The yield of Limulus amoebocyte lysate (LAL), which is used for endotoxin detection and adjunct diagnosis of invasive fungal infection, was optimized with caffeine collection buffer.

Background: Limulus amoebocyte lysate (LAL) is a major component of the Fungal Infection Diagnosis Kit for the adjunct diagnosis of invasive fungal infections. During the previous bleeding season, PBS-collection buffer was found to prevent degranulation during the blood collection procedure. In addition, PBS-derived Limulus Amoebocyte Lysate (LAL) was used in both the chromogenic assay and the turbidimetric test. A similar phenomenon was observed with the caffeine collection buffer. This collection buffer is easy to prepare. Methods: To further confirm these observations, we used caffeine collection buffer to collect blood from horseshoe crabs. Six crabs were bled per week, for a total of 60 crabs. Blood was collected from each crab via both caffeine collection buffer and 3% NaCl collection buffer. The cell pellets were then resuspended in LAL reagent water (LRW) or 5 mM CaCl Results: Caffeine collection buffer prevented degranulation for more than 1 h, and the yield of caffeine-derived LAL was much greater than that of the 3% NaCl solution. Notably, caffeine-derived LAL was found to work in both chromogenic tests and turbidimetric assays. The enzyme characteristics of the LAL were also determined. Conclusion: Caffeine collection buffer prevents amoebocyte degranulation during blood collection and processing. The activity of caffeine LAL is much greater. Caffeine LAL works in both chromogenic tests and turbidimetric assays.