Endotoxin Detection – from Limulus Amebocyte Lysate to Recombinant Factor C

Abstract

Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro-enzymatic endotoxin diagnostic assay for high-throughput screens of endotoxin. Using the rFC, Lonza Inc. has spawned the "PyroSense" which serves as checkpoints of the biotechnology production line. Thus, from cloning to commercial applications, the rFC has initiated a new era in endotoxin-testing for the quality assurance of biomedical products and for the healthcare industry, whilst sparing the endangered horseshoe crabs.

Probiotic supplementation attenuates increases in body mass and fat mass during high‐fat diet in healthy young adults

“…Interleukin (IL) 6 and tumor necrosis factor-a (TNFa) was measured with a BioPlexV R multiplex immunoassay (BioRad, Hercules, CA). LPS binding protein (LBP; Abnova, Taipei City, Taiwan) and soluble cluster of differentiation (sCD)14; (Minneapolis, MN) of the LPS-TLR4 signaling cascade were measured by ELISA as these proteins have been suggested to be more stable biological indicators of endotoxin exposure given the known difficulty of detecting circulating endotoxin (13,14). High-sensitivity Creactive protein (hsCRP; R&D Systems, Minneapolis, MN) was also measured with a commercially available ELISA.…”

“…Pyruvate oxidation was used to assess the activity of pyruvate dehydrogenase (PDH), the enzyme that catalyzes the oxidation of pyruvate and the provision of glucose-derived acetyl CoA to the TCA. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-pyruvate oxidation was assessed in a similar manner to fatty acid oxidation with the exception that pyruvate was substituted for BSA-bound palmitic acid. All samples were run in triplicate and data were normalized to total protein content and expressed in nmol/mg protein/h.…”

Metabolic endotoxemia with obesity: Is it real and is it relevant?

“…In patients with sepsis, the concentration of circulating endotoxin is often elevated a hundred fold or higher compared to age-matched, healthy controls (e.g., 581±49 vs. 5.1±7.3 pg/mL, respectively) 14 . The elevation in endotoxin in bacterial infection (endotoxemia) results in mass overproduction of pro-inflammatory cytokines, which can lead to shock, cell damage, and potentially multiple organ failure 15 . Conversely, Cani et al 4 described “metabolic endotoxemia” as a condition of chronically elevated plasma LPS at levels 10–50 times lower than during septic conditions.…”

Oxysterols exert proinflammatory effects in placental trophoblasts via TLR4-dependent, cholesterol-sensitive activation of NF- B

“…The limulus amoebocyte lysates (LAL) assay is commonly used to detect and quantify bacterial endotoxins in the quality assurance of medical devices, injectable drugs and biological preparations (Ding and Ho, 2010). To ensure that the proinflammatory effects of oxysterols were not the result of endotoxin contamination, the LAL assay was performed according to the manufacturer's instructions.…”