Patent ID: 6171809
Filing Date: 2001-01-09
Classification: C12Q

Abstract:
A method for detecting the presence of both firefly luciferase and renilla luciferase in a sample by measuring the luminescence of said sample comprising:(a) mixing said sample with a first reagent mixture containing firefly luciferin, adenosine triphosphate (ATP), co-factors necessary for firefly luciferase activity, dithiothreitol (DTT) or functional equivalents thereof, and adenosine monophosphate (AMP), the amounts of said firefly luciferin, ATP, co-factors, DTT or equivalent, and AMP being sufficient to produce luminescence having a duration of at least one hour and an intensity that varies substantially linearly with time; and(b) measuring the luminescence produced by the mixture of the sample and first reagent mixture of (a);(c) adding a second reagent mixture comprising a buffer solution containing coelenterazine and, as a free radical scavenger, EDTA or functional equivalents thereof, to the mixture resulting from (a) after said measuring of (b) in an amount sufficient to partially quench the luminescence measured in (b);(d) measuring the luminescence produced by the addition of (c), said luminescence having a greater intensity than measured in (b) an extended glow period of at least one hour and which varies substantially linearly with time.