Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs
Transcrição
For calibration standard preparation, use 900 microliters of Limulus Amebocyte Lysate or LAL-grade water, and 100 microliters of control standard endotoxin, to prepare as many intermediate dilutions as needed, to enable the preparation of a calibration standard with a concentration of one endotoxin-unit-per-milliliter.
Then, using 900 microliters of LAL-grade water and 100 microliters of the one endotoxin-unit-per-milliliter calibration standard, prepare a second calibration standard at a concentration of 0.1 endotoxin-unit-per-milliliter.
Then, repeat the serial 10-fold dilution to prepare two lower calibration standards, to obtain a total of four calibration standards ranging from 0.001 to 1 endotoxin-unit-per-milliliter.
To prepare a 0.05 endotoxin-unit-per-milliliter quality control, combine 50 microliters of the one endotoxin-unit-per-milliliter of control standard endotoxin solution with 950 microliters of LAL-grade water.
Next, in the software, select the 'Instrument settings', and create the template. Select 'Collect data', and enter the test ID and data group in the 'General' tab. Under the 'Hardware' tab, select the instrument type, and the LAL method, and verify that a serial number, the system ID, and the serial port information appear on the screen.
Click 'OK' two times to confirm the instrument selection and the communication with the instrument, and enter the sample IDs in the same order that the samples will be tested. Then, use the default buttons to enter the negative control, standard curve, and test samples.
Add the appropriate volume of negative control calibration standards and quality control into duplicate pre-labeled glass tubes, and add the appropriate volume of LAL to the first test vial. Vortex the vial briefly, and place the vial into the appropriate test slots in the instrument carousel.
Before loading the samples, perform several dilutions of the test nanomaterial within the maximum valid dilution as demonstrated.
