Understanding Limulus Amebocyte Lysate:
In 1977, the U.S. Food and Drug Administration approved the use of limulus amebocyte lysate (LAL) for detecting bacterial endotoxins in pharmaceutical products. Specifically, LAL is an aqueous extract of blood cells from horseshoe crabs which when reacting with bacterial endotoxins forms a gel-clot. Bacterial endotoxins are strongly regulated and strictly tested for in the pharmaceutical industry because when they are injected into the human body they can be extremely dangerous.
USP chapter <85> titled “The Bacterial Endotoxin Test” outlines three test methods for bacterial endotoxins which are the gel-clot method, the turbidimetric method and the chromogenic method. Although these three methods are different they are based on the same principle which is the fact that bacterial endotoxins react with LAL.
Understanding how LAL reacts with bacterial endotoxins:
The reaction of LAL with bacterial endotoxins is known as the LAL cascade, shown in figure 1. The LAL cascade can be explained as follows; when bacterial endotoxins react with LAL, it first reacts with Factor C, causing it to become activated. In turn, sequential enzymes within the cascade become activated. Ultimately, the proclotting enzyme turns into the clotting enzyme which cleaves the coagulgen into coagulin. The coagulin is the protein from horseshoe crab blood that forms a gel-clot when reacted with bacterial endotoxins. This is the basis behind the gel-clot method. For the turbidimetric method, a turbidimetric assay is introduced to the reaction which causes turbidity. Then the intensity of the turbidity is measured with a spectrophotometer at 405 nm. This is the basis behind the turbidimetric method. For the chromogenic method, a chromogenic substrate is introduced to the reaction. If bacterial endotoxins are present, the substrate is cleaved and a yellow color is produced. The intensity of the yellow color is then measured with a spectrophotometer at 405 nm. This is the basis behind the chromogenic method. Again, all three methods are based on the same principle which is the fact that bacterial endotoxins react with LAL (an aqueous extract from horseshoe crab blood).
In conclusion, the unique thing about horseshoe crabs is not just their extraordinary structure or historic existence but a chemical found in their blood cells that can detect mere traces of bacterial endotoxins. For over 30 years, pharmaceutical companies have been taking advantage of this phenomenon to detect contamination in their products. If there are dangerous bacterial endotoxins in a sample, even at trace amounts, the horseshoe crabs blood extract will work its magic, turning the sample into a gel!
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