Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs
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 the turbidimetric format of the Limulus amoebocyte lysate, or LAL, assay to estimate endotoxin contamination in PEGylated liposomal doxorubicin nano-formulations. The turbidity of the solution following the reaction of the endotoxin with the LAL reagent helps to estimate the endotoxin in the sample.
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 …
Divulgations
The authors have nothing to disclose.
Materials
| 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 Reagent | Associates of Cape Cod | T0051 | This reagent can be used with turbidity assay only |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| 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 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 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 | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
Tags
Citer Cet Article
Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs. J. Vis. Exp. (Pending Publication), e21174, doi: (2023).