Discrimination Between Endotoxin And (l->3)-b-d-glucan Using Turbidimetric Kinetic Assay With Limulus Amebocyte Lysate

Abstract

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (l->3)-B-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (l->3)-/?-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Z)max) and the reaction time required to obtain Z)max (Jp) were defined. The logarithmic plottings of Tp versus Z)max (Tp-Z)max plot) discriminated between endotoxin and (l-*3)-/?-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Z)max (ET-Z)max plot). The endotoxin calculated from Z>max was less influenced by (l->3)-/l-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (l->3)-/l-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (l->3)-/f-D-glucan. (1) Identify the main substance (endotoxin or (l-3)-/?-D-glucan) triggering the activation of LAL using the Fp-Anax plot. (2) Use the appropriate method to measure the main substance: the ET-Z)max plot for endotoxin or the LAL with polymyxin B for (l-3)-/J-D-elucan. © 1990, The Pharmaceutical Society of Japan. All rights reserved.