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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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.
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.
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.
Ilkka, L. and Takala, J. (1996) Plasma endototoxin levels in the early phase of septic shock. J. Intensive Care Med. 22, 1–9.
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.
Roth, R.I. and Levin, J. (1992) Purification of Limulus polyphemus proclotting enzyme. J. Biol. Chem. 267, 24097–24102.
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.
USP-NF (2007) Bacterial Endotoxins Test, volume 1. United States Pharmacopeia, Rockville, pp. 109–113.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Neun, B.W., Dobrovolskaia, M.A. (2011). Detection and Quantitative Evaluation of Endotoxin Contamination in Nanoparticle Formulations by LAL-Based Assays. In: McNeil, S. (eds) Characterization of Nanoparticles Intended for Drug Delivery. Methods in Molecular Biology, vol 697. Humana Press. https://doi.org/10.1007/978-1-60327-198-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-60327-198-1_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-197-4
Online ISBN: 978-1-60327-198-1
eBook Packages: Springer Protocols