Professional Documents
Culture Documents
Bacterial
Endotoxins
Test
Limulus Amebocyte Lysate
Test
A Reference Guide
By Bio Solutions.
B-14, Kanika Co-Op. Society, Sarvapally Radhakrishnan Marg, Off Nagardas Road,
Near Bhuta High School, Andheri (East),Mumbai – 400069. TELEFAX: 0091-22-26824494.
Phone: 9819973583. mail@biosolutions.co.in, biosolutions@rediffmail.com
Introduction
History,
Horseshoe
crab
&
Endotoxins
INTRODUCTION
The most significant application of the Limulus Amebocyte Lysate (LAL) test is evaluation
of parenteral drugs and medical devices for endotoxins [Lipopolysaccharides (LPS)] content.
The use of LAL to detect and control the presence of pyrogenic substances in
pharmaceuticals and medical devices is a relatively recent development.
Pyrogens, endotoxins and lipopolysaccharides are words that are interchangeably used when
referring to pyrogen testing. The word pyrogen applies to any material which induces fever.
It describes an in vivo characteristic. A solution that contains a fever producing substance is
said to be pyrogenic. The word endotoxin refers to a specific pyrogen associated with the
membrane of gram negative bacteria. Endotoxins are the most prevalent pyrogen found in
aqueous solutions. Lipopolysaccharides LPS describes the biochemical structure of
endotoxin. Endotoxins contain a fatty acid portion termed Lipid A and a long chain of
repeating sugars or polysaccharides. LPS are purified endotoxins and are used as standards
for in vivo pyrogen tests or in vitro assays.
Biochemistry
The biochemical basis for the use of Limulus amebocyte lysate in the detection of
endotoxin lies entirely in the coagulation reaction inherent in Limulus blood. The ability of
Limulus blood to from a gelatinous clot was initially described by Howell, Lobe, and
Blanchard. These early investigations showed that coagulation was induced by foreign
substance and that the circulating amebocyte (= granulocytes) was involved in the reaction.
The association between endotoxin and coagulation began in 1956, when Bang reported that
infecting the horseshoe crab, Limulus polyphemus, with Vibrio, a Gram-negative bacteria,
caused a fatal intravascular coagulation. By 1964, Levin and Bang had demonstrated the
extracts of the circulating amebocytes would gel in the presence of Gram-negative bacterial
endotoxin.
Limulus blood has always held a certain fascination because of its bluish appearance.
The blue color is due to hemocyanin, a copper-based oxygen acceptor. The hemocyanin
while produced by the cyanocytes, is extra-corporeal and remains in the plasma after
centrifugation of whole blood. The amebocyte is the only cell present in Limulus blood. It
represents a homogeneous population of nucleated, disk-shaped granular cells. The
amebocyte contains all the components of the entire coagulation systems. Cell-free
hemolymph does not gel in the presence of endotoxin and does not enhance the endotoxin-
mediated reaction associated with amebocyte extracts. Lysates from washed amebocytes
have been prepared by lysis in hypotonic solutions by physical disruption using mechanical
shear, or by freeze-thaw cycles. The sensitivity of amebocyte lysate to endotoxin can be
increased by subsequent extraction with organic solvents.
The clotting protein, coagulogen, is the last component of the coagulation cascade. It
comprises almost half of total protein present in amebocyte lysate. The active clotting
enzyme is a serine protease and hydrolyzes a single peptide bond within the coagulogen to
from a shorter peptide, coagulin. Once generated, the coagulin self-associates, forming a
three-dimensional lattice structure and eventually gels.
The catalytic nature of each activated component in the coagulation cascade serves to
amplify the next step in turn. This amplification most probably results in the extreme
sensitivity of Limulus amebocyte lysate to endotoxin. All commercial lysates are capable of
detecting picogram (pg) quantities (10 -12) of endotoxin.
Endotoxins
Coagulogen Coagulin
This section explains how an MBL scientist
discovered some amazing properties in
horseshoe crab blood that enable has
revolutionized ways to detect potentially Enter Limulus and an MBL scientist named Fred
lethal bacterial toxins and spawned a Bang.
multi-million dollar industry.
Limulus lives in an aquatic world; the sea. The sea is almost literally awash in
gram-negative bacteria. Millions can be found in a single gram of sediment.
Bacteria that are both harmless as well as pathogenic (disease-causing).
Why is horseshoe crab blood blue? Limulus is an
arthropod, a close
relative to spiders.
In fact more closely
related to spiders
than to true crabs.
Arthropods possess
a semi-closed
circulatory system.
We mammals have
literally thousands of miles of blood vessels that
The oxygen-carrying pigment in carry blood to our tissues through vast networks
horseshoe brab blood is a protein called
hemocyanin. It is very similar to the of capillaries. Bacteria entering our bodies
hemoglobinmolecule we have in our through these capillaries are initially limited in the
blood. Hemoglobin gets it's red color area they can infect, having to fight their way into
(which makes our blood red) from the
iron molecule in the center of the
the body through these narrow channels, all the
protein. Hemocyanin contains a copper while in contact with the white blood cells that are
molecule which results in a blue color. our first line of defense.
The circulatory system of Limulus is far more open. Large sinuses exist that
allow blood direct contact with tissues. There are many wide open spaces and
bacteria entering a crack in the shell of a horseshoe crab have easy access to
large internal areas of the crab, a potentially deadly scenario. Over the course of
it's hundreds of millions of years of interacting with the bacterial swarms it
coexists with, Limulus, like us, has developed exquisitely sensitive means for
detecting the presence of bacteria through the LPS they shed into their
environment.
It's a very sensible system. Imagine a horseshoe crab has sustained a small
injury. Seawater comes into contact with the tissue and bacteria come into
contact with the blood and begin to enter (ie infect) the body of the crab. Small
bits of the cell wall slough off as the bacteria propel itself through the blood. A
Limulus blood cells detects this tiny fragment and responds by releasing the
contents of the granules into the surround medium. These granules contain a
clotting factor, called coagulogen. The thought is that by clotting the
immediate surroundings very quickly, the invading
bacteria can become enmeshed and therefore
stopped! Larger clots may not only stop enmeshed
bacteria but serve as a barrier to the outside
environment in the case of a severed limb or large
incision. Bang found these clots to be very stable and
prevented even Brownian motion in trapped bacteria.
The bottom line to all this is that Limulus contains an exceeding sensitive means
to detect the presence of bacterial endotoxins that can be detected by the
formation of a gel-like clot. This may not strike one as significant until one
understands the impact of endotoxins on our health and healthcare systems.
Anything that goes into your body during surgery, by injection, or for therapy,
has to be free of bacteria. If not, the recipient will get an infection. Not only
must this material be sterile (meaning no living bacteria are present) but it must
be pyrogen-free.! As was demonstrated long ago, our bodies, like the bodies of
horseshoe crabs , respond to the presence or endotoxin, not just the bacteria.
The industry of ensuring that injectable drugs, irrigation fluids, surgical tubing,
etc are free of bacterial endotoxins is a big business. In the past, companies
maintained large rabbit colonies. Rabbits, like use, are sensitive to endotoxin
and if a suspect sample of saline injected into a rabbit caused a fever then it
was contaminated. No fever, no contamination. This method was not only
expensive (it isn't cheap to keep thousands of rabbits) it is also slow. A rabbit
test might require 48 hours to obtain a result. A Limulus amoebocyte lysate
(LAL) assay can take as little as 45 minutes. A suspect sample is mixed with
reconstituted LAL and allowed to sit in a small tube. After 45 minutes the tube is
inverted and if a clot has formed it will stick to the top of the inverted tube.
LAL is a multi-million dollar business. It received FDA approval in the 1970's for
use in the testing of drugs, blood products, intravenous fluids, and disposable
pharmaceutical devices and in 1983 was registered in the U.S. Pharmacopeia.
The lysate is produced by extracting blood from the crab. This is done using a
non-lethal method where blood is taken from a large dorsal blood sinus, the
pericardium. The crabs are returned to the water within 24 hours and
completely recover.
The blood is a milky-blue color due to the copper-
pigmented hemocyanin molecule which turns blue upon
contact with oxygen the same way our blood turns red.
The blood is a mixture of liquid serum and suspended
amoebocytes. Of course, the conditions for extracting
blood must be sterile and pyrogen-free else the
amoebocytes would immediately do their job and form a
clot. If these conditions are met, the blood can be
centrifuged and the result is a separation of blood cells
from the serum. A small whitish pellet forms at the
bottom of the tube. Technicians pour off the serum and
rinse the pellet with saline. It is then resuspended and
added to the collection of amoebocytes. Eventually
pyrogen-free, distilled water is mixed with the Jack Levin
suspension of blood cells. This causes the cells to absorb demonstrates the
fresh water and balloon until they eventually burst (or removal of blood from a
"lyse" - hence "lysate"). This releases the coagulogen Limulus. This
into solution. procedure does not
permanently harm the
The resultant solution is filtered to remove cellular debris animal.
and then freeze-dried to form a white powder of the
lysate. This lysate is then packaged and sold to be reconstituted as the assay
described above.
4. Mürer, E.H., Levin. J. and Holm, R., 1975. Isolation and studies of the granules of the
ameobocytes of Limulus polyphemus, the horseshoe crab. J. Cell Physiol., 86: 533-542
5. Armstrong, P.B. 1979, Motility of the Limulus Amebocyte, Biomedical Applications of the
Horseshoe Cran (Limulidae), 73-92.
Quigley, J.P., Corcoran, G., Armstrong, P.B., A Hemolytic Activity Secreted by the
Endotoxin-Challenged Horseshoe Crab: A Novel Immune System Operating at the Surface
of the Carapace. , Biological Bulletin, 193: 273 (October 1997)
8. Sargent, William., The Year of the Crab., W.W. Norton & Company 1987
THE HORSESHOE CRAB, Limulus polyphemus
A “living fossil”
Not a true crab, the horseshoe crab is actually more closely related to spiders,
scorpions, and ticks than to other crustaceans. This "living fossil" has been
around since well before the dinosaurs. Its large eyes and its unique blood have
made it uniquely valuable for biomedical research.
Spring high tides trigger a massive migration of the Atlantic genus, Limulus
polyphemus onto sheltered Delaware and New Jersey beaches. The sand can be
piled deep with spawning crabs, and females can lay as many as 88,000 eggs
each season. Migratory shorebirds rely on these abundant eggs to fuel their
journey from South America to the Arctic Circle.
Not a true crab, the horseshoe crab is actually more closely related to spiders,
scorpions, and ticks than to other crustaceans. This large marine arthropod gets
its common name from its shell, or carapace, which is U-shaped. Brownish-gray
in color, the carapace provides camouflage against the muddy or sandy seabed
where the horseshoe crab lives.
They haven't changed much since the Devonian era, some 360 million years
ago, well before dinosaurs. Fossil data suggests that there were never more
than twenty species of horseshoe crabs. Today only four species, grouped into
three genera, remain.
Two genera are found along the coast of Southeast Asia and nearby countries
such as Japan. The third is found along the entire Atlantic coast of the United
States and along the Gulf of Mexico as far as the Yucatan. Believed to have once
been far more widely distributed, Limulus is the only surviving species in its
genus, which indicates that its lineage is very ancient.
The scientific name for the Atlantic coast crab is Limulus polyphemus, after the
one-eyed giant of Greek myth.
Because its basic body design has changed so little over the ages, the horseshoe
crab is often referred to as a "living fossil.” They have as much genetic variation
as many other species, but are so well adapted to their ecological niche that
large-scale species radiation simply hasn't occurred. As biologist Mark Botton of
Fordham University puts it, "They seem to have hit upon a body plan that
worked a long time ago, and hasn't really changed."
A Curious Body
Unlike true crabs, which have five pairs of legs, horseshoe crabs have four. First
come a pair of appendages called "chelicerae," then a pair of "pedipalps,"
followed by the four pairs of legs. These eight legs have pincers, and are used
for walking and for grinding and manipulating food. To find its favorite foods--
worms, mollusks, and dead fish--the horseshoe crab crawls along the bay
bottom using its pedipalps as feelers to detect prey. When it comes upon a
worm or clam, the small claws pick it up and move it to the bristly areas near
the base of the walking legs where the mouth is located. The horseshoe crab
has no jaws and uses the bristles to crush the food as it moves its legs.
Behind the horseshoe crab’s mouth and the walking legs is its abdomen, which
is connected to the rest of the carapace by a flexible joint that allows it to move
up and down. The gills, which are called book gills and are also found in spiders,
are attached to the underside of the abdomen. Made up of about one hundred
thin leaves or plates, these respiratory organs enable the animal to get oxygen
from the water, and also from the air (if the crab is on the beach) as long as the
gills are wet. Last is a long tail, or telson, which helps the animal flip over if
turned upside down. The tail has no stinger and is not poisonous. The tail will
not regenerate if broken off, so a horseshoe crab should always be picked up by
the shell. Though they may look dangerous, horseshoe crabs are completely
harmless.
Young horseshoe crabs can swim upside down. To propel themselves, they flap
their gills and move their abdomens up and down.
During the cold months, Limulus lies buried in the bottom of the Delaware Bay
and the Atlantic Ocean. In the spring, a signal synchronized in some way with
spring high tides triggers a massive migration. By late May, millions of these
ancient creatures begin crawling shoreward in the Delaware Bay, seeking out
sandy beaches that are protected from the waves.
At peak times, beaches can be piled deep with crabs and the air filled with the
sound of shells clacking against each other. Peak spawning usually coincides
with the high tides that accompany the full and new moons in May and June,
and generally takes place at night. Water temperature and weather conditions
such as heavy surf can prevent spawning.
Males come in to shore attached to females, or alone, and accumulate at the
base of the beach. Studies have shown that males use their eyes to find mates.
The males, which are smaller than the females, have special pincers at the end
of their first pair of walking legs with which they attempt to hook onto the
female's abdomen. Sometimes a chain of several crabs is formed, as one male
clasps onto another behind a female in a tenacious embrace called "amplexus."
Once a male is in tow, the female slowly makes her way to the edge of the
water, where she scoops out a nest six to eight inches (about fifteen to twenty
centimeters) deep in the sand. There she deposits thousands of BB-sized eggs,
while the male passes over and fertilizes the eggs. As many as a dozen males
may jostle around the mating pair.
If nests are numerous, female crabs may disturb earlier nests when digging new
ones. The disturbed eggs accumulate on the surface of the beach, providing a
feast for shore birds, which cannot reach the buried eggs.
By spawning at full and new moons, when tides are highest, the female protects
her eggs from being washed away. Nests are usually located close enough to the
water to stay damp, but high enough for the sand to contain adequate oxygen.
These factors, along with the temperature, determine how long it takes for the
eggs to develop. Eggs usually hatch in about a month, and out come tiny
horseshoe crabs--minus tails--about three millimeters long. They're called
trilobite larvae because they look so much like ancient, extinct trilobites.
Molting
When a horseshoe crab outgrows its shell, it has to molt: leave the old shell and
grow a new one. The old shell splits around the front edge and the crab crawls
out. The new shell, which is about 25 percent larger, soon hardens. Shells found
on the beach are typically these outgrown ones.
Horseshoe crabs molt five times in their first year of life, two or three times
during their second year, twice in the third year, and then once a year until they
mature at nine or ten years of age.
For their first two years, juveniles tend to stay in the intertidal flats near where
they hatched, then gradually move to deeper waters. Feeble swimmers, adult
horseshoe crabs walk on the ocean floor. For most of the year they crawl along
the bottom of bays and along the continental shelf. Once they reach maturity,
they will crawl back to shore each year to spawn.
They have few natural predators, though loggerhead turtles sometimes tear out
the flapping gills and pieces of the horseshoe crab have been found inside
sharks. No one really knows how long horseshoe crabs can live. A few have been
kept in aquaria for as long as fifteen years.
Useful in many ways, the horseshoe crab has proven uniquely valuable to basic
biomedical science.
The blood (hemolymph) of Limulus turns blue when exposed to oxygen, and
turns out to be perhaps the most valuable part of this ancient creature, from a
human point of view. When a crab receives a wound, cells form a clot, and kill
certain kinds of bacteria which are also harmful to humans. This process was
discovered in the early 1950s by a scientist named Frederick Bang, who was
able to separate the chemical that caused the bacteria-sensitive clots to form.
Later, the extract was named LAL (Limulus Amoebocyte Lysate), and it is used
to detect whether things that go into the human body--including injectable
drugs, needles, and heart valves--are free of dangerous endotoxin producing
bacteria. Using LAL is more accurate, simpler, and less expensive than similar
tests for bacteria.
Blood is collected from horseshoe crabs taken out of the shallow waters off the
Atlantic coast, during the summer months.
They're checked for health and then bled through a stainless steel tube. It takes
around five minutes to extract about 20 percent of the animal's blood, after
which it is returned to the ocean. About 90 percent of horseshoe crabs survive
this procedure. Approximately two hundred thousand crabs are bled each year
for LAL.
Educational Eyes
For over fifty years, the eyes of horseshoe crabs have been used in eye
research. The crab's lateral compound eye has shown scientists a great deal
about how the human eye functions. It's easy to study because both the
horseshoe crab's eye and its optic nerve (which transmits signals from the eye
to the brain) are large, and because its organization is much simpler than a
human's. This has enabled scientists to analyze the electric signals that send
visual information to the brain, and therefore to understand many underlying
principles of all visual systems--how the human eye perceives lines, borders,
and contrasts, for example. Research on horseshoe crabs has also helped
researchers understand diseases such as retinitis pigmentosa, which causes
tunnel vision and can lead to blindness. In fact, the horseshoe crab is the only
animal for which we now understand the complete neural code for vision, which
should help us understand more complex visual systems in the future.
An Age-Old Survivor
The horseshoe crab, Limulus polyphemus, has been around for a very long time.
Fossils from British Columbia (situated at the time in warm, shallow tropical
seas) show that relatives lived in North America some 520 million years ago.
The horseshoe crab went on to survive several mass extinctions, including ones
that wiped out many other marine species. The habitat of this genus of the
ancient animal is now restricted to a little peninsula on the Eastern shore of the
United States, where it has been spawning since time immemorial in sequence
with the phases of the moon. It now faces arguably the most serious crisis ever,
one that is man-made.
. . . Until Now?
Between the 1880s and the 1920s about a million horseshoe crabs were
harvested each year for use as fertilizer and in hog fodder. Chemical
supplements replaced them, but fifty years passed before the crab population
rebounded.
Horseshoe crabs have again become a valuable commodity, this time as bait.
Traditionally fishermen picked crabs off the beaches by hand, for free, and the
harvest was small. However, the domestic and international market for eels and
whelks (also called conch) has been growing rapidly. Female, egg-bearing
horseshoe crabs make particularly good bait, so the demand for Limulus has
greatly increased as well. With blue crabs scarce and other fisheries in a slump,
horseshoe crabs have been selling for between eighty-five cents and a dollar a
piece, a price that has made it even more attractive as bait to commercial
fishermen and others. Some fishermen converted their boats to trawlers, which
drag nets across the ocean floor and can catch tens of thousands of horseshoe
crabs in one day.
A solution might lie in a synthetic horseshoe crab "scent" that would replace the
need to use actual animals as bait, which scientists are working on.
Since 1985, the commercial harvest of horseshoe crabs along the Atlantic coast
has greatly expanded. From 1990 to 1994, the National Marine Fisheries Service
reported the Delaware, Maryland, and New Jersey harvest increasing from
685,648 pounds (311,000 kilograms) to 1,386,367 (628,846 kilograms), which
translates to an annual haul estimated at half a million crabs. Dr. Mark. L.
Botton, associate professor of biology at Fordham University, cites a much
higher figure: "It's now becoming clear that the number taken is approaching a
million a year." Botton, who is studying the New Jersey crab population, notes
that "on the one hand, some of the watermen argue that there's absolutely no
indication that the numbers are going down. On the other hand, some of the
environmental spokespeople are talking about the population on the verge of
extinction. Neither of these extremes is correct, and we're trying to provide the
data that allow for proper management of the population."
This species matures slowly, and doesn't begin to reproduce until around age
ten. It's also long-lived. These factors lessen the effect of a single or several
poor spawning years, so horseshoe crab populations tend to be fairly stable. It
also means that serious, long-term impacts on the population will take a decade
to become fully apparent.
Habitat Degradation
It is the absence of inlets or other breaks in the Delaware Bay shorefront that
makes it an ideal habitat for horseshoe crabs. Environmentalists and researchers
now believe that much of the recent population decline can be attributed to
severe habitat degradation on the New Jersey side of the Delaware Bay, in the
form of man-made inlets and sandbars. These artificial breaks reduce beach
sand deposits, and large areas of mud flats form behind the breached
shorefronts. Huge numbers of horseshoe crabs--three hundred thousand by
some estimates--have been observed entrapped in these back-marsh mud
areas. The adult crabs are stranded and die, and their eggs do not develop in
the mud.
Ninety percent of the horseshoe crab stock is located between Virginia and New
Jersey, and only five years ago prime beaches would have been literally piled
meters deep with spawning horseshoe crabs. Spawning surveys are not always
reliable, but the effect of habitat degradation is becoming more and more
obvious. Four separate scientific studies conducted in Delaware Bay since 1990
have estimated that the horseshoe crab population has declined by more than
50 percent. By any count it is apparent that the Delaware Bay population of
Limulus polyphemus is swiftly declining.
Like all species, the horseshoe crab is part of a web of life, the disruption of
which affects many other kinds of animals:
It could take years for the crab numbers to rebound, during which time the bird
populations will be highly vulnerable.
• The highly nutritious eggs are also eaten by raccoons, foxes, diamondback
terrapins, moles, and even mollusks.
A steep decline in the horseshoe crab population will have a significant impact
on the fishing and medical industries, as well as on ecotourism. Birdwatchers
who flock to the Delaware Bay each spring to see the feeding shorebirds bring in
millions of dollars to local businesses.
What's Being Done
In Maryland, trawling and dredging are prohibited between April 1 and June 30
within Chesapeake Bay, coastal bays, and within one mile (1.6 kilometers) of
the Atlantic Ocean. Hand collection is also limited during this period. Virginia has
banned trawling with state waters and within three miles (4.8 kilometers) of the
coast, and requires commercial fishermen to report any crabs harvested as
bycatch.
New Jersey has dedicated $80,000 to research the population size of the local
horseshoe crab population, and also that of migrating shorebirds.
Maryland and Virginia are also taking steps to learn more about the species and
to protect it, especially during spawning. Maryland began a spawning survey in
1994, and also tags horseshoe crabs. The hope is that a balance--a sensible
harvest--can be arrived at. "While I'm concerned that the trends are downward,
I don't think it's cause for panic yet," says Dr. Botton. "What I do think is called
for is some prudent management strategy to conserve enough crabs to keep the
shorebirds fed, but yet permit the watermen to have a livelihood."
Efforts in Japan
Another genus of horseshoe crabs is native to Japan. Along with over a dozen
other aquatic species, it has been declared endangered. Land reclamation and
water pollution are the culprits. The animal used to thrive in large areas around
the Seto Inland Sea and northern Hyushu, in particular on a tidal flat along
Kasaoka Bay. Although it was designated a protected area, in 1966 the
Japanese government began a huge land reclamation project here. Completed in
1990, the project has completely wiped out the local crab population.
Most of the work on the chemical structure of endotoxin has been done with
species of Salmonella and E. coli. LPS can be extracted from whole cells by
treatment with 45% phenol at 90o. Mild hydrolysis of LPS yields Lipid A plus
polysaccharide.
• Region I. Lipid A
• Region II. Core (R) antigen
• Region III. Somatic (O) antigen or O polysaccharide
The structure of LPS in Salmonella typhimurium and E. coli is seen in Figure 3).
The elucidation of the structure of LPS relied heavily on the availability of
mutants each blocked at a particular step in LPS synthesis. The biosynthesis of
LPS is strictly sequential. The core sugars are added sequentially to Lipid A by
successive additions, and the O side chain is added last, one preassembled
subunit at a time. The properties of mutants producing incomplete LPS
molecules suggests the nature and biological functions performed by various
parts of the LPS molecule:
Both Lipid A (the toxic component of LPS) and the polysaccharide side chains
(the nontoxic but immunogenic portion of LPS) act as determinants of virulence
in Gram-negative bacteria. Virulence and the property of "smoothness"
(associated with an intact O polysaccharide) are regularly associated in many
bacterial infections. The polysaccharide chain must also be important for
virulence as shown by the fact that small changes in the sugar sequences in the
side chains of LPS, result in major changes in virulence. How are the
polysaccharide side chains involved in the expression of virulence? There are a
number of possibilities:
a. Smooth antigens could allow organisms to adhere specifically to certain
tissues, especially epithelial tissues.
Endotoxins are toxic to most mammals. Even though endotoxins are strong
antigens, they seldom elicit immune responses which gives full protection to the
animal against secondary challenge with the endotoxin. They cannot be
toxoided. Regardless of the bacterial source, all endotoxins produce the same
range of biological effects in the animal host.
Most of our knowledge of the biological activities of endotoxins derives not from
the study of natural disease but by challenge of experimental animals.
1. fever
2. changes in white blood cell counts
3. disseminated intravascular coagulation
4. tumor necrosis
5. hypotension
6. shock
7. lethality
Procedure:
37°C ± 1°C,
60 ± 2 min
10X75 mm tubes
Negative Test
100mL Sample Positive Test Viscous/ Cloudy,
+ 100mL LAL Gel formation No gel
Important criteria
Accessories
• Heating Block
• Vortex Mixer
• Depyrogenated glass test tubes for assay (10X75mm)
• Depyrogenated glass test tubes for dilutions
• Micro Pipette – 10 - 100µL, 20 - 200µL, 1000µL
• Sterile Micropipette Tips
• Timer
• Depyrogenated glass pipettes - 5mL, 2mL
REGULATORY INITIATIVES
After seven years of review, the FDA and the LAL industry agreed on the LAL TEST
Guideline (FDA 1987). A pharmaceutical or medical device producer could switch from
rabbit to LAL testing if the LAL Test Guideline were followed. The FDA approved LAL
because of concern about the relative insensitivity and unreliability of the rabbit test. There
was less concern about the remote possibility of missing non-endotoxin pyrogens or
materials-mediated pyrogens have not materialized.
The FDA’s LAL Test Guideline was the most influential document during the rapid
growth of LAL testing in the past two decades. It introduced the concept of the Endotoxin
Limits (EL) and provided formulas for dilution (MVD, Maximum Valid Dilution) and
concentration limits (MVC, Minimum Valid Concentration). The LAL Test Guideline has
detailed sections for testing parenteral drugs and medical devices. Three types of testing are
described. First, Initial QC is a set of assays designed to qualify analysts and confirm the
label-claim sensitivity of new LAL reagents with calibrated standards. These infrequent tests
require assaying one vial of reagent with four replicates from each endotoxin standard
dilution, 2λ through 1/4λ, which brackets the expected endpoint of the LAL reagent. For the
other two tests, the same standard dilution series (SDS) is required in quadruplicate for
validation and in duplicate for routine LAL test. In other words, the test for label claim is
accomplished with the SDS
Harmonized BET
A BET became effective in January 2001 in the 2nd supplement to USP 24 and the
European Pharmacopiea, which was produced by the International Conference of
Harmonization. The LAL Test Guideline and the new BET are now very similar. The new
text includes simplified procedures and inclusion of all LAL methods. In the unlikely case of
dispute, the gel-clot method is the referee test.
THE BACTERIAL ENDOTOXINS TEST
INTRODUCTION
The Bacterial Endotoxins Test can be performed using lysate extracted form the
Amoebocytes of Limulus polyphemus (LAL), Tachypleus tridentatus, Tachypleus gigas
(TAL), Carcinoscropius rotundicauda - CAL. USP 29 recognizes LAL and TAL from
T. tridentatus spp. The IP 2000 addendum recognizes all LAL, TAL and CAL.
BACTERIAL ENDOTOXINS
The test for bacterial endotoxins is used to detect or quantify endotoxins of gram-
negative bacterial origin using amoebocyte lysate from horseshoe crab (Limulus polyphemus
or Tachypleus tridentatus). There are 3 techniques for this test: the gel-clot technique, which
is based on gel formation; the turbidimetric technique, based on the development of turbidity
after cleavage of an endogenous substrate; and the chromogenic technique, based on the
development of colour after cleavage of a synthetic peptide-chromogen complex.
Proceed by any of the 6 methods for the test. In the event of doubt or dispute, the final
decision is made based upon method A unless otherwise indicated in the monograph.
Apparatus
Depyrogenate all glassware and other heat-stable apparatus in a hot-air oven using a
validated process. A commonly used minimum time and temperature is 30 minutes at 250 ºC.
If employing plastic apparatus, such as microtitre plates and pipette tips for automatic
pipetters, use apparatus shown to be free of detectable endotoxin and of interfering effects for
the test.
NOTE: In this chapter, the term ‘tube’ includes all type of receptacles, for example microtitre
plate well.
Preparation of the standard endotoxin stock solution
The standard endotoxin stock solution is prepared from an endotoxin reference
standard that has been calibrated against the International Standard, for example endotoxin
standard Biological Reference Preparation (BRP). (EP) or US Reference Standard (USP)
NOTE: One International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.)
Follow the specifications in the package leaflet and on the label for preparation and
storage of the standard endotoxin stock solution.
The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample
at which the endotoxin limit can be determined. Determine the MVD using the following
formulae:
The Endotoxin Limit for active substances administered parenterally, defined on the basis of
dose, is equal to:
Endotoxin Limit = K / M
The limit formula for radio pharmaceuticals is 175/V except for intrathecally
administered products. 14/V for intrathecal drugs.
V equals the maximum recommended dose, in mL, at the expiration date or time.
λ = the labeled lysate sensitivity in the gel-clot technique (IU/mL) or the lowest point
used in the standard curve of the turbidimetric or chromogenic techniques.
GEL-CLOT TECHNIQUE (METHODS A AND B)
1. PREPARATORY TESTING
Mix a volume of the lysate solution with an equal volume of 1 of the standard
solutions (such as 0.1 mL aliquots) in each tube. When single test vials or ampoules
containing lyophilized lysate are employed, add solutions directly to the vial or ampoule.
Incubate the reaction mixture for a constant period according to the recommendations of the
lysate manufacturer (usually at 37 +1 ºC for 60 + 2 min), avoiding vibration. Test the
integrity of the gel: for tubes, take each tube in turn directly from the incubator and invert it
through approximately 180º in one smooth motion. If a firm gel has formed that remain in
place upon inversion, record the result as positive. A result is negative if an intact gel is not
formed.
The test is not valid unless the lowest concentration of the standard solutions shows a
negative result in all replicate tests.
The end-point is the last positive result in the series of decreasing concentrations of
endotoxin. Calculate the mean value of the logarithms of the end-point concentrations and
then the antilogarithm of the mean value using the following expression:
ƒ = number of replicates.
Steps:
1. Reconstitute the Lyophilized material with required quantity of LRW (mentioned in the
CoA) to obtain mentioned concentration of EU/mL with the help of pyrogen free
pipette.
3. From 1 EU/mL prepare CSE of 4λ, 2λ,λ, 0.5λ,0.25λ dilutions. Where λ = Labelled
lysate sensitivity. e.g. λ = 0.125 EU/mL .Vortex each dilution for at least 1 min.
Labeled Claim sensitivity of Lysate (λ) is 0.125EU/mL. Following will be the dilutions one
needs to prepare for Confirmation of the labeled lysate sensitivity. From the Control Standard
Endotoxin vial prepare 1 EU/mL.
Control curve
Test carried out in clean depyrogenated 10 x 75 mm tubes only. Each dilution to be tested
in quadruplicate.
Total
LRW CSE Dilution in
Tube No. Dilution Volume
(µL) µL µL
1,2 Negative 100 - 100
Control
3,4,5,6 2λ - 100 ( of 2 λ ) 100
7,8,9,10 λ - 100 ( of λ ) 100
11,12,13,14 0.5 λ - 100 ( of 0.5 λ ) 100
15,16,17,18 0.25 λ - 100 ( of 0.25λ ) 100
To each tube, add 100 µl of LAL reagent. Mix gently & incubate in Heating Block at
37ºC ± 1ºC for 60±2 min. After incubation, remove the tubes gently from the Heating Block
& slowly invert through 180 º & scroll the result.
I II III
NEG Control –– –– ––
2λ ++++ ++++ ++++
λ ++++ –––– ++++
0.5 λ –––– –––– ++++
0.25λ –––– –––– ––––
End point at λ 2λ 1/2λ
Ideal Within + one twofold dilution
IV V VI
NEG Control –– –– ++
2λ –––– ++++ ++++
λ –––– ++++ ++++
0.5 λ –––– ++++ ++++
0.25λ –––– ++++ ++++
CSE Storage (?) Dilutions (?) Accessories
Dilutions (?) LRW (?)
Geometric Mean Calculation- Example
Formula:
Calculation:
= -4.214
4
= Antilog [(-1.0536)]
= 0.0883 EU/mL
Prepare solutions A, B, C and D as shown in Table 2.6.14-1, and use the test solutions
at a dilution less than the MVD, not containing and detectable endotoxins, operating as
described under 1. Preparatory testing, (i) Confirmation of the labeled lysate sensitivity.
The test for interfering factors is repeated when any changes are made to the
experimental conditions that are likely to influence the result of the test.
The test is not valid unless all replicates of solutions A and D show no reaction and
the result of solution C confirms the labelled lysate sensitivity.
If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not
greater than 2λ, the test solution does not contain interfering factors under the experimental
conditions used. Otherwise, the solution interferes with the test.
If the preparation being examined interferes with the test at a dilution less than the
MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD.
The use of a more sensitive lysate permits a greater dilution of the preparation being
examined and this may contribute to the elimination of interference.
Solution A: a sample solution of the preparation under test that is free of detectable
endotoxins.
Solution B: test for interference.
Solution C: control for labeled LAL Reagent sensitivity.
Solution D: negative control of LAL Reagent Water
SCREENING FOR INTERFERENCE:
Step I
The Least DILUTION / Highest CONC. of product for which PPC is Positive
and NPC is Negative is the Non Interfering Dilution (NID)/ Non Interfering
Concentration (NIC).
EXAMPLE
Drug : Cefotaxime Sodium Sterile 1000mg/4mL
Endotoxin Limit : NMT 0.2 EU/mg
Lysate sensitivity : 0.125 EU/mL
MVD = Potency x E.L
λ
= 250 mg/ mL X 0.2 EU/mg
0.125EU/mL
MVD = 400
If you get the following results –
Since the Non- interfering dilution is ⅛ MVD (1:50). Product can be validated
between ¼ MVD and MVD.
Sample
Test Sample LRW CSE LAL
(1:100)
Negative Water Control 100 µL − − 100 µL
GM CSE in Water = log (0.125) + log (0.125) + log (0.125) + log( 0.125)
4
= Anti[(-0.9030) + (-0.9030) +(-0.9030) + (-0.9030)]
4
= Antilog [(-0.9030)]
= 0.125 EU/mL
GM CSE in Product = log (0.0625) + log (0.0625) + log (0.125) + log( 0.125)
4
= Anti[(-1.2041)+ (-1.2041)]+(-0.9030) + (-9030)]
4
= Antilog [(-1.0536)]
= 0.0883 EU/mL
Since Geometric mean of CSE in water and CSE in product is between 2λ (0.25EU/mL) and
½ λ (0.0625EU/mL) product is validated at ½ MVD (1:200).
2. LIMIT TEST (METHOD A)
(i) PROCEDURE
Prepare solution A, B, C and D as shown in Table 2.6.14 and perform the test on
these solutions following the procedure described under 1. Preparatory testing,
(i) Confirmation of the labelled lysate sensitivity.
Prepare solution A and solution B (positive product control) using a dilution not
greater than the MVD and treatments as described in 1. Preparatory testing, (ii) Test for
interfering factors. Solutions B and C (positive controls) contain the standard endotoxin at a
concentration corresponding to twice the labelled lysate sensitivity. Solution D (negative
control) consists of water for BET.
(ii) Interpretation
The test is not valid unless both replicates of the 2 positive control solutions B and C
are positive and those of the negative control solution D are negative.
The preparation being examined complies with the test when a negative result is
found for both replicates of solution A.
- if the preparation being examined is diluted to the MVD, it does not comply with the
test,
- if the preparation being examined is diluted to a dilution less than the MVD, the test
is repeated at a dilution not greater than the MVD.
Repeat the test if a positive result is found for one replicate of solution A and a negative
result is found for the other. The preparation being examined complies with the test if a
negative result is found for both replicates of solution A in the repeat test.
Example :
MVD = 400
Sample CSE
LRW LAL
(1:200) (4λ)
NWC 100 µL − − 100 µL
PWC 50 µL − 50 µL 100 µL
NPC 50 µL 50 µL − 100 µL
PPC − 50 µL 50 µL 100 µL
In this method sample is directly prepared and tested at same dilution and CSE dilution is
prepared at 20λ = 20 x 0.125EU/mL = 2.5EU/mL
Sample CSE
LRW LAL
(1:400) (20λ)
NWC 100 µL − − 100 µL
PWC 100 µL − 10 µL 100 µL
NPC − 100 µL − 100 µL
PPC − 100 µL 10 µL 100 µL
Expected Results
NWC ––
PWC ++
Sample results
NPC –– ++ –– –+
PPC ++ ++ –– ++
Pass Fail Interference Repeat
Interference related problems are addressed separately later in this manual. Please refer to the
technical literature section also for a publication on Resolving LAL Test interferences and
The Impact of Non- Endotoxin LAL Reactive Material on LAL analyses.
3. SEMI – QUANTITATIVE TEST (METHOD B)
(i) Procedure
The test quantifies bacterial endotoxins in the test solution by titration to an end-point.
Prepare solution A, B, C and D as shown Table 2.6.14.-3, and test these solutions according to the
procedure described under 1. Preparatory testing, (i) confirmation of the labelled lysate sensitivity.
If none of the dilutions of the test solution is positive in a valid test, record the endotoxin
concentration as less than λ (or if a diluted sample was tested as less than λ x the lowest dilution
factor of the sample). If all dilutions are positive, the endotoxin concentration is recorded as equal to
or greater than the greatest dilution factor multiplied by λ .
The preparation meets the requirements of the test if the endotoxin concentration is less than
that specified in the individual monograph.
Example
MVD = 400
1
/16MVD ⅛MVD ¼ MVD ½ MVD MVD
(1:25) (1:50) (1:100) (1:200) (1:400)
NPC –– ++ –– –– ––
PPC –– ++ ++ ++ ++
= 50 x 0.125EU/mL
= 6.25 EU/mL
250 mg/mL
= 0.025 EU/mg
REFERENCE STANDARD ENDOTOXIN
CSE is the in-house standard. The Standard Testing Procedure and Standard Operating Procedure is
in compliance if it requires. 1. Acceptance of CoA from the CSE vendor
2. Completion of a label claim verification.
If need for USP reference to use CSE then one can refer to Pg. 1696 of USP 23 which gives the most
recent description of RSE / CSE ratio determination for Gel Clot Methods.
USP requires ALL CSE to have a ratio between 2 EU/ng and 50 EU/ng.
Every Manufacturer of LAL provides a Control Standard Endotoxin with a certificate of analysis that
contains the RSE to CSE ratio for specific LAL lot. All RSE to CSE ratios are Lot specific. Hence
before using LAL one should check whether they are using the correct combination of LAL and CSE
which is mentioned on the CoA.
Preparation of CoA for RSE/CSE:
USP requires ALL Control Standard Endotoxins (CSE) to have a ratio between
2 EU/ng and 50 EU/ng.
Hence with different batches of LAL of same sensitivity with same lot of CSE you will have
different RSE /CSE ratios. We can get different results which will result in different
RSE/CSE ratios. Some examples of the results and their calculations are listed below
Each CSE contains 100ng/vial. Each is assayed as follows. From each vial 0.025, 0.0125,
0.00625, 0.003125ng/mL is prepared and 0.1mL LAL is added to each tube. Assay is
performed in quadruplicate.
CSE contains 100ng /vial and is diluted to the below mentioned concentrations.
= 0.125 EU/mL
0.0125 ng/mL
= 10 EU/ng
Result 2
CSE Concentrations
Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + + + − 0.00625
2 + + − − 0.0125
3 + + − − 0.0125
4 + + − − 0.0125
= 0.0105 ng/mL.
= 0.0088 ng/mL.
= 0.0074 ng/mL.
Result 5
CSE Concentrations
Replicates 0.025 ng/mL 0.0125 ng/mL 0.00625 ng/mL 0.003125 ng/mL End point
1 + − − − 0.025
2 + − − − 0.025
3 + − − − 0.025
4 + − − − 0.025
= 0.025 ng/mL.
Reconstitute LAL after preparing and adding sample, CSE to the test tubes.
Steps :
1. Remove aluminum seal of the Lysate vial without opening rubber stopper.
2. Carefully remove the stopper. Keep the stopper in a clean surface without touching the
inner portion of stopper.
3. Rehydrate the lyophilized powder using required quantity of LRW with the help of
depyrogenated pipette.
With 1 mL LRW (for Limusate-1mL 10 test vial)
With 5 mL LRW (for Limusate-5 mL 50 test vial)
4. Stopper or use parafilm to seal the reconstituted vial. Do not shake. Mix gently without
formation of any air bubbles. AVOID TOUCHING OF LYSATE TO THE STOPPER.
DO NOT VORTEX.
Storage:
Store the reconstituted LAL reagent vial at 2 to 8° C for 8 hours. Reconstituted Lysate should
be preferably stored in the freezer below 0°C when it is to be used intermittently.
Freeze it & use on the next day. If not, can be frozen below - 20°C.up to 2 weeks, to be
frozen & thawed only once.
TESTING OF WATER FOR INJECTION USING HOT SPIKE
METHOD AND 50-50 METHOD.
If NPC −ve, PPC +ve report WFI contains less than 0.03 EU/mL.
If NPC +ve, PPC +ve report WFI contains 0.03 EU/mL
As per the USP 29, Chapter 1211 defines an acceptable cycle for depyrogenation as one
which gives a three log reduction in the endotoxin concentration at the end of the cycle.
PREPARATION
1. Remove seals.
2. Aseptically remove stoppers and discard.
3. Cover endotoxin vials with a double layer of aluminum foil.
1. Initiate recovery by rehydrating the endotoxin indicator vials including positive controls
with 1mL of LRW. Immediately vortex each container for 5 minutes.
2. Controls:
b. Standard Curve
Make a valid 4-point standard curve of the CSE using concentrations 2λ,λ,λ/2,λ/4 where
λ is the sensitivity of the Lysate or use a Lysate whose sensitivity is verified.
c. Negative Control
The LRW used above is the negative control.
CALCULATIONS
3. Log {Recovered EU/mL from PC} - Log {Recovered EU/mL from Sample}
= X - Log Reduction
INTERPRETATION OF RESULTS
1. The depyrogenation cycle was successful if there was a > 3-log reduction in endotoxin in
the heat exposed vials.
FLOW CHART FOR VALIDATION OF DRY HEAT STERILIZERS
OVENS / TUNNELS
Test all final dilutions in DUPLICATE with a LAL reagent of 0.125EU/mL sensitivity.
Results :
Test all final dilutions in DUPLICATE with a LAL reagent of 0.125 EU/mL sensitivity.
Results:
1 3
2
4
TOP 5
6 8
7
9
FRONT
Locations of Vials in Tunnel
3 7
6 8
2 4
1 5 9
Potency : 40 mg/mL
MVD : 1: 227
Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL
Results:
Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in
Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.125 EU/mL.
Preliminary Screening Test
MVD : 1:8
Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the
product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST
Results:
Test Endotoxin NEG NEG Test Log of Geometric
Material Concentration EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
2λ λ ½λ ¼λ
CSE + + − − − 0.0625 -1.2041
Water + + − − − 0.0625 -1.2041
Control + + − − 0.0625 -1.2041 0.0625 EU/mL
+ + − − 0.0625 -1.2041
Positive + + − − − 0.0625 -1.2041
Product + + − − − 0.0625 -1.2041 0.0625 EU/mL
Control + + − − − 0.0625 -1.2041
+ + − − − 0.0625 -1.2041
+ = Firm Gel − = No Gel or less than firm Gel
Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in
Product = 0.0625 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.06 EU/mL.
Preliminary Screening Test
MVD : 1:8
Conclusion: No inhibition observed. Test dilution of 1:4 to be chosen for validating the
product.
LIMULUS AMEBOCYTE LYSATE (LAL) TEST VALIDATION
AS AN END-PRODUCT ENDOTOXINS TEST
Preparation :
Results :
Test Endotoxin Concentration NEG NEG Test Log of Geometric
EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
Material 2λ λ 0.5λ 0.25λ
CSE + + − − − 0.0625 -1.2041
Water + + − − − 0.0625 -1.2041
Control + + − − 0.0625 -1.2041 0.0625
EU/mL
+ + − − 0.0625 -1.2041
Positive + + + − − 0.03125 -1.5051
Product + + + − − 0.03125 -1.5051 0.03125
Control + + + − − 0.03125 -1.5051 EU/mL
+ + + − − 0.03125 -1.5051
Comments : The end points of CSE in Water = 0.0625 EU/mL. The end point of CSE in
Product = 0.03125 EU/mL which is within ± one two fold dilution of the LAL labeled
sensitivity λ. λ = 0.0625 EU/mL.
Preliminary Screening Test
MVD : 1:320
Test Concentrations: 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, 0.2 mg/mL
Results :
Test Endotoxin NEG NEG Test Log of Geometric
Material Concentration EU/mL. Control Product Endpt. Endpt. Mean
Control EU/mL
2λ λ ½λ ¼λ
CSE + + − − − 0.125 -0.903
Water + + − − − 0.125 -0.903
Control + + − − 0.125 -0.903 0.125
EU/mL
+ + − − 0.125 -0.903
Positive + + + − − 0.0625 -1.2041
Product + + + − − 0.0625 -1.2041 0.0625
Control + + + − − 0.0625 -1.2041 EU/mL
+ + + − − 0.0625 -1.2041
+ = Firm Gel − = No Gel or less than firm Gel
Comments : The end points of CSE in Water = 0.125 EU/mL. The end point of CSE in
Product = 0.06 EU/mL which is within ± one two fold dilution of the LAL labeled sensitivity
λ. λ = 0.125 EU/mL