Bacterial endotoxin test (revision 2)

The bacterial endotoxin test detects lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria that provokes a febrile response when introduced parenterally. The test exploits a clotting cascade in amoebocyte lysate derived from horseshoe crab blood, which is activated by picogram quantities of endotoxin.^[1]

Endotoxin is not a living organism and is not removed by sterilisation. A preparation can be sterile and pyrogenic at the same time: autoclaving kills bacteria but leaves their lipopolysaccharide intact and biologically active. Endotoxin is the commonest but not the only pyrogen, and a separate rabbit pyrogen test exists for the wider class.^[2] This is why endotoxin and sterility are separate determinations answering separate questions.^[1]

Three variants are recognised. The gel-clot method observes whether a firm clot forms at a given dilution and gives a limit result rather than a number. The turbidimetric method follows the increase in turbidity as the cascade proceeds and is quantitative. The chromogenic method measures release of a coloured product from a synthetic substrate and is also quantitative.^[1]

Recombinant factor C assays, which use a recombinant version of the initiating enzyme rather than crab-derived lysate, have been introduced and are increasingly accepted. They avoid the animal-derived reagent and are more specific, since they respond only to endotoxin and not to β-glucans.