Detection of Bacterial Endotoxin - LAL Testing

The Limulus Amebocyte Lysate (LAL) or bacterial endotoxin test is now a well-
established method for the screening of parenteral medicines, irrigation fluids,
dialysis solutions, medical devices, as well as water quality.

The test is based on an extract from the blood (Amebocyte) of the horseshoe crab
(Limulus polyphemus), which has a primitive clotting mechanism triggered by
bacterial endotoxin present in gram negative bacterial cell walls. Most pyrogenic
responses in medicines are the result of bacterial contamination at some point in the
manufacturing process. The bacteria need not be living and the femeral (pyrogenic)
response may be triggered by fragments of bacterial cell wall. There are chemical
pyrogens which cannot be detected by this method, and some molecules (mainly
biological) are incompatible with the test system and still require testing by the
Rabbit Pyrogen Test.

Lysate is extremely sensitive and will detect very low levels of endotoxin. 10
endotoxin units (EU) are approximately equal to 1 ng. Standards for the test are
prepared from E. coli – derived endotoxin. After harmonisation the unit of measure is
the Endotoxin Unit (EU) for USP or International Unit (IU) for Ph Eur and the rest of
the world. Both units are interchangeable.

A standard curve is prepared and tested with each assay, with all reagents and
equipment used in the assay verified as being low endotoxin or endotoxin free. It is
preferable to use a matched endotoxin standard and batches of lysate to avoid
having to characterise the lysate using reference endotoxin.
Bacterial endotoxin (LAL) testing has been refined to give us the following three
methods;

Gel Clot method

This is still the reference method and is an end point assay giving a positive or
negative result at the sensitivity of the lysate used. 0.25, 0.125, 0.06 and 0.03EU
sensitivity are available.

A semi-quantitative method can be performed by preparing dilutions of the product,

which are then tested with known lysate. The endpoint on the dilution series can be
used to calculate the approximate endotoxin level by multiplying the dilution by the
lysate sensitivity.

Kinetic Chromogenic Method.

In this method the lysate is formulated with a chromaphore, which develops as the
gel forms. The test is conducted in a 96 well plate and the assay is read using an
automatic plate reader and suitable software. Each well on the plate is read every 30
seconds and compared with a standard curve. The rate of development of the colour
from the lysate is a measure of the amount of endotoxin in the sample. A value is
calculated in the programme using the standard curve and the dilution of the
product.

As the gel does not have to completely form, the sensitivity of this method is greater
than the gel clot and sensitivity to 0.005 EU/mL can be detected. This enables us to
test products that may interfere with the system by diluting out the interference.

Turbidimetric Method

A plate reader is also required for this method and it is very similar in operation to
the kinetic chromogenic method as above. In this method however, turbidity is
measured via spectrophotometry as opposed to measuring colour development. This
method is also very sensitive and can detect 0.005EU/mL, again enabling dilution to
test certain products.