A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan

Kambayashi, J.; Yokota, M.; Sakon, M.; Shiba, E.; Kawasaki, T.; Mori, T.; Tsuchiya, M.; Oishi, H.; Matsuura, S.

Journal of Biochemical and Biophysical Methods 22(2): 93-100


ISSN/ISBN: 0165-022X
PMID: 2061565
DOI: 10.1016/0165-022x(91)90022-o
Accession: 039092189

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The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.