Title : Phosphate buffer saline-caffeine: An environmentally friendly exocytosis inhibitor as a viable alternative to standard invasive fungal disease detection

Abstract:

Limulus Amebocyte Lysate (LAL) assays emerged as one of the most effective approaches for endotoxin and fungi detection in vitro since their preface 50 years prior. Although detailed protocols are publicly available, conventional LAL collection methods (3% sodium chloride) waste as much as 80% LAL loss during blood accumulation, ascertaining incompatibility with the lasting survival of the American Horseshoe crab. For this reason, new implementations of blood-collection-suspension buffer combinations are critical. Here, we aimed to assess the effectiveness of the PBS-caffeine solution on the degranulation during the bleed process.

As such, six crabs were bled once a week for 60 total crabs by the PBS-caffeine collection solution; cell pellets were resuspended into 5.0 mM CaCl2 to facilitate degranulation. Both the chromogenic test and turbid-metric assay evaluated the LAL enzyme activity. There was no observable impact on the activity output of crab size during the bleed season. This protocol substantially benefits prior processes, as the PBS-caffeine collection solution lessened amebocyte aggregation/clot formation during processing.

Furthermore, we detailed the prime biochemical parameters of PBS-caffeine-derived LAL. We develop an accessible, promising phosphate-caffeine-based blood collection buffer that deters exocytosis and amebocyte degranulation for over four hours amid the blood extraction, maximizing LAL yield. Moreover, our analysis showcases that phosphate-caffeine-derived LAL is uniquely adapted to be compatible with the chromogenic and turbidimetric assay techniques. By employing this method for LAL blood extraction, our same-cost approach fosters significantly higher LAL yields, simultaneously ensuring a healthy Limulus polyphemus population.