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).