Best Niche Antibody-based Biologics

Regarding Endotoxin

regarding endotoxins

03/31/2023 Updated

The interaction of endotoxins with TLR (Toll-like receptor)/CD14/MD2 receptor complexes and a variety of TRP (transient receptor potential) ion channels can activate a broad spectrum of cells which can be a source of significant variability of the experimental background. Protein reagents produced by ABB contain very low levels of endotoxin and are suitable for most in vitro and in vivo studies.

1. What is endotoxin

Endotoxin was considered to be a toxin retained inside the bacterial cell as in contrast to the exotoxin that is released into the surroundings by live bacteria. Although subsequent work showed that endotoxin can be released by live bacteria as well. Today endotoxin is commonly used as a synonym for lipopolysaccharides (LPS). LPS is the major component of the outer membrane of Gram-negative bacteria. Gram-negative bacteria do not retain crystal violet as they carry a thin cell wall which is made of a single layer of peptidoglycan surrounded by the outer membrane which invariably contains LPS. During Gram-negative sepsis, endotoxin binds to host macrophages and induces release of pro-inflammatory cytokines and excessive inflammation can lead to multiple organ failure and death.

2. Biochemical nature of endotoxin

LPS on the outer membrane of Gram-negative bacteria is made of three parts: the O-antigen, the core oligosaccharide and the lipid A. LPS is well known for being highly thermostable.

The O-antigen also called O polysaccharide is a repetitive glycan polymer comprised the outermost domain of the LPS. Variation in the exact sugar content of the O-antigen accounts for multiple serotypes. Together with Lipid A (see below) O-antigen contributes the virulence of Gram-negative bacteria.

The core oligosaccharide contains sugars such as heptose and Keto-deoxyoctulosonate (also known as KDO). In addition, the LPS core may also contain non-carbohydrate components such as phosphate, amino acids, and ethanolamine substitutes.

Lipid A is a phosphorylated glucosamine disaccharide decorated with multiple fatty acid chains. The fatty acids provide the hydrophobic anchors to the bacterial membrane and facilitate the rest of the LPS projects outward from the cell surface. While Lipid is quite conserved in general, its subtle structural variations among Gram-negative bacteria nonetheless determine the ultimate immune response of the infected host.

3. Biological effects of endotoxin

LPS is best known for its inflammation- inducing activity which result in fevers and pains. At high concentration in circulation, it can trigger septic shock.  

LPS binds to the canonical CD14/TLR4/MD2 receptor complex on the surface of many cell types including monocytes, dendritic cells, macrophages and B cells and triggers releasing of proinflammatory cytokines, nitric oxide, superoxide, and eicosanoids. Damaged endothelial barrier by these mediators can cause capillary leak syndromes, blood vessel dilation, low cardiac output and ultimately lead to sepsis shock. The loss of endothelial integrity can also trigger disseminated intravascular coagulation and result in thrombosis and is the main cause for loss-of-function of certain internal organs such as kidney, adrenal glands, and lungs. Similar vascular damage coupled with depletion of coagulating factors may be manifested as skin petechiae, purpura and ecchymoses.

Most recently, LPS was found to activate several members of the Transient Receptor Potential (TRP) family of cation channels. Mammalian TRP ion channel family consists of 28 members divided into 6 subgroups expressed in neuronal and non-neuronal cells. Even though it is not clear how many TRPs interact with LPS, at least TRPA1, TRPV1, TRPM3, and TRPM8 are responding to LPS with the first two members account for the majority of the LPS induced neuronal activation. For example, the pain and neurogenic inflammation induced upon exposure to LPS are TRPA1-dependent. In addition, the response of TRPV4 on the epithelial lining of the mucosal surface along the respiratory track to LPS is TLR4-independent which induces nitric oxide and serves as bactericidal defense mechanism.

The effects of LPS on TPR ion channels are not very well studied. Nonetheless, since TLRs and TRPs are co-expressed in many tissues, it would be of interest to explore the interplay of these two LPS-detecting pathways and the potential crosstalk of their intracellular signaling cascades.

4. Measuring endotoxin content.

The current FDA-approved assay for detecting the endotoxin content is the Limulus Amebocytes Lysate (LAL) assay which uses the blood from the Horseshoe crab (Limulus Polyphemus). LAL assay is quite sensitive and routinely detects down to picogram levels of LPS. However, limited supply of the horseshoe crabs has called for alternatives for endotoxin testing. Indeed, an ELISA based test has been developed using a recombinant factor C which is the initiating component of the coagulation cascade in LAL assay. Endotoxin content is expressed by Endotoxin Unit (EU) rather than by weight since its potency dependents on additional factors such as length and the linkage of the polysaccharide chains, material sources, solubility in the sampling fluid, associated non-LPS molecule(s), etc. Nonetheless, in general, 10 EU/ml solution contains approximately 1 ng/ml endotoxin

5. Endotoxin limits

FDA ‘s guideline calls for maximal endotoxin allowed for parenteral and intrathecal administration to be 5 EU/kg and 0.2 EU/kg, respectively. Accordingly,

the maximal endotoxin can be administered to a 30-gram mouse is 0.15 EU

(5.0 EU/kg x 0.03 kg/mouse = 0.15 EU/mouse); and for

an injection solution with of 1 EU/ml, the maximal injection volume is 0.15 ml/mouse

(0.15 EU/mouse ÷ 1 EU/ml = 0.15 ml/mouse);

Assuming the bolus dosing volume of 0.5 ml, the limits of the injecting solution is 0.3 EU/ml

 (0.15 EU/mouse ÷ 0.5ml/mouse = 0.3 EU/ml).

For intrathecal application, a 25-fold (5 EU/kg ÷ 0.2 EU/kg = 25) reduction of the endotoxin content is required.

6. Why the endotoxin content of protein reagents is important for your experiments

In light of the broad expression of the TLR receptors and the TRP ion channels, it is clear that endotoxins can affect both in vitro and in vivo cell growth and function and are a source of significant variability of experimental results. For studies in cell cultures, increasingly documented were the activation of leukocytes and macrophages which leads to production of cytokines and inflammatory mediators, inhibition of murine erythroid colony formation by very low levels (<1.0 ng/ml) of endotoxin. In animal studies, endotoxins often elicit an inflammatory response. The presence of endotoxin in products for injection (vaccines and injectable drugs) can result in pyrogenic responses ranging from fever and chills to irreversible and fatal septic shock.


  1. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. 282:2085-8, 1998
  2. TRP Channels as Sensors of Bacterial Endotoxins. Boonen B, Alpizar YA, Meseguer VM, Talavera K. Toxins (Basel) 10: 326,  2018
  3. Biochemical principle of Limulus test for detecting bacterial endotoxins. Iwanaga S. Proc Jpn Acad Ser B Phys Biol Sci. 83:110-9, 2007
  4. A new era in pyrogen testing. Ding JLHo B. Trends Biotechnol. 19:277-81, 2001
  5. Guidance for Industry. Pyrogen and endotoxins testing: questions and answers. June 2012 Compliance.