Applied Microbiology

1. Pyrogen Testing : Limulus Amebocyte
Lysate( LAL) Test
Department of pharmacy
University of Science and Technology Chittagong

2. Content
01. Introduction
 Pyrogen and Pyrogen
testing
02. Introduction
about LAL test
03. Types of LAL
Test
04. Process of
different LAL test
05. Conclusion
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 Gel-clot technique
 Turbidimetric technique
 Chromogenic technique

3. Objectives
1. To know about Pyrogen and Pyrogen testing
2. To know about the LAL test
3. To explain the process of different types of LAL test
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4. Pyrogen
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5. Pyrogen testing
Pyrogen testing defines a process used by drug manufacturers to determine if bacterial
toxins are present in vaccines and parenteral drugs that might cause fever when used
on humans.
Widely used pyrogen test methods are –
1. In-vivo testing: Animal testing. Detection of both endotoxin and non-endotoxin
pyrogenic contaminants.
1. Rabbit Pyrogen test( RPT )
2. In-vitro testing: Affordable non animal testing, highly sensitive and fast process.
1. Limulus Amebocyte lysate ( LAL) test
2. Recombinant Factor C (rFC)
3. Monocyte Activation Test (MAT)

6. Limulus Ameobosyte
Lysate(LAL) test
The LAL test is also known as Bacterial Endotoxin Test or
BET test.It is an in vitro assay used to detect the presence and
concentration of bacterial endotoxins in aqueous parenteral
preparation.
US FDA approved LAL test for detecting pyrogen on 1977
It is quantitative analysis method.
The reagent used in this test is amoebocyte lysate from RBC
extract of horseshoe crab, Limulus polyphemus
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7. “LAL test is preferred than other test because –
• It is in-vitro assay and does not require animal handling,
thus is more convenient.
• It is 10 times more sensitive than that of the in-vivo rabbit
test.
• Less time consuming
• Easier to perform
• It requires less laboratory facilities and minimum
equipment.

8. Types of LAL test
1.Gel – clot
technique
2. Turbidimetric
technique
3. Turbidimetric
technique
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9. Gel –clot technique
 The gel-clot technique is used for
detecting endotoxins based on clotting of
the lysate reagent in test tubes in the
presence of endotoxin.
The minimum concentration of
endotoxin required to cause the lysate to
clot under standard conditions.
 It is a qualitative method
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10. Method of Gel – clot
technique
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First of all, we have to prepare test
tubes of 4 sample dilutions.
Then LAL reagent is added to the equal
volume of the test tubes.
After that the test tubes are agitated and
then allowed to incubate for 1 hour at 37℃
After incubation the tubes are inverted
If the solid remain intact the product is
considered to contain endotoxin

11. Turbidimetric
technique
Method:-
 It is a photometric assay measuring the
increase in turbidity caused by the
reaction of endotoxin and the lysate.
 A spectrophotometer is used in this
regard.
 In this method turbidity is developed
after cleavage of an endogenous
substrate.
The sample is taken
LAL reagent is added to
the sample
produce gel-clot
This result in turbidity
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12. Continued……
The relationship between the appearance of the turbidity in the LAL test and the
endotoxin concentration is exponential.
By measuring the turbidity of the solution after certain periods of time using
spectrophotometric methods, it is possible to reveal the presence of endotoxin but also
the amount that is present.
Turbidimetric method is two types. These are-
Turbidimetric Endpoint method: The reading of the turbidity is taken at a
determined period of time. Which can cause error as only a single moment to
make the measurement.
Turbidimetric kinetic method: this method is design to provide accurate and
reliable results, as in this method reading of turbidity is taken within the test
procedure.

13. Chromogenic
technique
The chromogenic method is also a photometric assay measuring
the colour , developed by the chromophore and released by the
chromogenic substrate by the reaction with endotoxin lysate.
The resulting colour of the reaction is measured using
spectrophotometric method to reveal the concentration of the
endotoxin in the sample.
The chromogenic reagent used in this method is a peptide
connected to p-nitroaneline,a yellow clottable coagulant.
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14. Procedure
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First of all , the LAL reagent is mixed
with the chromogenic reagent.
The mixture is then added to the test
sample to create the test solution.
. The test solution is then allowed to
incubate.
If endotoxin is present in the sample the enzymatic
reaction of the LAL reagent break the peptide bond
connected to p-nitroaniline molecule, releasing the yellow
colour into the solution.

15. Continued….
If more endotoxin is present, the more yellow the solution will become.
The amount of time required for the colour change to occur is inversely
proportional to the amount of endotoxin present.
The chromogenic technique is usually two types. These are-
oEndpoint chromogenic technique : it involves taking the measurement of
colour once when the LAL enzymatic reaction has finished.
oKinetic chromogenic technique: it is based on the measurement of colour at
different intervals of time after the addition of the chromogenic substrate to
the test sample.
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16. Weakness of LAL Test
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Major drawbacks of LAL tests are
• Is not a human-specific assay
• Does not detect non-endotoxin pyrogens
• Cellular or blood products, proteins, lipids, aluminium hydroxide adjuvants
(common in vaccines), glucans (false positives) cannot be tested.
• Very susceptible to interference from conditions
• Horseshoe crabs die 15% of the time after their blood is drawn for LAL –
leading to endangerment of species.

17. Conclusion
Conclusion
• Pyrogen test is usually performed to check the presence or absence of pyrogens in
aqueous parenterals as a quality control (QC) test for parenteral preparation.
• Between two types of pyrogen test LAL test is preferable because it is an in-vitro
test and does not require animal handling, it is less time consuming and easy to
perform.
• Among all the three LAL test techniques , kinetic chromogenic test is the best as it
is highly sensitive and can detect down to 0.0002 EU/ml with the single test kit
whereas turbidimetric detector can detect as low as 0.001EU/ml and gel-clot
detector can detect 0.015-0.50 EU/ml.so it is the best choice when the highest
accuracy and sensitivity is needed.
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18. “
References:-
1.Pelczar JM, Chan E.C.S.Microbiology.5th edi. New Delhi: Tata McGraw-Hill Publishing
company Limited;2017 P.261.
2.Denyer SP , Gorman PS, Hodges AN. Hugo and Russell’s Pharmaceutical
Microbiology.8th edi.UK: A John Wiley @ Sons, Ltd ;2011 P.190.
3. Aulton EM, Ansel CH. Pharmaceutical Formulations.8th edi. New Delhi : Career
Publications;2017 P.194.
4.https://images.search.yahoo.com/search/i?p=limulus+amebocyte+lysate+picture&fr=mcaf
ee&fr2=sb-top-images.search&ei.

19. Thank you