JoVE Encyclopedia of Experiments
Biological Techniques
0 views • 4:38 min • July 8th, 2025
Endotoxin, a gram-negative bacterial toxin present on the outer membrane, is a lipopolysaccharide, LPS, composed mainly of lipids and polysaccharides, including lipid A and O-antigen. When released during infection, it is recognized by the immune system, triggering cytokine-based inflammatory responses.
To estimate endotoxin contamination in a liposomal drug nano-formulation using the Limulus amoebocyte lysate, LAL, assay, prepare reaction tubes, one containing the drug and the other containing the drug spiked with a known endotoxin concentration.
Add LAL reagent containing protein lysate obtained from horseshoe crab, Limulus polyphemus, blood cells, or amoebocytes. The lysate includes inactive serine proteases - factor B, factor C, proclotting enzyme - and a gel-clotting protein, coagulogen.
Vortex the tubes and load them into a turbidimeter to measure the turbidity of the solutions.
In the presence of contaminating LPS bound to liposomal drugs, the LPS-sensitive factor C gets autocatalytically activated, which then cleaves and activates factor B that converts the pro-clotting enzyme to clotting enzyme. This activated enzyme cleaves the coagulogen peptide bonds, forming coagulin m
View the full transcript and gain access to thousands of scientific videos