Enhanced Chemiluminescent Detection of Horseradish Peroxidase Labeled Probes

Enhanced chemiluminescence (ECL) is a generic detection system that has been applied to all standard, membrane-based molecular biology techniques (1 ). The technology is based on the well-characterized horseradish peroxidase (HRP) catalyzed oxidation of luminol in the presence of peroxide (2 ). In this reaction a cyclic scheme, involving the enzyme, leads to the formation of luminol radicals (3 ). These radicals decay through an excited intermediate compound (3-aminophthalate), and as this compound returns to the ground state light is emitted. The enhanced reaction offers a major improvement over the basic scheme in that the light output is increased over 1000 fold, whereas the background is reduced (4 ), The light emitted has a maximum wavelength of 428 nm and can be captured with high efficiency by blue-light sensitive X-ray film. Consequently, the final result is very similar in appearance to the results obtained with traditional procedures for the detection of radioactive labels; a hard copy is obtained, multiple exposures can be taken, and the results can be analyzed by densitometry and easily reproduced for presentations and publications.