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

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

1. Prepare the study sample in LAL-grade water.
2. If the sample pH is outside of the 6-8 range, adjust the pH by using pyrogen-free sodium hydroxide or hydrochloric acid.
3. 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

1. Dilute concentrated sodium hydroxide stock using pyrogen-free LAL reagent water to prepare a working solution at a concentration of 0.1 N.
2. Dilute concentrated hydrochloric acid....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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