A-294 Exocytosis Inhibitor Increases yield of Limulus Amebocyte Lysate (LAL), Used for adjunct diagnosis of Invasive Fungal infection

Abstract

Abstract Background The limulus amebocyte lysate (LAL) was widely used in endotoxin detection in vitro and the clinical diagnosis of invasive fungal infection. The previous data showed that a caffeine collection solution could prevent degranulation during the blood collection procedure, and caffeine-derived LAL could work in both the chromogenic test and turbidimetric method assays. However, the cell pellets collected by caffeine collection solution aggregates after re-suspension in 5 mM CaCl2. No similar phenomenon was observed with PBS collection buffer. The purpose of this study is to set up a PBS-caffeine bleed solution to improve the yield of LAL Methods During the 2023 bleed season, experiments were carried out to test the results of PBS-caffeine solution on the degranulation during bleed process. 6 crabs were bled once a week for a total of 60 crabs by the PBS-caffeine collection solution. The blood of each crab was collected by PBS caffeine collection solution. The cell pellets were finally re-suspended into 5 mM CaCl2 to promote degranulation. The LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The data demonstrates that PBS-caffeine collection solution could prevent degranulation for over 4 hours, and the activity yield of PBS-caffeine-derived LAL was higher. There was no discernable impact on the activity yield of the crab’s size during the bleed season. PBS-caffeine-collection solution could reduce amebocyte aggregation/clot formation during processing. Studies described the biochemical characteristics of PBS-caffeine-derived LAL. Moreover, PBS-caffeine-derived LAL could work in both the chromogenic and turbidimetric assays. Conclusions First, PBS-caffeine collection solutions prevent amebocyte degranulation during blood collection and processing. Next, a PBS-caffeine collection solution could reduce cell aggregation/clotting during processing, and the activity of PBS-caffeine LAL is higher. Finally, PBS LAL is functional in both the chromogenic test and turbidimetric assays