LIMULUS AMEBOCYTE LYSATE TEST LAL TEST

1. LIMULUS
AMEBOCYTE
LYSATETEST

2. ENDOTOXINS
• The toxic activity of LPS was first discovered and termed “endotoxin”
by Richard Friedrich Johannes Pfeiffer.
• Endotoxins are part of the outer membrane of the cell wall of Gram-
negative bacteria.
• These are invariably associated with Gram- negative bacteria whether
the organisms are pathogenic or not.
• These are important because it is a pyrogen and survive sterilization.
• Injectable drugs and medical devices which will contact blood or
spinal fluid are tested for endotoxins.
• They are thermostable, water- soluble, unaffected by the common
bactericides, non- volatile and liberated when bacteria die and cell
wall breaks apart.
• Consequences of endotoxin contamination:- Fever, Headache, Chills,
Nausea/ Vomiting, Hypotension, Acute lung injury, Miscarriage, Death.

3. LALTEST(BACTERIALENDOTOXINTEST)
• LIMULUS:- Genera of horseshoe crab ( Limulus polyphemus)
• AMEBOCYTE:- Crab blood cell from which active component is
derived.
• LYSATE:- Component is obtained by separating amebocytes
from the plasma and then lysing them.
• Developed in 1960’s by Drs. Bang and Levin.
• To measure the concentration of endotoxins of Gram- negative
bacterial origin.
• Based on clotting reaction of horseshoe crab blood to
endotoxin.

4. Principle
• The test is based on the primitive blood- clotting mechanism of the horseshoe crab.
• The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation or
gelation of the mixture.
• The rate of reaction depends on the concentration of endotoxin, the pH and the temperature.
• The reaction requires the presence of certain bivalent cations, a pro-clotting enzyme system and
clottable proteins all of which are provided by the lysate.
• Gram-negative bacterial endotoxin catalyzes the activation of proenzyme in the LAL. The initial rate of
activation is determined by the concentration of endotoxin present. The activated enzyme (coagulase)
hydrolyzes specific bonds within a clotting protein (coagulogen) also present in LAL. Once hydrolyzed,
the resultant coagulin self- associates and forms a gelatinous clot.

6. LYSATEREAGENT
● Bleeding adult crabs blood into
an anticlotting solution.
● Washing and centrifuging to
collect the amoebocytes.
● Lysing in 3% NaCl
● Lysate is washed and lyophilized
for storage.

7. 3DIFFERENTTECHNIQUES
GEL-CLOTTECHNIQUE
Gel formation
CHROMOGENIC
TECHNIQUE
The development of color
after cleavage of a
synthetic peptide-
chromogen complex.
01
02
03
TURBIDIMETRIC
TECHNIQUE
The development of
turbidity after cleavage of
an endogenous substrate

8. GELCLOTMETHOD
• A solid gel is formed in the
presence of endotoxins.
• This technique requires
positive and negative controls.
o Positive controls- a known
concentration of endotoxin
added to the lysate solution.
o Negative controls- water,
free from endotoxins, added
to the lysate solution.

9. TURBIDIMETRICMETHOD
• The test is based on the measurement
of opacity change due to the
formation of insoluble coagulin.
• Opacity is directly proportional to the
endotoxin concentration
• This technique is used for water
systems and simple pharmaceutical
products.
• The specimen is incubated with LAL
and either the rate of increase in
turbidity or the time taken to reach a
particular turbidity is measured
spectrophotometrically and compared
to standard curve.

10. CHROMOGENICMETHOD
• This is based on the measurement of
color change which is caused by the
release of the chromogenic chemical
p-nitroanilide.
• The quantity of the p-nitroanilide
produced is directly proportional to
the endotoxin concentration.
• Endotoxin catalyzes the activation of
a proenzyme in LAL which will cleave
a colorless substrate to produce a
colored end product which can be
measured spectrophotometrically and
compared to a standard curve

11. PROCEDURE
Formation of solid
gel clot that
withstands
inversion of the
tube constitutes a
positive test
Equal volume of
test solution and
LAL reagent are
mixed in glass test
tubes
After incubation
at 37° C for 1 h,
the tubes are
observed for clot
formation after
inverting them.
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12. RESULT

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