Endotoxin

What it determines

Endotoxin is the lipopolysaccharide component of gram-negative bacterial cell walls, and it persists through conditions that kill the organism itself. Limulus amebocyte lysate contains an enzyme cascade that endotoxin activates. The sample is reconstituted in endotoxin-free water, diluted, and combined with the lysate reagent in a microplate.

The plate reader follows the optical change in each well over time. The interval before a well reaches a set threshold varies with endotoxin concentration, and a standard curve prepared from a reference endotoxin converts that timing into a concentration. Each sample is run with a spiked control to confirm the peptide is compatible with the reaction.

Practical notes

• Everything the material touches carries endotoxin. Vials, stoppers, and reconstitution water all contribute, so samples for this assay benefit from depyrogenated containers and a dedicated unopened vial.
• Some peptides interfere with the enzyme cascade. Dilution usually relieves the interference, and the spiked control shows whether it did.