- 1977 FDA grants approval of first commercial LAL (Limulus Amybiocyte Lysate) reagent.
- Gel-clot technique based on the gel formation.
- Kinetic turbimetric based on the turbidity.
- Kinetic chromogenic based on the colour formation.
- "Frederick Bag" Gel-clot method is developed.
- Gel-clot method is pass or fail test.
- LAL (Limulus Amybiocyte Lysate) + Endotoxin = Clotting
- LAL test is in-vitro test for semi quantitative analysis of endotoxin ( it means given the results less than or more than of specified quantity).
- Depyrogenated dilution tubes(16 X 100 mm)
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Depyroginated Assay tubes(10 X 75 mm),
(polystyrene test tubes)
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Calibrated Micropipettes.
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Dipyrogenated Tips ( Endotoxin free pipette tips)
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pH Indicator
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calibrated stop watch
Equipment:
Calibrated Heating Block.
Materials:
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CSE (Controlled Standard Endotoxin)
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LAL ( Limulus Amybiocyte Lysate)
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LRW ( LAL Reagent Water)
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Test sample
Sample Preparation :
- Dispense 100 µL of sample and 100 µL of LAL in to 10 x 75 mm assay tubes ( Endotoxin free dilution tubes- Borosilicate glass or equivalent polystyrene plastic).
- Mix the sample solution.
pH Checking:
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The LAL reaction requires a neutral pH and is
time and concentration dependent.
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Sample and Lysate mixture take with the help
of the depyrogenated tip (Measure pH with one to one ratio of sample dilution and LAL to obtain an accurate pH reading).
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Put the one drop on pH indicator
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Read the pH
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Lysate having some pH adjustment buffers .
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Not maintain in the pH , please add the
sterile 0.1N Tri-NaoH, 0.1 NTri-Hcl.(Tri sodiumhydroxide, tri hydoxy chloride)
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The combination of LAL and prepared product
will have a neutral pH 6-8.
Observation:
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Temperature 37°C ±1°C
for 60 ± 2 min avoiding vibrations.
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Check the result 180° smoothly & gently
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Check the PPC results inverse position.
Select Lambda ( λ )
- Labelled LAL sensitivity.
- Choose one of the following LAL reagents (0.25 EU/mL, 0.125 EU/mL, 0.0625 EU/mL, 0.03125 EU/mL and 0.015625 EU/mL)
- Work in 2 fold dilutions for gel-clot tests.
- Run standard series from 2λ, λ, λ/2 and λ/4.
- 2λ - must always clot.
- λ/4 - must never clot.
- Negative controls must not clot.
PPC preparation 2 methods:
1. Hot Spike method :
In reaction Test tube 0.01 mL of 20λ + 0.1 mL (100 µL)of test Solution + 0.1 mL(100 µL) of LAL.
2. 50/50 method or Half strength method or Double Strength method:
- Use equal volumes of 4λ and sample.
- Sample should be 2x desired dilution/concentration.
- Accounts for 1: 2 dilution.
- It means 50 µL of 4λ + 50 µL of test solution + 100 µL LAL.
you can do 250 dilution, take 250 dilution of 50 µL sample and 50 µL LRW.
Routine Test Control:
- Each test has four controls.
- Negative Water Control (NWC)
- Positive Water Control (PWC)
- Negative Product Control (NPC)
- Positive Product Control (PPC)
Results observation:
End point sought by 180° inversion
Negative = anything other than firm gel.
Related : Pyrogen test ( Rabbit Test or Sham Test)