Limulus Amebocyte Lysate (LAL) Test Methods - PowerPoint PPT Presentation

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Limulus Amebocyte Lysate (LAL) Test Methods

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Limulus Amebocyte Lysate (LAL) Test Methods LAL Test Methods The gel-clot method The kinetic turbidimetric method The chromogenic methods (kinetic and endpoint ... – PowerPoint PPT presentation

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Title: Limulus Amebocyte Lysate (LAL) Test Methods


1
Limulus Amebocyte Lysate (LAL) Test Methods
2
LAL Test Methods
  • The gel-clot method
  • The kinetic turbidimetric method
  • The chromogenic methods
  • (kinetic and endpoint)

3
Gel-Clot
Turbidimetric
Chromogenic
4
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
5
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

6
Reading the Gel-Clot Test
7
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

8
Turbidimetric Methods
light
DETECTOR
9
Kinetic Data - OD vs time
10
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

11
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)

12
Standard Curve (Kinetic Test)
13
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

14
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

15
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

16
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

17
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)

18
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)

19
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)

20
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

21
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

16

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

64

l in EU/mL
0.03125 EU/mL
22
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
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