Endotoxin (lipopolysaccharide, LPS) is the major component of the outer membrane of gram-negative bacteria. Although there is a great compositional variation among endotoxins derived from different bacterial serotypes, they all share a common structural principle. Endotoxins are amphiphilic molecules consisting of a hydrophilic polysaccharide part and a covalently bound hydrophobic lipid component known as lipid A. The polysaccharide part can be divided into two subdomains, the core oligosaccharide and a heteropolysaccharide representing the surface antigen (O-antigen). Lipid A is the toxically active part of endotoxin, which does not act directly against cells or organs but by activating the immune system, especially the monocytes and macrophages. These cells release mediators, such as tumor necrosis factors, several interleukins, prostaglandins, colony stimulating factors, platelet activating factors and free radicals that have potent biological activities and are responsible for the adverse effects of endotoxin exposure. This affects structure and function of organs and cells, changes metabolic functions, raises body temperature, triggers the coagulation cascade, alters hemodynamics and causes shock.
Endotoxins are implicated in a number of pathophysiological conditions and diseases. The limulus amoebocyte lysate (LAL) assay based on horseshoe crab blood is the commonly used endotoxin detection method accepted by the pharmaceutical and medical device industries. In the LAL assay, coagulation cascade is initiated by the combination of endotoxins and zymogen factor C. The activated Factor C stimulates Factor B which converts the proclotting enzyme to the clotting enzyme. Finally, the two peptide bonds in the coagulogen are catalytically cleaved, resulting in the formation of the coagulin gel. All LAL assays are in principle based on this coagulation cascade, and they are modified to enable quantitative determination of endotoxins. The main LAL assay formats are available: gel-clot (semi-quantitative), turbidimetric, and chromogenic (quantitative). Recombinant Factor C (rFC) has been developed as non-animal-derived reagent for bacterial endotoxin detection, which allows more defined assay conditions and avoids the problem of relying on wild species.
Using LAL assays for detection of endotoxin in biological samples is limited because they are affected by components in the blood. In addition, heating causes morphological changes in fibrinogen, lipoproteins and platelets, which may alter their interaction with endotoxin, resulting in both false positive results and binding altered. Endotoxins have a net negative charge at physiological pH due to two phosphate groups on the disaccharide. And endotoxins contain long hydrophobic fatty acids chains. Thus, any molecule that is positively charged or contains a hydrophobic region is likely to bind to endotoxins and the binding is too strong to be diluted or removed by heating or extraction.
Endotoxin can bind to many factors in the blood, and enzymes in the cascade of LAL test may be bound, activated and inactivated by many components in the blood. Amerigo Scientific offers Endotoxin Sample Preparation (ESP™) kits to treat citrated human plasma to allow accurate endotoxin quantitation within 60 minutes. ESP™ is a sample treatment system to remove the interfering effects of blood plasma components on endotoxin and to achieve accurate detection and quantitation. Following the provided protocol, ESP™ components are compatible with recombinant Factor C endotoxin detection assays.
|Endotoxin Sample Preparation (ESP) Kit||ESP™ Buffer #1, ESP™ Buffer #2, ESP™ Protease Solution, and ESP™ Assay control buffer||Kit|
Figure 1. Removing effects of blood components on endotoxin by ESP™
Sub-nanogram levels of endotoxin can trigger immune responses and alter the phenotype and function of many cells including monocytes, neutrophils, dendritic cells, hepatocytes, vascular and respiratory epithelium, and arterial smooth muscle cells. Thus, strict guidelines for allowable endotoxin content have been established in the pharmaceutical and medical device fields. In addition, the presence of endotoxin affects many studies in life science, such as cell culture and animal model experiments. Thus, accurate endotoxin detection and quantitation is crucial for production of biological samples (such as protein/peptide). LAL and rFC assays are the main of endotoxin detection methods. They rely on enzyme reactions and are therefore susceptible to a range of factors that can inhibit or enhance enzyme performance. An accepted practice to address these issues is to dilute the sample until the inhibition/enhancement factor has no effect on the detection performance. However, sample dilution can reduce the detection sensitivity. For example, cationic proteins tightly bind to negative endotoxin molecules, and this binding cannot be dissociated by simple dilution. In these cases, it is inadequate and cumbersome if strategies such as perchloric acid treatment, phenol extraction, and dilution heating methods are used. There is limited success in using proteases to digest protein solutions prior to LAL or rFC assay. Factor C, the LAL enzyme that initiates the clotting cascade, is a serine protease as is the downstream enzyme Factor B and the proclotting enzyme. Therefore, using common serine proteases to digest protein samples can activate the LAL cascade. Amerigo Scientific offers a sample treatment kit that sufficiently degrades most proteins and peptides in 60 minutes without interfering with LAL and rFC assays for a more accurate endotoxin assay.
EndoPrep™ is a sample treatment system that removes the effects of proteins and peptides on endotoxin and enables accurate detection and quantitation. Following the provided protocol, the system components are compatible with both classical LAL and rFC endotoxin detection assays.
|EndoPrep™ Kit||SB™ Digestion Buffer and SB™ Protease Solution||Kit|
Figure 2. Effects of protein/peptide removal on endotoxin using EndooPrep™
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