Endotoxin Detection and Removal

Endotoxin is a complex lipopolysaccharide (LSP) present in the outer cell wall of gram-negative bacteria that contributes to the organization and stabilization of the cell membrane and is released into the circulation upon disruption of the membrane by cell death. Endotoxin is known to cause reactions in animals with symptoms of high fever, vasodilation, diarrhea, and in extreme cases, and fatal shock. It is composed of three regions: a core polysaccharide, a long chain polysaccharide, and a non-polar lipid called lipid A. The core polysaccharide has an outer hexose region and an inner heptose region, and the long chain polysaccharide is a strain specific surface antigen (O antigen) composed of repeated oligosaccharide subunits. Both core polysaccharide and O-antigen are hydrophilic, whereas lipid A is hydrophobic. Lipid A is composed of a hydrophilic, negatively charged bisphosphorylated diglucosamine backbone and a hydrophobic domain of acyl chains. Because of its unique structure, Lipid A is responsible for endotoxin biological function, specificity, and affinity to the relative proteins. Lipid A triggers the production of proinflammatory cytokines and activation of the coagulation cascade, leading to sepsis and septic shock. As little as one nanogram of endotoxin per kilogram of body weight per hour can cause a pyrogen reaction.

Although biological products are sterilized, the microbial endotoxin remains if gram-negative bacteria are present before sterilization. Because during sterilization, when organisms are killed, endotoxins are not eliminated but released. In the fields of biological products, medical devices, injectable drugs, food and water safety, the detection of endotoxin in finished products is an important part of ensuring the safety of sterilized products. Biological detection techniques include the rabbit pyrogen test (RPT), limulus amoebocyte lysate (LAL) assay, and the bovine whole blood test (BWBA), which are still in use today. But they are being replaced by newer and more accurate detection methods, such as biosensors with endotoxin affinity components as sensing elements. Because their inherent properties of fast response, easy operation, low cost, high sensitivity, and great specificity, biosensors satisfy the development requirements of endotoxin detection. Endotoxin has high stability, heat and pH resistance. In addition, endotoxin may form stable interactions with many biomolecules, which results in hiding endotoxin molecules, thus complicating endotoxin removal in downstream processes of protein purification. The removal of endotoxin is more challenging when it is associated with unstable proteins. Many methods are commonly used to reduce endotoxin contamination of protein preparations, including ion-exchange chromatography, affinity adsorbents (such as immobilized L-histidine, poly-l-lysine, poly (γ-methyl L-glutamic acid), and polymyxin B), gel filtration chromatography, ultrafiltration, sucrose gradient centrifugation, Triton X-114 phase separation, etc. Amerigo Scientific offers endotoxin assay products for accurate endotoxin detection or effective removal of endotoxin from aqueous samples.