Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

The outer membrane of Gram-negative bacteria contain endotoxins called lipopolysaccharides, or LPS. The LPS has a hydrophilic oligosaccharide portion and a hydrophobic lipid A region.

Once inside the host, LPS-binding proteins recognize endotoxins. These proteins then bind the receptors on circulatory immune cells. This binding activates the immune cells, which release inflammatory factors. The over-production of these factors exaggerates the immune response, causing cell death.

To detect endotoxins using the Limulus Amoebocyte Lysate, or LAL assay, take a formulation containing endotoxins bound to the lipophilic surface of nanoparticles via their hydrophobic lipid A portions.

Add LAL reagent to the tube. This reagent is an aqueous lysate containing inactive enzymes and proteins derived from the amebocytes - horseshoe crab blood cells.

Incubate the tube to facilitate the binding of endotoxins to the lysate-derived inactive clotting factor C proenzyme, activating it autocatalytically.

The activated factor C cleaves another proenzyme, factor B, forming a protease that cleaves a pro-clotting enzyme and converts it into an active form - limulus clotting enzyme. This enzyme cleaves coagulogen - an inactive clotting protein - into coagulin.

Coagulin monomers bind via their hydrophobic tails to the hydrophobic heads of other coagulin monomers. This self-polymerization results in the formation of a homopolymer that appears as a gelatinous clot at the tube's base. Invert the tube gently.

A firm clot that remains at the bottom of the tube confirms the presence of endotoxins.