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 ,-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 104 to 106 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 Alkaligenes faecalis and for laminarin from Laminaria digitata. We also found that the j-glucan-activated pathway could be totally blocked by laminarin (>1 ,ug/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 I-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the P-glucan pathway, but not the endotoxin pathway, of LAL.

Pivotal Advance: Activation of cell surface Toll-like receptors causes shedding of the hemoglobin scavenger receptor CD163

“…b Zymosan contains ␤-glucans, which are known to activate the LAL, potentially resulting in a false positive for endotoxin contamination [23]. before assaying for CD163 shedding.…”

House Dust Induces IL-6 and IL-8 Response in A549 Epithelial Cells

“…Lipopolysaccharide (LPS) and b-1-3 D-glucan originating respectively from Gram-negative bacteria and fungi are among the possible agents in dust responsible for the various symptoms and illnesses upon inhalation (Fogelmark et al, 1991;Fogelmark et al, 1992;Herbert et al, 1992;Michel et al, 1991;Rylander et al, 1989;Rylander et al, 1990;Rylander et al, 1992). A bioassay commonly used for such exposure assessments is the Limulus assay, which can be used to measure not only endotoxin but also b-1-3 D-glucan (Zhang et al, 1994); however, the reproducibility of this assay may be poor. Alternatively, determination of unique chemical structures, microbial markers, by gas chromatography-mass spectrometry or tandem mass spectrometry (GC-MSMS) can be applied.…”

Self-diagnosis of Vulvovaginal Candidiasis is Poor - A Comparison of Diagnostic Methods Introducing β-Glucan as a Complement

“…Several explanations for partly inhibitory effects of high amounts of yeast cells are plausible. One explanation could be that presence of excess amounts of β-glucan disturbs the binding to the protein participating in the activation of the proclotting enzyme system of the Limulus ameobocyte lysate [24]. Possibly, high numbers of yeast cells in vaginal fluids need to be diluted further or heat-inactivated, in order to counteract inhibitory activities on the assay system.…”