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 Turbidimetric Endotoxin Factor C
Activated Factor C
b-Glucan
Factor B
Activated Factor(s) B/G
Factor 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
positive
cloudy
negative

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
optical
density
time
threshold OD
0.25 EU/ml
0.125 EU/ml
EU/ml
EU/ml
0.5 EU/ml

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 (PPC’s) 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 (PPC’s) 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 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
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