Limulus Amebocyte Lysate (LAL) Assay
Unlike RPT, LAL assay developed in the 1960s does not involve live test
subjects. It does, however, rely on an extract from the blood of theLimulus polyphemus species of horseshoe crab
52,53. The extract is used in one of three ways. First
and simplest, the gel-clot. This test involves mixing equal parts of
extract and sample. If a gel has formed and the mixture remains intact
in the bottom of the tube, the test shows positive 54.
This means the sample has at least enough endotoxins to trigger a
positive reaction, the limit of this being around the range of 0.03
EU/mL or 0.003 to 0.006 ng/ml. The other two methods are turbidimetric
and chromogenic methods. Both are referred to as photometric tests as
they require an optical reader for analysis. The chromogenic assay is
performed by replacing a natural substrate, Coagulen, with a
chromogenic, or colored one. The chromogenic substrate is cleaved by an
endotoxin-activated enzyme coagulase, and the chromogenic molecule is
released from the substrate into the suspension measured by
spectrophotometry 55. The turbidimetric method is
similar to the chromogenic method, but instead measures the turbidity of
the solution 56. The rate of turbidity and absorbance
(color change) are proportional to the endotoxin concentration. All
three tests rely on the same protein, Factor C coagulation cascade found
in horseshoe crabs’ blood (Figure 3 ). The endotoxin activates
Factor C which goes onto activate Factor B following the formation of a
clotting enzyme 57,58. In gel clot and turbidity
assays, the clotting enzyme transforms Coagulen into Coagulin, creating
the gel in the gel clot test, as well as the clouding agent in the
turbidity test. The Chromogenic method follows the same pathway, but
instead of using Coagulen, it uses a complex of amino acids and
p-nitroaniline (pNA), as the chromogenic factor. The enzyme trims the
pNA off of the complex, turning the suspension a yellow color. This
color is too faint to discern by the naked eye so a spectrometer must be
used 23. These tests are widely accepted as the
official endotoxin test in the pharmaceutical community
59. Every drug and medical device that is tested by
the US Food and Drug Administration (FDA) must undergo and pass a LAL
test 60,61. As previously mentioned, this method is
much more accurate than RPT, particularly the photometric methods. This
method still has its drawbacks. For example, the protein cascade it
relies on is disrupted in samples with free metal ions, and similarly to
RPT, the method is subject to the same public outcry for its treatment
of horseshoe crabs. While the phlebotomy itself is not fatal, an
approximated 20% of the crabs fail to survive after being returned to
sea 23. Following the discovery of Factor C as
endotoxin-activated portion of the protein cascade, attempts have been
made to replace the conventional LAL test, with one using recombinant
Factor C 62. As technology improves, alternative
techniques are being developed to ease the pressure on the horseshoe
crab population.