Detection of bacterial endotoxins

Bacterial endotoxins, which are a component of the outer membrane of gram-negative bacteria, are strong pyrogens. When they come into contact with the bloodstream, endotoxins can cause fever, inflammation, endotoxemia, and even sepsis.¹ Therefore, parenteral pharmaceutical products are routinely screened for endotoxin contamination, highlighting the need for robust and accurate endotoxin assays. Moreover, the development of such assays aids research into the functions and mechanisms of action of endotoxins.

The Limulus amebocyte lysate (LAL) assay as a gold standard for endotoxin detection

Due to its high reliability and sensitivity, the LAL assay, which has been approved by the FDA, has become the most broadly used test for endotoxin detection. It relies on incubating a sample of interest with a protein extract from blood of horseshoe crabs. If the analyzed sample contains endotoxin, pro-clotting enzymes and the coagulation cascade are activated, and a gel clot is formed.²

Qualitative and quantitative LAL assays

The LAL assay can be carried out as a qualitative gel-clot test or as a quantitative turbidimetric or chromogenic test. The methods used to evaluate the assay findings also vary and may include visualization or quantitative measurements with a Toxinometer®️ or a microplate reader.

LAL gel clot assay – It relies on the visualization and qualitative assessment of a formed clot. The LAL gel clot assay enables a quick and sensitive detection of endotoxin. However, it does not allow endotoxin quantification and may not be suitable if numerous samples need to be evaluated simultaneously.

LAL turbidimetric assay (KTA) – It quantifies endotoxin by evaluating the turbidity (cloudiness) of a sample, which develops after enzymatic substrate cleavage but prior to gel formation. Moreover, the LAL turbidimetric assay (kinetic or end point) enables high-throughput analysis while retaining high sensitivity.

LAL chromogenic assay (KCA) – This assay detects chromogenic release caused by the cleavage of a chromogenic substrate. It can be performed as a kinetic or an end-point test that detects and photometrically quantifies changes in the color of the reaction mixture. In addition, the LAL chromogenic assay is characterized by both high sensitivity and capabilities for automated and high-throughput analysis.

Designing sensitive and specific LAL assays

There are factors that may cause sample interference or increase the risk of false positive or false negative results in the LAL assay. Examples include the presence of turbid or suspended products in the turbidimetric LAL test or components capable of changing their color in the chromogenic LAL test. Moreover, (1→3)-ß-D-glucans may mimic the actions of endotoxins and cross-react with the LAL reagent, leading to false positive results. Further, a pH outside of the optimal 6–8 range may cause protein denaturation and inactivation, enhancing the risk of false negative results.

PYROSTAR has developed endotoxin-specific ES-F reagents with ES-F buffer included in the formulation. The PYROSTAR™ ES-F reagents are unreactive to (1→3)-β-D-glucans, thus decreasing the risk of false positive findings. In addition, they include ES-F buffer, which uses Tris to neutralize both acids and bases and to maintain the pH of the reaction mixture within the optimal 6–8 range, thus reducing the risk of false negative findings.

Literature sources

1. Sampath VP. Bacterial endotoxin-lipopolysaccharide; structure, function and its role in immunity in vertebrates and invertebrates. Agriculture and Natural Resources 2018;52:115–120. https://doi.org/10.1016/j.anres.2018.08.002.
2. Mehmood, Y. What Is Limulus amebocyte lysate (LAL) and its applicability in endotoxin quantification of pharma products. Growing and Handling of Bacterial Cultures. IntechOpen 2019. Doi: 10.5772/intechopen.81331.