Development of an endotoxin-specific Limulus amebocyte lysate test blocking beta-glucan-mediated pathway by carboxymethylated curdlan and its application

Tsuchiya, M.; Takaoka, A.; Tokioka, N.; Matsuura, S.

Nihon Saikingaku Zasshi. Japanese Journal of Bacteriology 45(6): 903-911


ISSN/ISBN: 0021-4930
PMID: 2082026
DOI: 10.3412/jsb.45.903
Accession: 039792502

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We developed a simple new endotoxin-specific assay method that uses Limulus amebocyte lysate (LAL) containing a sufficient amount of a water-soluble (1----3)-beta-D-glucan derivative as a blocker of the (1----3)-beta-D-glucan-mediated coagulation pathway. The addition of 0.1 mg/ml or more of carboxymethylated (1----3)-beta-D-glucan completely blocked the activation of LAL by (1----3)-beta-D-glucan itself. The assay of endotoxin was unaffected by the presence of 1 mg/ml carboxymethylated (1----3)-beta-D-glucan. Spiked endotoxin was recovered well from beta-glucans by the turbidimetric kinetic method with LAL containing 1 mg/ml of carboxymethylated (1----3)-beta-D-glucan. Besides, this new LAL formulation was applied for an endotoxin-specific assay by the conventional gel-clot method or the chromogenic method. Gram-negative bacteria were specifically detected by the turbidimetric kinetic method with the LAL formulation. This LAL formulation may be used for an endotoxin-specific assay not only in pharmacology but also in clinical microbiology.