LAL Test (Limulus Amebocyte Lysate)
The standard test for detecting bacterial endotoxins in pharmaceutical products, using a reagent derived from horseshoe crab blood cells. LAL assays are exquisitely sensitive and can detect endotoxin levels far below those that would cause clinical harm. Results are reported in endotoxin units (EU).
Technical Context
LAL is prepared from the blood cells (amebocytes) of horseshoe crabs (Limulus polyphemus, Atlantic species; Tachypleus tridentatus, Asian species). Amebocytes contain a clotting cascade (Factor C → Factor B → proclotting enzyme → clotting enzyme) that is activated by endotoxin binding to Factor C. This cascade produces a clot (gel-clot method) or cleaves chromogenic/fluorogenic substrates (quantitative methods). Sensitivity: LAL can detect endotoxin at concentrations as low as 0.001 EU/mL (approximately 0.1 pg/mL LPS). Interference testing: sample-specific validation is required because peptides, formulation components, or pH may inhibit or enhance the LAL reaction — maximum valid dilution (MVD) calculations determine the appropriate sample dilution. Conservation concerns about horseshoe crab harvesting have driven development of recombinant alternatives (rFC assay), now accepted by EP and under evaluation by USP/FDA for routine use.