Bacterial Endotoxin Test

BACTERIAL ENDOTOXIN TEST PRINCIPLE BET measures the concentration of bacterial endotoxin that may be present in article to be tested. The detection of the Gram negative bacterial endotoxin are based on the use of a Limulus Amoebocytes Lysate (LAL) obtained from the aqueous extract of circulating amoebocyte of horse shoe crab (Limulus polyphemus) used as LAL Reagent

THEORY LAL assay is based on the observation that when an endotoxin contacts the clotable protein from circulating amoebocytes of Limulus, a gel clot forms. The assay kits contain calcium proclotting enzyme and procoagulogen. The proclotting enzyme is activated by bacterial endotoxin lipopolysaccharide and calcium to form active clotting enzyme. Active clotting then catalyzes the procoagulogen into polypeptide subunits (coagulogen). The subunit joins by disulfide bond to form a gel clot .

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Proclotting Enzyme (inactive)

Endotoxin Sample | | v -----------------------

Clotting Enzyme (active)

| | | v Procoagulogen -------------(soluble)

Coagulogen (insoluble) | | v Gel clot

DETECTION OF ENDOTOXIN A threshold concentration of endotoxin for the product to be tested should be set so as to ensure that as long as the endotoxin concentration in the product remain below this threshold even the maximal dose administered by the intended route per hour does not contain sufficient endotoxin to cause a toxic reaction . The quantities of endotoxin are expressed in defined Endotoxin unit (EU). Route of administration Intravenous Intrathecal

K (IU of endotoxin / kg of body mass/ hr 5 .2