Detection and Quantitative Evaluation of Endotoxin Contamination in Nanoparticle Formulations by LAL-Based Assays

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 697)

Abstract

Bacterial endotoxin or lipopolysaccharide (LPS) is a membrane component of all Gram-negative bacteria. The administration of products contaminated with bacterial endotoxin can cause fever, shock, and even death. Accordingly, the FDA sets limits on the number of endotoxin units (EU) that may be present in a drug or device product. Limulus amoebocyte lysate (LAL) is the extract from amoebocytes of the horseshoe crab Limulus polyphemus, which reacts with bacterial endotoxin. Detection of the products of this reaction is an effective means of quantifying the EU present in a drug formulation. However, nanoparticles frequently interfere with the reactivity of endotoxin, the LAL reaction, or the detection of the reaction products. This interference can be manifested as either an enhancement or an inhibition, causing a respective overestimation or underestimation of the EU in the sample. Here, we present two methods for the detection and quantification of endotoxin in nanoparticle preparations: one is based on an end-point chromogenic LAL assay, and the second approach is based on measuring the turbidity of the LAL extract.

Key words

endotoxin lipopolysaccharide Limulus amoebocyte lysate (LAL) 

Notes

Acknowledgments

This project has been funded in whole or in part by federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.

References

  1. 1.
    Uribe, R.M., Lee, S., and Rivier, C. (1999) Endotoxin stimulates nitric oxide production in the paraventricular nucleaos of hypothalamus through nitric oxide synthase: correlation with hypothalamic-pituitary-adrenal axis activation. Endocrinology 140(12), 5971–5981.CrossRefGoogle Scholar
  2. 2.
    Chan, E. and Murphy, J.T. (2003) Reactive oxygen species mediate endotoxin-induced human dermal endothelial NF-kB activation. J. Surg. Res. 111, 120–126.CrossRefGoogle Scholar
  3. 3.
    Henricson, B.E., Benjamin, W.R., and Vogel, S.N. (1990) Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance. Infect. Immun. 58(8), 2429–2437.Google Scholar
  4. 4.
    Ilkka, L. and Takala, J. (1996) Plasma endototoxin levels in the early phase of septic shock. J. Intensive Care Med. 22, 1–9.CrossRefGoogle Scholar
  5. 5.
    Shapira, L., Soskolne, W.A., Houri, Y., Barak, V., Halabi, A., and Stabholz, A. (1996) Protection against endotoxic shock and lipopolysaccharide induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion. Infect. Immun. 64(3), 825–828.Google Scholar
  6. 6.
    Roth, R.I. and Levin, J. (1992) Purification of Limulus polyphemus proclotting enzyme. J. Biol. Chem. 267, 24097–24102.Google Scholar
  7. 7.
    FDA (1987) Guideline on validation of the Limulus Amebocyte Lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices. December 1987.Google Scholar
  8. 8.
  9. 9.
    USP-NF (2007) Bacterial Endotoxins Test, volume 1. United States Pharmacopeia, Rockville, pp. 109–113.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Nanotechnology Characterization Laboratory, Advanced Technology ProgramSAIC – Frederick, Inc., National Cancer Institute at FrederickFrederickUSA

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