Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

Published: April 30, 2023

Abstract

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019).

This video describes a gel-clot assay to determine the presence of bacterial endotoxins in nano-formulations using Limulus Amoebocyte Lysate derived from the blood cells of horseshoe crabs. The assay helps test for endotoxin contamination of pharmaceutical products, including nano-based formulations.

Protocol

1. Preparation of Nanoparticle Samples Prepare the study sample in LAL-grade water. If the sample pH is outside of the 6-8 range, adjust the pH by using pyrogen-free sodium hydroxide or hydrochloric acid. Using LAL-grade water, prepare several dilutions of the study sample. Make sure that the highest dilution does not exceed maximum valid dilution (MVD). 2. Preparation of Reagents Common Between LAL Formats Dilute concentrated sodium hydroxide…

Disclosures

The authors have nothing to disclose.

Materials

LAL reagent Associates of Cape Cod G5003 This reagent is specific to the gel clot assay
Control endotoxin standard Associates of Cape Cod E0005 This reagent can be used with turbidity and gel clot assays
Sodium hydroxide Sigma S2770 When needed, it is used to adjust sample pH to be between 6-8
Hydrochloric acid Sigma H9892 When needed, it is used to adjust sample pH to be between 6-8
LAL grade water Associates of Cape Cod WP0501 This reagent can be used with any LAL format
Glucashield buffer Associates of Cape Cod GB051-25 Used to prevent false-positive response from beta-glucans
Disposable endotoxin-free glass dilution tubes 12 x 75 mm Associates of Cape Cod TB240 These tubes can be used with all three assays
Disposable endotoxin-free glass reaction tubes 10 x 75 mm Associates of Cape Cod TS050 These tubes are for use with the gel-clot assay
Pyrogen-free tips with volumes 0.25 and 1 mL RAININ PPT25, PPT10 Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed
Pyrogen-free microcentrifuge tubes, 2.0 mL Eppendorf 22600044 Other equivalent supplies can be used
Pyrogen-fee combitips, 5mL Eppendorf 30089669 Other equivalent supplies can be used
Repeat pipettor Eppendorf 4982000020 Other equivalent supplies can be used
Microcetrifuge Any brand Any brand can be used
Refrigerator, 2-8° C Any brand Any brand can be used
Vortex Any brand Any brand can be used
Freezer, -20 C Any brand Any brand can be used
Water bath, 37° C Any brand Any brand can be used, however, it is important either to switch off water circulation or use a non-circulating water bath because water flow will affect clot formation and lead to false-negative results

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Cite This Article
Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations. J. Vis. Exp. (Pending Publication), e21175, doi: (2023).

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