The Kinetic Limulus Amebocyte Lysate (LAL) Endotoxin Binding Assay
Lipopolysaccharides (LPS), also known as endotoxin, are an integral component of the gram-negative bacterial cell membrane and are responsible for many of the toxic effects that occur during bacterial sepsis. Endotoxin directly stimulates host monocytes and macrophages to secrete a wide panel of inflammatory cytokines.
The late-breaking enterohaemorrhagic Escherichia coli EHEC epidemy in Germany once more demonstrates the urgend need for novel and innovative approaches to fight systemic microbal attack.
The most applied ligand binding assay for the quantitative detection of endotoxins is the limulus amebocyte lysate (LAL) assay. This ligand binding assay is a typical liquid phase assay par excellence. The assay comes in a 2.0 mL tube format, and combines only limulus amebocyte lysate (LAL) with test sample. The appearance of turbidity is observed after a defined time period. Endotoxin is present in the sample if a cloudy gel has formed, this observation-based yes/no decision requires some experience of the lab staff (I did it once and found it quite difficult, but every assay result you can´t express in numbers is a sort of flubdub, anyway…).
The kinetic turbidimetric limulus amebocyte lysate (LAL) assay in a 96-well plate format allows for high throughput screening, the appearance of turbidity is permanently monitored in a multiwell plate reader.
A chromogenic assay variant comprises only two assay components: limulus amebocyte lysate (LAL), and a chromogenic substrate (peptide-p-nitroaniline). The mixture is combined with a sample containing endotoxins in a transparent microwell plate, and incubated. If endotoxin is present in the sample, it binds to Factor C, and the conformational change activates enzymatic activity on the chromogenic substrate. The cleavage of the peptide-p-nitroaniline conjugate releases p-nitroaniline and generates a detectable signal. The signal intensity increases with the amount of endotoxin present in the sample.
The rFC Liquid Phase Fluorogenic Endotoxin Binding Assay
One of the key patents was filed in 1994 (US 5,716,834) comprising recombinant Factor C (rFC) of the Singapore horseshoe crap Carcinoscorpius rotundicauda for endotoxin testing (1). Factor C is a serine protease being the first component of the horseshoe crap intra-vascular coagulation cascade.
The “methods and reagents for detecting endotoxin” patent application (US 2003/0054432 A1) comprising Carcinoscorpius rotundicauda horseshoe crap recombinant Factor C (rFC) in the use of a ligand binding assay was filed in 2001 by BioWhittaker Inc (2), a company formerly aquired by Cambrex Corp. in 1997. Camprex distributed a chromogenic recombinant Factor C (rFC) endotoxin detection assay under the PyroGene® trademark. In 2007 Lonza Group aquired Cambrex´s Bioproducts and Biopharma segment, also including the endotoxin assay product line, giving Lonza the right to market the PyroGene® rFC assay under its own name.
Said patent application used the Baculovirus expression system for rFC production, certainly because the preparations are expected to be endotoxin-free.
This ligand binding assay is a typical liquid phase assay par excellence. It comprises only three assay components: recombinant Factor C (rFC), a surfactant, and a fluorogenic substrate (DPR-coumarin). The mixture is combined with a sample containing endotoxins in a black microwell plate, and incubated. If endotoxin is present in the sample, it binds to rFC, and the conformational change activates enzymatic activity on the fluorogenic substrate (1). The cleavage of the DPR-coumarin conjugate generates a detectable signal. The signal intensity increases with the amount of endotoxin present in the sample.
By the way patent US 7,297,551 applied the 38 kDa LPS binding domain of recombinant Factor C (3) to the removal of endotoxin traces from injectable components (like glucose or xylitol solutions). And patent US 7,939,492 even anvanced the same rFC fragment as an anti-microbial agent for sepsis treatment (4).
(1) Ding JL and Bow H (1994), Patent US 5,716,834.
(2) Chen et al (2003), Patent Application US 2003/0054432 A1.
(3) Ding JL et al (2003), Patent US 7,297,551.
(4) Ding JL et al (2007), Patent US 7,939,492.
PyroGene® is a trademark owned by Lonza Group.