Abstract:
Endotoxin detection is essential for pharmaceutical safety and clinical diagnostics, but remains challenging due to interference with formulation components and biological matrices. This thesis investigates factors affecting detection, focusing on storage conditions, the effect of lipopolysaccharide (LPS) structural variants on low endotoxin recovery (LER), and masking mechanisms in human plasma.
First, the influence of container materials on (1→3)-β-D-glucan (BDG) recovery was assessed, as BDG interferes with endotoxin measurements by falsely activating the Limulus amebocyte lysate (LAL) cascade via factor G. While short-term storage had no effect, long-term storage at -20°C reduced recoveries, especially in polyethylene containers. This highlights the importance of storage material selection for reliable BDG detection.
Next, the effect of LPS structural diversity on endotoxin masking was analysed using rough LPS mutants (Ra – Re) from S. minnesota and E. coli. Polysaccharide length influenced masking susceptibility: rough mutants, with a lower hydrophilic/hydrophobic ratio and stronger negative charge, showed greater resistance to masking due to enhanced cation binding and stabilised supramolecular structures, resulting in higher recovery rates. Masking kinetics varied between detection systems, with LAL and recombinant Factor C (rFC) assays showing similar masking behaviour, while the Monocyte Activation Test (MAT) showed improved recovery.
Finally, endotoxin detection in human blood was investigated, where plasma proteins contribute to masking. While previous research has emphasised electrostatic interactions, this study found that highly charged LPS structures, such as Re LPS, are less susceptible to masking, suggesting additional contributing factors.
Overall, this dissertation advances the understanding of endotoxin masking, structural influences on detection and external factors affecting test reliability. These findings will help to optimise endotoxin testing, reduce false negatives and improve pharmaceutical and clinical safety.
Endotoxin