lal test

1. LAL: Choice of Test Method
By
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. 2.6.14 /
USP <85>

4. Introduction - LAL Test
 Main Test Methods:
Test Method Covered
Gel-clot
✓
Kinetic turbidimetric
✓
Kinetic end-point
✓*
Kinetic chromogenic
✓
Kinetic end-point
✓*
Non-EP / USP e.g. wet protein
colorimetric
X

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
Endotoxin
Factor C Active Factor C ß-(1,3)-D-Glucan
Factor B Active Factor B------------------------Active Factor G Factor G
Proclotting Enzyme Clotting Enzyme
Coagulogen Coagulin
Gel Formation

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
» Slide spot, micro-plate or tube
 Tube method
» Water bath or hot block (37oC +/- 1oC)
» Reaction tube: 0.1 ml lysate + 0.1 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
» 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?
Method Gel-clot Kinetic
Turbidimetric
Kinetic
Chrmogenic
Typical lower
detection limit
0.03 EU / mL 0.001 Eu / mL 0.005 EU / mL
Typical upper
detection limit
Fixed 100 EU / mL 50 EU / mL
Technician
involvement
High Medium Medium
Ease of use Low Medium High

23. Comparison
 How do they compare?
Method Gel-clot Kinetic
Turbidimetric
Kinetic
Chrmogenic
Test robustness Medium Medium High
Equipment cost Low High Medium
Reagent cost Medium Medium High

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