Bacterial Endotoxin

The Limulus Amebocyte Lysate (LAL) test is an in vitro assay used for detection of pyrogenic substances (endotoxin) in sterile parenteral drugs, in-process manufacturing samples, cleaning validation rinse samples, and medical devices. Limulus amebocyte lysate is an aqueous extract of blood cells (amebocytes) from the horseshoe crab, Limulus polyphemus. The LAL test can be performed by any of the following methods:

• Gel-Clot method
• Kinetic Turbidimetric
• Kinetic Chromogenic

Selection of the most suitable LAL test method is based on the assay results obtained during the assay development and validation.

Gel-Clot LAL Test

The Gel-Clot LAL test is performed by adding the lysate and the test specimen in a pyrogen-free reaction tube. The reaction solution is placed immediately in a dry block incubator or water bath at 37 ± 1°C for 60 ± 2 minutes. After 60 minutes the tube is removed from the incubator and inverted. A positive test is a firm gel (clot) that remains intact in the bottom of the reaction tube after inversion of 180°. The concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lysate. Lack of a gel indicates a negative test. The Gel-Clot LAL test can detect as little as 0.015 EU/per mL.

Kinetic Turbidimetric LAL Test

In the Kinetic Turbidimetric LAL method, either the rate of increase in turbidity or the time taken to reach a particular level of turbidity (the onset time) is determined. The assay requires a 96 well plate reader to incubate multiple samples at a controlled temperature (37°C) and takes optical density readings at regular intervals. Standard curves are prepared by plotting the log onset time against the log concentration of standard endotoxin and are used to calculate endotoxin concentrations in specimens. The sensitivity of this test is 0.01 EU/mL.

Chromogenic LAL Test

In the Chromogenic LAL test, the LAL reagent is mixed with test sample in a microplate and incubated in a reader at 37 ± 1°C. Absorbance measurements are collected with time after addition of Chromo-LAL and analyzed by the K-QCL software. The time (onset time) taken for a sample to reach a specified absorbance (onset OD) is calculated; and a standard curve, showing the linear correlation between the log onset time and the log concentration of standard endotoxin, is generated. The sensitivity of this test is 0.005 EU/mL.

Water, liquid, and powder samples are tested per USP <85> “Bacterial Endotoxin or EP 2.6.14 “Bacterial Endotoxin”. Devices are tested per USP <161>, Transfusion and Infusion Assemblies and Similar Medical Devices and USP <85>. Other methods such as ANSI/AAMI ST72:2002, Bacterial endotoxins- Test methodologies, routine monitoring, and alternatives to batch testing and USP are available. Samples are stored at 2-8°C for 24 hrs or -20°C if not tested in 24 hours.