Limulus Lysate Test

The LAL test is a method of the Bacterial Endotoxin Test (BET) for detecting the
presence, and to go some way to determining the level, of Gram-negative bacterial
endotoxins in a given sample or substance.

From: Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals, 2013

Related terms:

Gram-Negative Bacteria, Lipopolysaccharide, Enzyme, Protein, Pyrogen, Endotox-

in, Meningitis, Bacterium

View all Topics

RAPID METHODS FOR FOOD HY-

GIENE INSPECTION
Matthias Upmann, Christine Bonaparte, in Encyclopedia of Food Microbiology,
1999

Limulus Amoebocyte Lysate Test

The limulus amoebocyte lysate (LAL) test is a simple method for the detection of
viable and non-viable Gram-negative bacteria. Certain cell-wall lipopolysaccharides
(i.e. endotoxins) of this bacterial group lead to gelation of blood cell (amoebocytes)
lysates of the Limulus polyphemus crab. Using a dilution row and determining the
limit at which no more gel formation occurs, a semi-quantitative estimation of the
Gram-negative content is possible.

Several test-kits are available. Mostly used for pyrogen control of pharmaceutical
products the LAL-test is applicable for predominantly Gram-negative containing
foods such as fresh meat, milk and eggs. Another field of application may be the
retrospective assessment of the microbiological quality of heated products.

> Read full chapter

Handbook of Modern Pharmaceutical
Analysis
Gregory A. Birrer, ... Judy Estrada, in Separation Science and Technology, 2001

A. Basic LAL Test Procedure

The LAL test reagent (as well as complete test kits) can be purchased from various
commercial sources and are stored frozen. The reagent is combined with equal
volumes of the serially diluted test sample. After incubation at 37°C, the mixture
is checked for evidence of a clotting reaction (gel clot), and the test samples are
compared to parallel dilutions of a reference endotoxin. The formation of a gel
clot indicates the presence of bacterial endotoxins in the sample and the test is
considered positive. The test must be properly controlled, all testing materials
such as tubes must be pyrogen free, and the temperature, pH, and reaction time
must be tightly controlled per USP instructions. Additionally, for BET testing to be
valid, the laboratory must be qualified to conduct the LAL test and the amount of
variability of the LAL test performed in the laboratory must be determined. The
activity (sensitivity) of each lot of lysate must be verified. This is accomplished by
testing the lysate versus a range of concentrations of control standard endotoxin
(CSE). The CSE is a commercially available standard preparation of endotoxins from
Escherichia coli ATCC 0113 (also commercially available). The sensitivity of the lysate
is confirmed if the gel clot end point is within a 2-fold dilution of the CSE.

> Read full chapter

MICROBIOLOGICAL TECHNIQUES
G.L. Pettipher, ... H.H. Wang, in Encyclopedia of Analytical Science (Second Edition),
2005

Limulus test
The Limulus test can be used to determine rapidly and specifically the cumulative
content of Gram-negative bacteria in foods. Gram-negative bacteria produce a
lipopolysaccharide (LPS) (endotoxin), which is a high-molecular-weight complex; it is
not produced by Gram-positive bacteria. Present in the blue blood of the horseshoe
crab, Limulus polyphemus is a nucleated cell called an amoebocyte, the cytoplasm
of which is densely packed with granules. Limulus blood clots in the presence of
bacterial LPS. All the necessary clotting factors are contained in the extract of the
amoebocyte granules, called Limulus lysate. The Limulus test is specific for LPSs and
is very sensitive. As little as 10−12 g LPS per milliliter can be detected, occasionally
even 10−15 g ml−1. A single Gram-negative bacterium contains 10−14 g of LPS.

For the Limulus test, a 10-fold dilution series of the sample is prepared and equal
volumes of the Limulus lysate and diluted sample are mixed in a test tube. The
test tube is then incubated before being inverted and read. If the mixture remains
unchanged and runs out of the tube then that dilution of the sample does not
contain LPSs. If a firm opaque gel is formed that sticks to the bottom of the tube
then that dilution of the sample contains LPSs. Generally, a visual reading of the
tenfold or twofold dilutions gives sufficient information about the level of LPSs
present in the sample.

The limulus amoebocyte lysate (LAL) test with chromogenic substrate is faster
than the gelation method, and it can be automated. The chromogenic substrate is
attached to -nitroaniline, that is released when reacted with the endotoxin-activated
enzymes. The free -aniline is read at 405 nm.

> Read full chapter

Pyrogenicity and bacterial endotoxin

Tim Sandle, in Sterility, Sterilisation and Sterility Assurance for Pharmaceuticals,
2013

2.5 Alternative assays

Later developments with the LAL test have seen the growing use of recombinant
lysates, due to concerns with the prolonged ecological and economic use of horse-
shoe crabs. Other methods have also been developed, such as ELISA (Enzyme-Linked
Immunosorbent Assay) based methods. With these methods, endotoxin is bound to
a phage protein and detected by recombinant Factor C (rFC) and quantified through
the detection of a fluorescence substrate.

An alternative to both the LAL test and the classic rabbit pyrogen test is the Monocyte
Activation Test (MAT). The basis of the MAT is that pyrogens stimulate monocytes to
produce cytokines (IL-6, TNF-a) or lead to the formation of metabolites (neopterin,
nitrite) from cytokine-inducible pathway cells, which can then be measured in the
supernatants of the cultured cells by ELISA methods (IL-6, TNF-a, neopterin). Thus,
the test can detect non-endotoxin pyrogens as well as endotoxins [20]. Whilst the
method is limited by the need of blood from donors and the variability from one
donor to another, it is described in the European Pharmacopoeia, which confers it
legitimacy (chapter 2.6.30: Monocyte-activation Test). pharmacopoeia

> Read full chapter

Bacterial Sepsis and Meningitis

Victor Nizet, Jerome O. Klein, in Infectious Diseases of the Fetus and Newborn
(Seventh Edition), 2011

Rapid Techniques for Detection of Bacterial Antigens in Body

Fluid Specimens
In the 1970s, the limulus lysate assay for detection of endotoxin produced by
gram-negative bacteria based on a gelation reaction between lysates of Limulus
(horseshoe crab) amebocytes and bacterial endotoxin was investigated for diagno-
sis of neonatal meningitis with equivocal results [537–541]. Counterimmunoelec-
trophoresis also was used successfully for detecting the capsular polysaccharide
antigens of various pathogenic bacteria, including S. pneumoniae, N. meningitidis, H.
influenzae, and GBS (see Chapter 12) in CSF, serum, and urine. Less complex and
more rapid detection methods have replaced these two assays.

Latex agglutination detection now is preferred because of its speed, simplicity,

and greater sensitivity for selected organisms. Kits designed to detect cell wall or
capsular or cell wall antigen released into body fluids are commercially available.
Latex agglutination assays have been shown to be of potential benefit in early
detection of bacterial antigens in CSF of patients with acute meningitis, which may
be of increased importance in the era of intrapartum antibiotic prophylaxis and its
potential interference with culture yield. Among the prevalent bacterial pathogens
in neonatal infections, only GBS is routinely analyzed by latex agglutination. N.
meningitidis group B shares a common capsular antigen, however, with the neonatal
meningitis pathogen E. coli serotype K1, which should allow cross-identification
of the latter using a meningococcal latex reagent [542]. The sensitivity of latex
agglutination methods for identifying infants with group B streptococcal meningitis
ranges from 73% to 100% for CSF and 75% to 84% for urine [543]. Possible cross-re-
actions have occurred when concentrated urine was tested. GBS cell wall antigen can
occasionally cross-react with antigens from S. pneumoniae, CoNS, enterococci, and
gram-negative enteric bacteria, including P. mirabilis and E. cloacae.

False-positive results in urine for a positive latex agglutination test for GBS often
were caused by contamination of bag specimens of urine with the streptococci from
rectal or vaginal colonization [544]. The poor specificity of GBS antigen detection
methods used with urine led to the U.S. Food and Drug Administration (FDA)
recommendation in 1996 that these methods not be employed except for testing
of CSF and serum.

> Read full chapter

Endotoxin and pyrogen testing

Tim Sandle, in Pharmaceutical Microbiology, 2016

11.5 The limulus amebocyte lysate test

The most widespread endotoxin test is the LAL test. The principle of the LAL test
is a reaction between LPS and a substance (clottable protein) contained within
amoebocyte cells derived from the blood of the horseshoe crab, as illustrated in
Figure 11.4 (of which Limulus polyphemus is the most commonly used species,
although other species, such as Carcinoscorpius and Tachypleus demonstrate the
same effect). The reaction of the horseshoe crab to endotoxin (the formation of a
clot) has been known since the 1950s [13]. LAL is an aqueous extract obtained after
lysis of blood cells (amoebocytes).

Figure 11.4. Image of the Limulus “horseshoe crab.”Image from Creative Commons
Library.

When endotoxin comes into contact with LAL, it initiates a series of enzymatic
reactions that result in the activation of a pathway to the production of at least three
serine protease zymogens (factor C, factor B, and pro-clotting enzyme). This pathway
alters amoebocyte coagulogen (an invertebrate fibrinogen-like clottable protein) to
form coagulin gel.
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine
serves as the nucleophilic amino acid at the active site. They are found in humans
as wells as in horseshoe crabs (and indeed in all mammals). In humans, they are
responsible for co-ordinating various physiological functions, including digestion,
immune response, blood coagulation, and reproduction. It is the blood coagulation
reaction that is similar in both humans and horseshoe crabs [29]. It is on this basis
that LAL has become the sensitive test reagent made from the soluble protein extract
(lysate) of horseshoe crab blood cells (amoebocytes). All commercial lysates can
detect picograms (10− 12 g) of endotoxin.

The reference test in the pharmacopoeias is the gel clot (or gelation) and is conducted
on the end-point principle. The description of the test and the necessary validation
and accompanying controls is so detailed in both US Pharmacopeia (USP) and
European Pharmacopoeia (Ph. Eur.) (harmonized since 1999).

The clotting mechanism of the blood of the crab is designed to prevent the spread
of bacterial contamination throughout the horseshoe crab’s biochemical system.
When the endotoxin of Gram-negative bacteria contacts with the horseshoe crab’s
amoebocytes, a series of enzymatic reactions begin. The pathway alters amebocyte
coagulogen into a fibrinogen-like clottable protein, which forms a coagulin gel. The
defence mechanism is also effective against fungi, hence a similar reaction occurs in
response to a fungal infection, which triggers the clotting cascade. In the reactions,
-glucans trigger the protease enzyme factor G, whereas endotoxin triggers the
factor C enzyme, although the end result—coagulin—is the same [14].

A considerable amount more glucan (1000 times) is required to trigger the clotting
cascade than the equivalent amount of endotoxin. The glucan required to trigger
factor G can be of varying molecular weights (such weights range from 3 to 100 kDa)
[15]. The clotting reaction is illustrated in Figure 11.5.

Figure 11.5. LAL-clotting cascade.Adapted by the author.

Glucan as an interfering substance is explored below.

The LAL reagent used for the gel-clot is supplied with an identified sensitivity or
label claim ( ), for example, 0.03 EU/mL. This means that when mixed with an equal
volume of the material under test, a gel or clot will form if the material contains
0.03 EU/mL or greater. For the kinetic methods, the lysate does not come with a
label claim. The test sensitivity is determined by the lowest point of the standard
curve used with each assay [16].

When it is necessary to quantify endotoxin concentration in a material, it is usual

to test a series of doubling dilutions against the reagent in temperature-controlled
conditions. The greatest dilution that gives a positive result (formation of a gel or
clot) is the end point, and the concentration of endotoxin in the original material can
be calculated by multiplying the dilution factor at the end point by the sensitivity of
the LAL reagent.

LAL tests require validation for each technician and each product before being ap-
plied routinely and, even then, valid assays require careful internal standardization.
LAL tests require the use of standard endotoxin, termed a positive product control
(PPC), which is a known amount of endotoxin mixed with a test material to confirm
the absence of interference.

In the early days of the LAL test, endotoxin standards were variable. The potency
varied with the method of purification, the bacteria of its origin and how it was
formulated. Initially LAL test results were reported as units of weight. The problem
with this was that results between tests and laboratories were not comparable. This
was because different endotoxins, of the same weight and from different species of
bacteria, can have different potencies. This led to the need for an endotoxin standard.
This standard was based on Escherichia coli 0113: H10 K negative. This introduced
the EU, which is a measure of the activity (potency) of endotoxin against the LAL test,
irrespective of mass [17].

The units of measurement for the LAL test are EU. These are a measure of the
activity of the endotoxin. Endotoxins differ in their biological activity or potency; the
pyrogenicity or LAL reactivity of one endotoxin preparation may be very different
from that of another of the same weight. Conversely, two endotoxin molecules
may be different sizes and different weights but may have the same reactivity in an
LAL test. The potency of an endotoxin determined with one LAL reagent lot may
differ from that determined with another lot. Expressing endotoxin concentrations
in EUs avoids the issues of different potencies of different endotoxins and allows
us to compare results of different LAL tests performed in different laboratories.
Consequently, it is not the usual practice to convert results of an LAL test in EU/mL
to units of weight of endotoxin per milliliter.
The origin of the test standard is from when the USP and US Food and Drug
Administration (FDA) commissioned Rudbach to resolve the issue by preparing a
reference endotoxin that was stable, could be chemically characterized, could be
lyophilized without loss of activity and that was free from biologically active proteins
[18]. This is termed reference standard endotoxin (RSE). Since RSE is expensive and
potentially exhaustible, control standard endotoxin (CSE), also manufactured from
E. coli 0113, is normally used for routine work but not necessarily for fundamental
research. The potency of a given batch of CSE is determined relative to the current
Ph. Eur., USP, or FDA lot of RSE and is specific to particular batches of LAL reagent.
Thus, a purified form of LPS is used to manufacture a reference standard for the LAL
assay. The endotoxin control is lyophilized with lactose and polyethylene glycol.

> Read full chapter

Analysis of Glycans; Polysaccharide

Functional Properties
N. Ohno, in Comprehensive Glycoscience, 2007

2.17.3.5 Early Diagnosis of Deep Mycosis and BG

The number of deep mycosis patients tends to increase with time. It seems that
several high-risk factors, such as an increase in the number of patients with an
impaired immune system accompanying aging and the spread of highly advanced
medical care, are involved. Therefore, the availability of early diagnostic methods is
important. The typical causes of deep mycoses are Candida and Aspergillus. Since
1-3-glucan is present in their cell walls, it is useful for early diagnosis to measure
trace amounts of BG in the blood. BG can be measured at trace level by the
limulus test, in which the biodefense mechanism in limulus specifically reacts with
1-3-glucan in the patient's blood (Figure 3).75–80 A positive reaction in the limulus
test indicates that 1-3-glucan is actually released into the blood. However, as the
substance released could not be isolated, its real structure is unknown. When Ca.
albicans was grown in a complete synthetic medium, the soluble macromolecular
fraction, named CAWS, was released into the medium. CAWS was subjected to
the limulus test, and a positive result was obtained.81 Interestingly, this fraction
consisted not only of BG; rather, its main component was a mannoprotein complex.
Further analysis revealed that CAWS has a large molecular weight distribution and
was not a single population. When the biological activity of CAWS was examined,
various activities were found, including acute lethal toxicity and angiitis-inducing
effect. The question arises then if this is indeed the substance present in blood. No
conclusions have been drawn yet at this stage.

Figure 3. Outline of coagulation cascades of endotoxin and 1-3-glucan in limulus

lysate. Limulus lysate contained specific recognition proteins for bacterial endotoxin
(factor C) and 1-3-glucan (factor G). Binding of endotoxin or 1-3-glucan to
these proteins activates coagulation cascade of the lysate and produces ‘clot’. The
coagulation cascade include several specific proteinases showing very restricted sub-
strate specificity. For the quantitative determination of clinical materials, synthetic
substates are applied, as shown in this figure.

> Read full chapter

A Framework for Evaluating Nonclini-

cal Safety of Novel Adjuvants and Adju-
vanted Preventive Vaccines
P.E. Boucher, in Immunopotentiators in Modern Vaccines (Second Edition), 2017

In Vitro Tests for Pyrogens: The Limulus Amebocyte Lysate and

Monocyte Activation Test Assays
Pyrogens are fever-inducing substances usually derived from microorganisms [en-
dotoxins or lipopolysaccharide (LPS)] and when present systemically in sufficient
quantity can lead to severe signs of inflammation, shock, multiorgan failure, and
sometimes even death in humans. Testing for pyrogens is a requirement for all
parenteral products including injectable vaccines, and there have traditionally been
two tests for pyrogenicity: the rabbit pyrogen test (RPT) and the Limulus amebocyte
lysate (LAL) test. In keeping with the policy of the 3Rs, the FDA has in many
cases accepted the use of the in vitro test in lieu of the animal test for more
than 30 years. The LAL test is a highly quantitative way to measure contaminating
endotoxin from gram-negative bacteria and is based on the clotting reaction of the
hemolymph of the horseshoe crab. We note that the WHO guidance recommends
that vaccine developers should provide evidence that the adjuvant or adjuvanted
vaccine formulation does not interfere with LAL test. However, the high specificity
for bacterial LPS restricts the LAL test's ability to monitor pyrogenicity and potential
immune activation mediated by other product contaminants.18 The test is incapable
of detecting pyrogens such as lipoproteins, peptidoglycan, and lipoteichoic acids
from gram-positive bacteria9,70 and is thus an imperfect reflection of the human
febrile reaction. This precludes the complete dismissal of the RPT test in many cases.

The MAT is based on the in vitro activation of human monocytoid cells in whole
human blood or cell lines by pyrogens. The presence of pyrogens such as bacterial
endotoxin stimulates the release of proinflammatory cytokines interleukin (IL)-1-
, IL-6, and tumor necrosis factor (TNF)- , which are quantified by ELISA, flow
cytometry, or bead-array.46,49,75 Variants of the MAT have been standardized and
validated47,83 and were adopted by the European Pharmacopoeia in 2010. Depending
on the diverse immunization strategies and novel vaccines and adjuvants, the use
of both the MAT and the LAL test for routine lot release safety evaluation has been
recommended.69

It is noteworthy that despite the benefits offered by the use of validated in vitro
cell culture assays, discussions with regulatory authorities are highly recommended
to ensure that any MAT analysis would suffice to monitor pyrogen contamination.
For example, pyrogens may induce fever independent of cytokines, by acting at
TLRs.21 Studies in mice genetically engineered to lack pyrogenic cytokines and
clinical observations of fevers that frequently occur without a concomitant increase
of circulating cytokines11,43,62,72 collectively suggest that the MAT assay may not
always faithfully reflect a physiological febrile response in humans. In such scenarios,
the RPT may be an indispensable or at least complementary tool to monitor febrile
signals.

> Read full chapter

Bacterial and Fungal Intracranial Infec-

tions
Linda S. de Vries, Joseph J. Volpe, in Volpe's Neurology of the Newborn (Sixth
Edition), 2018
Identification of the Microorganism in Cerebrospinal Fluid.
Determination of the bacterial cause of meningitis obviously is made most readily
and decisively by culture of the CSF. The yield in the previously untreated patient
whose CSF is cultured promptly on the various media necessary to isolate the
different microorganisms responsible for neonatal bacterial meningitis (including
Listeria) approaches 100%. The result is usually available within 48 hours.

Faster techniques available for identifying microorganisms include Gram-stained

smears, countercurrent immunoelectrophoresis, limulus lysate assay, and latex par-
ticle agglutination. Stained smears demonstrated bacteria on the initial CSF evalua-
tion of 119 newborn infants with meningitis in 83% of patients with GBS meningitis
and in 78% of those with gram-negative bacterial meningitis.238 This universally
available, simple procedure should be performed on every CSF sample. Countercur-
rent immunoelectrophoresis is a sensitive and rapid technique that demonstrates
the presence of bacterial antigen within approximately 2 hours. The test was formerly
widely used, particularly in the previously treated patient with possible nonviable
bacteria in the CSF. Countercurrent immunoelectrophoresis requires specific an-
tiserum, which is available for type III GBS and K1 E. coli (among the common
pathogens for neonatal meningitis).30,233,241-245 Rapid detection of meningitis caused
by gram-negative enteric organisms was formerly based particularly on the limulus
lysate assay.30,246 These last two tests have been largely replaced by latex particle ag-
glutination tests,247 which is based on the agglutination of specific antibody-coated
latex particles by bacterial antigen. The test is particularly useful for GBS and E. coli
K1. The assay can provide a result in minutes.

> Read full chapter

Limulus Coagulation Factor G

Shun-ichiro Kawabata, ... Sadaaki Iwanaga, in Handbook of Proteolytic Enzymes
(Third Edition), 2013

Name and History

Limulus coagulation factor G is a component of the serine protease cascade that
leads to coagulation of the horseshoe crab hemolymph. The system and its compo-
nents are introduced in the chapter on limulus coagulation factor C (Chapter 672).

In the course of the diagnostic application of the limulus test, it was pointed out that
positive reactions are observed with plasma of some patients even in the absence of
 bacterial lipopolysaccharide (LPS) [1]. Since some of these patients were suffering
from fungus infection or were undergoing hemodialysis with cellulosic dialyzers,
this pseudo-positive reaction had been suggested to be at least in part caused
by -1,3-glucans. Eventually, the -1,3-glucan-sensitive protease was found in the
hemocyte lysate [2,3] and purified, and termed factor G [4,5].

> Read full chapter

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