Endotoxin assessment

Endotoxins are lipopolysaccharide molecules located in the cell wall of gram-negative bacteria. Endotoxins are not secreted by the bacteria, but they are released to the environment after cell lysis. The molecules may be toxic to humans and animals, and may cause feverm diarrhea, sepsis and altered resistance to bacterial infections. Endotoxins are very stable and can withstand very high temperature. Because endotoxins are toxic to individual cells, it is important in cell based laboratory work that all materials (comsumables, containers, reagents, liquids, etc.) are free of endotoxins. Even small amounts of the molecules can have severe effects in all experiments that usue cell lines or primary cells.

Endotoxin assays are therefore commonly used for quality control purposes to confirm that materials are endotoxin-free and safe to be used. The bacterial endotoxin assayiss based on using the LAL reagent. The reagent is an amebocyte extract from the Horseshoe Crab (Limulus polyphemus).

Researchers have demonstrated that when a gram-negative bacterium infects a Horseshoe Crab, the immune response is an intravascular clotting reaction. This coagulation results from a reaction between endotoxin and a clottable protein secreted by amebocytes. The same endotoxin clotting reaction is utilized in commercial LAL endotoxin assays. The formation of clot causes increase in turbidity of the sample. This process can be measured in kinetic format on a microplate photometer by Multiskan FC.