Differential blocking of coagulation-activating pathways of Limulus amebocyte lysate

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.