Lal presentation
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Overview of bacterial endotoxin testing methods (LAL)
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Lal presentation
- 1. LAL: Choice of Test MethodLAL: Choice of Test Method ByBy Tim SandleTim Sandle Bio Products LaboratoryBio Products Laboratory
- 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Thank you for your timeThank you for your time
Hinweis der Redaktion
- Who am I? Tim Sandle Manager of Microbiology at Bio Products Laboratory (BPL) BPL is the NHS’ manufacturer of blood products - derived from human plasma - for England and Wales I have a long history of using the LAL test, starting in the 1980s
- This is what we will be covering Please can questions be saved to the end or until the discussion period Because time is limited I can only touch on this subject. This talk won’t conclude with a recommendation of which method - it is only guidance I won’t be covering regulatory issues or future directions of the test (it’s possible that five years from now we won’t be using LAL at all to detect endotoxins)
- By LAL test - methods designed to meet the Bacterial Endotoxin Test monographs in USP, EP and JP Methods which detect the LPS component from cell wall of Gram negative bacteria, where a one millionth of a gram can be detected (using 0.03 sensitivity lysate) LAL test emerged as an alternative to the rabbit pyrogen test for the pharamceutical industury in the 1980s (although used to test medical devices in 1970s) However, work began on LAL reactions in the 1960s LAL is far more sensitive than pyrogen test - 1000s of fold. Today 0.001 EU can be detected using the photometric methods, which is aprox. Total endotoxin extracted from 10 E.coli bacteria The first method to appear in the pharmacopeias was the gel-clot - others appeared later - and with most pharmacopeial changes took a while to gain acceptance
- I will covering the gel-clot, kinetic turbdimetric and kinetic chromogenic. Although I won’t be specifically covering the end-point versions of the two “photometric” methods a lot of what I will say will apply An end-point method = read at a fixed time interval kinetic method = read continuously (collecting data points every so many seconds). Advantage over end point is no set period of incubation (technician terminates the assay) and reduces what are three step processes into a single step assay
- The three main methods work on the same principle The use the same reagent - LAL LAL is extracted from the horse shoe crab - mainly Limulus polyphemus (although Tachypleus and Carcinoscorpius are sometimes used world wide) It is a lysate extracted from the crab’s amoeboyctes The lysate detects endotoxin produced from Gram negative bacteria Detection is based on the natural clotting mechanism of the crab; part of defence against bacteria and fungi (because it will reacr against other things other than endotoxin) Based on Levin and Bang’s research
- The LAL test takes the clotting cascade by utilising the fact that the crab’s amoeboyctes contain various proteins, co-factors and ions which all interact to initiate coagulation This talk isn’t designed to go into detail here - but as all the methods use this clotting cascade (some in a more natural way than others) it is worth pausing Endotoxin catalyses the activation of coagulase which hydrolyses bonds within a clotting protein called coagulogen to form hydrolysed coagulin This forms an insouluble, gelatinous clot
- LAL can be purified to a particular sensitivity so that the reaction - gel formation - equates to a particular value of endotoxin. Therefore gel clot lysate is often available as 0.25, 0.125, 0.06 and at its most sensitive 0.03 EU Therefore a clot at 0.03 lysate = probability of 0.03 EU or greater in a sample - say probability because of margin of error in the test
- Tube method is the accepted method re: regulatory compliance Low level of equipment Hot block is better than water bath - stability, vibration, evaporation Tube is typically 10 x 75 mm; soda lime glass - equal volume of lysate and sample Fixed temperature and incubation time
- To move beyond the limit test, by making two-fold dilutions the test can become semi-quantitative Can become semi-quantitative through a dilution series (or ‘titration’ according to EP) e.g.using 0.03 sensitive lysate a neat sample gives a clot but a 1/2 dilution gives no clot, the result is somewhere between 0.03 and 0.06 (or 0.03125 and 0.0625 to be precise). Calculations using geometric means
- Easy to perform once trained Qualitative - pass or fail = quick and simple for product release Low equipment cost Regulatory / reference test advantage is less of an issue these days - now more understand the other methods / other methods more wide spread
- Although it is semi-quantitative - this needs lots of dilutions for a sample with high level of endotoxin or need to over come interference; a lot when doing it in duplicate or quadruplicate! Fixed incubation time - other methods are quicker; gives no idea of what is happening until final read e.g. test controls may fail Interference - at every step of the clotting cascade from various factors like pH; alcohols…..many pharmaceutical products are increasingly complex Limited sensitivity - the test dilution to overcome interference might be so great that the result looses meaning or conflicts with MVD….also less good for trending Margin of error is high - any result can be with one-two fold dilution of the one obtained. E.g. test at 1/8th dilution and get a gel, result could be at 1/4 or 1/16th Subjective: open to operator interpretation Very reliant on paper records etc. for compliance / auditing…no print-outs etc.
- Called photometric because they use spectrophotometers and measure threshold of absorbance The kinetic methods use a standard curve of endotoxin concentrations, from which logs are taken and it is made linear and can be used to measure the endotoxin content of samples if it has a correlation coefficient of 0.980 or greater Margin of error is about 50% (down from either side of a two-fold dilution for gel-clot)
- Uses same basic reaction as the gel-clot Called a photometric method in EP Instead of going for final gelation it looks at the rate of gelation - as turbidity The test - using appropriate software - plots the rate / time taken to reach a level of turbidity against a series of endotoxin standards When an unknown is run, the time taken for it to become turbid can be measured against the onset times of a standard series and the endotoxin concentration extrapolated from the curve. Kinetic method is by far the most common these days
- Tube reader or plate reader = depends on personal preference / manufacturer used Spectrophotometer measures (typically at 340 nm) the rate of reaction in milli-absorbance units and the time taken to reach a particular onset time. Once the onset time threshold is crossed the amount of endotoxin can be measured Computer software performs all the calculations - construction of a standard curve; converting the curve into logs; measuring Sensitivity of the lysate is determined by the standard curve used - typically 100 EU to 0.001 EU (much more sensitive than the gel-clot)
- Results can be seen - if a sample is going to fail or controls are not working, action can be taken reading is automated = removes subjectivity Software allows all sorts of calculations and trending Good for compliance etc Less prone to vibration Allows greater dilutions - overcoming interference Can be less expensive as an in use method than gel-clot - once validation - because less dilutions involved
- Difficult to test samples that a re turbid or have a degree of ppt without large dilutions - then comes the MVD issue again Equipment set up is expensive Prone to vibration - bubbles in tubes can mess up tests Arguably need more skilled technicians in preparation….although some would argue that the automated part can be used to deskill labs!
- The second of the photometric methods Uses pNA (para-nitroaniline). pNA is a colourless peptide but becomes yellow when dia-associated. It is artificially introduced into the cascade reaction by substituting the synthetic pNA substrate from the enzymatic reaction to work on. If endotoxin is present the yellow colour is produced The intensity of the yellow colour is linearly related to endotoxin concentration because the quantity released is directly proportional to the amount of endotoxin released during the cascade reaction An azo method was developed (with a purple colour) but this is less common place
- Similar in equipment to the turbidimetric (now some machines are available that do both) Tube reader or plate reader = depends on personal preference / manufacturer used Spectrophotometer at 405 nm measuring the rate of colour development at a pre-determined level of absorbance Computer software performs all the calculations - construction of a standard curve; converting the curve into logs; measuring Sensitivity of the lysate is determined by the standard curve used - typically 50 EU to 0.005 EU (again much more sensitive than the gel-clot)
- Advantages similar to the turbidimetric Arguably a faster method Same visual advantages - track results on computer, removal of subjectivity, software advanatages Allows greater dilutions - overcoming interference; can test turbid samples
- Difficult to test samples that a coloured without dilutions - then comes the MVD issue again Equipment set up is expensive / the reagent cost is generally higher Arguably needs the highest level of skill from technicians out of the three methods considered
- All use or are based on the clotting cascade reaction for the horse shoe crab All use purified lysate from the crab - as a lyophilised reagent Use tube or plate reader Need endotoxin standards derived from internationally accepted Reference Standard of E.coli Importantly - meet the expectations of Regulatory Authorities
- This table and the next one is a little subjective, especially ease of use and robustness The different manufacturers here today can discuss with you equipment and reagent cost!
- This is where you won’t get any easy answers! Gel-clot - simple, easy to use; if you want a limit test and aren’t too worried about trending; OK with lots of a paper work. It is still the most widely used method in the world! But….if you need to test more complicate products; want trending; want to demonstrate compliance (spike recoveries / remove subjectivity etc) etc……consider the photometric methods But….they are easier to apply these days by people who understand assays…so the microbiologist needs to meet with the biochemist! Much of the turbidimetric vs. chromogenic debate is down to personal choice But…if you are testing lots of water or non-tubrid samples…consider turbidimetric If you are testing a lot of final prodicts or have samples with turbidity or have slight ppt consider chromogenic The photometric kinetic methods are generally easier than their end-point versions re: less manipulations