Limulus Amebocyte Lysate LAL Test Methods

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Limulus Amebocyte Lysate (LAL) Test Methods
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LAL Test Methods

• The gel-clot method
• The kinetic turbidimetric method
• The chromogenic methods
• (kinetic and endpoint)

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Gel-Clot
Turbidimetric
Chromogenic
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Biochemical Reaction
Endotoxin
Factor C
Activated Factor C
b-Glucan
Factor B
Factor G
Activated Factor(s) B/G
Clotting Enzyme
Activated Clotting Enzyme
Coagulogen
Coagulin
Gelation
Turbidimetric
Modified from Iwanaga et al., 1985
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The Gel-Clot Method

• Simplest and most widely used
• The USP referee method
• The labeled gel-clot reagent sensitivity (l) is
  the least concentration of endotoxin to cause a
  solid clot under standard conditions

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Reading the Gel-Clot Test
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Turbidimetric Methods

• As coagulin molecules coalesce forming particles,
  the reaction mixture becomes turbid
• The rate of increase in turbidity is a function
  of endotoxin concentration

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Turbidimetric Methods
light
DETECTOR
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Kinetic Data - OD vs time
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Kinetic Turbidimetric Method

• The threshold OD (onset OD) is used as a point of
  reference for data collection
• The greater the endotoxin concentration, the
  shorter the time taken to reach the onset OD

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Kinetic Turbidimetric Method

• The onset time is the time that it takes (in
  seconds) for the reaction to reach the onset OD
• Standard curves are constructed by plotting
  log10(onset time) on log10(endotoxin
  concentration)

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Standard Curve (Kinetic Test)
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Kinetic Turbidimetric Method

• Calculate sample endotoxin content by comparing
  with standards
• Take sample onset time and reference against
  standard curve to determine its endotoxin content

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Interference

• Most samples, at some concentration, interfere
  with the LAL reaction
• Interference is caused by
• sample interaction with the LAL reagent
• sample interaction with endotoxin

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Inhibition

• Inhibition is a reduction in sensitivity of the
  assay which causes an underestimation of the
  concentration of endotoxin
• Inhibition controls (PPCs) prevent
  misinterpretation of negative results

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Enhancement

• Enhancement is an increase in the sensitivity of
  the assay which causes an overestimation of the
  concentration of endotoxin
• Positive product controls (PPCs) prevent
  misinterpretation of positive results in the
  photometric methods

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False Positives

• Enhancement is not a false positive!
• A false positive test is a positive in the
  absence of endotoxin
• False positives are rare
• trypsin (all methods)
• activated serine proteases (chromogenic)
• beta-glucans (suspected, all methods)

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Positive Product Control

• All LAL tests must have a control to demonstrate
  that the sample itself does not cause a false
  negative result
• A known quantity of endotoxin is added to a
  portion of the sample under test to provide an
  inhibition or positive product control (PPC)

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Remove Interference

• Dilute with LRW first
• Use a more sensitive LAL reagent or method to
  increase the MVD
• Reconstitute LAL with Pyrosol (strongly buffered
  products outside the pH range, highly
  concentrated electrolytes, or for
  sample/endotoxin interactions)

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Maximum Valid Dilution

• The maximum valid dilution (MVD) is the greatest
  possible dilution at which the limit can be
  detected
• This is the dilution used for the pass/fail test
• The MVD increases with increasing test sensitivity

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Maximum Valid Dilution

• If l is 0.125 EU/mL and the unknown has an
  endotoxin limit of 2 EU/mL, calculate the MVD

Limit in EU/mL
2 EU/mL

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l in EU/mL
0.125 EU/mL
2 EU/mL
Limit in EU/mL

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l in EU/mL
0.03125 EU/mL
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Select a Sensitivity

• Sensitivity is lowest point on curve
• Consider the endotoxin limit and MVD
• Perform preliminary tests
• If interference cannot be overcome without
  exceeding the MVD of a product, go to a more
  sensitive reagent or method