Choosing the Best Detection Method for Your LAL Assay8th August 2019
The Limulus Amebocyte Lysate (LAL) assay is the most widely-used method for quantifying levels of bacterial endotoxins. Endotoxins are lipopolysaccharides found in the cell walls of Gram-negative bacteria. Since endotoxins can trigger dangerous inflammatory reactions upon contact with the human body, the LAL test is a vital part of quality control in numerous product sectors, including pharmaceuticals, medical devices, and laboratory cell cultures.
The LAL assay relies on a protein from the blood of horseshoe crabs that undergoes a clotting reaction upon contact with endotoxins. There are three principle methodologies that can be used for this assay: gel-clot, kinetic-chromogenic, and kinetic-turbidimetric. This article will discuss the differences between these three detection methods, as well as, their pros and cons for various applications.
Gel-clot is the simplest and most economical type of LAL assay. It is a qualitative detection method wherein the formation of a gel indicates the presence of endotoxins in the sample. Using this method, a positive result can be determined at a single glance without requiring any detection equipment.
The gel-clot method cannot be used in a quantitative manner. In addition, it has lower sensitivity than the other two methods, with a detection range between 0.015 and 0.50 endotoxin units (EU) per mL of sample. Despite these drawbacks, the gel-clot method remains the ideal choice for rapid, informal testing early in the product development pipeline.
The PYROSTAR ES-F Series from FUJIFILM Wako offers a series of kits that can be used with the gel-clot detection method, including specialized gel-clot reaction tubes. Simply incubate the reaction for one hour at 37 degrees Celsius and then rotate the tubes to check for gel formation.
The kinetic-turbidimetric method is a quantitative LAL test. It is more sensitive than the gel-clot method, with a detection range as low as 0.001 EU/mL. The same PYROSTAR ES-F Series may be used for the gel-clot or the kinetic-turbidimetric assay.
The principle behind this method is that the cloudiness (turbidity) of the test solution increases prior to gel formation, and a faster increase in turbidity indicates a higher level of endotoxins. Thus, by reading kinetically using a photometric instrument such as the Toxinometer®, one can accurately quantify endotoxin levels with a high degree of sensitivity.
The kinetic-turbidimetric detection method is a useful option to pair with the gel-clot method. Together, these two methodologies allow for both qualitative and quantitative testing at various stages of the product development process using only a single kit.
The kinetic-chromogenic detection method utilizes a colored dye to visualize the clotting reaction. The same chemical cascade that induces clotting also results in hydrolysis of a chromogenic substrate. This releases a yellow chromogen that can be visualized using a Toxinometer® tube reader or a microplate reader.
The Limulus Color KY Series, which rely on the kinetic-chromogenic method, are highly sensitive, and can detect down to 0.0002 EU/ml with the single test kit and 0.0005 EU/mL with the multi test kit. Despite requiring additional detection equipment, the test is straight-forward to use and allows for a quantitative readout. It is the best choice when the highest accuracy and sensitivity are needed.
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