Comparison of PBS-Caffeine and Caffeine Buffers for Inhibiting Exocytosis During Horseshoe Crab Blood Collection and Improving the Yield of Limulus Amebocyte Lysate (LAL) for Endotoxin Detection

Limulus amebocyte lysate (LAL) detects bacterial endotoxin through a serine-protease coagulation cascade in which Factor C responds to lipopolysaccharide and Factor G to (1,3)-β-D-glucan. Sustainable LAL production depends on collection buffers that prevent amebocyte degranulation while preserving these clotting factors. We previously showed that caffeine buffer inhibits degranulation, but caffeine-collected pellets aggregated upon resuspension in 5 mM CaCl2, unlike phosphate-buffered saline (PBS). We therefore developed a PBS-caffeine collection solution and compared it with caffeine buffer. Over one bleeding season, 121 crabs were bled; blood was collected in caffeine, PBS-caffeine, or PBS-caffeine supplemented with EDTA, EGTA, or both, and LAL activity was measured by chromogenic and turbidimetric assays. Both buffers prevented degranulation and gave comparable LAL activity, but PBS-caffeine reduced aggregation and clotting. Treating PBS-caffeine LAL with 10% PEG-8000 selectively abolished endotoxin-sensitive Factor C activity while preserving (1,3)-β-D-glucan–sensitive Factor G activity, and the resulting Factor G lysate, formulated in 20 mM acetate (pH 5.6), remained stable for 27 months. These results define an improved collection buffer and identify conditions that selectively stabilize Factor G zymogen in liquid form.