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A method for removing interleukin-1- and tumor necrosis factor-inducing substances from bacterial cultures by
1Department of Medicine, Tufts University School of Medicine, Boston, MA.
Abstract:
The ability of human mononuclear cells (MNC) to produce cytokines is a highly sensitive and biologically relevant test system for the presence of microbial products. The safety of parenteral fluids is presently determined by gelation of the limulus amebocyte lysate (LAL) to endotoxin. In the present study, crude bacterial culture supernatants from Escherichia coli were subjected to ultrafiltration using polysulfone and the ultrafiltrates were tested for their ability to stimulate human MNC. Total interleukin-1 (IL-1) and tumor necrosis factor (TNF) produced by MNC were measured by radioimmunoassay. Endotoxin-like substances in E. coli cultures are rejected by a factor of at least 100,000. Rejection takes place by molecular size exclusion and by absorption. The sensitivity of the LAL and MNC cytokine production were comparable. These studies demonstrate a wide margin of safety for the production of parenteral fluids using ultrafiltration for endotoxin-containing materials.
Insights
Human mononuclear cells (MNC) cytokine production is a sensitive test for microbial products. Ultrafiltration effectively removes endotoxins from bacterial cultures, ensuring parenteral fluid safety with comparable sensitivity to the Limulus amebocyte lysate (LAL) test.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Cytokine production by human mononuclear cells (MNC) serves as a sensitive indicator for microbial products.
- Current parenteral fluid safety assessment relies on the Limulus amebocyte lysate (LAL) test for endotoxin detection.
Purpose of the Study:
- To evaluate the efficacy of ultrafiltration in removing endotoxins from bacterial cultures.
- To compare the sensitivity of MNC cytokine production assay with the LAL test for endotoxin detection.
Main Methods:
- Crude bacterial culture supernatants from Escherichia coli were subjected to polysulfone ultrafiltration.
- Ultrafiltrates were tested for their ability to stimulate human MNC cytokine production (Interleukin-1 and Tumor Necrosis Factor).
- Radioimmunoassay was used to quantify cytokine levels.
Main Results:
- Ultrafiltration demonstrated a rejection factor of at least 100,000 for endotoxin-like substances.
- Endotoxin removal occurred via molecular size exclusion and absorption.
- MNC cytokine production assay showed comparable sensitivity to the LAL test.
Conclusions:
- Ultrafiltration is a highly effective method for removing endotoxins from materials used in parenteral fluid production.
- This study demonstrates a significant safety margin for producing parenteral fluids using ultrafiltration, even with endotoxin-containing materials.