A Limulus Amoebocyte Lysate Activating Activity (LAL Activity) That Lacks Biological Activities of Endotoxin Found in Biological Products

Abstract

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test. Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF‐α) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS‐binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IHA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.

IFNγ primes macrophages for inflammatory activation by high molecular weight hyaluronan

“…In contrast, polymyxin B failed to neutralize the activity detected by LAL assay with HMW-HA (Figure 1H). Non-endotoxin LAL assay activity of biological products is not uncommon, but require LPS inhibitors, such as polymyxin B, to validate LAL assay results[13] Taken together, results from this series of studies show that LAL activity and TNF induction by HMW-HA are endotoxin-independent..…”