Abstract
Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active. We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. Furthermore, whereas masked endotoxin cannot be detected in Factor C based assays, it is still detectable in a cell-based TLR4-NF-κB-luciferase reporter gene assay. Moreover, in primary human monocytes, masked LPS induces the expression of pro-inflammatory cytokines and surface activation markers even at very low concentrations. We therefore conclude that masked LPS is a potent trigger of immune responses, which emphasizes the potential danger of masked LPS, as it may pose a health threat in pharmaceutical products or compromise experimental results.
Highlights
Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins
The term “low endotoxin recovery” (LER) describes the inability of the limulus amebocyte lysate (LAL)-based assay to detect endotoxin due to a “masking effect”, which occurs as a result of formation of a macromolecular complex that prevents Factor C from binding to endotoxin, even while positive controls show no evidence of test interference
Even commercially prepared proteins expressed by Gram-negative species may be contaminated by endotoxin up to several EU/ml[21]
Summary
Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. The term “low endotoxin recovery” (LER) describes the inability of the limulus amebocyte lysate (LAL)-based assay to detect endotoxin due to a “masking effect”, which occurs as a result of formation of a macromolecular complex that prevents Factor C (the main component of the LAL coagulation cascade) from binding to endotoxin, even while positive controls show no evidence of test interference. CD14 transports LPS to myeloid differentiation protein 2 (MD2) in the TLR4-MD2 complex[8,9]
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