This article discusses the role of lipopolysaccharides (LPS) in Gram-negative bacteria and their interaction with the immune system. It details the Limulus Amoebocyte Lysate (LAL) assay used for detecting endotoxins and the biochemical processes involved in this detection.
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.
To perform a gel-clot LAL, prepare control standard endotoxin to a final concentration of 4 lambda, and combine 100 microliters of standard with 100 microliters of water, or test sample, to achieve a final concentration of control standard endotoxin 2 lambda.
Add 100 microliters of lysate to each lambda dilution. Vortex the tubes briefly, and place the rack of tubes into a 37-degree Celsius water bath for 1 hour.
At the end of the incubation, invert the tubes with a smooth motion, and manually record the results using plus for a firm clot, and minus for no clot or a loose clot.
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