limulus amebocyte lysate lal test methods
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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). Gel-Clot. Turbidimetric. Chromogenic. Biochemical Reaction. Endotoxin. Factor C. Activated Factor C. b - Glucan.

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lal test methods
LAL Test Methods
  • The gel-clot method
  • The kinetic turbidimetric method
  • The chromogenic methods

(kinetic and endpoint)





biochemical reaction
Biochemical Reaction


Factor C

Activated Factor C


Factor B

Activated Factor(s) B/G

Factor G

Clotting Enzyme

Activated Clotting Enzyme





Modified from Iwanaga et al., 1985

the gel clot method
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
turbidimetric methods
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
kinetic data od vs time

0.5 EU/ml

0.25 EU/ml

0.125 EU/ml



0.0625 EU/ml

0.03125 EU/ml

threshold OD


Kinetic Data - OD vs time
kinetic turbidimetric method
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 method1
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)
kinetic turbidimetric method2
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
  • 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 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 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
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
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
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
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 dilution1
Maximum Valid Dilution
  • If l is 0.125 EU/mL and the unknown has an endotoxin limit of 2 EU/mL, calculate the MVD:

2 EU/mL

Limit in EU/mL




l in EU/mL

0.125 EU/mL

2 EU/mL

Limit in EU/mL




l in EU/mL

0.03125 EU/mL

select a sensitivity
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