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

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.