Automating Liquid Handling for Bacterial Endotoxin Testing Using ENDOZYME^® II GO and Andrew+

First published in OneLab - June 2026
By Paola Vigilanza, Global Solutions Manager, bioMérieux and Luca di Bello, Application Specialist, bioMérieux  

Bacterial Endotoxin Testing (BET) is a mandatory quality control step in the release of injectable pharmaceuticals, biologics, and medical devices. Endotoxins are lipopolysaccharides (LPS) found in the outer membrane of Gram-negative bacteria. If they enter a patient's bloodstream even at very low concentrations via contaminated parenteral medications or medical devices, they can trigger severe immune reactions which could lead to death.

Figure 1: Bacterial Endotoxins are released by the membrane of Gram-negative bacteria

For over 40 years, the test for bacterial endotoxins has been performed by conventional Limulus Amoebocyte Lysate (LAL) which rely on the blood of the horseshoe crab (Limulus polyphemus), an ancient marine animal whose hemolymph contains a cascade of serine proteases that react specifically to bacterial endotoxins. The dependence on a wild-harvested animal raises concerns around supply security, lot-to-lot variability, and ethical acceptability in the context of the 3Rs principles (Replace, Reduce, Refine).

For each linear standard curve range (5 – 0.05; 20 min incubation time & 5 – 0.005 EU/mL; 60 min incubation time), 10 GOPLATEs were prepared by the Andrew+ liquid handling system and measured afterwards with a Synergy HTX reader.

In the following figures, the distribution of the standard curve parameters is shown:

Figure 2: Distribution of standard curve parameter for the 3-point linear standard curve (5 - 0.05 EU/mL) measured with 20 min total incubation time. n = 10

Figure 3: Distribution of standard curve parameter for the 4-point linear standard curve (5 - 0.005 EU/mL) measured with 60 min total incubation time. n = 10

All 10 plates were filled with endotoxin-free water as samples. To evaluate the accuracy of the setup, 210 samples were evaluated In the following figures the distribution of the acquired Positive Product Control (PPC) recoveries is shown:

Figure 4: Spike recovery distribution of 210 samples measured on 10 plates for each standard curve range. Left: 5 - 0.005 EU/mL with 60 min incubation time. Right: 5 - 0.05 EU/mL with 20 min incubation time

As the instructions indicate, all materials used for assay execution must be free of detectable endotoxins. A clean environment is strongly recommended to execute these assays.