Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans

Abstract

The sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans (LRGs) and lipid A was tested by using commercially available and experimentally formulated LAL reagents. The glucans included two kinds of beta-(1,3)-D-glucans, laminarin and curdlan, and cellulosic material, LAL-reactive material (LAL-RM), extracted from a hollow-fiber (Cuprophan) hemodialyzer. LAL-RM loses its LAL activity when it is digested with cellulase and thus appears to be a beta-(1,4)-D-glucan or a mixed glucan containing a substantial proportion of beta-(1,4) linkages. All LAL reagents tested were at least 1,000-fold more sensitive to endotoxin than to LRGs. The presence of the surfactant Zwittergent was shown to interfere with reactivity to LRGs; LAL reagents without added Zwittergent reacted more strongly to LRGs than did the same reagents containing Zwittergent. Chloroform extraction of LAL increased the reagents' sensitivity to both endotoxin and LRGs, but it was not responsible for LRG reactivity. The addition of Zwittergent significantly reduced the sensitivity of LAL reagents to lipid A. LAL without the surfactant was equally sensitive to endotoxin and lipid A. Both curdlan and LAL-RM amplified or enhanced the LAL response to endotoxin. Kinetic turbidimetric studies demonstrated that the enhancement was dependent on the glucan concentration.