1. LAL: Choice of Test MethodBy
Tim Sandle
Bio Products Laboratory

2. Introduction How did we get here?
Main test methods - Gel-Clot, Turbidimetric and Chromogenic
Advantages and disadvantages of each method
Comparison between the main methods

3. Introduction - LAL Test
LAL Test - for performing BET
Alternative to Pyrogen (Rabbit) during 1980s
USP 1980; FDA Guide 1987; Ph. Eur. 1988; BP 1989 (Gel-clot test)
To meet the requirements for the Bacterial Endotoxin Test in Ph. Eur / USP <85>

4. Introduction - LAL Test
Main Test Methods:

5. Introduction - LAL Test
Different methods, same principles
Limulus amebocyte lysate reagent from horse shoe crabs
LAL detecting endotoxin
Detection based on natural clotting mechanism (Levin and Bang, 1968)
Tests utilise the clotting cascade

6. Introduction - LAL Test
Different methods, same principles

7. Gel -clot LAL Test Principle
LAL can be purified to be of different sensitivities so a clot = probability of a number of Endotoxin Units (EU) in a given sample
Limit or semi-quantitative through dilution series

8. Gel -clot LAL Test Method Tube method Slide spot, micro-plate or tube
Water bath or hot block (37oC +/- 1oC)
Reaction tube: 0.1 ml lysate ml sample
One hour incubation (+/- 2 minutes)
Invert tube through 180o - check for gelation

9. Gel -clot LAL Test EP / USP testing More complicated Positive controls
Negative controls
Endotoxin standard curve (confirm label claim)
Positive product controls (spiked samples)
Semi-quantitative through two-fold dilutions

10. Gel -clot LAL Test Advantages of the test Easy to perform
As a qualitative test - quick and simple
Inexpensive
Low equipment costs
Good for simple products or water
Reference method for USP / EP

11. Gel -clot LAL Test Disadvantages to the test Quantitation is difficult
Fixed incubation time
Interference
Limited ‘limit of detection’
Margin of error
No automation
Vibration
Subjective
Compliance issues

12. Photometric methods Turbidimetric and Chromogenic Techniques
Many similarities
Use spectrophotometry
Use a standard curve (r = 0.980)
Increased throughput
Wider ranges of quantitation
As kinetic methods - single steps
Reduced margins of error

13. Turbidimetric LAL Test
Principle:
Links the rate of gelation (as turbidity) to determine endotoxin content
Plotting turbidity (optical density) against endotoxin concentration from a series of standards
End-point or kinetic method

14. Turbidimetric LAL Test
Method
Heated plate reader or heated tube reader (37oC +/-1oC)
Spectrophotometer
Computer software
Lysate sensitivity determined via curve

15. Turbidimetric LAL Test
Advantages of the test
Real time measurement
Results can be ‘seen’
Reading is automated: objectivity
Software
Less dilutions cf Gel-clot (in-use costs)
Over-coming interference

16. Turbidimetric LAL Test
Disadvantages of the test
Turbid samples
Samples with precipitation
Some biologicals
Equipment cost
Vibration, bubbles and background noise
Technician expertise

17. Chromogenic LAL test Principle End-point or kinetic method
Uses the clotting cascade, but in a modified way
Synthetic chromogenic substrate - pNA - in the presence of LAL and endotoxin produces a yellow colour. Intensity of colour = relates to amount of endotoxin
End-point or kinetic method

18. Chromogenic LAL test Method
Heated plate reader or heated tube reader (37oC +/-1oC)
Spectrophotometer
Computer software
Lysate sensitivity determined via curve

19. Chromogenic LAL test Advantages of the test
Fast, real time measurement
Results can be ‘seen’
Reading is automated: objectivity
Software
Less dilutions cf Gel-clot (in-use costs)
Over-coming interference

20. Chromogenic LAL Test Disadvantages of the test Coloured samples
Equipment and reagent cost
Technician expertise / variability

21. Comparison What’s similar? Use the same / similar principle Use LAL
Use tube or micro-plate
Require endotoxin standards
Meet Regulatory requirements - FDA, MCA, EP, USP, JP

22. Comparison
How do they compare?

23. Comparison
How do they compare?

24. Comparison How do I choose? Gel-clot method? Turbidimetric method?
Chromogenic method?
End point or kinetic?

25. Summary Brief introduction to the LAL test The three main methods
Advantages and disadvantages of each
Comparison between them

26. Summary Final choice… It’s up to you: Your budget
Your application - water, raw materials, in-process samples or final products
Volumes to be tested
Material to be tested - turbid, coloured, precipitate, inhibition
Endotoxin limit
Degree of compliance

27. Thank you for your time