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LAL: Choice of Test MethodLAL: Choice of Test Method
ByByTim SandleTim Sandle
Bio Products LaboratoryBio Products Laboratory
IntroductionIntroduction
How did we get here?How did we get here? Main test methods - Gel-Clot, Turbidimetric Main test methods - Gel-Clot, Turbidimetric
and Chromogenicand Chromogenic Advantages and disadvantages of each Advantages and disadvantages of each
methodmethod Comparison between the main methodsComparison between the main methods
Introduction - LAL TestIntroduction - LAL Test LAL Test - for performing BETLAL Test - for performing BET Alternative to Pyrogen (Rabbit) during 1980sAlternative to Pyrogen (Rabbit) during 1980s USP 1980; FDA Guide 1987; Ph. Eur. 1988; USP 1980; FDA Guide 1987; Ph. Eur. 1988;
BP 1989 (Gel-clot test)BP 1989 (Gel-clot test) To meet the requirements for the Bacterial To meet the requirements for the Bacterial
Endotoxin Test in Ph. Eur. 2.6.14 / USP Endotoxin Test in Ph. Eur. 2.6.14 / USP <85><85>
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 proteincolorimetric
X
Introduction - LAL Test Introduction - LAL Test
Different methods, Different methods, same principlessame principles» LimulusLimulus amebocyte lysate reagent from horse shoe amebocyte lysate reagent from horse shoe
crabscrabs» LAL detecting endotoxinLAL detecting endotoxin» Detection based on natural clotting mechanism Detection based on natural clotting mechanism
(Levin and Bang, 1968)(Levin and Bang, 1968)» Tests utilise the clotting cascadeTests utilise the clotting cascade
Introduction - LAL TestIntroduction - LAL Test
Different methods, Different methods, same principlessame principlesEndotoxin
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
Gel -clot LAL TestGel -clot LAL Test
PrinciplePrinciple» LAL can be purified to be of different sensitivities LAL can be purified to be of different sensitivities
so a clot = probability of a number of Endotoxin so a clot = probability of a number of Endotoxin Units (EU) in a given sampleUnits (EU) in a given sample
Limit or semi-quantitative through dilution Limit or semi-quantitative through dilution seriesseries
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 (37ooC +/- 1C +/- 1ooC)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
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
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» Inexpensive Inexpensive » 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
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
Photometric methodsPhotometric methods Turbidimetric and Chromogenic TechniquesTurbidimetric and Chromogenic Techniques 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
Turbidimetric LAL TestTurbidimetric LAL Test
Principle:Principle:» Links the rate of gelation (as turbidity) to determine Links the rate of gelation (as turbidity) to determine
endotoxin contentendotoxin content» Plotting turbidity (optical density) against endotoxin Plotting 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
Turbidimetric LAL TestTurbidimetric LAL Test
MethodMethod» Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37ooC C
+/-1+/-1ooC)C)» SpectrophotometerSpectrophotometer» Computer softwareComputer software» Lysate sensitivity determined via curveLysate sensitivity determined via curve
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
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
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 the Synthetic chromogenic substrate - pNA - in the
presence of LAL and endotoxin produces a yellow presence of LAL and endotoxin produces a yellow colour. Intensity of colour = relates to amount of colour. Intensity of colour = relates to amount of endotoxinendotoxin
End-point or kinetic methodEnd-point or kinetic method
Chromogenic LAL testChromogenic LAL test
MethodMethod» Heated plate reader or heated tube reader (37Heated plate reader or heated tube reader (37ooC C
+/-1+/-1ooC)C)» SpectrophotometerSpectrophotometer» Computer softwareComputer software» Lysate sensitivity determined via curveLysate sensitivity determined via curve
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
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
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
ComparisonComparison
How do they compare?How do they compare?Method Gel-clot Kinetic
TurbidimetricKineticChrmogenic
Typical lowerdetection limit
0.03 EU / mL 0.001 Eu / mL 0.005 EU / mL
Typical upperdetection limit
Fixed 100 EU / mL 50 EU / mL
Technicianinvolvement
High Medium Medium
Ease of use Low Medium High
ComparisonComparison
How do they compare?How do they compare?Method Gel-clot Kinetic
TurbidimetricKineticChrmogenic
Test robustness Medium Medium High
Equipment cost Low High Medium
Reagent cost Medium Medium High
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?
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
SummarySummary
Final choice…Final choice…– It’s up to you:It’s up to you:
Your budgetYour budget Your application - water, raw materials, in-process Your 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