The Limulus amebocyte lysate (LAL) assay is the most popular method for detection and quantification of bacterial endotoxins. Care must be taken to avoid common mistakes with this assay that may lead to incorrect results. This article will outline four tips to avoid these common pitfalls and ensure accuracy in your LAL data.
The LAL relies on a clotting reaction that occurs in the presence of endotoxin; however, studies have shown that a fungal polysaccharide called (1→3)-β-D-glucan also induces clotting, which can lead to false positive results. To avoid this problem, it is important to use reagents which contain high levels of (1→3)-β-D-glucan in order to minimize its interference with the assay. Kits from FUJIFILM Wako now include high levels of this compound.
For endotoxin testing on medical devices, the surface of the device is rinsed with a solution in order to extract the endotoxins. The choice of extracting solution can have a considerable impact on the results of the LAL assay. For example, research has found that using water as an extracting solution can allow certain other substances to interfere with the LAL reaction. Metals, amino acids, antibiotics, and enzymes are all potential sources of interference. FUJIFILM Wako’s Endotoxin Extracting Solution for LAL Test is highly effective for extracting endotoxins while avoiding materials that interfere with this assay. This solution is recommended for testing devices that may come into contact with fluids containing blood or protein.
Disposable labware can be a problematic source of endotoxin contamination during LAL testing. All consumables used for the assay should be certified free of detectable endotoxin. Some products may be labeled as “apyrogenic,” but it is important to check the specific endotoxin limit used for testing, and in some cases, it can be prudent to test a batch of labware prior to use. The BioClean® series, which includes pipette tips, test tubes, and caps, has been tested to ensure endotoxin levels below 0.005 EU per product. The tips specifically are sterilized by gamma radiation and come individually packed in dust-protecting film.
Dilution errors during the construction of a standard curve can lead to considerable errors in LAL assay results. To avoid these errors, the American Pharmaceutical Review recommends that prior to the release of a lysate lot for routine use, the dilution series should be quantified three times each by three separate technicians. In addition, the starting concentration and dilution type should be kept consistent between multiple routine tests. A typical standard curve starts at 1000 EU/mL.
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