Spectral characteristics of bacterial endotoxin from different sources and their effects on the immune response of monocytes
Objective To compare the differences in spectral characteristics and monocyte immune effects for bacterial endotoxin from different sources.Methods Ultraviolet absorption spectrum and three-dimensional fluorescence spectrum were used to identify excipients(polyethylene glycol 8000 and α-lactose)and biological impurities(protein and nucleic acid)in 5 kinds of control standard endotoxin.Biological contamination was further confirmed by the detection of Toll-like receptors(TLRs)in HL-60 cells.The monocyte activation test was used to compare the release of the inflammatory cytokines interleukin 6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)caused by 5 kinds of endotoxins on HL-60 cells.Results As for control standard endotoxin,absorption peaks related to excipients were observed in ultraviolet absorption spectrum,while fluorescence peaks related to excipients and proteins were shown in three-dimensional fluorescence spectrum.The expression levels of TLRs of HL-60 cells indicated that protein contamination was more significant than nucleic acid contamination.All endotoxins induced improvement in cellular IL-6 and IL-1β gene relative expression levels and their protein mass concentrations in HL-60 cells.However,the TNF-α gene relative expression levels and its protein concentrations were not elevated.Endotoxins from the same or different bacterial strains had significant differences in their ability to induce inflammation under the same endotoxin activities.Conclusion Excipients and protein contamination of endotoxins are identified by spectral detection,while proteins and nucleic acids in biological impurity are confirmed by the TLRs assay of HL-60 cells.Molecular structure of endotoxins,as well as protein contamination influence the ability of endotoxins to induce the release of the inflammatory cytokines.
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