How Endotoxin-specific (ES) Buffer Enhances the Specificity of Endotoxin Detection in the LAL Assay
Endotoxins are released by gram-negative bacteria during their lysis and are ubiquitous in the environment. However, if endotoxins gain access to the bloodstream, they can lead to severe health consequences, including fever, sepsis, and septic shock. Therefore, routine endotoxin testing of parenteral drugs and medical devices has been established to prevent their endotoxin contamination.
The Limulus amebocyte lysate (LAL) assay has been widely accepted as the gold-standard method for endotoxin detection.1 In the LAL assay, amoebocyte extract from the horseshoe crab is incubated with the sample, which is being assessed for endotoxin presence. If the sample contains endotoxin, clot formation occurs. The LAL assay was initially developed as a qualitative gel-clot technique. Quantitative LAL assays, which rely on chromogenic (chromogenic LAL assay) and turbidimetric assessments (turbidimetric LAL assay), have also been established.
The LAL assay is characterized with high reliability and accuracy. However, sample interference may occur and may be caused by a number of different factors, including components of the tested sample or assay mixture. Examples of substances that may cause sample interference include certain chemical inhibitors, denaturing agents, and base alcohol molecules.1 Suboptimal pH values, endotoxin masking, and endotoxin absorption may also lead to misleading results.
(1,3)-ß-D-glucans, which may mimic endotoxins and cross-react with the LAL reagent, are a major factor that may cause false-positive findings.2,3 They activate a different pathway (Factor G) than endotoxins do (Factor C activating Factor B), which can lead to clot formation and the delivery of positive results. (1,3)-beta-D-glucan contamination of samples or reagents may originate from fungal or cellulose products, since (1,3)-beta-D-glucans are present in the cell wall of fungi.
Fujifilm Wako has created an ES buffer that prevents the fungal activation cascade by (1,3)-beta-D-glucans and only permits the bacterial activation cascade by endotoxins. This effect is achieved by the addition of large quantity of carboxymethylated curdlan (CMC) to the buffer’s composition. As CMC is closely related to (1,3)-beta-D-glucans, its presence in the reaction mixture prevents the activation of Factor F by (1,3)-beta-D-glucans. Thus, the ES buffer enhances the specificity of LAL reagents and reduces or even eliminates the risk of false-positive results in the LAL assay.
In addition, all of Fujifilm Wako’s LAL reagents are endotoxin-specific, with the ES buffer already integrated in their formulation. The inclusion of the ES buffer renders Fujifilm Wako’s LAL reagents unreactive to (1,3)-β-D-glucans, decreasing the likelihood of false-positive findings. Therefore, no further addition of ES buffer to Fujifilm Wako’s LAL reagents is required, and the reagents are easy and simple to use.
Further, the inclusion of ES buffer in LAL reagents reduces the risk of false-negative results in the LAL assay. As the ES buffer contains Tris, it neutralizes bases and acids and helps maintain the pH of the reaction mixture in the 6–8 range, which enables the endotoxin reaction and decreases the likelihood of false-negative results.
The LAL assay is the golden standard for endotoxin determination, but risks for false-positive and false-negative results may still exist. However, the development of Fujifilm Wako’s ES buffer has further enhanced the specificity and reliability of the LAL assay for endotoxin detection.
Literature sources:
- Wheeler, A. Comparing endotoxin detection methods. Pharmaceutical Technology 2017;41:58–62.
- Dawson ME. Interference with the LAL test and how to address it. LAL Update 2005; 22(3): 1–5.
- Interfering factors in the LAL test. Fujifilm. https://www.wakopyrostar.com/blog/kit-lal/post/interfering-factors-in-the-lal-test/