limulus amebocyte lysate (lal) test methods
DESCRIPTION
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. b - Glucan. - PowerPoint PPT PresentationTRANSCRIPT
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
Modified from Iwanaga et al., 1985
Gelation
Coagulogen Coagulin
Endotoxin
Factor B Factor G
Clotting Enzyme
Activated Factor(s) B/G
Activated Clotting Enzyme
Factor C Activated Factor C -Glucan
Turbidimetric
The Gel-Clot Method
• Simplest and most widely used
• The USP referee method
• The labeled gel-clot reagent sensitivity () is the least concentration of endotoxin to cause a solid clot under standard conditions
Reading the Gel-Clot Test
positive
negative
cloudy
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
opticaldensity
timethreshold OD
0.25 EU/ml0.125 EU/ml0.0625 EU/ml
0.03125 EU/ml
0.5 EU/ml
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
• If is 0.125 EU/mL and the unknown has an endotoxin limit of 2 EU/mL, calculate the MVD:
Maximum Valid Dilution
Limit in EU/mL in EU/mL
= 2 EU/mL0.125 EU/mL
= 16
Limit in EU/mL in EU/mL
= 2 EU/mL0.03125 EU/mL
= 64
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