Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate.

Abstract:

:The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.

journal_name

J Clin Microbiol

authors

Zhang GH,Baek L,Buchardt O,Koch C

doi

10.1128/JCM.32.6.1537-1541.1994

subject

Has Abstract

pub_date

1994-06-01 00:00:00

pages

1537-41

issue

6

eissn

0095-1137

issn

1098-660X

journal_volume

32

pub_type

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