Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination

Carbon Nanotubes Activate Limulus Amebocyte Lysate Coagulation by Interface Adsorption

“…It is known that LAL zymogen can be inactivated by heat treatment, whereas coagulogen can remain its ability under this condition. 20 Hence we treated the gel-clotting LAL reagent at 70 °C for 20 min to prepare inactivated LAL (I-LAL). Then endotoxin or O-MWCNT-aq was incubated with I-LAL at 37 °C for 1 h. SDS-PAGE results showed that O-MWCNT-aq at each tested concentration (0.001−10 μg/mL) fail to induce the conversion of coagulogen to coagulin in I-LAL, indicating that only by activating certain enzymes in the gel-clotting LAL reagent so could O-MWCNT induce clot formation (Figure 3a).…”

“…LAL clots are composed of coagulin, therefore, the other enzymes would be left in the aqueous phase when the clots formed, and the supernatants contain the excess enzymes that can induce further coagulogen conversion. 20 Therefore, we supplemented O-MWCNT-aq in the gel-clotting LAL reagent to induce clot formation. After that, the clot was centrifuged and the supernatant was collected, which was then added into the I-LAL (Figure 3b).…”

“…Next we investigated whether the other proteins in LAL assay was required in the process of O-MWCNT-aq inducing LAL activation in addition to coagulogen. It is known that LAL zymogen can be inactivated by heat treatment, whereas coagulogen can remain its ability under this condition . Hence we treated the gel-clotting LAL reagent at 70 °C for 20 min to prepare inactivated LAL (I-LAL).…”