Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies

Coen Govers*, Monic M.M. Tomassen, Anne Rieder, Simon Ballance, Svein H. Knutsen, Jurriaan J. Mes

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

6 Citations (Scopus)

Abstract

The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5 h incubation in 0.1 M NaOH) at 56 °C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.

Original languageEnglish
Pages (from-to)15-25
JournalBioactive Carbohydrates and Dietary Fibre
Volume8
Issue number1
DOIs
Publication statusPublished - 2016

Fingerprint

Alkalies
alkalis
lipopolysaccharides
Polysaccharides
Lipopolysaccharides
inactivation
polysaccharides
Assays
assays
In Vitro Techniques
Limulus
Pyrogens
Horseshoe Crabs
analytical kits
Cell culture
cell viability
Cell Survival
cell culture
Contamination
Ethanol

Keywords

  • Alkaline-ethanol
  • HEK-Blue hTLR4
  • Immune-modulation
  • In vitro studies
  • LPS quantification
  • LPS removal

Cite this

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title = "Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies",
abstract = "The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5 h incubation in 0.1 M NaOH) at 56 °C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.",
keywords = "Alkaline-ethanol, HEK-Blue hTLR4, Immune-modulation, In vitro studies, LPS quantification, LPS removal",
author = "Coen Govers and Tomassen, {Monic M.M.} and Anne Rieder and Simon Ballance and Knutsen, {Svein H.} and Mes, {Jurriaan J.}",
year = "2016",
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language = "English",
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Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies. / Govers, Coen; Tomassen, Monic M.M.; Rieder, Anne; Ballance, Simon; Knutsen, Svein H.; Mes, Jurriaan J.

In: Bioactive Carbohydrates and Dietary Fibre, Vol. 8, No. 1, 2016, p. 15-25.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies

AU - Govers, Coen

AU - Tomassen, Monic M.M.

AU - Rieder, Anne

AU - Ballance, Simon

AU - Knutsen, Svein H.

AU - Mes, Jurriaan J.

PY - 2016

Y1 - 2016

N2 - The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5 h incubation in 0.1 M NaOH) at 56 °C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.

AB - The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5 h incubation in 0.1 M NaOH) at 56 °C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.

KW - Alkaline-ethanol

KW - HEK-Blue hTLR4

KW - Immune-modulation

KW - In vitro studies

KW - LPS quantification

KW - LPS removal

U2 - 10.1016/j.bcdf.2016.09.001

DO - 10.1016/j.bcdf.2016.09.001

M3 - Article

VL - 8

SP - 15

EP - 25

JO - Bioactive Carbohydrates and Dietary Fibre

JF - Bioactive Carbohydrates and Dietary Fibre

SN - 2212-6198

IS - 1

ER -