Choosing the Best Detection Method for Your LAL Assay

Choosing the Best Detection Method for Your LAL AssayThe 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 Detection

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.

Kinetic-Turbidimetric Detection

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.

Kinetic-Chromogenic Detection

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.


LAL Reagent Kit LAL Accessories for Bacterial Endotoxin Test
LAL Reagent Kit LAL Accessories for Bacterial Endotoxin Test