Experimental application of a synthetic luminescent substrate assay using endotoxin-specific limulus amebocyte lysate to human blood

Abstract

A synthetic luminescent substrate method, using a mutant-type luciferase whose luminescence intensity is more than ten times as intense as the wild type, was developed recently. We conducted the first basic studies on clinical application of the novel endotoxin measurement method. We assessed and established measurement conditions, including reagent concentrations and reaction time, so that it would be possible to apply the luminescent synthetic substrate method proposed by Noda et al. to measurements in human blood. When we added lipopolysaccharide (LPS) to water, it was possible to measure LPS at a concentration of 0.1pg/ml, whereas it was possible to measure LPS in tenfold diluted and heated plasma at a concentration of 1pg/ml. When plasma was further diluted, inhibiting activity decreased considerably. Thus, it will be necessary to completely eliminate the inhibitor present in plasma. However, the shortest time after collecting the specimen in which it was possible to make measurements was 30-40min, suggesting that if an assay is established, it will be possible to use the method as a novel blood endotoxin assay.