BL or CL assay, the intensity or the total light emission is measured and related to the concentration of the unknown analyte. Light is measured quantitatively using a luminometer (photomultiplier tube or charge-coupled device) as a detector or qualitatively by means of photographic or X-ray film. The main advantages of these reactions are simplicity and an analytical sensitivity capable of obtaining results from minute amounts of analyte [attomole–zeptomole (10−18–10−21 mol) range]. In immunoassay and nucleic acid hybridization tests, these reactions or their components are used in two ways: as labels and as detection systems for labels of other types (e.g. enzymes). Acridinium esters, acridinium sulfonyl carboxamides, isoluminol derivatives, the photoprotein aequorin, and luciferases from the firefly, marine bacteria, Vargula (formerly Cypridina), and Renilla are the principal luminescent labels. CL and BL detection schemes have been developed for assaying alkaline phosphatase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, and xanthine oxidase labels. In addition, simple homogeneous assays are possible with CL acridinium ester and isoluminol labels, as well as with glucose 6-phosphate dehydrogenase labels. CL and BL reactions can be adapted for the analysis of enzymes, substrates, cofactors, inhibitors, and metal ions, for the study of cellular processes (e.g. phagocytosis), and for toxicity testing. Luciferase genes (luc, lux) are now used as reporter genes, and the gene expression is quantitated by measuring the expressed luciferase in a BL assay.