Monomeric lipopolysaccharide (LPS) activity affected by dimerization of receptors is the key for the similarity between the Limulus amebocyte lysate (LAL) test and the rabbit pyrogen test

Abstract

Endotoxin or lipopolysaccharide (LPS) triggers inflammatory cytokine production of mammal cells through Toll-like receptor 4 (TLR4) activation. This reaction causes pyrogenicity in rabbits. The rabbit pyrogen test has been used for the pyrogen detection in parenteral drugs and medical devices. Limulus amebocyte lysate (LAL) test, adopted as an alternative test for the rabbit pyrogen test in pharmacopoeias, has been safely used for more than 40 years. Even though the mechanisms of these biological assays are different, there should be similarity between the LAL test and the rabbit pyrogen test. Biological activity relates to the aggregation states of LPS, and there is still controversy whether the biologically active form of LPS is monomers or aggregates. Previous studies showed inconsistent results in biological activities of monomeric LPS. This suggests the difficulty to prepare monomeric LPS. Therefore, a simulation can be helpful to understand the activity of different status of LPS. In this study, the TLR4 activation was simulated by using simplified models, and compared the activity of LPS in different forms. The activation of TLR4 and the LAL activation require dimerization of receptors, which reduces the activity of monomeric LPS. The inert monomeric LPS caused by the dimerization of the receptors is the key to the similarity between the LAL test and the rabbit pyrogen test.