The discovery of the horseshoe crab's
most significant biological role in
recent medicine was made by Frederick
Bang in the early 1950's. Bang discovered
that the horseshoe crab's blood cells,
called amoebocytes, contain a clotting
agent that attaches to dangerous endotoxins
produced by gram negative bacteria. The
test was accepted by the United States
Food and Drug Administration (FDA)
in 1983 as a standard test for endotoxins. In
1987, the FDA established guidelines
for LAL testing of pharmaceuticals
and medical devices.
Principle of the Test:
LAL Chromogenic endotoxin assay utilizes
a modified Limulus Amoebocyte Lysate
and a synthetic color-producing substrate
to detect endotoxin presence. This
assay is quantitative and the color
intensity developed upon addition
of the sample to the LAL supplied
with the kit is proportional to the
amount of endotoxin present in the
sample and can be calculated from a
standard curve. The kinetic chromogenic
quantitative assay for bacterial
endotoxin can be performed at the low
assay range (0-5 EU/ml) or the higher
range (5-50 EU/ml) depending on sponsor's
request. The assay is performed using
a Molecular Devices Microplate reader
controlled by Softmax Pro software. During
the 60 minute incubation, 241 readings
are collected from all wells. The
standard curve is deduced from the
241 readings and sample concentrations
are calculated automatically based
on the standard curve. The assay is
highly sensitive.
Sample Requirements:
USP requires 3% of the production
lot with a minimum of 3 and maximum
of 10 devices to be pooled and tested. For
liquid samples, a minimum of 5 ml
is required and powder sample require
enough material to reconstitute into
a minimum of 5 ml pyrogen-free water. Initial
validation on 3 lots must be performed
in order to validate the method of
testing for a given product.
Samples containing B-Glucans (carbohydrates,
mold, starch, plant material, food
supplements) interfere with the pyrochrome
and produce falsely elevated endotoxin
levels. Please inform the laboratory
if your sample contains carbohydrates
or B-glucans. A special buffer is
used to resuspend the pyrochrome in
order to overcome the nonspecific
interference. There is an additional
$30 per sample for samples requiring
the special re suspension buffer.
Turnaround time :
24-72 hours for LAL performed under
cGMP conditions and 48-96 hours for
LAL tests performed under GLP conditions.
Background:
