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

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 stock using pyrogen-free LAL reagent water and prepare a working solution at a final concentration of 0.1 N.
3. Preparation of the Control Standard Endotoxin (CSE)
   1. Reconstitute the CSE according to the certificate of analysis supplied by the manufacturer.
      NOTE: Refer to the Table of Materials for the details regarding catalog number and application of a given CSE formulation in different LAL formats.
4. Preparation of the LAL Reagent
   1. Reconstitute the LAL reagent according to the certificate of analysis provided by the manufacturer.
      NOTE: Refer to the Table of Materials for the details regarding catalog number and application of a given LAL reagent formulation in different LAL formats.

3. Gel-Clot LAL

NOTE: This assay identifies the presence of endotoxins in the sample based on the visual observation and detection of a clot in the reaction tube. The experimental steps are described below. Use a bench sheet to record the results. This bench sheet is not mandatory, and other ways of recording the assay results are also acceptable. An example of such a bench sheet is provided in supplementary materials for the convenience of a reader. Lambda (l) is the sensitivity of the gel-clot assay and is 0.03 EU/mL.

1. Label as many reaction tubes as needed to accommodate the number of analyzed test samples. Refer to the bench sheet for details about the number of replicates used in step 1, step 2, and step 3 of the assay.
2. Aliquot 100 μL of water, controls, or test sample per tube.
3. Prepare CSE such that the final concentration is equal to 4λ.
4. Combine 100 μL of the standard mentioned above with 100 μL of water or test sample to achieve the final concentration of CSE of 2λ. Repeat three more times to achieve lambda and half-lambda and a quarter lambda.
5. Make sure that the temperature in the water bath is 37 °C.
6. Add 100 μL of lysate per test tube, vortex briefly, and place the rack with all the tubes into the water bath for 1 h.
7. Invert the tube with a smooth motion.
8. Manually record results using "+" (firm clot) or "-" (no clot or loose clot) on the bench sheet.
9. Proceed with the analysis according to the USP BET 85; use the bench sheet as supporting material.