Endotoxin assay by bioluminescence using mutant firefly luciferase
AnalBiochem_397_152.pdf 643 KB
Ahmed, Abo Bakr F
Limulus amebocyte lysate
The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005 EU/ml of endotoxin within 15 min.
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