DSPMFJO -ZTJOF EEVDU$PNQFUJUJWF &*,JU Manual/MK150_e.v1608.pdfThe kit is a competitive EIA that uses a monoclonal antibody that reacts specifically with the formyl-dehydropiperidino

Cat. # MK150

Product Manual

Acrolein-Lysine Adduct Competitive

EIA Kit

For Research Use

v201608

2 URL:http://www.takara-bio.com

Acrolein-Lysine Adduct Competitive EIA KitCat. #MK150

v201608

Table of Contents

I. Description.......................................................................................................... 3

II. Components....................................................................................................... 4

III. MaterialsRequiredbutnotProvided........................................................ 4

IV. Storage.................................................................................................................. 4

V. IntendedUse....................................................................................................... 4

VI. Principles.............................................................................................................. 5

VII. Protocol................................................................................................................. 7

VIII. Performance......................................................................................................10

IX. ExperimentalExamples................................................................................12

X. Precautions........................................................................................................15

XI. References..........................................................................................................15

XII. RelatedProducts.............................................................................................15


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3URL:http://www.takara-bio.com

I. Description Acrolein(CH2=CHCHO)isachemicalsubstancethatisproducedbyburningofpetroleum,coal,wood,andplastic,andalsobycigarettesmoke,exhaustgas,andtheheatingofcookingoil.Acroleinishighlycytotoxicandisaby-productoflipidperoxidationinthehumanbody.Thebiologicalandphysiologicaleffectsofacroleinarebeingactivelyinvestigated.1)-5)

TheAcrolein-LysineAdductCompetitiveEIAKitcanbeusedformeasurementofacrolein-proteinadductsinblood,urine,ortissuesamples.ThekitisacompetitiveEIAthatusesamonoclonalantibodythatreactsspecificallywiththeformyl-dehydropiperidino(FDP)-lysine(Lys)structuresthatarecreatedwhenacroleinbindstothelysineresiduesofproteins.QuantitativemeasurementofacroleinadductsinasampleiscalculatedusingthequantityofFDP-Lys.

Figure1.Mechanismofadductformation.(Fromreference2)

NC

N

RO

HR'

O

Nε-(3-formyl-3,4-dehydropiperidino)-lysine

N-acetyl-FDP-lysine(Acrolein adduct): R=OH R’=H

Acrolein Lysine and other amino acids,

peptides, polypeptides and

proteins



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II. Components1. AntigenCoatedMicrotiterPlate 1plate

Acrolein-LysineAdductImmobilizedplates(96wells:8wellsx12strips)

2. SampleDiluent 50mlBuffersolutionforsampledilution

3. Standard 350μlx7N-acetyl-FDP-lysine(7differentconcentrations)

4. AntiAcroleinMonoclonalAntibody(Lyophilized) For10mlPrimaryantibodyforacroleinadduct

5. AntibodyDiluent 11mlSolutionforreconstitutingtheprimaryantibody

6. WashBuffer(20X) 50mlBuffersolutionwithTween20

7. POD-LabeledAntiMouseIgGConjugate(100Xconc.) 120μlPOD-labeledmouseIgGsecondaryantibody

8. ConjugateDiluent 12mlDiluentforlabeledsecondaryantibody

9. SubstrateSolution(TMBZ) 12ml3,3',5,5'-Tetramethylbenzidinesolution

10. StopSolutionwithoutSulfuricAcid 12mlReactionstopsolution(Containsnosulfuricacid)

11. PlateSeal 2pieces

III. Materials Required but not Provided• Pipettes,micropipettes,andtips• Tubesorflasksforpreparationofbuffers• Plate-washingdevice;dispensingburetteandaspiratorformanualwashingNote: PersonalMicroplateWasher(Cat.#MK950)＊isrecommended.• Platemixer• Temperature-controlledincubator(20-30℃)• Microplatereader(capableofmeasuringabsorbanceofupto3.5whensetto450nm)

＊: Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.

IV. Storage 4℃

V. Intended Use• Detectionofacroleinadductsinenvironmentalsamples• Detectionofacroleinadductsproducedasaby-productofindustrialproductmanufacturing• Detectionofacroleinadductsinbiologicalsamples

Note: Thiskitisforresearchuseonly.Itcannotbeusedfordiagnosticpurposes.


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VI. PrinciplesThiskitisasingle-antibodycompetitiveEIA.Thequantityofantigeninthesampleismeasuredbyaddingtheprimaryantibodyinthepresenceofbothsolid-phaseantigen(acrolein-lysineadduct)andthetestsample.Anenzyme-labeledsecondaryantibodydetectsprimaryantibodyboundonthesolid-phaseplate.Whenthereisalargeamountofantigen(acroleinadduct)inthesample,themajorityoftheprimaryantibodywillbeboundinsolution,resultinginadecreaseintheamountoftheprimaryantibodyboundtothesolid-phaseantigenandaproportionaldecreaseintheabsorbance.

1. BringtheAntigenCoatedMicrotiterPlatetoroomtemperature.

2. DilutetheStandardorsampleappropriatelyandaddtotheAntigenCoatedMicrotiterPlate.

3. AddthereconstitutedAntiAcroleinMonoclonalAntibody.

Add50μlofthesampleorStandardperwell.

4. Theantibodywillreactwithacroleinadductinthesampleandboundtotheplate.(Ifthereisalargequantityofacroleinadductinthesample,lessoftheprimaryantibodywillbindtothesolidphase.)

Add50μloftheAntiAcroleinMonoclonalAntibodyperwell.

Allowtostandfor30minutesatroomtemperature(20-30℃).

NH2

Coating antigen

O

Coating antigen

N

O



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5. Washtheplatetoremoveexcessreactants.

6. AddthePOD-LabeledAntiMouseIgGConjugatetotheplate.

7. WashtheplatetoremoveunboundPOD-labeledAntiMouse,IgGConjugate.

8. AddtheSubstrateSolutionandallowcolortodevelop(blue).

9. Add100μloftheStopSolutionperwell.Thecolorofthesolutionwillchangefrombluetoyellow.Measureabsorbanceat450nm.

Wash4times(200-300μl/well).

Add100μlofthePOD-LabeledAntiMouseIgGConjugateperwell.Incubateatroomtemperature(20-30℃)for60minutes.

Wash4times(200-300μl/well).

Add100μloftheSubstrateSolution(TMBZ)perwell.Incubateatroomtemperature(20-30℃)for5-15minutestoallowthesubstratecolor-developmentreaction.

Absorbance(450nm) 2.5

2.0

0.5

1.0

0

1.5

1 10 100 1,000FDP-Lys(nmol/ml)


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VII. Protocol1. Samples

• Urine,bloodserum,bloodplasma,asciticfluid,orcellsupernatant/extracts,fromanysourcecanbeusedformeasurement.Inaddition,intermediateproductscanbemeasured.• Storesamplesat2-10℃;storefrozenifmeasurementwilltakeplace>12hoursaftersamplecollection.• Ifdilutionisnecessary,diluteusingSampleDiluent.• Whenmeasuringbiologicalspecimens(i.e.,urine),diluteatleast10timesusingSampleDiluentbeforemeasurement.• Forbloodspecimens,measurementofconcentratedspecimensmayresultinpoorlinearity.Preliminarytestingofthedilutionratioisnecessarywhenmeasuringbloodsamples.• ThePOD-labeledsecondaryantibodyisananti-mouseIgGconjugate.Whenmeasuringmousebloodsamples,non-specificbackgroundmaybeobtained.Therefore,itisnecessarytodiluteatleast10timeswithSampleDiluent,andtoperformrelativeevaluationusingsamplesfromacontrolgroup.• Becausethismeasurementwiththiskitreliesonanantigen/antibodyreaction,thepHofthereactionsystemshouldbemaintainedwithinaneutralrange.Inaddition,proteindenaturantsmayinhibitthereaction.• Impuritiesandturbidityinthesampleshouldberemovedbeforemeasurementbycentrifugation(3,000rpm,10minutes)orfiltration.• Hemolyzedbloodserummayaffecttheresults.

2. Reagent Preparation• AntigenCoatedMicrotiterplatePriortouse,bringtoroomtemperatureandopen.ThePlateSealmaybeusedtopreventdrying.

• PrimaryAntibodySolutionAdd10mlofAntibodyDiluenttoAntiAcroleinMonoclonalAntibody(Lyophilized)anddissolvecompletelyforuse.Thereagentisstablefor2weeksat4℃afterpreparation.Roughly5.5mlofsolutionisneededperplate.

• POD-LabeledSecondaryAntibodySolutionDilutePOD-LabeledAntiMouseIgGConjugate(100Xconc.)100timeswithConjugateDiluent.11mlofthesolutionisneededperplate.DilutedPOD-labeledsecondaryantibodysolutionshouldbeusedwithin24hoursafterpreparation.

• WashSolutionDilutetheWashBuffer(20X)withdistilledwatertoobtaina1Xsolution.TheresultingsolutionwillbePBSwith0.05%Tween20.

• N-acetyl-FDP-lysineStandardSolution(Standard)Theseconsistofsevenconcentrationsstandards(200,100,50,25,12.6,6.25,and3.13nmol/ml),andcanbeusedasis.Toavoidlossduetonon-specificadsorption,donottransfertoadifferenttubeorcontainer.UseSampleDiluentforblankmeasurements.Storeat4℃protectedfromlightintheglasscontainerprovided.



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• SubstrateSolution(TMBZ)Bringtoroomtemperaturebeforeuse.Priortouse,confirmthattheSubstrateSolutionhasnotturneddarkblue.Avoidmixingwithtapwater,asreactionwithmetalionsmaychangethecolor.Whenonlyusingapartialamount,aliquotthenecessaryquantity.

• StopSolutionwithoutSulfuricAcidThisisaperoxidasereactionstopsolutionthatcontainsnosulfuricacid.Becausethisisahighly-concentratedsolution,mixwellusingaplatemixerafteraddingtotheplate.

3. ProcedureAssaysamplesinduplicate.Allowreagentsinthekitandsamplestoreturntoroomtemperatureandmixallsolutionsuniformlywithoutcreatingbubblesbeforeuse.

(1) Useaseparate96wellplatetopreparedilutionsofthesampleandadd50μltoeachwelloftheAntigenCoatedMicrotiterplateusingan8-channelpipette.Also,directlyadd50μlofeachconcentrationofStandardintotheplatefromthespecializedglasscontainers(avoidtransferringthestandardtodifferentplates/tubestoavoidadsorption).Thenadd50μlofAntiAcroleinMonoclonalAntibodysolutiontoeachwell.Completeadditionoftheprimaryantibodysolutionwithin5minutesusingequipmentsuchasacontinuousdispensingburetteoran8-channelpipette.Lightlyagitatetheentireplatetoensurethatthesolutionuniformlymixed.Sealtheplateandallowtostandfor30minutesatroomtemperature(20-30℃).Performthereactionatroomtemperature(20-30℃).Incubationat37℃maycompromiseantigenicity.[Firstreaction]

(2) Discardthereactionsolution,andwash4timeswiththeWashSolution(PBSwith0.05%Tween20).Thenadd100μlofthePOD-LabeledAntiMouseIgGConjugatetoeachwellusingan8-channelpipette,sealtheplate,andallowtostandatroomtemperature(20-30℃)for60minutes.[Secondreaction]

(3) Discardthereactionsolution,wash4timeswiththeWashSolution(PBSwith0.05%Tween20),andcompletelyremovethealloftheliquid.Then,add100μloftheSubstrateSolution(TMBZ)toeachwellusingan8-channelpipetteandallowtostandatroomtemperature(20-30℃)forapproximately5-15minutes.[Thirdreaction]

(4) Add100μlofStopSolutiontoeachwellinthesameorderthattheSubstrateSolution(TMBZ)wasadded,andagitatewellusingaplatemixerafterthereactionhasbeenstopped.

(5)Measureabsorbanceatawavelengthof450nm. Thecolorisstableforatleast6hoursafterthereactionisstopped.

(6) PrepareastandardcurvebyplottingtheconcentrationofeachStandardonthex-axisandthecorrespondingabsorbanceonthey-axis.Usetheabsorbanceofthespecimentocalculatethecorrespondingconcentrationofacroleinadduct.


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Unknown sample: Pretreatment and dilution

Unknown sample:50 μl/well

Wash solution

Dilute 20 times with distilled water

Wash solution stock

Antigen-coated plate

Primary reaction: Room temperature, 30 min

Wash 4 times

Secondary reaction: Room temperature, 60 min

Wash 4 times

Color-development reaction: Room temperature, 15 min

Stop reaction

Measure absorbance at 450 nm

Prepare standard curve; quantitative determination

Reconstitutionof antibody solution

100X dilution

Standard solution:50 μl/well

Primary antibody solution:50 μl/well

POD-labeled secondaryantibody solution:100 μl/well

Substrate solution: 100 μl/well

Stop solution:100 μl/well

<Workflow>



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VIII. Performance1．Standard Curve (Acrolein-Lysine Adduct Competitive EIA Kit)

Thefollowingisatypicalstandardcurve.Prepareastandardcurveforeachexperiment.

RangeofMeasurement: 3.13-200nmol/mlRangeofConfidence: 3.13-100nmol/mlLimitofDetection: 3.13nmol/ml

(CurveFit:4-Parameter)

N-acetyl-FDP-Lysine(nmol/ml) 200.0 100.0 50.0 25.0 12.5 6.25 3.13

A450 0.120 0.160 0.210 0.401 0.679 1.375 2.083

Colordevelopment:15minutes

1 10 100 10000

0.5

1

1.5

2

4-P Fit: y = (A - D)/( 1 + (x/C)B ) + D : A B C D R22.99 1.53 5.2 0.125 0.999

StandardCurve

MeanValue

Conc


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(CurveFit:Log-Log)

1 10 1000.1

1

10

Log-Log Fit: Log(y) = A + B * Log(x) : A B R20.713 -0.783 0.99

StandardCurve

MeanValue

Conc

N-acetyl-FDP-Lysine(nmol/ml) 100.0 50.0 25.0 12.5 6.25 3.13 0.0

A450 0.160 0.210 0.401 0.679 1.375 2.083 2.303

2．Reproducibility

<Intra-assayprecisiontest>ReproducibilitytestingwasperformedwiththreeconcentrationsofacontrolcontainingN-acetyl-FDP-Lysine.

Sample(n=4) Mean(nmol/ml) SD CV(%)

ControlA 10.491 0.472 4.5ControlB 7.367 0.470 6.4ControlC 5.777 0.401 6.9

<Inter-assayprecisiontest(n=3)>Threeconcentrationsofacontrolwereassayedonthreeseparatedays.

Sample Mean（nmol/ml） SD CV(%)

ControlD 10.457 0.842 8.1ControlE 7.349 0.431 5.9ControlF 5.226 0.395 7.6



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<SpikeandRecoveryTest>Equalamountsofvariousconcentrationsofacrolein-containingsamplesweremixedandtherecoveryratewascalculatedbycomparingtheanticipatedtheoreticalvaluewiththeactualmeasurement.

SampleA SampleBTheoreticalValue(A+B)/2

AssayResult RecoveryRate(％)

11.471 11.471 11.471 7.874 68.611.471 7.868 9.670 7.860 81.311.471 5.028 8.250 7.515 91.111.471 1.637 6.554 7.254 110.77.868 5.028 6.448 5.911 91.77.868 1.637 4.753 4.570 96.212.500 6.250 9.375 8.444 90.16.250 3.130 4.690 5.141 109.6

Units:nmol/ml

IX. Experimental Examples1. Dilution Linearity of Biological SamplesAcrolein-lysineadductsweremeasuredinurineandstool(waterextracted)samplesfrom8-9weekoldmice(BALB/candC57BL6).Forstoolsamples,500μlofSampleDiluentand1standard-sizedfecalpelletwerehomogenizedwiththeTaKaRaBioMasherStandard(Sterile)(Cat.#9791).Thesampleswerecentrifugedandthesupernatantwasusedforanalysis.Forurinesamples,spoturinewasused.Whenperformingcomparativeevaluationbetweenindividuals,itisadvisabletousepooledurinefromametaboliccage.Dilutionbyatleast10-20foldisnecessarytoobtainadilutioncurvefromurinesamples.

MeasuredConcentrationofAcrolein-LysineAdducts

MouseStrain SampleDilutionRatio

5X 10X 20X 40X 80XBALB/cNo.1 Urine over over 172.7 66.5 30.5BALB/cNo.2 Urine over over 182.5 56.6 26.3BALB/cNo.3 Urine over over 149.6 55.1 27.6C57BL6No.1 Urine over over over 84.3 35.8C57BL6No.2 Urine over over 150.1 47.8 20.5

C57BL6No.3StoolWaterExtracted

16.8 7.7 4.3 2.8 1.0

Units:nmol/ml


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<BALB/cNo.1-3,Urine>

200

180

160

140

100

120

60

80

40

0.01 0.02 0.03 0.04 0.05 0.0600

Measurement(nmol/ml)

No.1

No.3No.2

Measurement(nmol/ml)

<C57BL6No.3,Stool>

20

Dilution

Dilution

0.05 0.1 0.15 0.2 0.250

1816

1412

810

46

2

0



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2. Relative Measurement of Urine ProteinAcroleinandIgGweremeasuredintheurinefromnormal(female)mice.Kits:Acrolein-LysineAdductCompetitiveEIAKit(Cat.#MK150)andMouseIgGEIAKit(Cat.#MK137;discontinued)Samples:Spoturinefrom8-9weekoldmice(BALB/candC57BL6strains)Foracrolein,40Xsampledilutionwasused.FormouseIgG,20Xsampledilutionwasused.

UrineAcrolein(nmol/ml)

UrineIgG(ng/ml)

Acrolein/IgG（nmol/ng）

MouseStrain Sample 40X

ValueConvertedforUndilutedSolution

20X

ValueConvertedforUndilutedSolution

per1ml

BALB/cNo.1 Urine 66.5 2,661.9 62.0 1,239.6 2.1BALB/cNo.2 Urine 56.6 2,262.2 50.4 1,007.3 2.2BALB/cNo.3 Urine 55.1 2,203.4 44.2 884.0 2.5C57BL6No.1 Urine 84.3 3,372.8 122.5 2,450.3 1.4C57BL6No.2 Urine 47.8 1,913.0 100.9 2,017.2 0.9

Results: Comparativeevaluationwithaproteinintheurinecanbeusedtonormalizeacroleinmeasurements.Albumins,etc.maybeusedfornormalization.

3. Measurement from Tissue ExtractsExtractspreparedfromvariousnormalmousetissueswerepreparedbysolubilizingwithlysisbufferusingaTaKaRaBioMasherStandard(Sterile)homogenizer.Measurementwasperformedonserialdilutions(10,20,40,and80-folddilutions).Thevaluemeasuredforthe10-folddilutionwasused,andvalueswerenormalizedtothevalueoflysisbufferalone.SamplesB,C,andDwereisolatedsimultaneouslyfromthesameindividual.

Sample Tissue WetWeight

BufferVolume

MeasuredValue(10X)(nmol/ml)

StockSolutionConvertedValue

(nmol/ml)

Acrolein-LysineAdductpermgofTissue(nmol/mg)

A ICRMouseLiver 420mg 1ml 10.6 71 0.169

B BALB/cMouseSpleen 40mg 1ml 4.37 9 0.225

C BALB/cMouseBrain 250mg 1ml 7.75 43 0.172

D BALB/cMouseKidney 50mg 1ml 5.5 20 0.400

E LysisBuffer － 1ml 3.5 0 －

Result: Acroleinadductaccumulationwasobservedinkidneytissue. Thissuggeststhepossibilitythatacroleinisexcretedintheurine.


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X. Precautions1. Donotmixreagentsorsolutionsfromkitswithdifferentlotnumbers.2. Donotexposereagentstostronglightduringstorageorreaction.3. Thepipettes,etc.usedforthesubstratesolution(TMBZ)andstopsolutionshouldnotcontainmetal.

4. Preventthesubstratesolution(TMBZ)andstopsolutionfromcomingintocontactwithhandsandmucousmembranes.

5. Donotusesubstratesolution(TMBZ)thathaschangedcolor.6. Eachreactioncanbeaffectedbytimeandtemperature;prepareastandardcurveforeachexperiment.

7. Handlebloodspecimenswithsufficientcare.

XI. References1) K.Uchida.Productionofacroleinbyproteinperoxidationreactionsandproteinmodification.JournalofOleoScience. (1998)47:1207-1215.

2) K.Uchida,etal .Acroleinisaproductoflipidperoxidation:formationoffreeacroleinanditsconjugatewithlysineresiduesinoxidativelowdensitylipoproteins.JBiolChem.(1998)273:16058-16066.

3) K.Uchida,etal .Protein-boundacrolein:Potentialmarkersforoxidativestress.ProcNatlAcadSci USA. (1998)95:4882-4887.

4) N.Y.Calingasar,etal .Protein-boundacrolein:AnovelmarkerofoxidativestressinAlzheimer’ sdisease.JNeurochem.(1999)72:751-756.

5) K.Sato,etal .Aone-hourELISAforquantitationofAcroleinandhydroxynonenal-Modifiedproteinsbyepitope-boundcaseinmatrixmethod.AnalBiochem.(1999)270:323-328.

XII. Related ProductsTaKaRaBioMasherStandard(Sterile)(Cat.#9791)WashandStopSolutionforELISAwithoutSulfuricAcid(Cat.#MK021)PersonalMicroplateWasher(Cat.#MK950)＊

ELISAkitsthatmeasureurineproteins:FibronectinEIAKit(Cat.#MK115)Laminin(LN)EIAKit(Cat.#MK107)E-cadherinEIAKit(Cat.#MK117)HumanAlbuminEIAKit(Cat.#MK132)HumanIgGEIAKit(Cat.#MK136)

＊ :Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.



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NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTAKARABIOINC.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656973orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

Documents

DSPMFJO -ZTJOF EEVDU$PNQFUJUJWF &*,JU Manual/MK150_e.v1608.pdfThe kit is a competitive EIA that uses a monoclonal antibody that reacts specifically with the formyl-dehydropiperidino