Fact Sheet: Overview Of Bacterial Endotoxin

Bacterial Endotoxin Overview:
•    Bacterial endotoxins, also known as lipopolysaccharides, are hydrophobic molecules that form the majority of the out membrane of Gram-negative bacteria.
•    Endotoxin is released when the bacteria dies and the outer membrane disintegrates.
•    Endotoxins have a high heat stability so are not destroyed through regular sterilisation methods, and must be removed through depyrogenation.
•    Endotoxin is a pyrogenic molecule and when entered into the blood stream, releases cytokines such as interleukin 1,  produces tumor necrosis factor, and triggers an immune response, usually inflammation
•    Bacterial endotoxin is known to cause fever, sepsis, and has even been linked to Sudden Infant Death Syndrome (SIDS).

History of Lysate Testing:
•    Pyrogenicity testing was first performed using the rabbit pyrogen test (RPT) however after years of research an in vitro method known as Limulus Amebocyte Lysate (LAL) or Tachypleus Amebocyte Lysate (TAL) was discovered.
•    This lysate is an aqueous extract of amebocyte cells found in certain types of horsehoe crabs as their blood has evolved to clot whenever exposed to pathogens; blood is removed from the pericardium of the crab, after which it is returned to the water.
•    These clotting capabilities were first observed in 1885 at Johns Hopkins University, then in 1953 Frederik B. Bang observed that clotting in the horsehoe crab’s blood only occurred when exposed to Gram-negative bacteria.
•    The first detailed modern description of clotting taking place in Limulus / Tachypleus occurred in 1964, where it was also discovered that endotoxin is a key factor in the clotting of the blood.

In the Lab / at Wickham Micro Ltd
•    LAL was the first used in the diagnosis of human disease in 1970. By 1977, the FDA permitted this method to be used as a replacement for RPT for detecting endotoxin in finished pharmaceutical, raw materials, and medical devices.
•    This testing, now known as bacterial endotoxin testing (BET) due to intertnational harmonisation, is an established pharmacopeial method (Ph. Eur 2.6.14, USP <85>, JP 4.0.1) for the screening of parental medicines, irrigation fluids, dialysis solutions, and purified water.
•    It is also used to access the safety of medical devices designed to come into direct or indirect contact with blood and body tissues by testing for endotoxins via an extraction process used to flush material off the article ready for combining with the lysate reagent ().
•    There are three standard methods of BET which include gel clot (assay using dilutions), chromogenic (a quantitative method using chromophores)  and  turbidmetric (a quantitative method measuring turbidity via spectrophotometry)
•    BET is only valid for detecting endotoxins and not the presence or absence of other pyrogenic compounds; the RPT or the newer in vitro method, Monocyte Activation Test (MAT), must still be used in testing for the presence of other types of pyrogens.