Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

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

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 format.

3. Turbidity LAL Assay

1. Preparation of the Calibration Standards
   1. Using 900 µL of LAL grade water and 100 µL of CSE, prepare as many intermediate dilutions as needed to enable the preparation of a calibration standard with a concentration range from 0.001 to 1 EU/mL.
   2. First label tubes and add 900 μL of LAL-grade water into each tube. Then add 100 μL of 10 EU/mL solution to prepare calibration standard with concentration of 1EU/mL.
   3. Repeat the serial 10-fold dilution as described above to prepare three lower calibration standards. Verify that four calibration standards ranging from 0.001 to 1 EU/mL have been prepared.
2. Preparation of the Quality Controls
   1. Prepare a 0.05 EU/mL quality control by combining 50 µL of the 1 EU/mL CSE solution with 950 µL of LAL-grade water.
3. Preparation of Inhibition/Enhancement (IEC) Controls
   1. Prepare IEC with concentration of 0.05 EU/mL by combining 25 µL of the 1 EU/mL CSE solution and 475 µL of the test nanomaterial at a given dilution.
4. Experimental procedure
   1. Allow the instrument to warm up by turning it on approximately 30 min in advance. Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL.
   2. Sign in by typing the username and password.
   3. Open the software (Table of Materials) by clicking on the corresponding icon on a computer screen.
   4. Select Collect data on the software home screen. Enter the test ID and data group information into the corresponding space in the General tab on the home screen.
   5. Click the Hardware tab. Choose the instrument type from a dropdown menu.
   6. Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL by choosing the LAL turbidity method.
   7. Verify that a serial number, system ID and serial port information appear on the screen. Click OK. Click OK one more time to confirm.
   8. Enter the sample ID in the same order the sample is tested. Use default buttons to enter the negative control, standard curve and test samples.
   9. Prepare duplicate tubes for each sample and add 200 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of negative control (water), calibration standards, quality control, IEC and test nanoparticles into pre-labeled glass tubes.
   10. Add 50 µL (test ratio 4:1) or 100 µL (test ratio 1:1) of LAL reagent to first test vial, vortex it briefly, and insert into test slot in the instrument carousel. If the 1:1 ratio is used, the volume of the LAL reagent is 100 µL.
   11. Repeat the procedure described above for other samples. Process samples one at a time.
        

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