Limulus Amebocyte Lysate Test Using Chromogenic Methods Of Endotoxin Detection

By Lisa Komski, M.T., ASCP Senior Manager, Export And LAL; Wako Chemicals USA

The bacterial endotoxin detection method using Limulus Amebocyte Lysate (LAL) was developed in the 1960s. Since then, it has served as a highly useful tool in both research and industry. Since the beginning of the century, it was known that the hemolymph of the crab Limulus Polyphemus went through a process of coagulation. Later, it was discovered that the coagulation of the hemolymph was caused by the Gram-negative bacteria, which triggered a series of enzymatic chain reactions. These enzymatic reactions occur in amobecytes due to the presence of the endotoxins that constitute the cellular wall of the Gram-negative bacteria and they result in the formation of the coagulant, the protein causing the process of coagulation.

Later on, research into the enzymatic chain reactions that are produced in the amebocytes of Limulus indicated that these reactions are capable of breaking the peptide bonds that are found connected to the p-nitroaniline molecules. Upon the release of the p-nitroaniline, which is a yellow colourant, the solution becomes colored. By adding a peptide connected to p-nitroaniline to the LAL reagent in the presence of endotoxins, color is built up in the solution and therefore, the detection of endotoxins via a chromogenic method can take place. In this case, a spectrophotometer is needed to quantify the concentration of the released colourant, which is the product of the enzymatic reaction. This detection method could seem expensive due to the fact that it requires a spectrophotometer; however, it is not costly if you take into account that the spectrophotometer has various uses in any laboratory. Therefore, any equipment that is specific for this test would not be needed.