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

Protocolo

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.

Materiais

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