Transcript ACC 2001 New Product Highlight

Limulus Amebocyte Lysate (LAL)
Test Methods
LAL Test Methods
• The gel-clot method
• The kinetic turbidimetric method
• The chromogenic methods
(kinetic and endpoint)
Gel-Clot
Turbidimetric
Chromogenic
Biochemical Reaction
Endotoxin
Factor C
Activated Factor C
Factor B
b-Glucan
Activated Factor(s) B/G
Clotting Enzyme
Factor G
Activated Clotting Enzyme
Coagulogen Coagulin
Gelation
Modified from Iwanaga et al., 1985
Turbidimetric
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
Reading the Gel-Clot Test
positive
cloudy
negative
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
Turbidimetric Methods
light
DETECTOR
Kinetic Data - OD vs time
0.5 EU/ml
0.25 EU/ml
0.125 EU/ml
0.0625 EU/ml
optical
density
0.03125 EU/ml
threshold OD
time
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
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)
Standard Curve (Kinetic Test)
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
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
Inhibition
• Inhibition is a reduction in sensitivity
of the assay which causes an
underestimation of the concentration
of endotoxin
• Inhibition controls (PPC’s) prevent
misinterpretation of negative results
Enhancement
• Enhancement is an increase in the
sensitivity of the assay which causes
an overestimation of the
concentration of endotoxin
• Positive product controls (PPC’s)
prevent misinterpretation of positive
results in the photometric methods
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)
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)
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)
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
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
Limit in EU/mL = 2 EU/mL
= 64
l in EU/mL
0.03125 EU/mL
Select a Sensitivity
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•
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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