Endotoxin Testing Services
The Native Antigen Company offers services to quantitate endotoxin in samples using the LAL assay.
About Endotoxin Testing
What are endotoxins?
Endotoxins are small (~10 kDa) lipopolysacchride (LPS) molecules associated with the outer membranes of gram-negative bacteria. They are released from bacteria at low levels during normal cell growth but also in much larger quantities after cell death, when their cell membranes rupture and disintegrate. The released LPS is composed of three distinct regions: O antigen, a core domain and Lipid A. O antigen is found on the outermost domain of the LPS molecule and is associated with immunogenicity; Core domain is composed of sugars and other non-carbohydrates and attaches directly to Lipid A; Lipid A anchors the LPS into the bacterial membrane and it is this part of the molecule which is responsible for the toxicity of gram-negative bacteria. Indeed, endotoxin is known to be a potent inflammatory mediator and pyrogen in vivo and can result in toxic shock, tissue injury, and death.
Why are endotoxins a problem in bioproduction?
Recombinant proteins, particularly those manufactured in E. coli, are often contaminated with endotoxin. In fact, a single E. coli cell can release up to 2 million LPS molecules. These contaminants are hydrophobic giving them a strong affinity for laboratory plasticware and are stable at high temperature, making it impossible to destroy them by sterilization. Water used to make routine buffers and solutions can be a source of endotoxins as can be reagents such as sera and media. Poor aseptic technique is another potential source of LPS. During the bioproduction process, the host cells will normally be removed by centrifugation or filtration; however, any endotoxin contamination will remain and a significant proportion is likely to be carried through the purification process. As such, endotoxins are frequent contaminants of recombinant DNA and protein solutions. Unfortunately, the presence of these toxins, even at relatively low levels, can significantly affect the results of both in vivo and in vitro biological assays causing non-specific cellular responses and false readings in cell-based assays. Endotoxin-free production may be carried out to try and reduce the LPS burden but ultimately this is no guarantee of success, and endotoxin levels above acceptable thresholds may still be present within the final preparation.
Endotoxin is measured in endotoxin units per milliliter (EU/mL) where 1 EU equals approximately 0.1-0.2 ng endotoxin/ml of solution. Generally, endotoxin levels below 0.1 EU/ml are considered acceptable for in vivo studies. However, even lower amounts of endotoxin have been shown to cause changes in physiology in vivo and it is likely that acceptable levels will vary across different assays and experimental conditions.
About Endotoxin Testing
Testing for endotoxins
We offer a service to detect and quantitate the amount of endotoxin in your sample using a kinetic chromogenic assay, called the Limulus Amebocyte Lysate (LAL) test. LAL comprises blood cells (amebocytes) from the Atlantic horseshoe crab (Limulus polyphemus) which clot when they encounter bacterial endotoxin lipopolysaccharides. This clotting can be detected using a chromogenic substrate where the enzymatic reaction between the endotoxin and lysate produces a yellow colour. The intensity of this colouration is directly linked to the quantity of endotoxin present in the sample.
What we offer
+ We use a chromogenic method that has been FDA approved for all stages of therapeutic product development.
+ Multiple samples types can be tested, including DNA, recombinant proteins and antibodies, with the sensitivity to detect endotoxin levels down to 0.005 EU/ml.
+ Routine test results are completed in 1-2 days.
Test samples are compared to a standard curve prepared from known endotoxin concentrations and all tests are performed in duplicate alongside a positive and negative control, as recommended by the harmonized USP Bacterial Endotoxins Test (BET) and the FDA guideline for LAL testing.