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Updated: Jul 1, 2026
Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients
Published on: June 24, 2019
[Study of in vitro endotoxin neutralization effect and antibacterial activity of limulus anti-lipopolysaccharide
Hong-Fu Gao1, Guang-Xia Xiao, Pei-Yuan Xia
1Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing 400038, PR China.
Objective:
To investigate the role of REMP2 derived from limulus anti-lipopolysaccharide factor in neutralizing endotoxin in vitro and its antibacterial activity.
Methods:
(1) REMP2 and PMB in the concentrations of 100.00, 10.00, 1.00, 0.10, 0.01 micromol/L were respectively mixed with LPS (lEU/mL), with PMB as positive control. The LPS concentrations in different specimens were determined by routine method, and the neutralizing percentage was respectively calculated. (2) After adding isotonic saline (NS), the final concentrations of REMP2 and PMB were 10, 20, 40, 80 micromol/L, and the concentration of LPS was 100 microg/L. The murine monocytic macrophages were stimulated with LPS, then cultured with REMP2 and PMB, with NS in culture as negative control. The content of tumor necrosis factor (TNF)-alpha was determined by ELISA kit. (3) The morphologic changes of Escherichia coli. was observed under electron microscope at 10, 20 and 40 minutes after addition of REMP2 to Escherichia coli suspension (with terminal concentration of REMP2 at 40 micromol/L).
Results:
There were no significant difference in endotoxin-neutralizing percentages between PMB and REMP2 in concentrations of 0.10, 10.00, 100.00 micromol/L (P > 0.05). The contents of TNF-alpha were 1175 +/- 162, 859 +/- 122, 645 +/- 142, 489 +/- 102 ng/L, respectively,after treatment of 10, 20, 40, 80 micromol/L REMP2, which were obviously lower than that of NS (3463 +/- 218 ng/L, P < 0.01). Under transmission electron microscope, the outer and interior membranes of Escherichia coli were obscure and rough, bacterial bodies were swollen with vacuoles in cytoplasm after treatment with REMP2.
Conclusion:
REMP2 has ability of neutralizing endotoxin and also antibacterial activity.
Insights
The limulus anti-lipopolysaccharide factor-derived REMP2 protein effectively neutralizes endotoxin and exhibits antibacterial activity against Escherichia coli. This suggests REMP2
Area of Science:
- Biochemistry
- Microbiology
- Immunology
Background:
- Endotoxins, potent activators of inflammatory responses, pose significant health risks.
- Limulus anti-lipopolysaccharide factor (LALF) is known for its endotoxin-neutralizing properties.
- REMP2 is a protein derived from LALF, with its functional role requiring further investigation.
Purpose of the Study:
- To evaluate the in vitro endotoxin-neutralizing capacity of REMP2.
- To assess the antibacterial activity of REMP2 against Escherichia coli.
- To investigate REMP2's effect on inflammatory mediator production.
Main Methods:
- Endotoxin neutralization assay comparing REMP2 and Polymyxin B (PMB) against lipopolysaccharide (LPS).
- ELISA to measure tumor necrosis factor-alpha (TNF-α) levels in LPS-stimulated macrophages treated with REMP2.
- Transmission electron microscopy to observe morphological changes in Escherichia coli after REMP2 treatment.
Main Results:
- REMP2 demonstrated comparable endotoxin-neutralizing percentages to PMB across various concentrations.
- REMP2 significantly reduced TNF-α production in LPS-stimulated macrophages in a dose-dependent manner.
- Electron microscopy revealed structural damage to Escherichia coli, including membrane disruption and cytoplasmic vacuolation, upon REMP2 exposure.
Conclusions:
- REMP2 possesses significant endotoxin-neutralizing capabilities.
- REMP2 exhibits direct antibacterial activity against Escherichia coli.
- REMP2 holds potential as a therapeutic agent for endotoxin-related conditions and bacterial infections.
