Extraction and Quantification of Bacterial Endotoxin

Extraction and Quantification of Bacterial EndotoxinEndotoxins are lipopolysaccharide molecules found in the outer membrane of Gram-negative bacteria. Endotoxins that enter the human body can illicit a dangerous inflammatory reaction. For this reason, FDA guidelines impose strict limits on endotoxin levels in medical device extracts. Devices that contact the cardiovascular or lymphatic system must be below 0.5 EU/mL, while devices contacting cerebrospinal fluid must be below 0.06 EU/mL.

The Limulus Amebocyte Lysate (LAL) test is the most popular method for testing endotoxin levels. This method uses amebocyte lysate from the Limulus Polyphemus crab, wherein the presence of endotoxin triggers a defense mechanism that induces coagulation. The LAL test is commonly used in many industries including pharmaceuticals, medical devices, hospitals, and research laboratories.

The sample solution used with LAL testing can have a substantial impact on the efficiency of the endotoxin reaction. Generally, sample solutions are prepared in water; however research has found that there could also be certain other substances present in these solutions that can interfere with the LAL coagulation reaction. Common sources of interference include metals, amino acids, antibiotics, and enzymes.

The Endotoxin Extracting Solution, manufactured by FUJIFILM Wako, enables extraction of endotoxins, which cannot be extracted in water or saline. The solution is particularly recommended for medical devices that may come into contact with fluids containing blood or protein. One kit includes four 10 mL vials.

The extracting solution is straight-forward and easy to use: simply dilute a single vial of the product 1:20 in endotoxin-free water and either add it to the sample or dip the sample in it. After gentle agitation and a one-hour incubation at room temperature, the extracted endotoxin is ready to be measured.

Following extraction, the Limulus Color KY Series provides a robust method for quantifying endotoxin levels. The presence of endotoxin triggers a color development cascade involving the activation of several serine protease precursors. In the final reaction, the LAL clotting enzyme hydrolyzes a chromogenic substrate, releasing a yellow chromogen.

The kit relies on the Kinetic-Chromogenic Assay, which monitors the time for absorbance to reach a threshold value, known as activation time (Ta). Measuring Ta for known endotoxin samples enables preparation of a standard curve, which allows for endotoxin concentrations for an unknown sample to be determined from its Ta.

 The Limulus Color KY kit is highly sensitive, with a quantitative detection limit of 0.0002 EU/mL (single-type) or 0.0005 EU/mL (multi-type). Additionally, since a fungal polysaccharide called (1→3)-β-D-glucan has been shown to activate the LAL, the reagent contains high levels of (1→3)-β-D-glucan, thereby inhibiting its effects. This allows endotoxins to be detected with high specificity, avoiding false positives.

The Limulus Color KY kit is compatible with a Toxinometer® tube reader or microplate reader format. It is available as a single-test or multi-test kit and can be purchased with control standard endotoxin (CSE). CSE contains lyophilized endotoxin purified from E. coli strain UKT-B and can be used as a standard for LAL testing.


Lysate Reagent Water Wako Bioclean Plates BioClean® Series
Lysate Reagent Water Wako Bioclean Plates BioClean® Series