Endotoxin Quantitation, Removal, and Detection

Endotoxins are heat-stable lipopolysaccharide (LPS) molecules associated with the outer membranes of certain gram-negative bacteria, such as E. coli. Endotoxins are routinely released by bacteria as they grow and divide in their normal life cycle, but large volumes of endotoxins can be released during cell lysis and have been associated with disease. Endotoxins in large quantities are toxic and can trigger endotoxic shock, inflammation, or sepsis in animals. Given these compounds are heat-stable and can survive sterilization procedures, sensitive endotoxin detection testing is essential in life science and medical research due to the low endotoxin levels required for in vitro and in vivo research.

Endotoxins are released into their surrounding environment when gram-negative bacteria undergo normal cell division. Because endotoxins are pyrogenic (fever-inducing) in mammals, the identification and measurement of endotoxin levels is extremely important in many research fields.

Endotoxins have been associated with non-alcoholic fatty liver disease (NAFLD), meningococcal disease, and various types of sepsis. Endotoxins can be problematic in burn victims due to the translocation of endogenous bacteria and loss of fluids, resulting in GI-related disease such as bowel ischemia or intestinal damage. Within the field of microbiome analysis, there have been reported cases of leaky gut syndrome due to an impaired intestinal barrier, leading to increased endotoxin, chronic inflammation, and hepatic damage. The severity of symptoms in Crohn's disease, ulcerative colitis, and cystic fibrosis has also been linked to LPS levels in the gut.

Whether your research requires endotoxin quantitation, removal, or detection, we are here to support all your experimental needs, including key assays, endotoxin-free consumables, and pyrogen-free accessories. Endotoxin testing of raw materials, intermediates, and final drug products is essential for patient safety. Our products are not licensed by the FDA and should not be used for the end-product release of pharmaceutical drugs, devices, or biologics that are FDA regulated and are intended for research use only.

Learn more about endotoxin quantitation, removal, and detection products below.

The Pierce Chromogenic Endotoxin Quant Kit uses the amebocyte lysate method to measure the interaction of endotoxins with proenzyme Factor C, which is sequestered from the amebocytes of horseshoe crabs. The proteolytic activity of this proenzyme is activated in the presence of LPS. Endotoxin levels are determined by measuring the activity of Factor C in the presence of the colorless chromogenic synthetic peptide substrate (Ac-Ile-Glu-Ala-Arg-pNA) that releases p-nitroaniline (pNA) after proteolysis, producing a yellow color that can be measured at an absorbance of 405 nm. The developed color intensity is directly proportional to the amount of endotoxin present in the sample and is calculated using a standard curve of a known amount of endotoxin. The Chromogenic Endotoxin Quantitation Kit is a highly sensitive quantitative assay that accurately measures and detects endotoxins in a protein, peptide, nucleic acid, or antibody sample. This assay has become the industry standard for endotoxin detection in pharmaceuticals, medical devices, food, and water testing, as well as life science and medical research.

Benefits of the Pierce Chromogenic Endotoxin Quant Kit include:

• Highly sensitive with a broad range—detect as little as 0.01 EU/mL to 1 EU/mL
• Specific—no interference from ß-glucans and suitable for a wide range of samples, including plasma, protein, vaccine, plasmid, or nucleic acids (DNA and RNA)
• Fast—perform assay in as little as 20 minutes
• Flexible—available in 30 to 240 reaction-size kits

Endotoxin assessment has become an important part of quality testing. Selecting the optimal resins for endotoxin removal is key, considering binding capacity and protein compatibility (see tables below). Thermo Scientific Pierce High-Capacity Endotoxin Removal Resin selectively binds and removes endotoxins from protein, peptide and antibody samples using a modified ε-poly-L-lysine [poly(ε-lysine)] affinity ligand. Endotoxin levels in biological samples are reduced by up to 99% in as little as 1 hour using our spin column format, and protein recovery is ≥ 85%. Pierce High-Capacity Endotoxin Removal Resin is available as a slurry to pack a custom column or in convenient pre-packed, single-use spin columns optimized for different sample volumes.

• Fast—endotoxin depletion within 1 hour
• High capacity—bind up to 2,000,000 EU/mL to eliminate >99% of endotoxins
• High performance—obtain endotoxin levels <5 EU/mL
• Clean—single-use of Endotoxin Removal Spin Columns avoid cross contamination
• Flexible—spin columns are optimized for different volumes
• Economical—reuse Pierce High-Capacity Endotoxin Removal Resin up to 10 times

Table 1. Efficiently remove endotoxins from heavily contaminated samples. Thermo Scientific Pierce High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were challenged for 1 hour with protein (2 mL of 1 mg/mL BSA) containing 5,000 to 500,000 EU/mL from E. coli strain 055:B5. Even at the highest endotoxin levels clean-up was >98%.

Table 2. Endotoxin removal and protein recovery of various proteins at different molecular weights and isoelectric points. Pierce High Capacity Endotoxin Removal Spin Columns, 0.5 mL, were challenged for 1 hour with different proteins of various molecular weight and charge. In all samples endotoxin removal was >99% and protein recovery was >85%.

Endotoxins are a natural component of the bacterial systems widely used to manufacture therapeutic proteins. When manufacturing therapeutic proteins or gene therapies, endotoxin detection and quantification must occur during the processing of biological samples to control endotoxin levels and prevent subsequent contamination. Endotoxin detection is particularly important for the biopharmaceutical industry to monitor endotoxin limits and adhere to endotoxin regulations given their pyrogenic properties and role in reduced transfection rates. When using endotoxin-sensitive cell lines, the presence of endotoxins can significantly reduce transfection efficiencies, thus emphasizing the importance of endotoxin detection.

Rapid Gel Clot Endotoxin Assay Kits use the Limulus amebocyte lysate (LAL) assay and provide a quick and easy positive (clot) or negative (no clot) reaction, with each kit detecting its range of sensitivity in EU/mL. LAL is widely used as a simple and sensitive assay for the detection of endotoxins. The Rapid Gel Clot Endotoxin Assay Kits have a fast, efficient protocol, are reproducible, and contain all the reagents and supplies needed. Each kit supplies positive control tubes, endotoxin-free tubes, and pipette tips required for each test. Each kit is sufficient for 10 tests, using 0.5mL of test sample.

Endotoxins are characterized by a net negative charge in solution and high heat stability. They tend to form large aggregates, given their hydrophobic properties. The characteristics of LPS molecules allow them to adhere to glassware, even after sterilization. Therefore, ensuring that your cell culture glassware and plastic accessories are endotoxin-free is critical. Maintaining these safeguards in which reagents and accessories used in cell culture workflows will achieve low levels of endotoxins to circumvent unwanted inflammatory responses in vitro and downstream contamination or cytotoxicity. Creating a clean experimental environment using endotoxin-free consumables and pyrogen-free accessories is imperative for cell culture or gene therapies. It will improve experimental success and prevent in vivo pyrogenic side effects.

Thermo Scientific provides high-quality, endotoxin-free accessories such as essential buffers, assay test tubes and plates, and beta-glucan blocking solutions, all of which help minimize invalid results and the need for repeating tests.