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.
Zhang
G H
GH
Department of Immunology, State Serum Institute, Copenhagen, Denmark.
Baek
L
L
Buchardt
O
O
Koch
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D060829:Arthropod Proteins; D001773:Blood Cells / Q000737:chemistry*; D002457:Cell Extracts / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004731:Endotoxins / Q000032:analysis* / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005189:False Positive Reactions; D005936:Glucans / Q000032:analysis* / Q000494:pharmacology; D006458:Hemolymph / Q000187:drug effects / Q000502:physiology*; D006737:Horseshoe Crabs / Q000737:chemistry / Q000502:physiology*; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D011112:Polymyxin B / Q000494:pharmacology; D011134:Polysaccharides / Q000032:analysis / Q000494:pharmacology; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000276:immunology; D047071:beta-Glucans
D000911:Antibodies, Monoclonal; D060829:Arthropod Proteins; D002457:Cell Extracts; D004731:Endotoxins; D004792:Enzyme Precursors; D005936:Glucans; D008070:Lipopolysaccharides; D011134:Polysaccharides; D047071:beta-Glucans; C038459:curdlan; C008247:laminaran; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C; D011112:Polymyxin B; D004121:Dimethyl Sulfoxide