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 .
 Endotoxin Sample
|
|
o Proclotting Clotting Enzyme
Enzyme v
-----------------------
(active)
(inactive)
|
|
|
v
Procoagulogen -------------- Coagulogen
(soluble) (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 K (IU of endotoxin / kg of body mass/ hr

Intravenous 5

Intrathecal .2