This document describes a protocol for a quantitative detection of Gram negative bacterial endotoxin in nanoparticle preparations using an end-point Limulus Amebocyte Lysate (LAL) assay. The protocol is based on QCL-1000 kit insert manufactured by BioWhittaker/Cambrex Corporation (10.1) and the US FDA Guideline “Validation of the LAL test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices” (10.2). Gram negative bacterial endotoxin catalyzes the activation of proenzyme in the Limulus Amebocyte Lysate. The activated
enzyme then catalyzes the splitting of p-nitroanilin from the colorless substrate. The released p-nitroanilin is measured photometrically at 405 nm after reaction is terminated with a stop reagent. Concentration of endotoxin in a sample is in direct proportion with absorbance and is calculated from a standard curve. The assay requires approximately 1.0 mg of test nanomaterial.