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LAL: Choice of Test MethodLAL: Choice of Test Method
ByBy
Tim SandleTim Sandle
Bio Products LaboratoryBio Products Laborat...
IntroductionIntroduction
• How did we get here?How did we get here?
• Main test methods - Gel-Clot, TurbidimetricMain test...
Introduction - LAL TestIntroduction - LAL Test
• LAL Test - for performing BETLAL Test - for performing BET
• Alternative ...
Introduction - LAL TestIntroduction - LAL Test
• Main Test Methods:Main Test Methods:
Test Method Covered
Gel-clot

Kinet...
Introduction - LAL TestIntroduction - LAL Test
• Different methods,Different methods, same principlessame principles
» Lim...
Introduction - LAL TestIntroduction - LAL Test
• Different methods,Different methods, same principlessame principles
Endot...
Gel -clot LAL TestGel -clot LAL Test
• PrinciplePrinciple
» LAL can be purified to be of different sensitivitiesLAL can be...
Gel -clot LAL TestGel -clot LAL Test
• MethodMethod
» Slide spot, micro-plate or tubeSlide spot, micro-plate or tube
• Tub...
Gel -clot LAL TestGel -clot LAL Test
• EP / USP testingEP / USP testing
– More complicatedMore complicated
» Positive cont...
Gel -clot LAL TestGel -clot LAL Test
• Advantages of the testAdvantages of the test
» Easy to performEasy to perform
» As ...
Gel -clot LAL TestGel -clot LAL Test
• Disadvantages to the testDisadvantages to the test
» Quantitation is difficultQuant...
Photometric methodsPhotometric methods
• Turbidimetric and ChromogenicTurbidimetric and Chromogenic
TechniquesTechniques
•...
Turbidimetric LAL TestTurbidimetric LAL Test
• Principle:Principle:
» Links the rate of gelation (as turbidity) to determi...
Turbidimetric LAL TestTurbidimetric LAL Test
• MethodMethod
» Heated plate reader or heated tube reader (37Heated plate re...
Turbidimetric LAL TestTurbidimetric LAL Test
• Advantages of the testAdvantages of the test
» Real time measurementReal ti...
Turbidimetric LAL TestTurbidimetric LAL Test
• Disadvantages of the testDisadvantages of the test
» Turbid samplesTurbid s...
Chromogenic LAL testChromogenic LAL test
• PrinciplePrinciple
» Uses the clotting cascade, but in a modified wayUses the c...
Chromogenic LAL testChromogenic LAL test
• MethodMethod
» Heated plate reader or heated tube reader (37Heated plate reader...
Chromogenic LAL testChromogenic LAL test
• Advantages of the testAdvantages of the test
» Fast, real time measurementFast,...
Chromogenic LAL TestChromogenic LAL Test
• Disadvantages of the testDisadvantages of the test
» Coloured samplesColoured s...
ComparisonComparison
• What’s similar?What’s similar?
» Use the same / similar principleUse the same / similar principle
»...
ComparisonComparison
• How do they compare?How do they compare?
Method Gel-clot Kinetic
Turbidimetric
Kinetic
Chrmogenic
T...
ComparisonComparison
• How do they compare?How do they compare?
Method Gel-clot Kinetic
Turbidimetric
Kinetic
Chrmogenic
T...
ComparisonComparison
• How do I choose?How do I choose?
– Gel-clot method?Gel-clot method?
– Turbidimetric method?Turbidim...
SummarySummary
• Brief introduction to the LAL testBrief introduction to the LAL test
• The three main methodsThe three ma...
SummarySummary
• Final choice…Final choice…
– It’s up to you:It’s up to you:
Your budgetYour budget
Your application - wat...
Thank you for your timeThank you for your time
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Lal presentation

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Overview of bacterial endotoxin testing methods (LAL)

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Lal presentation

  1. 1. LAL: Choice of Test MethodLAL: Choice of Test Method ByBy Tim SandleTim Sandle Bio Products LaboratoryBio Products Laboratory
  2. 2. IntroductionIntroduction • How did we get here?How did we get here? • Main test methods - Gel-Clot, TurbidimetricMain test methods - Gel-Clot, Turbidimetric and Chromogenicand Chromogenic • Advantages and disadvantages of eachAdvantages and disadvantages of each methodmethod • Comparison between the main methodsComparison between the main methods
  3. 3. Introduction - LAL TestIntroduction - LAL Test • LAL Test - for performing BETLAL Test - for performing BET • Alternative to Pyrogen (Rabbit) duringAlternative to Pyrogen (Rabbit) during 1980s1980s • USP 1980; FDA Guide 1987; Ph. Eur.USP 1980; FDA Guide 1987; Ph. Eur. 1988; BP 1989 (Gel-clot test)1988; BP 1989 (Gel-clot test) • To meet the requirements for the BacterialTo meet the requirements for the Bacterial Endotoxin Test in Ph. Eur. 2.6.14 /Endotoxin Test in Ph. Eur. 2.6.14 / USP <85>USP <85>
  4. 4. Introduction - LAL TestIntroduction - LAL Test • Main Test Methods: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. 5. Introduction - LAL TestIntroduction - LAL Test • Different methods,Different methods, same principlessame principles » LimulusLimulus amebocyte lysate reagent from horse shoeamebocyte lysate reagent from horse shoe crabscrabs » LAL detecting endotoxinLAL detecting endotoxin » Detection based on natural clotting mechanismDetection based on natural clotting mechanism (Levin and Bang, 1968)(Levin and Bang, 1968) » Tests utilise the clotting cascadeTests utilise the clotting cascade
  6. 6. Introduction - LAL TestIntroduction - LAL Test • Different methods,Different methods, same principlessame 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. 7. Gel -clot LAL TestGel -clot LAL Test • PrinciplePrinciple » LAL can be purified to be of different sensitivitiesLAL can be purified to be of different sensitivities so a clot = probability of a number of Endotoxinso a clot = probability of a number of Endotoxin Units (EU) in a given sampleUnits (EU) in a given sample • Limit or semi-quantitative through dilutionLimit or semi-quantitative through dilution seriesseries
  8. 8. Gel -clot LAL TestGel -clot LAL Test • MethodMethod » Slide spot, micro-plate or tubeSlide spot, micro-plate or tube • Tube methodTube method » Water bath or hot block (37Water bath or hot block (37oo C +/- 1C +/- 1oo C)C) » Reaction tube: 0.1 ml lysate + 0.1 ml sampleReaction tube: 0.1 ml lysate + 0.1 ml sample » One hour incubation (+/- 2 minutes)One hour incubation (+/- 2 minutes) » Invert tube through 180Invert tube through 180oo - check for gelation- check for gelation
  9. 9. Gel -clot LAL TestGel -clot LAL Test • EP / USP testingEP / USP testing – More complicatedMore complicated » Positive controlsPositive controls » Negative controlsNegative controls » Endotoxin standard curve (confirm label claim)Endotoxin standard curve (confirm label claim) » Positive product controls (spiked samples)Positive product controls (spiked samples) » Semi-quantitative through two-fold dilutionsSemi-quantitative through two-fold dilutions
  10. 10. Gel -clot LAL TestGel -clot LAL Test • Advantages of the testAdvantages of the test » Easy to performEasy to perform » As a qualitative test - quick and simpleAs a qualitative test - quick and simple » InexpensiveInexpensive » Low equipment costsLow equipment costs » Good for simple products or waterGood for simple products or water » Reference method for USP / EPReference method for USP / EP
  11. 11. Gel -clot LAL TestGel -clot LAL Test • Disadvantages to the testDisadvantages to the test » Quantitation is difficultQuantitation is difficult » Fixed incubation timeFixed incubation time » InterferenceInterference » Limited ‘limit of detection’Limited ‘limit of detection’ » Margin of errorMargin of error » No automationNo automation » VibrationVibration » SubjectiveSubjective » Compliance issuesCompliance issues
  12. 12. Photometric methodsPhotometric methods • Turbidimetric and ChromogenicTurbidimetric and Chromogenic TechniquesTechniques • Many similaritiesMany similarities » Use spectrophotometryUse spectrophotometry » Use a standard curve (r = 0.980)Use a standard curve (r = 0.980) » Increased throughputIncreased throughput » Wider ranges of quantitationWider ranges of quantitation » As kinetic methods - single stepsAs kinetic methods - single steps » Reduced margins of errorReduced margins of error
  13. 13. Turbidimetric LAL TestTurbidimetric LAL Test • Principle:Principle: » Links the rate of gelation (as turbidity) to determineLinks the rate of gelation (as turbidity) to determine endotoxin contentendotoxin content » Plotting turbidity (optical density) against endotoxinPlotting turbidity (optical density) against endotoxin concentration from a series of standardsconcentration from a series of standards • End-point or kinetic methodEnd-point or kinetic method
  14. 14. Turbidimetric LAL TestTurbidimetric LAL Test • MethodMethod » Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37oo CC +/-1+/-1oo C)C) » SpectrophotometerSpectrophotometer » Computer softwareComputer software » Lysate sensitivity determined via curveLysate sensitivity determined via curve
  15. 15. Turbidimetric LAL TestTurbidimetric LAL Test • Advantages of the testAdvantages of the test » Real time measurementReal time measurement » Results can be ‘seen’Results can be ‘seen’ » Reading is automated: objectivityReading is automated: objectivity » SoftwareSoftware » Less dilutions cf Gel-clot (in-use costs)Less dilutions cf Gel-clot (in-use costs) » Over-coming interferenceOver-coming interference
  16. 16. Turbidimetric LAL TestTurbidimetric LAL Test • Disadvantages of the testDisadvantages of the test » Turbid samplesTurbid samples » Samples with precipitationSamples with precipitation » Some biologicalsSome biologicals » Equipment costEquipment cost » Vibration, bubbles and background noiseVibration, bubbles and background noise » Technician expertiseTechnician expertise
  17. 17. Chromogenic LAL testChromogenic LAL test • PrinciplePrinciple » Uses the clotting cascade, but in a modified wayUses the clotting cascade, but in a modified way » Synthetic chromogenic substrate - pNA - in theSynthetic chromogenic substrate - pNA - in the presence of LAL and endotoxin produces a yellowpresence of LAL and endotoxin produces a yellow colour. Intensity of colour = relates to amount ofcolour. Intensity of colour = relates to amount of endotoxinendotoxin • End-point or kinetic methodEnd-point or kinetic method
  18. 18. Chromogenic LAL testChromogenic LAL test • MethodMethod » Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37oo CC +/-1+/-1oo C)C) » SpectrophotometerSpectrophotometer » Computer softwareComputer software » Lysate sensitivity determined via curveLysate sensitivity determined via curve
  19. 19. Chromogenic LAL testChromogenic LAL test • Advantages of the testAdvantages of the test » Fast, real time measurementFast, real time measurement » Results can be ‘seen’Results can be ‘seen’ » Reading is automated: objectivityReading is automated: objectivity » SoftwareSoftware » Less dilutions cf Gel-clot (in-use costs)Less dilutions cf Gel-clot (in-use costs) » Over-coming interferenceOver-coming interference
  20. 20. Chromogenic LAL TestChromogenic LAL Test • Disadvantages of the testDisadvantages of the test » Coloured samplesColoured samples » Equipment and reagent costEquipment and reagent cost » Technician expertise / variabilityTechnician expertise / variability
  21. 21. ComparisonComparison • What’s similar?What’s similar? » Use the same / similar principleUse the same / similar principle » Use LALUse LAL » Use tube or micro-plateUse tube or micro-plate » Require endotoxin standardsRequire endotoxin standards » Meet Regulatory requirements - FDA, MCA, EP,Meet Regulatory requirements - FDA, MCA, EP, USP, JPUSP, JP
  22. 22. ComparisonComparison • How do they compare?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. 23. ComparisonComparison • How do they compare?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. 24. ComparisonComparison • How do I choose?How do I choose? – Gel-clot method?Gel-clot method? – Turbidimetric method?Turbidimetric method? – Chromogenic method?Chromogenic method? – End point or kinetic?End point or kinetic?
  25. 25. SummarySummary • Brief introduction to the LAL testBrief introduction to the LAL test • The three main methodsThe three main methods • Advantages and disadvantages of eachAdvantages and disadvantages of each • Comparison between themComparison between them
  26. 26. SummarySummary • Final choice…Final choice… – It’s up to you:It’s up to you: Your budgetYour budget Your application - water, raw materials, in-processYour application - water, raw materials, in-process samples or final productssamples or final products Volumes to be testedVolumes to be tested Material to be tested - turbid, coloured, precipitate,Material to be tested - turbid, coloured, precipitate, inhibitioninhibition Endotoxin limitEndotoxin limit Degree of complianceDegree of compliance
  27. 27. Thank you for your timeThank you for your time

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